Flow Cytometry-Aided Early Diagnosis and Alectinib Response in Advanced ALK-Positive Large B-Cell Lymphoma: A Case Report

Flow Cytometry-Aided Early Diagnosis and Alectinib Response in Advanced ALK-Positive Large B-Cell Lymphoma: A Case Report | 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 Case Report Flow Cytometry-Aided Early Diagnosis and Alectinib Response in Advanced ALK-Positive Large B-Cell Lymphoma: A Case Report Yudai Hayashi, Masao Tsukada, Marina Matsui, Daisuke Shinoda, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8042084/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 Anaplastic lymphoma kinase (ALK)-positive large B-cell lymphoma (LBCL) is an exceptionally rare subtype of B-cell lymphoma that typically arises independent of immunosuppression. Patients with stage IV disease have a particularly poor prognosis, with a median overall survival of approximately 1 year (95% confidence interval [CI], 0.8–1.5) and a 5-year survival rate of 5% (95% CI, 0–18%). Early diagnosis is often difficult because its morphological features may mimic those of solid tumors. In addition, no standard therapeutic regimen has been established, and many patients present with advanced disease and poor performance status that limit treatment options. We describe an elderly patient with ALK + LBCL who developed the disease while receiving immunosuppressive therapy for rheumatoid arthritis and systemic lupus erythematosus. Flow cytometry-aided early diagnosis by characterizing the distinct immunophenotype of the tumor. To our knowledge, this is the first reported case of an elderly patient (> 60 years) with ALK + LBCL treated with alectinib. Despite relapsed/refractory disease, the patient achieved tumor control for 10 months, suggesting that alectinib may represent a promising therapeutic option for this rare lymphoma. B-cell lymphoma anaplastic lymphoma kinase alectinib flow cytometry immunosuppression Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Anaplastic lymphoma kinase (ALK)-positive large B-cell lymphoma (LBCL) is an exceptionally rare subtype of B-cell lymphoma first described by Delsol et al. in 1997 [ 1 ]. It affects a wide range, from young to elderly patients, and typically occurs independent of immunosuppression [ 2 ]. The disease follows an aggressive clinical course, with a median overall survival (OS) of 1.8 years (95% confidence interval [CI], 1.2–2.2) and a 5-year survival rate of 28% (95% CI, 17–40%). Patients with stage IV disease have particularly poor outcomes, with a median OS of approximately 1 year (95% CI, 0.8–1.5) and a 5-year survival rate of 5% (95% CI, 0–18%) [ 2 ]. Morphologically, ALK + LBCL consists of large, cohesive cells that can closely resemble epithelial malignancies, such as lung carcinomas, and poorly differentiated variants may display plasmacytoid features. Immunohistochemically, tumor cells are typically negative for B-cell-associated antigens (CD19, CD20, CD79a, and PAX5) and T-cell-associated markers, while exhibiting aberrant ALK expression. Because ALK expression is also seen in certain lung carcinomas and neuroendocrine tumors, accurate diagnosis can be difficult. Flow cytometry (FCM) further complicates the diagnostic process, as these tumors often lack both B- and T-cell antigens and show variable cell size, weak to negative CD45 expression, and frequent CD38 negativity [ 3 ]. Consequently, most cases are diagnosed at an advanced stage (III–IV) [ 2 , 4 ], reflecting both the biological aggressiveness of the disease and the diagnostic challenges it presents. Consequently, diagnosis is often delayed, and many patients present with advanced disease and poor performance status that preclude intensive therapy. To date, no standard treatment has been established for ALK + LBCL. Conventional combination chemotherapy with cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) or CHOP-like regimens has shown limited efficacy, particularly in advanced-stage disease. Although long-term survival has occasionally been reported in transplant-eligible pediatric patients, therapeutic options remain extremely limited for relapsed disease, elderly patients, or those unfit for chemotherapy. We describe a case of advanced-stage ALK + LBCL that developed in an elderly patient receiving immunosuppressive therapy for rheumatoid arthritis and systemic lupus erythematosus. FCM successfully characterized the tumor immunophenotype, facilitating early diagnosis and prompt treatment. Upon relapse, the patient was treated with the ALK inhibitor alectinib and achieved durable disease control for 10 months, leading to prolonged survival. To our knowledge, this is the first reported case of an elderly patient (> 60 years) with ALK + LBCL treated with alectinib. This case highlights alectinib as a potential therapeutic option for refractory ALK + LBCL and underscores the importance of early diagnostic application of FCM in this rare entity. Case Presentation An Asian male was diagnosed with elderly-onset