Mutation profiling in differential diagnosis between TdT-positive high grade/large B-cell lymphoma and B-lymphoblastic leukaemia/lymphoma

Mutation profiling in differential diagnosis between TdT-positive high grade/large B-cell lymphoma and B-lymphoblastic leukaemia/lymphoma | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Mutation profiling in differential diagnosis between TdT-positive high grade/large B-cell lymphoma and B-lymphoblastic leukaemia/lymphoma Ming-Qing Du, Maria-Myrsini Tzioni, Francesco Cucco, Lívia Rásó-Barnett, and 24 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6172652/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract TdT is occasionally expressed in large B-cell lymphoma (LBCL), and this causes difficulty in differential diagnosis from B-lymphoblastic leukaemia/lymphomas (B-ALL/LBL). We reviewed 31 cases of TdT-positive LBCL and B-ALL/LBL, and their final diagnosis included 19 diffuse large/high-grade BCL with MYC and BCL2 rearrangements (DLBCL/HGBCL- MYC / BCL2 ), 3 DLBCL-NOS, 3 HGBCL-NOS, 4 B-ALL/LBL and 2 unclassifiable cases. TdT was variably expressed in all these cases, without any clear demarcation among different groups. Loss or partial loss of CD20 expression was seen in 13/17 DLBCL/HGBCL- MYC / BCL2 , 2/3 HGBCL-NOS, 2/2 unclassified, albeit not in DLBCL-NOS. Expression of BCL6 and/or MUM1 was seen in 3/4 B-ALL/LBL and 2/2 unclassified. Next generation sequencing revealed characteristic mutations associated with follicular lymphoma and its high-grade transformation in each DLBCL/HGBCL- MYC / BCL2 , and also frequent variants in genes targeted by somatic hypermutation (SHM) in almost all DLBCL/HGBCL- MYC / BCL2 , DLBCL-NOS and HGBCL-NOS but one case. In contrast, such mutations were absent in B-ALL/LBL. There were no pathognomonic mutations in the two unclassifiable cases although one showed a moderate level of somatic mutations in its rearranged IGHV . In conclusion, mutation profiling analysis including the SHM target genes is highly valuable in differential diagnosis between TdT-positive LBCL and B-ALL/LBL. Health sciences/Medical research/Translational research Health sciences/Pathogenesis/Clinical genetics/Cancer genetics Health sciences/Diseases/Cancer/Haematological cancer/Lymphoma/Non-hodgkin lymphoma/B-cell lymphoma TdT expression High grade B-cell lymphoma B-lymphoblastic leukaemia/lymphoma somatic hypermutation differential diagnosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 INTRODUCTION Immunophenotyping is pivotal in the diagnosis and classification of haematolymphoid malignancies. By comparing the immunophenotype of malignant cells with those of normal lymphoid cells at various stages of their development and maturation, it is possible to determine the tumour cell-of-origin, hence enabling its diagnosis. However, some of the immunophenotypic markers may be expressed aberrantly in lymphoma cells, and this may cause challenges in their application to lymphoma diagnosis. One of such immunophenotypic markers is terminal deoxynucleotidyl transferase (TdT). TdT is typically expressed in pro- and pre-B cells in the bone marrow, but downregulated and not expressed in mature B cells in peripheral lymphoid tissues. Thus, TdT is regarded as a marker of precursor B-cells and their derived leukaemias and lymphomas, i.e. B-lymphoblastic leukaemias/lymphomas (B-ALL/LBL). However, TdT is also expressed in rare cases of diffuse large B-cell lymphoma (DLBCL) with dual MYC and BCL2 rearrangements ( 1 ). In the revised 4th edition of the World Health Organization Classification of Haematolymphoid Tumours ( 2 ), rare TdT-positive B-cell lymphoma occurring with a history of follicular lymphoma (FL) and both MYC and BCL2 translocations have been designated as “lymphoblastic transformation”. As these cases are CD34 negative, often bear a mature B-cell phenotype and show a mutation profile similar to those of transformed FL ( 3 – 6 ). These findings together with their exclusive occurrence in adults support their origin from mature B-cells with TdT expression being aberrant. Accordingly, these cases are now designated as a subtype of diffuse large / high-grade B-cell lymphoma with MYC / BCL2 rearrangement (DLBCL/HGBCL- MYC / BCL2 , or DLBCL/HGBCL- MYC / BCL2 / BCL6 when presence of additional BCL6 rearrangement) in the 5th edition of the World Health Organization Classification of Haematolymphoid Tumours (WHO-HAEM5) ( 7 ). In general, DLBCL/HGBCL- MYC / BCL2 can be readily diagnosed by combining histopathological/immunophenotypic assessment with interphase FISH analysis for these translocations. In addition, DLBCL/HGBCL- MYC / BCL2 bears characteristic mutation signatures including pathogenic changes associated with classic FL as well as those associated with high grade transformation, serving as a robust genetic basis for differential diagnosis ( 8 – 12 ). However, aberrant TdT expression may also be seen in DLBCL/HGBCL without MYC and BCL2 translocation ( 1 ), and such cases may also bear features of immaturity such as loss of CD20, BCL6 and/or immunoglobulin light chain expression, posing a real challenge in their differential diagnosis from B-ALL/LBL due to a lack of pathognomonic genetic changes in the former ( 13 ). On the other hand, B-ALL/LBL, including rare cases with MYC and MYC/BCL2 translocations, occur commonly in children ( 13 ), but are also seen in young adults, thus overlapping with the age range of patients with DLBCL/HGBCL ( 14 – 16 ). Given the distinct cell of origins between TdT-positive DLBCL/HGBCL and B-ALL/LBL and potential differences in their mutation profiles, we investigated somatic mutations in both rearranged IGHV genes and lymphoma genes (n = 187) by targeted next generation sequencing and performed integrated analysis to explore their utility in differential diagnosis. We also investigated the evolutionary history of TdT-positive transformed FL in cases where biopsies from paired metachronous lymphomas were available. MATERIALS AND METHODS Case materials The study was performed in accordance with local ethical guidelines for the research use of tissue materials with the approval of the ethics committees of the involved institutions (05-Q1604-10). A total of 31 cases of TdT-positive aggressive B-cell lymphoma were retrieved from the authors’ institutions, and their histological diagnosis was reviewed with a final diagnosis assigned according to WHO-HAEM5. Interphase fluorescence in situ hybridisation (FISH) MYC , BCL2 and BCL6 translocation status was available in the majority of cases from routine haematopathological diagnosis. Any missing translocation data were completed retrospectively together with further interphase FISH with MYC / IGH (Abbott), MYC / IGK and MYC / IGL (Cytocell) dual fusion probes. Immunohistochemistry Majority of the immunophenotypic data were available from routine haematopathological diagnosis, and any missing data on MYC and CD34 were collected retrospectively under the same conditions as routine histological diagnosis (Table S1 ). Additional TdT immunocytochemistry was performed on formalin-fixed paraffin-embedded cell clots of DLBCL cell lines (both MYC and BCL2 translocation positive: OCI-Ly4, OCI-LY18, Su-DHL6, SC1, DB; MYC translocation positive: RIVA; MYC translocation negative: OCI-Ly3, OCI-Ly10, Karpas-422, Su-DHL2) and the Burkitt lymphoma cell line BJAB. DNA extraction and quality assessment Histology was reviewed and the areas containing confluent lymphoma cells (> 40%) in each tissue specimen were microdissected on consecutive tissue sections. DNA was extracted using the QIAamp DNA Micro Kit (QIAGEN, Crawly, UK), quantified with a Qubit® Fluorometer (Life Technologies, Carlsbad, CA, USA) and assessed for quality by PCR ( 17 ). Mutation analysis by targeted next generation sequencing This was carried out using a customised panel of 187 genes that are recurrently mutated in FL and DLBCL/HGBCL (Table S2 ). Apart from TdT-positive DLBCL/HGBCL, four cases of TdT-positive B-ALL/LBL were included for comparative analysis to depict their difference in mutation pattern among the panel genes investigated. A total of 80-200ng of FFPE tissue derived DNA was fragmented using the Covaris E220 Focused Ultrasonicator (Covaris, Brighton, UK). For each DNA sample, an indexed library was prepared with the xGen™ UDI-UMI indexes (IDT, Coralville, IA, USA) using the TWIST protocol, and then pooled for target enrichment using the TWIST probes (TWIST Biosciences, South San Francisco, CA, USA). The enriched DNA targets were amplified by PCR and pooled libraries were sequenced using the Illumina NextSeq 2000 platform (2x100bp paired-end sequencing protocol). The sequence data analysis, variant calling, and filtering were performed as described in our previous studies ( 8 , 17 , 18 ). For DNA samples with suboptimal quality (PCR amplification of genomic fragments ≤ 300bp), targeted sequencing was performed in duplicates and only variants detected in both replicates were considered as a true alteration (Figure S1 ). Clonality analysis of the rearranged immunoglobulin heavy chain genes (IGH) This was performed by adopting the BIOMED-2 assays, followed by NGS using 2x250bp paired-end sequencing protocol (Illumina NovaSeq X sequencer) (Supplementary materials and methods, Table S3 ). Statistical analysis The comparison of mutation load among different groups was assessed using Wilcoxon Rank-Sum test with two-sided P values. RESULTS Histopathological features and their value in differential diagnosis For each case, the histological diagnosis was reviewed and where possible a final diagnosis was made according to WHO-HAEM5. The final diagnosis included 5 DLBCL- MYC / BCL2 (including 1 with additional BCL6 rearrangement), 14 HGBCL- MYC / BCL2 (including 1 with additional BCL6 rearrangement), 3 DLBCL-NOS, 3 HGBCL-NOS, 4 B-ALL/LBL and 2 cases unclassifiable due to equal evidence for both immature (strong TdT expression with partial loss of both CD20 and CD79a expression) and mature (expression of both BCL6 and MUM1 together with no CD34 expression) immunophenotype (Table 1, Fig. 1 ). Among the 19 cases of DLBCL/HGBCL- MYC / BCL2 , 11 patients were male and 8 were female, aged between 34–79 years (median: 63 years). TdT positivity by immunohistochemistry varied considerably, ranging from scattered positive cells to diffuse positivity in variable proportions of lymphoma cells ( 70% in 5 cases), while CD34 was negative in each of the 19 cases examined. CD10 was positive in 16 of the 17 cases where data were available. CD20 was negative in 5, partially positive in 8 and diffusely positive in 4 cases among the 17 cases where data were available. Among the 3 cases of DLBCL-NOS, 2 patients were female (58 & 69 years) with one carrying IGH :: BCL2 and the other harbouring IGH :: MYC and BCL6 translocation, while the third patient was a male (89 years). TdT was positive in 20–90% of the tumour cells, while CD34 was negative in the 2 cases examined. CD10 was positive in all three cases. None of these cases showed any loss of CD20 by immunohistochemistry. The 3 cases of HGBCL-NOS were from male patients, aged between 77–81 years, and each carried an isolated MYC translocation. TdT was positive in 50–90% of tumour cells, while CD34 was negative in all 3 cases. CD10 was diffusely positive in two, and partially positive in the third case. Two cases showed a partial loss of CD20 expression, while the third case was CD20 positive. The 4 cases of B-ALL/LBL were from one child (4 years, female) and 3 adults (63–80 years, 1 male & 2 female). The paediatric case carried an IGL :: MYC translocation, and was TdT-positive in 40% of the tumour cells, but CD34 negative. The three adult cases lacked MYC translocation, two showed diffuse positivity for both TdT and CD34, and the remaining case was TdT positive in ~ 80% of tumour cells but lacked any CD34 expression. All four cases were CD10 positive. Two cases (the paediatric case, and one adult case) showed partial staining for CD20 but diffuse positivity for CD79a, with the paediatric case expressing MUM1 in 40% tumour cells. However, the remaining two cases displayed diffuse positivity for CD20, but variable CD79a expression (variable intensity in 1 case and partial expression in the other), and also expressed MUM1 in majority of the tumour cells. Lastly, in 2 cases, the final diagnosis remained as unclassifiable between B-ALL/LBL and HGBCL-NOS despite careful histological review due to equal evidence for both immature and mature immunophenotypic features. One patient was a 74-year-old male and the other a 70-year-old female. Neither of them carried a MYC , BCL2 or BCL6 translocation. One case showed diffuse TdT positivity (Fig. 2 ), while the other displayed TdT expression in 50% of tumour cells (Figure S2 ). Both were CD34 negative, and CD10 positive. One case showed variable staining of CD20 and CD79a in 90% tumour cells, and was positive for BCL6 (weak) and MUM1 (strong) (Fig. 2 ). The other case displayed partial expression of CD20 (~ 30%) and CD79a (90%, variable intensity) and diffuse positivity for BCL6, and focal positivity for MUM1 (Figure S2 ). Genetic features and their value in differential diagnosis Comparative analysis among various TdT-positive DLBCL/HGBCL, together with B-ALL/LBL as a reference, revealed distinct mutation features (Fig. 3 A, Figure S3 , Table S4 ). The TdT-positive DLBCL/HGBCL- MYC / BCL2 group was characterised by the mutation signature associated with FL ( BCL2 , KMT2D , CREBBP , TNFRSF14 ), and those associated with high grade transformation ( MYC , TP53 , CCND3 , PIM1 , B2M , DDX3X , S1PR2 ). These cases also showed frequent mutations in the genes known or predicted to be the targets of the somatic hypermutation (SHM) machinery, including BCL2 and MYC when involved in translocation( 8 , 19 , 20 ) (Fig. 3 A). In addition, 12 of the 13 cases of DLBCL/HGBCL- MYC / BCL2 successfully investigated showed frequent somatic mutations in their rearranged immunoglobulin heavy chain variable genes ( IGHV ) (Fig. 3 A, Table S5 ). These genetic changes are consistent with the GC B-cell origin of DLBCL/HGBCL- MYC / BCL2 . In contrast, none of the B-ALL/LBL cases showed these mutational features, but displayed TP53 , KRAS and PAX5 mutations that are frequently seen in B-ALL/LBL( 14 – 16 ) (Fig. 3 A). Paradoxically, one B-ALL/LBL (Case-28) showed a high level of SHM (86% identity to the germline) in its rearranged IGHV gene despite the absence of mutations in all SHM target genes investigated (Fig. 3 A, Table S5 ). Among the 3 DLBCL-NOS, one case (Case-21) with IGH :: MYC and BCL6 translocation showed mutation in several SHM target genes ( MYC , CXCR4 , HIST1H1C , HIST1H2BK ) and also evidence of SHM in its rearranged IGHV gene (Fig. 3 A). The remaining two cases were BCL2 translocation positive as shown by the presence of multiple BCL2 mutations (Case-20, FISH data unavailable) or interphase FISH investigation (Case-22). Case-20 showed the classic mutation pattern associated with FL and also mutations in several SHM target genes. While the other case (Case-22) showed TP53 and NRAS mutations, but not the above mutations of transformed FL (Fig. 3 A). Unfortunately, it was impossible to further investigate this case due to a lack of tissue materials. All three HGBCL-NOS carried a MYC translocation and showed mutations in several SHM target genes including MYC (Fig. 3 A). One case successfully investigated also showed frequent mutations in its rearranged IGHV gene. These genetic data, together with CD10 expression in each case, suggest their origin from GC B cells. Lastly, the two cases unclassifiable between B-ALL/LBL and HGBCL-NOS were negative for MYC , BCL2 and BCL6 translocation, but each harboured a TP53 mutation (Fig. 3 A). One case successfully investigated showed a moderate level of somatic mutations in its rearranged IGHV gene (Table S5 ), and this case also displayed a variant in a known SHM target gene ( CIITA ). Significant difference in mutation load of SHM target genes between TdT-positive LBCL and B-ALL/LBL As the number of B-ALL/LBL investigated in the present study was small, we compared the mutation load of a common set of SHM target genes between our TdT-positive LBCL (19 DLBCL/HGBCL- MYC / BCL2 , 6 DLBCL/HGBCL-NOS) and a larger cohort of B-ALL cases (n = 1716) from a previous study ( 14 ). As expected, the mutation load of SHM target genes was significantly higher in DLBCL/HGBCL- MYC / BCL2 and DLBCL/HGBCL-NOS than B-ALL ( p = 2.2E-16, p = 2.98E-05 respectively) (Fig. 3 B), with the vast majority (23/25 = 92%) of the DLBCL/HGBCL- MYC / BCL2 and DLBCL/HGBCL-NOS harboured a mutation load above the mean plus 2 standard deviations of the B-ALL group. Similarly, the overall mutation load among the 187 genes investigated was significantly higher in DLBCL/HGBCL- MYC / BCL2 and DLBCL/HGBCL-NOS than B-ALL ( p = 2.2E-16, p = 3.30E-06 respectively) (Fig. 3 B). Clonal evolution of TdT-positive HGBCL from FL or IGH :: BCL2 positive premalignant B-cells Among the TdT-positive DLBCL/HGBCL investigated, two cases (Case-06 & 07) had metachronous lymphomas with biopsies available for comparative mutation analysis, and one further case (Case-16) with a single microscopic nodule of FL component, thus allowing investigation of their evolutionary history. Case-06 (45-year-old, male) had a 1-year history of FL and presented with malaise and a right axillary lymph node enlargement. An excision biopsy (Case-06-DLBCL) showed a DLBCL with a GC phenotype (CD10+, BCL6+), TdT-, and was negative for MYC translocation. The patient was treated with 6 cycles of O-CHOP (obinutuzumab, plus cyclophosphamide, doxorubicin, prednisolone and vincristine) and achieved complete metabolic remission (CMR). Two years later, the patient was suspected with a lymphoma relapse, a core biopsy of the right inguinal lymph node (not analysed by NGS due to insufficient material) and a further core biopsy of the left neck lymph node (Case-06-FL) a month later displayed classic FL grade 1–2, and managed by active surveillance. Two months later, bone marrow trephine biopsy (Case-06-HGBCL) revealed infiltration of medium to large-sized immature lymphoid cells that were positive for CD19, CD79a, CD43, BCL2 and TdT (moderate to strong positivity in ~ 80% tumour cells), CD20 (partial) and MUM1 (partial), but negative for CD10, CD34, CD117 and BCL6. The patient was then treated with UKALL14 induction phase 1 but showed no response. Further investigations by interphase FISH revealed IGH :: MYC , IGH :: BCL2 and BCL6 rearrangement, hence supporting a diagnosis of DLBCL/HGBCL- MYC / BCL2 / BCL6. The patient was subsequently treated with 2 cycles of R-ICE, leading to complete cytogenetic response and followed by LACE autologous stem cell transplant. Ten months later, the patient showed lymphocytosis (20% lymphoblasts) in peripheral blood by flow cytometry despite no evidence of lymphadenopathy by CT imaging. The patient was treated with R-BP (rituximab, bendamstine, polatuzumab) and showed a significant clinical response, but died a month later. Targeted NGS analysis revealed both common and distinct variants among the DLBCL, FL and HGBCL- MYC / BCL2 / BCL6 (Fig. 4 A). These metachronous lymphomas shared 16 common clonal variants including 11 potentially pathogenic and 5 benign/synonymous/UTR changes, hence confirming their clonal relationship. The DLBCL had 17 private clonal variants including 10 potentially pathogenic changes, while the FL and HGBCL- MYC / BCL2 / BCL6 harboured different private variants. Intriguingly, the FL and HGBCL- MYC / BCL2 / BCL6 had both common (6 potentially pathogenic and 5 benign/synonymous/UTR changes) and unique variants (5 variants in FL including 2 potentially pathogenic changes, 5 variants in HGBCL- MYC / BCL2 / BCL6 including 2 potentially pathogenic changes). These mutation patterns indicated divergent evolution of these different lymphomas from a common lymphoma precursor (CLP) cell population, with the FL and HGBCL- MYC / BCL2 / BCL6 derived from an intermediate subclone of the CLP cell population (Fig. 4 A). Case-07 (42-year-old, male) was a referral, a lymph node excision biopsy (Case-07-FL) showed a FL grade 2, which was BCL2+, CD10+, but TdT-. The patient was treated with 3 cycles of R-CHOP and then switched to 2 cycles of R-ICE due to disease progression but showed no response. A further biopsy of an inguinal lymph node 4 months later showed a high-grade transformation with the large cell component being CD20-, BCL2+, CD10+ (weak), BCL6-, MUM1+, Ki67+ (100%), TdT- and TP53+. The patient was treated with 1 cycle of R-DHAP and venetoclax, and then switched to R-GemOx + Polivy (polatuzumab-vedotin) due to disease progression. Subsequently, the patient underwent abdominal exploration due to suspected splenic rupture, which instead revealed a peri-gastric tumour mass. A biopsy of the tumour mass (Case-07-HGBCL) showed a transformed lymphoma with large cell component being BCL2+ (50%), TdT+ (60%), MYC+ (80%). Interphase FISH showed both IGH :: MYC and BCL2 translocation, thus supporting a diagnosis of HGBCL- MYC / BCL2. The patient died one week later after diagnosis. Targeted sequencing analysis of the initial FL and peri-gastric HGBCL- MYC / BCL2 -DH revealed 16 common clonal variants (5 potentially pathogenic and 11 benign/synonymous/UTR changes), and also 4 and 7 private variants respectively. These, together with identical IGH rearrangements between the two lymphomas, indicated their divergent evolution from a CLP cell population (Fig. 4 B). Case-16 (77-year-old, male) presented with left leg swelling with multiple enlarged lymph nodes in the left groin. Multiple core biopsies of the lymph nodes showed HGBCL with a single microscopic nodule of FL component. The HGBCL cells were positive for PAX5, CD20 (~ 50%), CD10, BCL2, MUM1, MYC (100%) and TdT (~ 40%), but negative for CD34, BCL6, while the FL cells were diffusely positive for PAX5, CD20, CD10, BCL2, BCL6, partially expressed MUM1 (~ 30%) and MYC (~ 40%), but were negative for TdT and CD34 (Fig. 5 ). Interphase FISH showed both MYC and BCL2 translocation in HGBCL and FL, with MYC translocation in the FL component only seen in scattered large nuclei. Clonal analysis of the rearranged IG genes based on microdissected HGBCL and FL components showed identical sized IGH and IGK products (Figure S4 ), thus confirming their clonal relationship. Together, these findings suggest that the HGBCL originated from the FL following acquisition of MYC translocation. In line with this speculation, the HGBCL harboured characteristic mutations associated with FL, high-grade transformation and SHM activities (Fig. 3 A). Aberrant TdT expression and MYC translocation Since majority of TdT-positive DLBCL/HGBCL originate from IGH :: BCL2 positive FL or its precursor cell population following acquisition of a MYC translocation, this raises the question of whether the aberrant TdT expression might be due to dysregulated transcriptional activity of highly expressed MYC. To investigate this and identify appropriate cell lines for further in vitro studies, we examined TdT expression in 10 DLBCL (including 5 cell lines with MYC/BCL2- DH and an additional line with MYC translocation) and 1 Burkitt lymphoma cell line by immunocytochemistry. None of these cell lines showed any positive TdT staining. Additionally, extensive bioinformatics search did not reveal any evidence that TdT might be a potential MYC transcriptional target. DISCUSSION The differential diagnosis of TdT positive DLBCL/HGBCL and B-ALL/LBL may pose profound problems due to overlapping features of these two diagnostic categories/entities especially when MYC rearrangements are encountered in a CD34-negative aggressive B-cell neoplasm in an adolescent or young adult patient. By investigation of a spectrum of TdT-positive B-cell lymphomas including DLBCL/HGBCL- MYC / BCL2 , DLBCL-NOS, HGBCL-NOS, B-ALL/LBL and cases unclassified, the present study has highlighted several points that would aid differential diagnosis between TdT-positive DLBCL/HGBCL and B-ALL/LBL. Loss of expression of pan B-cell antigens, such as CD20, has been used to argue for a diagnosis of B-ALL/LBL, while the expression of mature B-cell antigens such as BCL6 and MUM1 favours the diagnosis of mature rather than precursor B-cell lymphoma. However, none of these immunophenotypic features is specific enough for reliable differential diagnosis between TdT-positive LBCL/HGBCL and B-ALL/LBL. Our data clearly demonstrate that a high proportion (15/23 = 65%) of TdT-positive LBCL/HGBCL show a complete or partial loss of CD20 expression, while MUM1 can also be positive in B-ALL/LBL ( 1 ). Nonetheless, CD34 is consistently negative in TdT-positive LBCL, but a lack of CD34 expression can occur in a high proportion of B-ALL/LBL as shown in the present and previous study ( 21 – 24 ). Apart from the detection of chromosomal translocations, mutation analysis is highly valuable in differential diagnosis between TdT-positive LBCL and B-ALL/LBL. Like conventional DLBCL/HGBCL- MYC / BCL2 , the TdT-positive cases with MYC / BCL2 translocation harboured a characteristic mutation profile, including mutation signatures associated with FL and also its high-grade transformation ( 8 – 12 ). TdT-positive DLBCL-NOS and HGBCL-NOS also show frequent mutations commonly seen in these entities albeit less striking as those of DLBCL/HGBCL- MYC / BCL2 . Furthermore, except for one case (Case-22), all other DLBCL-NOS and HGBCL-NOS exhibit frequent mutations in the SHM target genes, along with CD10 expression indicating their origin from GC B cells. This is also supported by findings of frequent mutations in their rearranged IGHV genes in each case successfully investigated. In contrast, B-ALL/LBL had few mutations, lacking somatic variants associated SHM activities as they originate from precursor B cells not yet undergone antigen affinity maturation in the peripheral lymphoid tissues ( 13 ). As targeted sequencing by NGS is increasingly used in diagnosis and sub-classification of B-cell lymphoma, it is important to include genes targeted by the SHM machinery and document their somatic changes as discussed above. It is necessary to report all somatic variants identified including both synonymous and nonsynonymous variants, as well as those present in the 5’UTR and intronic regions to maximise their utility as biomarkers in delineating the dichotomy of cell-of-origin between pre-GC and GC/post-GC B cells. This study serves as a proof of principle. Future studies are required to standardise the list of genes and their genomic regions to be covered by NGS, and also to establish the optimal method for quantifying somatic variants and their threshold value for reliable prediction of lymphoma cell-of-origin. Of the two cases with diagnostic ambiguity between B-ALL/LBL and HGBCL-NOS, the genetic data obtained failed to provide any strong evidence for their definitive diagnosis. Apart from TP53 mutation, both cases lack the other