The Intersection of Lynch Syndrome and Hematological Malignancies: A Rare Short Report.

A 75‐year‐old woman was admitted to our hospital with multiple myeloma (MM) and AML that had progressed from MDS. She had a history of multiple tumors. She had several colorectal adenomatous polyps removed by annual colonoscopy since the age of 40. She underwent hysterectomy for endometriosis at the age of 50. She was diagnosed with bile duct cancer at the age of 60 and ureteral cancer at the age of 73, and underwent surgery for each cancer. At the age of 72, she was diagnosed with MM and received chemotherapy (daratumumab, melphalan, bortezomib, and lenalidomide). Two years later, she also received chemotherapy (azacitidine) for MDS. MDS was refractory and progressed to AML after several months. In addition, she was recently diagnosed with gastric cancer and received palliative local radiotherapy. She had a family cancer history of young‐onset colorectal cancer, fulfilling Amsterdam Criteria II [ 4 ] (Figure  1 ). Pedigree chart. The case is indicated with II‐8. A large number of family members succumbed to young‐onset colorectal cancer or brain tumors. Her niece (III‐11) was diagnosed with LS. Based on her medical history and family history of cancer, LS was suspected. Immunohistochemistry (IHC) for MMR proteins was performed on previously resected bile duct and ureter cancer specimens and showed the loss of staining for the MSH2 and MSH6 proteins, suggesting LS. After genetic counseling and written informed consent, the patient underwent germline genetic testing, including MLH1 , MSH2 , MSH6 , PMS2 , and EPCAM . During testing, the percentage of myeloblasts in peripheral blood was 2%.

Tomomi Oka was responsible for study conception, drafting the article, and genetic counseling of the patient and family members. Takeshi Nakajima was responsible for proofreading the article and genetic counseling of the patient and family members. Makoto Iwasaki was responsible for the analysis and interpretation of data and drawing figures. June Takeda was responsible for the analysis and interpretation of data and drawing figures. Masako Torishima was responsible for the genetic counseling of family members. Maki Sakurada was responsible for the analysis of data. Takuya Shimizu was responsible for the analysis of data. Takero Shindo was responsible for the analysis of data and reviewing the article. Masakazu Fujimoto was responsible for the analysis and interpretation of data. Shinya Otsuki was responsible for reviewing the article. Hironori Haga was responsible for reviewing the article. Kokichi Sugano was responsible for the analysis and interpretation of data and reviewing the article. Miho Ando was responsible for the analysis of data. Chisaki Mizumoto was responsible for management of the patient. Junya Kanda was responsible for reviewing the article. Yasuhito Nannya was responsible for reviewing the article. Seishi Ogawa was responsible for reviewing the article. Akifumi Takaori‐Kondo was responsible for reviewing the article. Shinji Kosugi was responsible for reviewing the article.

The authors confirm that ethical approval was not required for this study.

Informed consent was obtained from the patient for the publication of this case report and any related images.