mutations seen in HGBCL-NOS. Case-27 showed a single mutation in a SHM target gene ( CIITA ) and a moderate level (95%) of SHM in its rearranged IGHV , while the other case showed no mutation in the SHM target genes investigated. Such low level of somatic mutations in the rearranged IGHV genes has been reported in B-ALL previously ( 25 ). These, together with their variable expression of pan-B-cell (CD20) and mature B-cell (BCL6, MUM1) markers may argue for the existence of grey zone cases between B-ALL/LBL and HGBCL-NOS. The mechanism for aberrant TdT expression in LBCL is unclear. Given that aberrant TdT expression is often associated with partial or complete loss of expression of pan-B cell antigen (CD20), it is possible that these cases develop a defect in the transcriptional network that controls the B-cell programme. In line with this speculation, there is no evidence of loss of CD20 expression in conventional DLBCL/HGBCL, and this is further reinforced by a retrospective review of 15 TdT-negative and MYC translocation-positive DLBCL/HGBCL. Among the transcriptional factors, PAX5 genetic changes including mutation p.P80R are frequently seen in B-ALL/LBL and these genetic changes are thought to affect B-cell programming, thereby contributing to leukaemia development ( 26 , 27 ). PAX5 is a SHM target and multiple mutations were observed in a case of HGBCL- MYC / BCL2 -DH (Case-08), affecting the paired box domain and also the highly conserved octapeptide motif (Fig. 3 A, Figure S5 ). In this context, it is worth noting mutations in several other transcriptional factors that regulate early B-cell development, such as ETS1 (SHM target), FOXO1 (SHM target), TCF3 (encoding E2A) and POU2F2 (encoding OCT2)( 28 ) (Fig. 3 A, Figure S5 ). It remains to be investigated whether mutations in these transcriptional factors and related regulators may dysregulate the B-cell programme, causing aberrant TdT expression and also perturbing B-cell antigen expression. In conclusion, our findings demonstrate that mutation profile analysis, including SHM target genes, is highly valuable in differential diagnosis between TdT-positive DLBCL/HGBCL and B-ALL/LBL. Declarations Acknowledgements: The authors would like to thank Dr Ana Toribio and Jessica Ferreira Gouveia for their assistance with Illumina sequencing. Authors contributions: FISH, targeted NGS, data collection and analyses: MMT, FC, ZC, EM, JM, AES, FG, MQD; Case contribution and pathology: LRB, AW, KSK, CE, ES, LH, LK, JTG, CL, LX, LM, LP, LMRG, NLH, TG, JK, WK, IO, AR and GO; Study conception, coordination and research funding: MQD, LRB, GO; Manuscript writing and preparation: MQD & MMT with contributions from all authors. All authors read and approved the final manuscript. Conflict of interest: The authors declare no conflict of interest. Additional Information: All core data generated or analysed during this study are included in this published article, and additional raw data are available from the corresponding author on reasonable request. References Duffield AS, Dogan A, Amador C, Cook JR, Czader M, Goodlad JR, et al. 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Leongamornlert D, Gutierrez-Abril J, Lee SW, Barretta E, Creasey T, Gundem G, et al. Diagnostic utility of whole genome sequencing in adults with B-other acute lymphoblastic leukemia. Blood Adv. 2023;7:3862. Ueno H, Yoshida K, Shiozawa Y, Nannya Y, Iijima-Yamashita Y, Kiyokawa N, et al. Landscape of driver mutations and their clinical impacts in pediatric B-cell precursor acute lymphoblastic leukemia. Blood Adv. 2020;4:5165–73. Cucco F, Clipson A, Kennedy H, Sneath Thompson J, Wang M, Barrans S, et al. Mutation screening using formalin-fixed paraffin-embedded tissues: a stratified approach according to DNA quality. Laboratory Investigation. 2018;98:1084–92. Zhang C, Stelloo E, Barrans S, Cucco F, Jiang D, Tzioni MM, et al. Non-IG::MYC in diffuse large B-cell lymphoma confers variable genomic configurations and MYC transactivation potential. Leukemia. 2024;38:621–9. Schuetz JM, Johnson NA, Morin RD, Scott DW, Tan K, Ben-Nierah S, et al. BCL2 mutations in diffuse large B-cell lymphoma. Leukemia. 2012;26:1383–90. Schmitz R, Wright GW, Huang DW, Johnson CA, Phelan JD, Wang JQ, et al. Genetics and Pathogenesis of Diffuse Large B-Cell Lymphoma. New England Journal of Medicine. 2018;378:1396–407. Garg N, Gupta R, Kotru M. CD34 is not Expressed by Blasts in a Third of B-ALL Patients and its Negativity is associated with Aberrant Marker Expression: A Retrospective Analysis. Asian Pac J Cancer Prev. 2021;22:919–25. Ali Shah M, Ahmad U, Tariq Mahmood M, Ahmad AH, Abu Bakar M. Frequency of CD34 and CD10 Expression in Adolescent and Young Adult Patients Having Precursor B-cell Acute Lymphoblastic Leukemia and Its Correlation With Clinical Outcomes: A Single-Center Study. Cureus. 2022;14:e21261. Qiu L, Xu J, Lin P, Cohen EN, Tang G, Wang SA, et al. Unique pathologic features and gene expression signatures distinguish blastoid high-grade B-cell lymphoma from B-acute lymphoblastic leukemia/lymphoma. Haematologica. 2023;108:895–9. Wood B, Gujral S. 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Summary of clinical and laboratory results of cases 26-31 investigated. Case-26 Case-27 Case-28 Case-29 Case-30 Case-31 Diagnosis Unclassified Unclassified B-ALL/LBL B-ALL/LBL B-ALL/LBL B-ALL/LBL Age /sex 70 year / F 74 year / M 63 year / F 4 year / F 80 year /F 75 year / M Clinical history pancytopenia B symptoms, splenomegaly, anaemia, right thyroid nodule with histopathology consistent with B-ALL/LBL pancytopenia thrombocytopenia and leukoerythroblastic picture pancytopenia and lytic bone lesion Nodal & splenic involvement N/A Splenomegaly, no lymphadenopathy by CT stage IV, wide lymphadenopathy, plus pancreas, nasopharynx, uterine and peritoneal lesions splenomegaly, no lymphadenopathy by CT N/A N/A Biopsy site bone marrow bone marrow lymph node bone marrow bone marrow bone marrow Histopathology diffuse infiltration by lymphoblastoid cells diffuse infiltration by lymphoblastoid cells diffuse infiltration by lymphoblastoid cells diffuse infiltration by lymphoblastoid cells diffuse infiltration by large atypical lymphocytes with moderate pleomorphism diffuse infiltration by lymphoblastoid cells Immunophenotype PAX5 diffuse positive diffuse positive N/A N/A diffuse positive N/A CD20 positive in ~90% positive in ~30% positive in ~50% weak, patchy positive diffuse positive diffuse positive CD79a diffuse positive with variable intensity diffuse positive diffuse positive diffuse positive diffuse positive with variable intensity positive in ~30% CD10 diffuse positive diffuse positive diffuse positive diffuse positive diffuse positive diffuse positive BCL6 weak -moderate positive positive negative negative weak partial positive negative MUM1 strong positive focal positive negative positive in ~40% weak positive moderate to strong positive in ~80% Ki67 90% 90% 60-70% 100% 60% ~90% MYC 60% 90% 60% 80% 60% 60% TDT strong positive in ~100% variable positivity in ~50% moderate positive in ~95% moderate to strong positivity in ~40% strong positive in 100% 80% CD34 negative negative diffuse positive negative diffuse positive negative Translocation MYC negative negative negative IGL::MYC negative negative BCL2 negative negative negative negative negative negative BCL6 negative negative negative negative negative negative Others N/A no FISH evidence of BCR::ABL1, ETV6::RUNX1, TCF3::PBX1, TCF3::HLF & KMT2A translocation N/A N/A N/A N/A Mutation load much lower than DLBCL/HGBCL much lower than DLBCL/HGBCL much lower than DLBCL/HGBCL much lower than DLBCL/HGBCL much lower than DLBCL/HGBCL much lower than DLBCL/HGBCL Any pathognomonic mutations no no no no no no SHM driven mutation no single synonymous change no no no no IGHV mutation N/A 95% identity to germline 86% identity to germline no no N/A Treatment & outcome N/A treated with UKALL60+ protocol and showed a short period of morphological and cytogenetic remission, then bone marrow relapse and treated with 3 cycles of Inotuzumab and achieved morphological and cytogenetic remission. treated with RCHOP, RDHAC then autografted. 2 years later, lymphoma relapsed with 15% of circulant blast and 98% in bone marrow aspiration. treated as Burkitt’s leukaemia with chemotherapy, achieved a short period of remission, then relapsed and allografted, further disease relapse and followed by palliative chemotherapy, died two years after initial diagnosis. patient died shortly after diagnosis treated with 2 cycles of R-mini-CVD, then transferred to hospice. B-ALL/LBL: B-lymphoblastic leukaemia/lymphomas; DLBCL: diffuse large B-cell lymphoma; HGBCL: high grade B-cell lymphoma; F: female; M: male; N/A: not available. Additional Declarations There is NO conflict of interest to disclose. Supplementary Files SUPPLEMENTARYFiguresandTableslegends.docx SupplementaryFigureS1.tif SupplementaryFigureS2.tif SupplementaryFigureS3.tif SupplementaryFigureS4.tif SupplementaryFigureS5.tif SupplementaryTables.xlsx SupplementaryMaterialsandMethods.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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TdT, CD34 and MYC immunohistochemistry was centrally performed or reviewed in the Cambridge lab and their expression level is semi-quantified, while CD20, CD10, BCL6 and MUM1 immunostaining results are from a referral laboratory and recorded in a non-quantitative manner. Except exclusive CD34 expression in two cases of B-ALL/LBL, there is no clear demarcation among the expression of other immunophenotypic markers between different groups. B-ALL/LBL: B-lymphoblastic leukaemia/lymphomas; DLBCL: diffuse large B-cell lymphoma; HGBCL: high grade B-cell lymphoma; tr+ve: translocation positive, tr-ve: translocation negative; IHC: immunohistochemistry; N/A: not available.\u003c/p\u003e","description":"","filename":"MainFigure1.png","url":"https://assets-eu.researchsquare.com/files/rs-6172652/v1/73376ffe33ea8804dbf8346f.png"},{"id":78659910,"identity":"ac2a91a0-80d1-41a9-b734-67ffb9bcb560","added_by":"auto","created_at":"2025-03-17 10:00:53","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":46200105,"visible":true,"origin":"","legend":"\u003cp\u003eHistological and immunophenotypic presentation of Case-26 with differential diagnosis between HGBCL-NOS and B-ALL/LBL. The bone marrow trephine biopsy shows infiltration by sheets of lymphoblastoid tumour cells that display diffuse PAX5 expression, but CD20 expression in ~90% tumour cells. The lymphoma cells are diffuse positive for CD10, BCL6, MUM1, MYC and TdT, but negative for CD34.\u003c/p\u003e","description":"","filename":"MainFigure2.png","url":"https://assets-eu.researchsquare.com/files/rs-6172652/v1/c45e66cd4dab4ec91e376b85.png"},{"id":78660921,"identity":"297f8487-42c9-4162-9eef-0db17e7b86f9","added_by":"auto","created_at":"2025-03-17 10:08:52","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":5995802,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of mutation profiles among different groups of TdT positive lymphomas. B-ALL/LBL: B-lymphoblastic leukaemia/lymphomas; FL: follicular lymphoma; DLBCL: diffuse large B-cell lymphoma; HGBCL: high grade B-cell lymphoma; tr+ve: translocation positive, tr-ve: translocation negative; IHC: immunohistochemistry; SNV: single nucleotide variation; SHM: somatic hypermutation; N/A: not available.\u003c/p\u003e\n\u003cp\u003eA: Heatmap presentation of genetic data (please refer to Figure S3 for complete mutation heatmap presentation). The mutated genes are grouped according to their association with lymphoma entity and the SHM process. \u0026nbsp;*Indicates \u003cem\u003eIG\u003c/em\u003e::\u003cem\u003eMYC\u003c/em\u003e translocations confirmed through FISH , **denotes the genes such as \u003cem\u003eBCL2\u003c/em\u003e and \u003cem\u003eMYC\u003c/em\u003e that are also target of SHM, but grouped according to their association with disease entity.\u003c/p\u003e\n\u003cp\u003eB: Comparison of mutation load among the 49 SHM target genes (left panel) and all 187 genes (right panel) among DLBCL/HGBCL-\u003cem\u003eMYC\u003c/em\u003e/\u003cem\u003eBCL2\u003c/em\u003e, DLBCL/HGBCL-NOS and B-ALL. The red dotted line denotes the mean plus 2 standard deviation of mutation load from B-ALL/LBL.\u003c/p\u003e","description":"","filename":"MainFigure3.png","url":"https://assets-eu.researchsquare.com/files/rs-6172652/v1/8b7e8cfc497a26e0d81ebe54.png"},{"id":78659883,"identity":"8a734f3a-8f81-405b-aa68-0c1967c85b14","added_by":"auto","created_at":"2025-03-17 10:00:52","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":5802947,"visible":true,"origin":"","legend":"\u003cp\u003eClonal evolution of TdT-positive HGBCL as revealed by mutation profiling. \u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eThe number of all clonal variants [including pathogenic (in bold), benign, synonymous variants and those in UTR regions] that occurred at each stage of lymphoma development are provided, and only nonsynonymous variants are presented in detail. The shared and distinct clonal variants in paired lesions and their predicted evolutionary trajectory in each case are illustrated. CLP: the predicted clonally related lymphoma precursor cells; FL: follicular lymphoma; DLBCL: diffuse large B-cell lymphoma; tr+ve: translocation positive; SHM: somatic hypermutation; IHC: immunohistochemistry; TH: triple-hit; DH: double-hit.\u003c/p\u003e\n\u003cp\u003eA: Case-06 shows divergent evolution of DLBCL, FL and TdT-positive HGBCL-TH (\u003cem\u003eBCL2\u003c/em\u003e/\u003cem\u003eMYC\u003c/em\u003e/\u003cem\u003eBCL6 \u003c/em\u003etranslocation) from a common CLP cell population, with the latter two lesions originating from a CLP subclone.\u003c/p\u003e\n\u003cp\u003eB: Case-07 displays divergent evolution of FL and TdT-positive HGBCL-DH (\u003cem\u003eBCL2\u003c/em\u003e/\u003cem\u003eMYC \u003c/em\u003etranslocation) from a common CLP cell population.\u003c/p\u003e","description":"","filename":"MainFigure4.png","url":"https://assets-eu.researchsquare.com/files/rs-6172652/v1/8f6c891bf69f0236dd67085e.png"},{"id":78659908,"identity":"396cce31-2c57-4cff-8d18-ad44b21d571d","added_by":"auto","created_at":"2025-03-17 10:00:53","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":36214264,"visible":true,"origin":"","legend":"\u003cp\u003eHistological and immunophenotypic presentation of Case-16 with HGBCL-\u003cem\u003eMYC\u003c/em\u003e/\u003cem\u003eBCL2\u003c/em\u003e and a single microscopic nodule of follicular lymphoma (FL) component. The HGBCL cells show diffuse PAX5 positivity (not shown), but CD20 positivity in ~50% of tumour cells, and are positive for MUM1, MYC (100%) and TdT (~40%), while the FL component shows scattered positivity for MUM1 (~30%) and MYC (~40%), but negative TdT expression.\u003c/p\u003e","description":"","filename":"MainFigure5.png","url":"https://assets-eu.researchsquare.com/files/rs-6172652/v1/134e3a1117b43323ca3e8784.png"},{"id":79827581,"identity":"4bcf0ea9-fd84-4cab-a854-f9b9b32d7c8e","added_by":"auto","created_at":"2025-04-03 09:50:55","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":252736083,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6172652/v1/ae0b771a-df3b-46b3-b03c-c4b1d06ea942.pdf"},{"id":78659881,"identity":"777f7454-fd7b-469b-99ff-abeb550f7d31","added_by":"auto","created_at":"2025-03-17 10:00:52","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":171839,"visible":true,"origin":"","legend":"","description":"","filename":"SUPPLEMENTARYFiguresandTableslegends.docx","url":"https://assets-eu.researchsquare.com/files/rs-6172652/v1/9611111f1d791b0c58cda8ae.docx"},{"id":78661408,"identity":"84ffb532-3e19-4da0-8801-31312651f133","added_by":"auto","created_at":"2025-03-17 10:16:53","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":7279442,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigureS1.tif","url":"https://assets-eu.researchsquare.com/files/rs-6172652/v1/58ac5319a95d967b8932e876.tif"},{"id":78659915,"identity":"d56938f3-3a94-4be1-9c58-0538edf9f0ad","added_by":"auto","created_at":"2025-03-17 10:00:54","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":40806522,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigureS2.tif","url":"https://assets-eu.researchsquare.com/files/rs-6172652/v1/422062a37d321741827df53c.tif"},{"id":78660919,"identity":"18cd1a06-4ce1-492c-ba2f-d28d7d9c8715","added_by":"auto","created_at":"2025-03-17 10:08:52","extension":"tif","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":10236642,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigureS3.tif","url":"https://assets-eu.researchsquare.com/files/rs-6172652/v1/b9af72fb5554357eefe0ad79.tif"},{"id":78659892,"identity":"9cadb185-94b2-450a-8462-91f05986fcc9","added_by":"auto","created_at":"2025-03-17 10:00:52","extension":"tif","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":6850250,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigureS4.tif","url":"https://assets-eu.researchsquare.com/files/rs-6172652/v1/0a2b27b96ce1bf016c5ce4df.tif"},{"id":78661406,"identity":"096ff7a4-81a2-4e84-a0f3-f54bac19a808","added_by":"auto","created_at":"2025-03-17 10:16:52","extension":"tif","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":5519876,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigureS5.tif","url":"https://assets-eu.researchsquare.com/files/rs-6172652/v1/b59d338bf9351567fbe5061d.tif"},{"id":78659884,"identity":"38c148b7-944d-47d6-9d17-395719c5b600","added_by":"auto","created_at":"2025-03-17 10:00:52","extension":"xlsx","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":78998,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTables.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6172652/v1/f0d03cdbdeb39db005f9204f.xlsx"},{"id":78660908,"identity":"0fba9042-1f8c-4fc1-b19e-ce9149fe19ab","added_by":"auto","created_at":"2025-03-17 10:08:52","extension":"docx","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":15879,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterialsandMethods.docx","url":"https://assets-eu.researchsquare.com/files/rs-6172652/v1/45ed890303befbc8611e1dbb.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e conflict of interest to disclose.","formattedTitle":"Mutation profiling in differential diagnosis between TdT-positive high grade/large B-cell lymphoma and B-lymphoblastic leukaemia/lymphoma","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eImmunophenotyping is pivotal in the diagnosis and classification of haematolymphoid malignancies. By comparing the immunophenotype of malignant cells with those of normal lymphoid cells at various stages of their development and maturation, it is possible to determine the tumour cell-of-origin, hence enabling its diagnosis. However, some of the immunophenotypic markers may be expressed aberrantly in lymphoma cells, and this may cause challenges in their application to lymphoma diagnosis. One of such immunophenotypic markers is terminal deoxynucleotidyl transferase (TdT).\u003c/p\u003e \u003cp\u003eTdT is typically expressed in pro- and pre-B cells in the bone marrow, but downregulated and not expressed in mature B cells in peripheral lymphoid tissues. Thus, TdT is regarded as a marker of precursor B-cells and their derived leukaemias and lymphomas, i.e. B-lymphoblastic leukaemias/lymphomas (B-ALL/LBL). However, TdT is also expressed in rare cases of diffuse large B-cell lymphoma (DLBCL) with dual \u003cem\u003eMYC\u003c/em\u003e and \u003cem\u003eBCL2\u003c/em\u003e rearrangements (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). In the revised 4th edition of the World Health Organization Classification of Haematolymphoid Tumours (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e), rare TdT-positive B-cell lymphoma occurring with a history of follicular lymphoma (FL) and both \u003cem\u003eMYC\u003c/em\u003e and \u003cem\u003eBCL2\u003c/em\u003e translocations have been designated as \u0026ldquo;lymphoblastic transformation\u0026rdquo;. As these cases are CD34 negative, often bear a mature B-cell phenotype and show a mutation profile similar to those of transformed FL (\u003cspan additionalcitationids=\"CR4 CR5\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). These findings together with their exclusive occurrence in adults support their origin from mature B-cells with TdT expression being aberrant. Accordingly, these cases are now designated as a subtype of diffuse large / high-grade B-cell lymphoma with \u003cem\u003eMYC\u003c/em\u003e/\u003cem\u003eBCL2\u003c/em\u003e rearrangement (DLBCL/HGBCL-\u003cem\u003eMYC\u003c/em\u003e/\u003cem\u003eBCL2\u003c/em\u003e, or DLBCL/HGBCL-\u003cem\u003eMYC\u003c/em\u003e/\u003cem\u003eBCL2\u003c/em\u003e/\u003cem\u003eBCL6\u003c/em\u003e when presence of additional \u003cem\u003eBCL6\u003c/em\u003e rearrangement) in the 5th edition of the World Health Organization Classification of Haematolymphoid Tumours (WHO-HAEM5) (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn general, DLBCL/HGBCL-\u003cem\u003eMYC\u003c/em\u003e/\u003cem\u003eBCL2\u003c/em\u003e can be readily diagnosed by combining histopathological/immunophenotypic assessment with interphase FISH analysis for these translocations. In addition, DLBCL/HGBCL-\u003cem\u003eMYC\u003c/em\u003e/\u003cem\u003eBCL2\u003c/em\u003e bears characteristic mutation signatures including pathogenic changes associated with classic FL as well as those associated with high grade transformation, serving as a robust genetic basis for differential diagnosis (\u003cspan additionalcitationids=\"CR9 CR10 CR11\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). However, aberrant TdT expression may also be seen in DLBCL/HGBCL without \u003cem\u003eMYC\u003c/em\u003e and \u003cem\u003eBCL2\u003c/em\u003e translocation (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e), and such cases may also bear features of immaturity such as loss of CD20, BCL6 and/or immunoglobulin light chain expression, posing a real challenge in their differential diagnosis from B-ALL/LBL due to a lack of pathognomonic genetic changes in the former (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). On the other hand, B-ALL/LBL, including rare cases with \u003cem\u003eMYC\u003c/em\u003e and \u003cem\u003eMYC/BCL2\u003c/em\u003e translocations, occur commonly in children (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e), but are also seen in young adults, thus overlapping with the age range of patients with DLBCL/HGBCL (\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eGiven the distinct cell of origins between TdT-positive DLBCL/HGBCL and B-ALL/LBL and potential differences in their mutation profiles, we investigated somatic mutations in both rearranged \u003cem\u003eIGHV\u003c/em\u003e genes and lymphoma genes (n\u0026thinsp;=\u0026thinsp;187) by targeted next generation sequencing and performed integrated analysis to explore their utility in differential diagnosis. We also investigated the evolutionary history of TdT-positive transformed FL in cases where biopsies from paired metachronous lymphomas were available.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCase materials\u003c/h2\u003e \u003cp\u003eThe study was performed in accordance with local ethical guidelines for the research use of tissue materials with the approval of the ethics committees of the involved institutions (05-Q1604-10).\u003c/p\u003e \u003cp\u003eA total of 31 cases of TdT-positive aggressive B-cell lymphoma were retrieved from the authors\u0026rsquo; institutions, and their histological diagnosis was reviewed with a final diagnosis assigned according to WHO-HAEM5.\u003c/p\u003e \u003cp\u003e \u003cb\u003eInterphase fluorescence\u003c/b\u003e \u003cb\u003ein situ\u003c/b\u003e \u003cb\u003ehybridisation (FISH)\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cem\u003eMYC\u003c/em\u003e, \u003cem\u003eBCL2\u003c/em\u003e and \u003cem\u003eBCL6\u003c/em\u003e translocation status was available in the majority of cases from routine haematopathological diagnosis. Any missing translocation data were completed retrospectively together with further interphase FISH with \u003cem\u003eMYC\u003c/em\u003e/\u003cem\u003eIGH\u003c/em\u003e (Abbott), \u003cem\u003eMYC\u003c/em\u003e/\u003cem\u003eIGK\u003c/em\u003e and \u003cem\u003eMYC\u003c/em\u003e/\u003cem\u003eIGL\u003c/em\u003e (Cytocell) dual fusion probes.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eImmunohistochemistry\u003c/h3\u003e\n\u003cp\u003eMajority of the immunophenotypic data were available from routine haematopathological diagnosis, and any missing data on MYC and CD34 were collected retrospectively under the same conditions as routine histological diagnosis (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAdditional TdT immunocytochemistry was performed on formalin-fixed paraffin-embedded cell clots of DLBCL cell lines (both \u003cem\u003eMYC\u003c/em\u003e and \u003cem\u003eBCL2\u003c/em\u003e translocation positive: OCI-Ly4, OCI-LY18, Su-DHL6, SC1, DB; \u003cem\u003eMYC\u003c/em\u003e translocation positive: RIVA; \u003cem\u003eMYC\u003c/em\u003e translocation negative: OCI-Ly3, OCI-Ly10, Karpas-422, Su-DHL2) and the Burkitt lymphoma cell line BJAB.\u003c/p\u003e\n\u003ch3\u003eDNA extraction and quality assessment\u003c/h3\u003e\n\u003cp\u003eHistology was reviewed and the areas containing confluent lymphoma cells (\u0026gt;\u0026thinsp;40%) in each tissue specimen were microdissected on consecutive tissue sections. DNA was extracted using the QIAamp DNA Micro Kit (QIAGEN, Crawly, UK), quantified with a Qubit\u0026reg; Fluorometer (Life Technologies, Carlsbad, CA, USA) and assessed for quality by PCR (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eMutation analysis by targeted next generation sequencing\u003c/h3\u003e\n\u003cp\u003eThis was carried out using a customised panel of 187 genes that are recurrently mutated in FL and DLBCL/HGBCL (Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). Apart from TdT-positive DLBCL/HGBCL, four cases of TdT-positive B-ALL/LBL were included for comparative analysis to depict their difference in mutation pattern among the panel genes investigated. A total of 80-200ng of FFPE tissue derived DNA was fragmented using the Covaris E220 Focused Ultrasonicator (Covaris, Brighton, UK). For each DNA sample, an indexed library was prepared with the xGen\u0026trade; UDI-UMI indexes (IDT, Coralville, IA, USA) using the TWIST protocol, and then pooled for target enrichment using the TWIST probes (TWIST Biosciences, South San Francisco, CA, USA). The enriched DNA targets were amplified by PCR and pooled libraries were sequenced using the Illumina NextSeq 2000 platform (2x100bp paired-end sequencing protocol). The sequence data analysis, variant calling, and filtering were performed as described in our previous studies (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFor DNA samples with suboptimal quality (PCR amplification of genomic fragments\u0026thinsp;\u0026le;\u0026thinsp;300bp), targeted sequencing was performed in duplicates and only variants detected in both replicates were considered as a true alteration (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eClonality analysis of the rearranged immunoglobulin heavy chain genes (IGH)\u003c/h3\u003e\n\u003cp\u003eThis was performed by adopting the BIOMED-2 assays, followed by NGS using 2x250bp paired-end sequencing protocol (Illumina NovaSeq X sequencer) (Supplementary materials and methods, Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe comparison of mutation load among different groups was assessed using Wilcoxon Rank-Sum test with two-sided \u003cem\u003eP\u003c/em\u003e values.