Multiplex ligation‐dependent probe amplification (MLPA) revealed extensive homozygous deletions ranging from Exon 9 of EPCAM to Exons 1–6 of MSH2 (Figure  2a , bottom). EPCAM :c.(?_904−1)_(*415_?)del and MSH2 :c.(?_ −125)_(1076+1_1077−1)del were considered to be present in both alleles based on the presence of only 3%–4% of the wild‐type sequence at this site. There were no mutations in MLH1 , MSH6 , and PMS2 . (a) Genetic test results of the patient at MM and AML (MLPA). An extensive deletion ranging from exon 9 of EPCAM to exons 1–6 of MSH2 was heterozygous at MM (the top). The deletion then changed to homozygous at AML (the bottom). (b) Results of target sequencing at MM and MDS (CN analysis). No CN abnormality in chromosome 2 was observed at MM (top). However, a CN abnormality in the short arm region of chromosome 2 (including EPCAM and MSH2 ) was not noted at MDS, where an allele imbalance was detected, suggesting uniparental disomy (bottom). (c) Genetic test results of the niece (MLPA, middle). Heterozygous deletions were noted at exon 9 of EPCAM and exons 1–6 of MSH2 , which confirmed the diagnosis of LS. The deletion range was exactly the same as that of the patient. AML, acute myeloid leukemia; CN, copy number; LS, Lynch syndrome; MLPA, multiplex‐ligation dependent probe amplification; MM, multiple myeloma. At the time of genetic testing, the patient had received multiple blood transfusions for severe anemia and thrombocytopenia. However, cell‐free DNA from blood transfusions was not considered to have affected the results obtained because isolated lymphocytes were used in genetic testing. Constitutional mismatch repair deficiency (CMMRD) is an autosomal recessive disorder caused by homozygous or compound heterozygous pathogenic germline variants in one of the four MMR genes [ 5 ]. It is rare and is associated with an increased risk of brain tumors, hematologic malignancies, colorectal cancer, and a number of other cancers in children, adolescents, and young adults. In the present case, genetic testing did not show the complete loss of MSH2 . IHC for MMR proteins showed positive staining for MSH2 in normal tissues. Therefore, our case was not considered to be CMMRD. Samples were screened for major driver mutations associated with myeloid neoplasms [ 6 , 7 , 8 ], and 1216 SNP sites for copy number (CN) detection [ 9 ] using targeted capture sequencing as previously described [ 10 ]. Multiple well‐known driver mutations of MDS, including KRAS (p.Q61H), DNMT3A (p.C351fs), SF3B1 (p.R625C), RUNX1 (p.463fs), BCOR (p.P1587fs), and BCORL1 (p.S1679fs), were detected in bone marrow cells during the development of MDS. In addition, uniparental disomy (UPD) in the short arm region of Chromosome 2 was identified by a CN analysis (Figure  2b , bottom). It was subsequently discovered that her niece had developed colorectal cancer at a young age and had been diagnosed with LS by genetic testing. Her niece's genetic test results showed extensive heterozygous deletions ranging from Exon 9 of EPCAM to Exons 1–6 of MSH2 , the exact same variant as the patient (Figure  2c ). Based on her niece's results, LS was strongly suspected. To reach an accurate diagnosis, we considered to examine germline genetic testing with another sample of normal tissue from the patient. However, the patient died 1 week later, and it was impossible to obtain another sample. Fortunately, DNA samples from the peripheral blood of the patient at the time of the MM diagnosis had been preserved and were used to investigate whether the loss of EPCAM and MSH2 was heterozygous or homozygous. Since there were no plasma cells in peripheral blood at the time of testing, the stored sample was treated as normal blood. As a precaution, targeted capture sequencing was also performed on the stored sample to confirm whether any of the variants present at the onset of MDS were detected. DNMT3A (p.R882H) was identified, which was different from the variant present at the onset of MDS. This indicated that MDS had not yet developed and that tumor clones had been replaced. Genetic testing results showed heterozygosity in known pathologic variants (Figure  2a , top), which confirmed the diagnosis of LS. We isolated cells that co‐expressed CD38 and CD56 from bone marrow at the onset of MM (Figure S1 ), and analyzed them using targeted capture sequencing. The results obtained revealed a KRAS mutation (p.Q61H). UPD in the short arm region of Chromosome 2 was not detected (Figure  2b , top). IHC for MMR proteins was performed on bone marrow specimens at the onset of MM and MDS. The former indicated that all cells were stained for the MSH2 and MSH6 proteins. The latter showed the loss of staining for the MSH2 and MSH6 proteins in myeloid cells (Figure S2a,b ). To examine the relationship between the deficiency of MSH2 and this hematologic malignancy, we assessed the MSI of blood tumor cells at MDS. We compared five loci (BAT‐25, BAT‐26, NR‐21, NR‐21, NR‐24, and MONO‐27) in the MDS sample with those in the MM sample and a control using MSI‐analysis‐system 1–2 (Promega). The results obtained showed that MSI was negative (Figure S3 ).