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eHistopathological features and their value in differential diagnosis\u003c/h2\u003e \u003cp\u003eFor each case, the histological diagnosis was reviewed and where possible a final diagnosis was made according to WHO-HAEM5. The final diagnosis included 5 DLBCL-\u003cem\u003eMYC\u003c/em\u003e/\u003cem\u003eBCL2\u003c/em\u003e (including 1 with additional \u003cem\u003eBCL6\u003c/em\u003e rearrangement), 14 HGBCL-\u003cem\u003eMYC\u003c/em\u003e/\u003cem\u003eBCL2\u003c/em\u003e (including 1 with additional \u003cem\u003eBCL6\u003c/em\u003e rearrangement), 3 DLBCL-NOS, 3 HGBCL-NOS, 4 B-ALL/LBL and 2 cases unclassifiable due to equal evidence for both immature (strong TdT expression with partial loss of both CD20 and CD79a expression) and mature (expression of both BCL6 and MUM1 together with no CD34 expression) immunophenotype (Table\u0026nbsp;1, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAmong the 19 cases of DLBCL/HGBCL-\u003cem\u003eMYC\u003c/em\u003e/\u003cem\u003eBCL2\u003c/em\u003e, 11 patients were male and 8 were female, aged between 34\u0026ndash;79 years (median: 63 years). TdT positivity by immunohistochemistry varied considerably, ranging from scattered positive cells to diffuse positivity in variable proportions of lymphoma cells (\u0026lt;\u0026thinsp;40% in 7, 40\u0026ndash;70% in 7, \u0026gt;\u0026thinsp;70% in 5 cases), while CD34 was negative in each of the 19 cases examined. CD10 was positive in 16 of the 17 cases where data were available. CD20 was negative in 5, partially positive in 8 and diffusely positive in 4 cases among the 17 cases where data were available.\u003c/p\u003e \u003cp\u003eAmong the 3 cases of DLBCL-NOS, 2 patients were female (58 \u0026amp; 69 years) with one carrying \u003cem\u003eIGH\u003c/em\u003e::\u003cem\u003eBCL2\u003c/em\u003e and the other harbouring \u003cem\u003eIGH\u003c/em\u003e::\u003cem\u003eMYC\u003c/em\u003e and \u003cem\u003eBCL6\u003c/em\u003e translocation, while the third patient was a male (89 years). TdT was positive in 20\u0026ndash;90% of the tumour cells, while CD34 was negative in the 2 cases examined. CD10 was positive in all three cases. None of these cases showed any loss of CD20 by immunohistochemistry.\u003c/p\u003e \u003cp\u003eThe 3 cases of HGBCL-NOS were from male patients, aged between 77\u0026ndash;81 years, and each carried an isolated \u003cem\u003eMYC\u003c/em\u003e translocation. TdT was positive in 50\u0026ndash;90% of tumour cells, while CD34 was negative in all 3 cases. CD10 was diffusely positive in two, and partially positive in the third case. Two cases showed a partial loss of CD20 expression, while the third case was CD20 positive.\u003c/p\u003e \u003cp\u003eThe 4 cases of B-ALL/LBL were from one child (4 years, female) and 3 adults (63\u0026ndash;80 years, 1 male \u0026amp; 2 female). The paediatric case carried an \u003cem\u003eIGL\u003c/em\u003e::\u003cem\u003eMYC\u003c/em\u003e translocation, and was TdT-positive in 40% of the tumour cells, but CD34 negative. The three adult cases lacked \u003cem\u003eMYC\u003c/em\u003e translocation, two showed diffuse positivity for both TdT and CD34, and the remaining case was TdT positive in ~\u0026thinsp;80% of tumour cells but lacked any CD34 expression. All four cases were CD10 positive. Two cases (the paediatric case, and one adult case) showed partial staining for CD20 but diffuse positivity for CD79a, with the paediatric case expressing MUM1 in 40% tumour cells. However, the remaining two cases displayed diffuse positivity for CD20, but variable CD79a expression (variable intensity in 1 case and partial expression in the other), and also expressed MUM1 in majority of the tumour cells.\u003c/p\u003e \u003cp\u003eLastly, in 2 cases, the final diagnosis remained as unclassifiable between B-ALL/LBL and HGBCL-NOS despite careful histological review due to equal evidence for both immature and mature immunophenotypic features. One patient was a 74-year-old male and the other a 70-year-old female. Neither of them carried a \u003cem\u003eMYC\u003c/em\u003e, \u003cem\u003eBCL2\u003c/em\u003e or \u003cem\u003eBCL6\u003c/em\u003e translocation. One case showed diffuse TdT positivity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), while the other displayed TdT expression in 50% of tumour cells (Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). Both were CD34 negative, and CD10 positive. One case showed variable staining of CD20 and CD79a in 90% tumour cells, and was positive for BCL6 (weak) and MUM1 (strong) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The other case displayed partial expression of CD20 (~\u0026thinsp;30%) and CD79a (90%, variable intensity) and diffuse positivity for BCL6, and focal positivity for MUM1 (Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eGenetic features and their value in differential diagnosis\u003c/h2\u003e \u003cp\u003eComparative analysis among various TdT-positive DLBCL/HGBCL, together with B-ALL/LBL as a reference, revealed distinct mutation features (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, Figure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e, Table \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe TdT-positive DLBCL/HGBCL-\u003cem\u003eMYC\u003c/em\u003e/\u003cem\u003eBCL2\u003c/em\u003e group was characterised by the mutation signature associated with FL (\u003cem\u003eBCL2\u003c/em\u003e, \u003cem\u003eKMT2D\u003c/em\u003e, \u003cem\u003eCREBBP\u003c/em\u003e, \u003cem\u003eTNFRSF14\u003c/em\u003e), and those associated with high grade transformation (\u003cem\u003eMYC\u003c/em\u003e, \u003cem\u003eTP53\u003c/em\u003e, \u003cem\u003eCCND3\u003c/em\u003e, \u003cem\u003ePIM1\u003c/em\u003e, \u003cem\u003eB2M\u003c/em\u003e, \u003cem\u003eDDX3X\u003c/em\u003e, \u003cem\u003eS1PR2\u003c/em\u003e). These cases also showed frequent mutations in the genes known or predicted to be the targets of the somatic hypermutation (SHM) machinery, including \u003cem\u003eBCL2\u003c/em\u003e and \u003cem\u003eMYC\u003c/em\u003e when involved in translocation(\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). In addition, 12 of the 13 cases of DLBCL/HGBCL-\u003cem\u003eMYC\u003c/em\u003e/\u003cem\u003eBCL2\u003c/em\u003e successfully investigated showed frequent somatic mutations in their rearranged immunoglobulin heavy chain variable genes (\u003cem\u003eIGHV\u003c/em\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, Table \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003e). These genetic changes are consistent with the GC B-cell origin of DLBCL/HGBCL-\u003cem\u003eMYC\u003c/em\u003e/\u003cem\u003eBCL2\u003c/em\u003e. In contrast, none of the B-ALL/LBL cases showed these mutational features, but displayed \u003cem\u003eTP53\u003c/em\u003e, \u003cem\u003eKRAS\u003c/em\u003e and \u003cem\u003ePAX5\u003c/em\u003e mutations that are frequently seen in B-ALL/LBL(\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Paradoxically, one B-ALL/LBL (Case-28) showed a high level of SHM (86% identity to the germline) in its rearranged \u003cem\u003eIGHV\u003c/em\u003e gene despite the absence of mutations in all SHM target genes investigated (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, Table \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAmong the 3 DLBCL-NOS, one case (Case-21) with \u003cem\u003eIGH\u003c/em\u003e::\u003cem\u003eMYC\u003c/em\u003e and \u003cem\u003eBCL6\u003c/em\u003e translocation showed mutation in several SHM target genes (\u003cem\u003eMYC\u003c/em\u003e, \u003cem\u003eCXCR4\u003c/em\u003e, \u003cem\u003eHIST1H1C\u003c/em\u003e, \u003cem\u003eHIST1H2BK\u003c/em\u003e) and also evidence of SHM in its rearranged \u003cem\u003eIGHV\u003c/em\u003e gene (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). The remaining two cases were \u003cem\u003eBCL2\u003c/em\u003e translocation positive as shown by the presence of multiple \u003cem\u003eBCL2\u003c/em\u003e mutations (Case-20, FISH data unavailable) or interphase FISH investigation (Case-22). Case-20 showed the classic mutation pattern associated with FL and also mutations in several SHM target genes. While the other case (Case-22) showed \u003cem\u003eTP53\u003c/em\u003e and \u003cem\u003eNRAS\u003c/em\u003e mutations, but not the above mutations of transformed FL (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Unfortunately, it was impossible to further investigate this case due to a lack of tissue materials.\u003c/p\u003e \u003cp\u003eAll three HGBCL-NOS carried a \u003cem\u003eMYC\u003c/em\u003e translocation and showed mutations in several SHM target genes including \u003cem\u003eMYC\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). One case successfully investigated also showed frequent mutations in its rearranged \u003cem\u003eIGHV\u003c/em\u003e gene. These genetic data, together with CD10 expression in each case, suggest their origin from GC B cells.\u003c/p\u003e \u003cp\u003eLastly, the two cases unclassifiable between B-ALL/LBL and HGBCL-NOS were negative for \u003cem\u003eMYC\u003c/em\u003e, \u003cem\u003eBCL2\u003c/em\u003e and \u003cem\u003eBCL6\u003c/em\u003e translocation, but each harboured a \u003cem\u003eTP53\u003c/em\u003e mutation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). One case successfully investigated showed a moderate level of somatic mutations in its rearranged \u003cem\u003eIGHV\u003c/em\u003e gene (Table \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003e), and this case also displayed a variant in a known SHM target gene (\u003cem\u003eCIITA\u003c/em\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eSignificant difference in mutation load of SHM target genes between TdT-positive LBCL and B-ALL/LBL\u003c/h2\u003e \u003cp\u003eAs the number of B-ALL/LBL investigated in the present study was small, we compared the mutation load of a common set of SHM target genes between our TdT-positive LBCL (19 DLBCL/HGBCL-\u003cem\u003eMYC\u003c/em\u003e/\u003cem\u003eBCL2\u003c/em\u003e, 6 DLBCL/HGBCL-NOS) and a larger cohort of B-ALL cases (n\u0026thinsp;=\u0026thinsp;1716) from a previous study (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). As expected, the mutation load of SHM target genes was significantly higher in DLBCL/HGBCL-\u003cem\u003eMYC\u003c/em\u003e/\u003cem\u003eBCL2\u003c/em\u003e and DLBCL/HGBCL-NOS than B-ALL (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2.2E-16, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2.98E-05 respectively) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB), with the vast majority (23/25\u0026thinsp;=\u0026thinsp;92%) of the DLBCL/HGBCL-\u003cem\u003eMYC\u003c/em\u003e/\u003cem\u003eBCL2\u003c/em\u003e and DLBCL/HGBCL-NOS harboured a mutation load above the mean plus 2 standard deviations of the B-ALL group. Similarly, the overall mutation load among the 187 genes investigated was significantly higher in DLBCL/HGBCL-\u003cem\u003eMYC\u003c/em\u003e/\u003cem\u003eBCL2\u003c/em\u003e and DLBCL/HGBCL-NOS than B-ALL (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2.2E-16, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3.30E-06 respectively) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003cb\u003eClonal evolution of TdT-positive HGBCL from FL or\u003c/b\u003e \u003cb\u003eIGH\u003c/b\u003e::\u003cb\u003eBCL2\u003c/b\u003e \u003cb\u003epositive premalignant B-cells\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAmong the TdT-positive DLBCL/HGBCL investigated, two cases (Case-06 \u0026amp; 07) had metachronous lymphomas with biopsies available for comparative mutation analysis, and one further case (Case-16) with a single microscopic nodule of FL component, thus allowing investigation of their evolutionary history.\u003c/p\u003e \u003cp\u003eCase-06 (45-year-old, male) had a 1-year history of FL and presented with malaise and a right axillary lymph node enlargement. An excision biopsy (Case-06-DLBCL) showed a DLBCL with a GC phenotype (CD10+, BCL6+), TdT-, and was negative for \u003cem\u003eMYC\u003c/em\u003e translocation. The patient was treated with 6 cycles of O-CHOP (obinutuzumab, plus cyclophosphamide, doxorubicin, prednisolone and vincristine) and achieved complete metabolic remission (CMR). Two years later, the patient was suspected with a lymphoma relapse, a core biopsy of the right inguinal lymph node (not analysed by NGS due to insufficient material) and a further core biopsy of the left neck lymph node (Case-06-FL) a month later displayed classic FL grade 1\u0026ndash;2, and managed by active surveillance. Two months later, bone marrow trephine biopsy (Case-06-HGBCL) revealed infiltration of medium to large-sized immature lymphoid cells that were positive for CD19, CD79a, CD43, BCL2 and TdT (moderate to strong positivity in ~\u0026thinsp;80% tumour cells), CD20 (partial) and MUM1 (partial), but negative for CD10, CD34, CD117 and BCL6. The patient was then treated with UKALL14 induction phase 1 but showed no response. Further investigations by interphase FISH revealed \u003cem\u003eIGH\u003c/em\u003e::\u003cem\u003eMYC\u003c/em\u003e, \u003cem\u003eIGH\u003c/em\u003e::\u003cem\u003eBCL2\u003c/em\u003e and \u003cem\u003eBCL6\u003c/em\u003e rearrangement, hence supporting a diagnosis of DLBCL/HGBCL-\u003cem\u003eMYC\u003c/em\u003e/\u003cem\u003eBCL2\u003c/em\u003e/\u003cem\u003eBCL6.\u003c/em\u003e The patient was subsequently treated with 2 cycles of R-ICE, leading to complete cytogenetic response and followed by LACE autologous stem cell transplant. Ten months later, the patient showed lymphocytosis (20% lymphoblasts) in peripheral blood by flow cytometry despite no evidence of lymphadenopathy by CT imaging. The patient was treated with R-BP (rituximab, bendamstine, polatuzumab) and showed a significant clinical response, but died a month later.\u003c/p\u003e \u003cp\u003eTargeted NGS analysis revealed both common and distinct variants among the DLBCL, FL and HGBCL-\u003cem\u003eMYC\u003c/em\u003e/\u003cem\u003eBCL2\u003c/em\u003e/\u003cem\u003eBCL6\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). These metachronous lymphomas shared 16 common clonal variants including 11 potentially pathogenic and 5 benign/synonymous/UTR changes, hence confirming their clonal relationship. The DLBCL had 17 private clonal variants including 10 potentially pathogenic changes, while the FL and HGBCL-\u003cem\u003eMYC\u003c/em\u003e/\u003cem\u003eBCL2\u003c/em\u003e/\u003cem\u003eBCL6\u003c/em\u003e harboured different private variants. Intriguingly, the FL and HGBCL-\u003cem\u003eMYC\u003c/em\u003e/\u003cem\u003eBCL2\u003c/em\u003e/\u003cem\u003eBCL6\u003c/em\u003e had both common (6 potentially pathogenic and 5 benign/synonymous/UTR changes) and unique variants (5 variants in FL including 2 potentially pathogenic changes, 5 variants in HGBCL-\u003cem\u003eMYC\u003c/em\u003e/\u003cem\u003eBCL2\u003c/em\u003e/\u003cem\u003eBCL6\u003c/em\u003e including 2 potentially pathogenic changes). These mutation patterns indicated divergent evolution of these different lymphomas from a common lymphoma precursor (CLP) cell population, with the FL and HGBCL-\u003cem\u003eMYC\u003c/em\u003e/\u003cem\u003eBCL2\u003c/em\u003e/\u003cem\u003eBCL6\u003c/em\u003e derived from an intermediate subclone of the CLP cell population (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCase-07 (42-year-old, male) was a referral, a lymph node excision biopsy (Case-07-FL) showed a FL grade 2, which was BCL2+, CD10+, but TdT-. The patient was treated with 3 cycles of R-CHOP and then switched to 2 cycles of R-ICE due to disease progression but showed no response. A further biopsy of an inguinal lymph node 4 months later showed a high-grade transformation with the large cell component being CD20-, BCL2+, CD10+ (weak), BCL6-, MUM1+, Ki67+ (100%), TdT- and TP53+. The patient was treated with 1 cycle of R-DHAP and venetoclax, and then switched to R-GemOx\u0026thinsp;+\u0026thinsp;Polivy (polatuzumab-vedotin) due to disease progression. Subsequently, the patient underwent abdominal exploration due to suspected splenic rupture, which instead revealed a peri-gastric tumour mass. A biopsy of the tumour mass (Case-07-HGBCL) showed a transformed lymphoma with large cell component being BCL2+ (50%), TdT+ (60%), MYC+ (80%). Interphase FISH showed both \u003cem\u003eIGH\u003c/em\u003e::\u003cem\u003eMYC\u003c/em\u003e and \u003cem\u003eBCL2\u003c/em\u003e translocation, thus supporting a diagnosis of HGBCL-\u003cem\u003eMYC\u003c/em\u003e/\u003cem\u003eBCL2.\u003c/em\u003e The patient died one week later after diagnosis. Targeted sequencing analysis of the initial FL and peri-gastric HGBCL-\u003cem\u003eMYC\u003c/em\u003e/\u003cem\u003eBCL2\u003c/em\u003e-DH revealed 16 common clonal variants (5 potentially pathogenic and 11 benign/synonymous/UTR changes), and also 4 and 7 private variants respectively. These, together with identical \u003cem\u003eIGH\u003c/em\u003e rearrangements between the two lymphomas, indicated their divergent evolution from a CLP cell population (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eCase-16 (77-year-old, male) presented with left leg swelling with multiple enlarged lymph nodes in the left groin. Multiple core biopsies of the lymph nodes showed HGBCL with a single microscopic nodule of FL component. The HGBCL cells were positive for PAX5, CD20 (~\u0026thinsp;50%), CD10, BCL2, MUM1, MYC (100%) and TdT (~\u0026thinsp;40%), but negative for CD34, BCL6, while the FL cells were diffusely positive for PAX5, CD20, CD10, BCL2, BCL6, partially expressed MUM1 (~\u0026thinsp;30%) and MYC (~\u0026thinsp;40%), but were negative for TdT and CD34 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Interphase FISH showed both \u003cem\u003eMYC\u003c/em\u003e and \u003cem\u003eBCL2\u003c/em\u003e translocation in HGBCL and FL, with \u003cem\u003eMYC\u003c/em\u003e translocation in the FL component only seen in scattered large nuclei. Clonal analysis of the rearranged \u003cem\u003eIG\u003c/em\u003e genes based on microdissected HGBCL and FL components showed identical sized \u003cem\u003eIGH\u003c/em\u003e and \u003cem\u003eIGK\u003c/em\u003e products (Figure \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e), thus confirming their clonal relationship. Together, these findings suggest that the HGBCL originated from the FL following acquisition of \u003cem\u003eMYC\u003c/em\u003e translocation. In line with this speculation, the HGBCL harboured characteristic mutations associated with FL, high-grade transformation and SHM activities (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eAberrant TdT expression and MYC translocation\u003c/h2\u003e \u003cp\u003eSince majority of TdT-positive DLBCL/HGBCL originate from \u003cem\u003eIGH\u003c/em\u003e::\u003cem\u003eBCL2\u003c/em\u003e positive FL or its precursor cell population following acquisition of a \u003cem\u003eMYC\u003c/em\u003e translocation, this raises the question of whether the aberrant TdT expression might be due to dysregulated transcriptional activity of highly expressed MYC. To investigate this and identify appropriate cell lines for further \u003cem\u003ein vitro\u003c/em\u003e studies, we examined TdT expression in 10 DLBCL (including 5 cell lines with \u003cem\u003eMYC/BCL2-\u003c/em\u003eDH and an additional line with \u003cem\u003eMYC\u003c/em\u003e translocation) and 1 Burkitt lymphoma cell line by immunocytochemistry. None of these cell lines showed any positive TdT staining. Additionally, extensive bioinformatics search did not reveal any evidence that TdT might be a potential MYC transcriptional target.\u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThe differential diagnosis of TdT positive DLBCL/HGBCL and B-ALL/LBL may pose profound problems due to overlapping features of these two diagnostic categories/entities especially when \u003cem\u003eMYC\u003c/em\u003e rearrangements are encountered in a CD34-negative aggressive B-cell neoplasm in an adolescent or young adult patient. By investigation of a spectrum of TdT-positive B-cell lymphomas including DLBCL/HGBCL-\u003cem\u003eMYC\u003c/em\u003e/\u003cem\u003eBCL2\u003c/em\u003e, DLBCL-NOS, HGBCL-NOS, B-ALL/LBL and cases unclassified, the present study has highlighted several points that would aid differential diagnosis between TdT-positive DLBCL/HGBCL and B-ALL/LBL.\u003c/p\u003e \u003cp\u003eLoss of expression of pan B-cell antigens, such as CD20, has been used to argue for a diagnosis of B-ALL/LBL, while the expression of mature B-cell antigens such as BCL6 and MUM1 favours the diagnosis of mature rather than precursor B-cell lymphoma. However, none of these immunophenotypic features is specific enough for reliable differential diagnosis between TdT-positive LBCL/HGBCL and B-ALL/LBL. Our data clearly demonstrate that a high proportion (15/23\u0026thinsp;=\u0026thinsp;65%) of TdT-positive LBCL/HGBCL show a complete or partial loss of CD20 expression, while MUM1 can also be positive in B-ALL/LBL (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Nonetheless, CD34 is consistently negative in TdT-positive LBCL, but a lack of CD34 expression can occur in a high proportion of B-ALL/LBL as shown in the present and previous study (\u003cspan additionalcitationids=\"CR22 CR23\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eApart from the detection of chromosomal translocations, mutation analysis is highly valuable in differential diagnosis between TdT-positive LBCL and B-ALL/LBL. Like conventional DLBCL/HGBCL-\u003cem\u003eMYC\u003c/em\u003e/\u003cem\u003eBCL2\u003c/em\u003e, the TdT-positive cases with \u003cem\u003eMYC\u003c/em\u003e/\u003cem\u003eBCL2\u003c/em\u003e translocation harboured a characteristic mutation profile, including mutation signatures associated with FL and also its high-grade transformation (\u003cspan additionalcitationids=\"CR9 CR10 CR11\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). TdT-positive DLBCL-NOS and HGBCL-NOS also show frequent mutations commonly seen in these entities albeit less striking as those of DLBCL/HGBCL-\u003cem\u003eMYC\u003c/em\u003e/\u003cem\u003eBCL2\u003c/em\u003e. Furthermore, except for one case (Case-22), all other DLBCL-NOS and HGBCL-NOS exhibit frequent mutations in the SHM target genes, along with CD10 expression indicating their origin from GC B cells. This is also supported by findings of frequent mutations in their rearranged \u003cem\u003eIGHV\u003c/em\u003e genes in each case successfully investigated. In contrast, B-ALL/LBL had few mutations, lacking somatic variants associated SHM activities as they originate from precursor B cells not yet undergone antigen affinity maturation in the peripheral lymphoid tissues (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAs targeted sequencing by NGS is increasingly used in diagnosis and sub-classification of B-cell lymphoma, it is important to include genes targeted by the SHM machinery and document their somatic changes as discussed above. It is necessary to report all somatic variants identified including both synonymous and nonsynonymous variants, as well as those present in the 5\u0026rsquo;UTR and intronic regions to maximise their utility as biomarkers in delineating the dichotomy of cell-of-origin between pre-GC and GC/post-GC B cells. This study serves as a proof of principle. Future studies are required to standardise the list of genes and their genomic regions to be covered by NGS, and also to establish the optimal method for quantifying somatic variants and their threshold value for reliable prediction of lymphoma cell-of-origin.\u003c/p\u003e \u003cp\u003eOf the two cases with diagnostic ambiguity between B-ALL/LBL and HGBCL-NOS, the genetic data obtained failed to provide any strong evidence for their definitive diagnosis. Apart from \u003cem\u003eTP53\u003c/em\u003e mutation, both cases lack the other mutations seen in HGBCL-NOS. Case-27 showed a single mutation in a SHM target gene (\u003cem\u003eCIITA\u003c/em\u003e) and a moderate level (95%) of SHM in its rearranged \u003cem\u003eIGHV\u003c/em\u003e, while the other case showed no mutation in the SHM target genes investigated. Such low level of somatic mutations in the rearranged \u003cem\u003eIGHV\u003c/em\u003e genes has been reported in B-ALL previously (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). These, together with their variable expression of pan-B-cell (CD20) and mature B-cell (BCL6, MUM1) markers may argue for the existence of grey zone cases between B-ALL/LBL and HGBCL-NOS.\u003c/p\u003e \u003cp\u003eThe mechanism for aberrant TdT expression in LBCL is unclear. Given that aberrant TdT expression is often associated with partial or complete loss of expression of pan-B cell antigen (CD20), it is possible that these cases develop a defect in the transcriptional network that controls the B-cell programme. In line with this speculation, there is no evidence of loss of CD20 expression in conventional DLBCL/HGBCL, and this is further reinforced by a retrospective review of 15 TdT-negative and \u003cem\u003eMYC\u003c/em\u003e translocation-positive DLBCL/HGBCL. Among the transcriptional factors, \u003cem\u003ePAX5\u003c/em\u003e genetic changes including mutation p.P80R are frequently seen in B-ALL/LBL and these genetic changes are thought to affect B-cell programming, thereby contributing to leukaemia development (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). \u003cem\u003ePAX5\u003c/em\u003e is a SHM target and multiple mutations were observed in a case of HGBCL-\u003cem\u003eMYC\u003c/em\u003e/\u003cem\u003eBCL2\u003c/em\u003e-DH (Case-08), affecting the paired box domain and also the highly conserved octapeptide motif (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, Figure \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003e). In this context, it is worth noting mutations in several other transcriptional factors that regulate early B-cell development, such as \u003cem\u003eETS1\u003c/em\u003e (SHM target), \u003cem\u003eFOXO1\u003c/em\u003e (SHM target), \u003cem\u003eTCF3\u003c/em\u003e (encoding E2A) and \u003cem\u003ePOU2F2\u003c/em\u003e (encoding OCT2)(\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, Figure \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003e). It remains to be investigated whether mutations in these transcriptional factors and related regulators may dysregulate the B-cell programme, causing aberrant TdT expression and also perturbing B-cell antigen expression.