The relationship between MMR genes and the development of hematologic malignancies has not yet been clarified. The feature of CMMRD suggests that the loss of function of both MMR genes may be associated with hematologic malignancies. Furthermore, previous studies reported that relapsed and refractory MDS/AML as well as t‐MDS/AML were more closely associated with MSI than de novo MDS/AML [ 2 , 3 ]. MMR gene mutations may have occurred in some hematologic malignancies. On the other hand, the relationship between MSI and the development of AML is still under active discussion [ 11 , 12 ]. In addition, CMMRD appears to be mainly associated with malignancies of T‐cell origin [ 13 ]. In blood cells, the function of MMR genes may be differentiated by lineage. Findings such as high MSI or MMR protein loss may also be caused by other factors, including MMR gene methylation. Furthermore, some patients with AML may show the extensive methylation of MLH1 , but not MSH [ 14 ]. The confirmation of MMR gene dysfunctions in blood cells in patients with hematologic malignancies is very rare. Therefore, the role of MMR gene dysfunctions in the development of MDS/AML remains a subject of ongoing debate. In the present case, the function of MSH2 in myeloid cells was not lost in spite of the large homozygous deletion of MSH2 . However, we cannot completely rule out the possibility of an issue with the accuracy of the testing. A study suggests that BAT‐25 and BAT‐26 could not be sensitive markers of MSI in AML [ 15 ]. In conclusion, this case originally had a deletion of MSH2 in one allele of all blood cells due to LS. At the onset of MM, normal and myeloma cells were still both hetero‐deleted, while at the onset of MDS, the majority of blood cells were homo‐deleted. The MSH2 homo‐deletion occurred after the onset of MM (Table  1 ), suggesting that this mutation was not involved in the development of MM. Chemotherapies may have caused the other allele of blood cells to have the deletion of MSH2 ; however, it remains unclear whether the homo‐deletion of MSH2 is involved in the development of MDS. In the present case, we observed several mutations in driver genes for the development of MDS, which may play a significant role in its development. However, the LOH of MSH2 may also be involved in the pathogenesis of this disease, such as MDS being refractory and progressing to AML in the present case, even if it was not the direct cause of the myeloid malignancy. Furthermore, it may accelerate disease progression when combined with the co‐occurrence of changes in DNMT3A , RUNX1 , BCOR , and KRAS . Statements of MSH2 deletion, MMR protein, MSI, and gene mutations in every sample. BM (isorated MM cells) PB (normal cells) DNMT3A (p.C351fs) SF3B1 (p.R625C) RUNX1 (p.P463fs) KRAS (p.Q61H) BCOR (p.P1587fs) BCORL1 (p.S1679fs) Abbreviations: AML, acute myeloid leukemia; BM, bone marrow; CNV, copy number; MDS, myelodysplastic syndrome; MM, multiple myeloma; MMR, mismatch repair; MSI, microsatellite instability; PB, peripheral blood; UPD, uniparental disomy. The interplay between LS and hematologic malignancies represents a critical area for further research. Future studies are warranted to confirm the impact of MMR variants on the pathogenesis/chemoresistance of myeloid neoplasms.

Lynch syndrome (LS) is an autosomal dominant disorder caused primarily by germline pathogenic variants of mismatch repair (MMR) genes. It causes a number of malignancies (related tumors), including colorectal, endometrial, ovarian, gastric, small intestinal, biliary tract, pancreatic, renal pelvis/ureter, and brain tumors [ 1 ]. Hematologic malignancies are not included as related tumors of LS because it has not yet been established whether the carcinogenesis of hematologic malignancies is associated with MMR genes. On the other hand, relapsed and refractory myelodysplastic syndrome (MDS)/acute myeloid leukemia (AML) and therapy‐related MDS/AML (t‐MDS/AML) were shown to be more closely associated with microsatellite instability (MSI) or dysfunctions in MMR proteins than de novo MDS/AML [ 2 , 3 ]. We encountered one LS case that developed hematologic malignancies with the loss of heterozygosity (LOH) in MMR genes. This rare LS case with AML had a large homozygous deletion in MMR genes.

Masako Torishima: Employment: KONICA MINOLTA JAPAN, INC. Seishi Ogawa: Funding: Japan Agency for Medical Research and Development, Japan Society for the Promotion of Science (JSPS), Chordia Therapeutics Inc., Eisai Co. Ltd., and Otsuka Pharmaceutical Co. Ltd. Shinji Kosugi: Employment: KONICA MINOLTA JAPAN, INC. The other authors declare no conflicts of interest.

Figure S1 : Gating positions for flow cytometry. Myeloma cells (DAPI−, CD3−, CD19‐ CD38+, CD56+) were isolated. Figure S2 : (a)Histopathological images of a bone marrow clot at MM (original magnification ÷600). Hematoxylin and eosin staining (left). Immunohistochemical staining for MSH6 (middle) and PMS2 (right), respectively.(b)Histopathological images of a bone marrow clot at MDS (original magnification ÷600). Hematoxylin and eosin staining (left). Immunohistochemical staining for MSH6 (top and middle), PMS2 (top and right), MSH2 (bottom and left), and MLH1 (bottom and right), respectively. Figure S3 : Results showing MSI at MM and MDS with bone marrow. Blue lines are the positions of the maximal peak.