\u003c/p\u003e \u003cp\u003eIn conclusion, our findings demonstrate that mutation profile analysis, including SHM target genes, is highly valuable in differential diagnosis between TdT-positive DLBCL/HGBCL and B-ALL/LBL.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements:\u003c/strong\u003e The authors would like to thank Dr Ana Toribio and Jessica Ferreira Gouveia for their assistance with Illumina sequencing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors contributions:\u003c/strong\u003e\u0026nbsp; \u0026nbsp;FISH, targeted NGS, data collection and analyses: \u0026nbsp;MMT, FC, ZC, EM, JM, AES, FG, MQD; Case contribution and pathology: LRB, AW, KSK, CE, ES, LH, LK, JTG, CL, LX, LM, LP, LMRG, NLH, TG, JK, WK, IO, AR and GO; Study conception, coordination and research funding: MQD, LRB, GO; \u0026nbsp; Manuscript writing and preparation: MQD \u0026amp; MMT with contributions from all authors. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest:\u0026nbsp;\u003c/strong\u003e The authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional Information:\u0026nbsp;\u003c/strong\u003eAll core data generated or analysed during this study are included in this published article, and additional raw data are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eDuffield AS, Dogan A, Amador C, Cook JR, Czader M, Goodlad JR, et al. Progression of follicular lymphoma and related entities: Report from the 2021 SH/EAHP Workshop. Am J Clin Pathol. 2023;159:572\u0026ndash;97. \u003c/li\u003e\n\u003cli\u003eSwerdlow SH, Campo E, Harris NL, Jaffe ES, Pileri SA, Stein H, Thiele J (Eds.): World Health Organization classification of Tumours of Haematopoietic and Lymphoid Tissues. Revised 4th ed. Lyon: IARC; 2017.\u003c/li\u003e\n\u003cli\u003eBhavsar S, Liu YC, Gibson SE, Moore EM, Swerdlow SH. Mutational Landscape of TdT+Large B-cell Lymphomas Supports Their Distinction from B-lymphoblastic Neoplasms: A Multiparameter Study of a Rare and Aggressive Entity. American Journal of Surgical Pathology. 2022;46:71\u0026ndash;82. \u003c/li\u003e\n\u003cli\u003eKhanlari M, Medeiros LJ, Lin P, Xu J, You MJ, Tang G, et al. Blastoid high-grade B-cell lymphoma initially presenting in bone marrow: a diagnostic challenge. Mod Pathol. 2022;35:419\u0026ndash;26.\u003c/li\u003e\n\u003cli\u003eOk CY, Medeiros LJ, Thakral B, Tang G, Jain N, Jabbour E, et al. High-grade B-cell lymphomas with TdT expression: a diagnostic and classification dilemma. Mod Pathol. 2019;32:48\u0026ndash;58. \u003c/li\u003e\n\u003cli\u003eMoench L, Sachs Z, Aasen G, Dolan M, Dayton V, Courville EL. Double- and triple-hit lymphomas can present with features suggestive of immaturity, including TdT expression, and create diagnostic challenges. Leuk Lymphoma. 2016;57:2626\u0026ndash;35. \u003c/li\u003e\n\u003cli\u003eTooze R, Barrans S, Davies A, Dunleavy K, Gopal A, Lenz G, et al. Diffuse large B-cell lymphoma/high-grade B-cell lymphoma with \u003cem\u003eMYC\u003c/em\u003e and \u003cem\u003eBCL2\u003c/em\u003e rearrangements. The 5th Edition of WHO classification of tumours: Haematolymphoid tumours: IARC; 2024:476-80. \u003c/li\u003e\n\u003cli\u003eCucco F, Barrans S, Sha C, Clipson A, Crouch S, Dobson R, et al. Distinct genetic changes reveal evolutionary history and heterogeneous molecular grade of DLBCL with MYC/BCL2 double-hit. Leukemia. 2019;34:1329\u0026ndash;41. \u003c/li\u003e\n\u003cli\u003ePasqualucci L, Khiabanian H, Fangazio M, Vasishtha M, Messina M, Holmes AB, et al. Genetics of Follicular Lymphoma Transformation. Cell Rep. 2014;6:130\u0026ndash;40. \u003c/li\u003e\n\u003cli\u003eBouska A, Zhang W, Gong Q, Iqbal J, Scuto A, Vose J, et al. Combined copy number and mutation analysis identifies oncogenic pathways associated with transformation of follicular lymphoma. Leukemia. 2017;31:83\u0026ndash;91. \u003c/li\u003e\n\u003cli\u003eKridel R, Chan FC, Mottok A, Boyle M, Farinha P, Tan K, et al. Histological Transformation and Progression in Follicular Lymphoma: A Clonal Evolution Study. PLoS Med. 2016;13:e1002197. \u003c/li\u003e\n\u003cli\u003eOkosun J, B\u0026ouml;d\u0026ouml;r C, Wang J, Araf S, Yang CY, Pan C, et al. Integrated genomic analysis identifies recurrent mutations and evolution patterns driving the initiation and progression of follicular lymphoma. Nat Genet. 2014;46:176\u0026ndash;81. \u003c/li\u003e\n\u003cli\u003eWagener R, L\u0026oacute;pez C, Kleinheinz K, Bausinger J, Aukema SM, Nagel I, et al. IG- MYC + neoplasms with precursor B-cell phenotype are molecularly distinct from Burkitt lymphomas. Blood. 2018;132:2280\u0026ndash;5. \u003c/li\u003e\n\u003cli\u003eBrady SW, Roberts KG, Gu Z, Shi L, Pounds S, Pei D, et al. The genomic landscape of pediatric acute lymphoblastic leukemia. Nature Genetics. 2022;54:1376\u0026ndash;89. \u003c/li\u003e\n\u003cli\u003eLeongamornlert D, Gutierrez-Abril J, Lee SW, Barretta E, Creasey T, Gundem G, et al. Diagnostic utility of whole genome sequencing in adults with B-other acute lymphoblastic leukemia. Blood Adv. 2023;7:3862. \u003c/li\u003e\n\u003cli\u003eUeno H, Yoshida K, Shiozawa Y, Nannya Y, Iijima-Yamashita Y, Kiyokawa N, et al. Landscape of driver mutations and their clinical impacts in pediatric B-cell precursor acute lymphoblastic leukemia. Blood Adv. 2020;4:5165\u0026ndash;73. \u003c/li\u003e\n\u003cli\u003eCucco F, Clipson A, Kennedy H, Sneath Thompson J, Wang M, Barrans S, et al. 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CD34 is not Expressed by Blasts in a Third of B-ALL Patients and its Negativity is associated with Aberrant Marker Expression: A Retrospective Analysis. Asian Pac J Cancer Prev. 2021;22:919\u0026ndash;25. \u003c/li\u003e\n\u003cli\u003eAli Shah M, Ahmad U, Tariq Mahmood M, Ahmad AH, Abu Bakar M. Frequency of CD34 and CD10 Expression in Adolescent and Young Adult Patients Having Precursor B-cell Acute Lymphoblastic Leukemia and Its Correlation With Clinical Outcomes: A Single-Center Study. Cureus. 2022;14:e21261.\u003c/li\u003e\n\u003cli\u003eQiu L, Xu J, Lin P, Cohen EN, Tang G, Wang SA, et al. Unique pathologic features and gene expression signatures distinguish blastoid high-grade B-cell lymphoma from B-acute lymphoblastic leukemia/lymphoma. Haematologica. 2023;108:895\u0026ndash;9. \u003c/li\u003e\n\u003cli\u003eWood B, Gujral S. B-lymphoblastic leukaemia/lymphoma. The 5th Edition of WHO classification of tumours: Haematolymphoid tumours: IARC; 2024:330\u0026ndash;5.\u003c/li\u003e\n\u003cli\u003eLi A, Rue M, Zhou J, Wang H, Goldwasser MA, Neuberg D, et al. Utilization of Ig heavy chain variable, diversity, and joining gene segments in children with B-lineage acute lymphoblastic leukemia: implications for the mechanisms of VDJ recombination and for pathogenesis. Blood. 2004;103:4602\u0026ndash;9. \u003c/li\u003e\n\u003cli\u003eBastian L, Schroeder MP, Eckert C, Schlee C, Tanchez JO, K\u0026auml;mpf S, et al. PAX5 biallelic genomic alterations define a novel subgroup of B-cell precursor acute lymphoblastic leukemia. Leukemia. 2019;33:1895\u0026ndash;909. \u003c/li\u003e\n\u003cli\u003eGruenbacher S, Jaritz M, Hill L, Sch\u0026auml;fer M, Busslinger M. Essential role of the Pax5 C-terminal domain in controlling B cell commitment and development. J Exp Med. 2023;220:e20230260.\u003c/li\u003e\n\u003cli\u003eLaidlaw BJ, Cyster JG. Transcriptional regulation of memory B cell differentiation. Nature Reviews Immunology. 2020;21:209\u0026ndash;20. \u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table 1","content":"\u003cp\u003e\u003cstrong\u003eTable 1. Summary of clinical and laboratory results of cases 26-31 investigated.\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"740\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 103px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCase-26\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCase-27\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14.7096%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCase-28\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCase-29\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCase-30\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13.8442%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCase-31\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 103px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDiagnosis\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eUnclassified\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eUnclassified\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14.7096%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eB-ALL/LBL\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eB-ALL/LBL\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eB-ALL/LBL\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13.8442%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eB-ALL/LBL\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 103px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAge /sex\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003e70 year / F\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e74 year / M\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14.7096%;\"\u003e\n \u003cp\u003e63 year / F\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003e4 year / F\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003e80 year /F\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13.8442%;\"\u003e\n \u003cp\u003e75 year / M\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 103px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eClinical history\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003epancytopenia\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eB symptoms, splenomegaly, anaemia, right thyroid nodule with histopathology consistent with B-ALL/LBL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14.7096%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003epancytopenia\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003ethrombocytopenia and leukoerythroblastic picture\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13.8442%;\"\u003e\n \u003cp\u003epancytopenia and lytic bone lesion\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 103px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNodal \u0026amp; splenic involvement\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003eN/A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eSplenomegaly, no lymphadenopathy by CT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14.7096%;\"\u003e\n \u003cp\u003estage IV, wide lymphadenopathy, plus pancreas, nasopharynx, uterine and \u0026nbsp;peritoneal lesions\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003esplenomegaly, no lymphadenopathy by CT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003eN/A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13.8442%;\"\u003e\n \u003cp\u003eN/A\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 103px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBiopsy site\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003ebone marrow\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003ebone marrow\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14.7096%;\"\u003e\n \u003cp\u003elymph node\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003ebone marrow\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003ebone marrow\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13.8442%;\"\u003e\n \u003cp\u003ebone marrow\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 103px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eHistopathology\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003ediffuse infiltration by lymphoblastoid cells\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003ediffuse infiltration by lymphoblastoid cells\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14.7096%;\"\u003e\n \u003cp\u003ediffuse infiltration by lymphoblastoid cells\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003ediffuse infiltration by lymphoblastoid cells\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003ediffuse infiltration by large atypical lymphocytes with moderate pleomorphism\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13.8442%;\"\u003e\n \u003cp\u003ediffuse infiltration by lymphoblastoid cells\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"7\" style=\"width: 740px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eImmunophenotype\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 103px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePAX5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003ediffuse positive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003ediffuse positive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14.7096%;\"\u003e\n \u003cp\u003eN/A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003eN/A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003ediffuse positive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13.8442%;\"\u003e\n \u003cp\u003eN/A\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 103px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCD20\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003epositive in ~90%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003epositive in ~30%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14.7096%;\"\u003e\n \u003cp\u003epositive in ~50%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003eweak, patchy positive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003ediffuse positive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13.8442%;\"\u003e\n \u003cp\u003ediffuse positive\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 103px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCD79a\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003ediffuse positive with variable intensity\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003ediffuse positive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14.7096%;\"\u003e\n \u003cp\u003ediffuse positive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003ediffuse positive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003ediffuse positive with variable intensity\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13.8442%;\"\u003e\n \u003cp\u003epositive in ~30%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 103px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCD10\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003ediffuse positive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003ediffuse positive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14.7096%;\"\u003e\n \u003cp\u003ediffuse positive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003ediffuse positive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003ediffuse positive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13.8442%;\"\u003e\n \u003cp\u003ediffuse positive\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 103px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBCL6\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003eweak -moderate positive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003epositive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14.7096%;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003eweak partial positive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13.8442%;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 103px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMUM1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003estrong positive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003efocal positive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14.7096%;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003epositive in ~40%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003eweak positive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13.8442%;\"\u003e\n \u003cp\u003emoderate to strong positive in ~80%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 103px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eKi67\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003e90%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e90%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14.7096%;\"\u003e\n \u003cp\u003e60-70%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003e100%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003e60%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13.8442%;\"\u003e\n \u003cp\u003e~90%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 103px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMYC\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003e60%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e90%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14.7096%;\"\u003e\n \u003cp\u003e60%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003e80%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003e60%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13.8442%;\"\u003e\n \u003cp\u003e60%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 103px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTDT\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003estrong positive in ~100%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003evariable positivity in ~50%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14.7096%;\"\u003e\n \u003cp\u003emoderate positive in ~95%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003emoderate to strong positivity in ~40%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003estrong positive in 100%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13.8442%;\"\u003e\n \u003cp\u003e80%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 103px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCD34\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14.7096%;\"\u003e\n \u003cp\u003ediffuse positive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003ediffuse positive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13.8442%;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"7\" style=\"width: 740px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTranslocation\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 103px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMYC\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14.7096%;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003eIGL::MYC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13.8442%;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 103px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBCL2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14.7096%;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13.8442%;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 103px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBCL6\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14.7096%;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13.8442%;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 103px;\"\u003e\n \u003cp\u003eOthers\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003eN/A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eno FISH evidence of BCR::ABL1, ETV6::RUNX1, TCF3::PBX1, TCF3::HLF \u0026amp; KMT2A translocation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14.7096%;\"\u003e\n \u003cp\u003eN/A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003eN/A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003eN/A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13.8442%;\"\u003e\n \u003cp\u003eN/A\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 103px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 14.7096%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 102px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 13.8442%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 103px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMutation load\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003emuch lower than DLBCL/HGBCL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003emuch lower than DLBCL/HGBCL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14.7096%;\"\u003e\n \u003cp\u003emuch lower than DLBCL/HGBCL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003emuch lower than DLBCL/HGBCL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003emuch lower than DLBCL/HGBCL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13.8442%;\"\u003e\n \u003cp\u003emuch lower than DLBCL/HGBCL\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 103px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAny pathognomonic mutations\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003eno\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eno\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14.7096%;\"\u003e\n \u003cp\u003eno\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003eno\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003eno\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13.8442%;\"\u003e\n \u003cp\u003eno\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 103px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSHM driven mutation\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003eno\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003esingle synonymous change\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14.7096%;\"\u003e\n \u003cp\u003eno\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003eno\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003eno\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13.8442%;\"\u003e\n \u003cp\u003eno\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 103px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eIGHV mutation\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003eN/A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e95% identity to germline\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14.7096%;\"\u003e\n \u003cp\u003e86% identity to germline\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003eno\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003eno\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13.8442%;\"\u003e\n \u003cp\u003eN/A\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 103px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 14.7096%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 102px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 13.8442%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 103px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTreatment \u0026amp; outcome\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003eN/A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003etreated with UKALL60+ protocol and showed a short period of morphological and cytogenetic remission, then bone marrow relapse and treated with 3 cycles of Inotuzumab and achieved morphological and cytogenetic remission.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14.7096%;\"\u003e\n \u003cp\u003etreated with RCHOP, \u0026nbsp;RDHAC then autografted. \u0026nbsp;\u003cbr\u003e\u0026nbsp;2 years later, lymphoma relapsed with 15% of circulant blast and 98% in bone marrow aspiration.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003etreated as Burkitt\u0026rsquo;s leukaemia with chemotherapy, achieved a short period of remission, then relapsed and allografted, further disease relapse and followed by palliative chemotherapy, died two years after initial diagnosis.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003epatient died shortly after diagnosis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13.8442%;\"\u003e\n \u003cp\u003etreated with 2 cycles of R-mini-CVD, then transferred to hospice.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eB-ALL/LBL: B-lymphoblastic leukaemia/lymphomas; DLBCL: diffuse large B-cell lymphoma; HGBCL: high grade B-cell lymphoma; F: female; M: male; N/A: not available.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"TdT expression, High grade B-cell lymphoma, B-lymphoblastic leukaemia/lymphoma, somatic hypermutation, differential diagnosis","lastPublishedDoi":"10.21203/rs.3.rs-6172652/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6172652/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTdT is occasionally expressed in large B-cell lymphoma (LBCL), and this causes difficulty in differential diagnosis from B-lymphoblastic leukaemia/lymphomas (B-ALL/LBL). We reviewed 31 cases of TdT-positive LBCL and B-ALL/LBL, and their final diagnosis included 19 diffuse large/high-grade BCL with \u003cem\u003eMYC\u003c/em\u003e and \u003cem\u003eBCL2\u003c/em\u003e rearrangements (DLBCL/HGBCL-\u003cem\u003eMYC\u003c/em\u003e/\u003cem\u003eBCL2\u003c/em\u003e), 3 DLBCL-NOS, 3 HGBCL-NOS, 4 B-ALL/LBL and 2 unclassifiable cases. TdT was variably expressed in all these cases, without any clear demarcation among different groups. Loss or partial loss of CD20 expression was seen in 13/17 DLBCL/HGBCL-\u003cem\u003eMYC\u003c/em\u003e/\u003cem\u003eBCL2\u003c/em\u003e, 2/3 HGBCL-NOS, 2/2 unclassified, albeit not in DLBCL-NOS. Expression of BCL6 and/or MUM1 was seen in 3/4 B-ALL/LBL and 2/2 unclassified. Next generation sequencing revealed characteristic mutations associated with follicular lymphoma and its high-grade transformation in each DLBCL/HGBCL-\u003cem\u003eMYC\u003c/em\u003e/\u003cem\u003eBCL2\u003c/em\u003e, and also frequent variants in genes targeted by somatic hypermutation (SHM) in almost all DLBCL/HGBCL-\u003cem\u003eMYC\u003c/em\u003e/\u003cem\u003eBCL2\u003c/em\u003e, DLBCL-NOS and HGBCL-NOS but one case. In contrast, such mutations were absent in B-ALL/LBL. There were no pathognomonic mutations in the two unclassifiable cases although one showed a moderate level of somatic mutations in its rearranged \u003cem\u003eIGHV\u003c/em\u003e. In conclusion, mutation profiling analysis including the SHM target genes is highly valuable in differential diagnosis between TdT-positive LBCL and B-ALL/LBL.\u003c/p\u003e","manuscriptTitle":"Mutation profiling in differential diagnosis between TdT-positive high grade/large B-cell lymphoma and B-lymphoblastic leukaemia/lymphoma","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-17 10:00:47","doi":"10.21203/rs.3.rs-6172652/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8a18ef5e-50ca-44ec-9145-56eb3860f036","owner":[],"postedDate":"March 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":45511811,"name":"Health sciences/Medical research/Translational research"},{"id":45511812,"name":"Health sciences/Pathogenesis/Clinical genetics/Cancer genetics"},{"id":45511813,"name":"Health sciences/Diseases/Cancer/Haematological cancer/Lymphoma/Non-hodgkin lymphoma/B-cell lymphoma"}],"tags":[],"updatedAt":"2025-04-03T09:41:09+00:00","versionOfRecord":[],"versionCreatedAt":"2025-03-17 10:00:47","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6172652","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6172652","identity":"rs-6172652","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}