rheumatoid arthritis at age 70, presenting with polyarthritis, high rheumatoid factor titers, and elevated C-reactive protein levels. His symptoms were well controlled with methotrexate (4 mg weekly), prednisolone (PSL; 3 mg daily), and iguratimod (50 mg daily). One year later, he developed worsening pericardial effusion and pleuritis. Laboratory findings revealed high antinuclear antibody titers (1:1,280, homogeneous/speckled pattern) and positive anti-double-stranded DNA antibodies, along with arthralgia, leading to a diagnosis of systemic lupus erythematosus. PSL was increased to 30 mg daily and gradually tapered as his condition stabilized with maintenance PSL (4 mg daily) and hydroxychloroquine. Two years after the diagnosis of systemic lupus erythematosus, the patient developed lateral chest and back pain accompanied by a 3-kg weight loss and night sweats. Physical examination revealed right supraclavicular lymphadenopathy. Laboratory tests showed the following: white blood cell count, 8.9 × 10³/µL; red blood cell count, 5.04 × 10 6 /µL; hemoglobin, 15.2 g/dL; platelet count, 323 × 10³/µL; lactate dehydrogenase, 1,279 U/mL; alkaline phosphatase, 1,062 U/mL; creatinine, 1.34 mg/dL; uric acid, 8.5 mg/dL; and C-reactive protein, 24.49 mg/dL. Immunoglobulin levels were IgG 1,043 mg/dL, IgA 441 mg/dL, and IgM 78 mg/dL. Free κ and λ light chain concentrations were 12.2 mg/L and 13.6 mg/L, respectively, with a κ/λ ratio of 0.90. Serum immunoelectrophoresis detected an IgA-κ monoclonal protein, whereas urine immunoelectrophoresis was negative for Bence Jones protein. HIV testing was negative. Tumor marker levels were within normal limits: carcinoembryonic antigen, 2.2 ng/mL (< 5.0); cytokeratin-19 fragment, 1.8 ng/mL (< 3.5); squamous cell carcinoma antigen, 0.8 ng/mL (< 2.5); and pro-gastrin-releasing peptide, 78.6 pg/mL (< 81.0). The soluble interleukin-2 receptor level was markedly elevated at 5,965 U/mL. Computed tomography (CT) demonstrated right supraclavicular and mediastinal lymphadenopathy. A biopsy of the right supraclavicular lymph node revealed atypical large cells with aberrant surface markers on FCM lymphocyte gating. Histopathological examination with hematoxylin and eosin staining suggested plasmacytoma based on morphology; however, immunohistochemistry for epithelial, B-cell, and plasma cell markers was unremarkable, precluding a definitive diagnosis. Bone marrow examination revealed 70.4% large atypical tumor cells, with a nucleated cell count of 3.9 × 10 4 /µL, megakaryocyte count of 9.0/µL, and a myeloid-to-erythroid ratio of 3.8 (Fig. 1 ). FCM using CD45 blast gating did not detect atypical cells within the lymphocyte gate. However, a large population of cells was identified above the weakly CD45-positive region and was characterized as CD3⁻CD19⁻CD20⁻CD79a^(dull) + CD138 + κ>>λ (Fig. 2 A, 2 B). Immunohistochemical analysis showed the following profile: CD20⁻CD79a⁻CD138 + AE1/AE3⁻CAM5.2⁻TTF-1⁻p63⁻CD4⁻CD8⁻CD30⁻CD34⁻TdT⁻S-100⁻desmin⁻p40⁻PAX5⁻EBER⁻MUM1 + Oct2 + BoB1 + IgA + κ + λ⁻ALK+ (Fig. 3 ). Fluorescence in situ hybridization demonstrated a 10% split signal pattern, confirming the diagnosis of ALK + LBCL. Positron emission tomography/CT (PET/CT) revealed fluorodeoxyglucose (FDG) uptake in both supra- and infradiaphragmatic lymph nodes and in multiple skeletal sites, including the spine, thoracic cage, pelvis, and bilateral proximal femurs (Fig. 4 ). The patient was staged as IVB with a high-risk International Prognostic Index score of 5. Cytogenetic analysis identified a complex karyotype: 46,XY,add(2)(p13),add(8)(p11.2),add(16)(p13.1). After a 3-day course of PSL (1 mg/kg daily), CHOP chemotherapy—cyclophosphamide 750 mg/m² (day 1), doxorubicin 50 mg/m² (day 1), vincristine 1.4 mg/m² (day 1), and prednisolone 100 mg (days 1–5)—was initiated. Following six cycles of CHOP, PET/CT performed demonstrated complete metabolic response. Approximately one year after achieving complete remission, the patient developed abdominal pain accompanied by elevated lactate dehydrogenase and soluble interleukin-2 receptor levels. Bone marrow examination confirmed relapsed ALK + LBCL. The patient received two cycles of gemcitabine (1,000 mg/m² on days 1 and 8), cisplatin (25 mg/m² on days 1–3), and dexamethasone (39.6 mg daily on days 1–3) (GDP regimen). However, PET/CT demonstrated new FDG uptake in the sternum and pelvis, indicating refractory disease. Alectinib was initiated at 300 mg twice daily, resulting in a progressive decline in soluble interleukin-2 receptor levels. At ten months after the initiation of alectinib, PET/CT performed demonstrated a partial metabolic response. The patient received three additional cycles of GDP, but PET/CT revealed progressive metabolic disease. One cycle of CHOP was administered, after which palliative care was initiated at the patient’s request. The patient died, achieving an OS of 3 years and 7 months. Discussion ALK + LBCL accounts for < 1% of diffuse LBCL and is recognized as a rare, aggressive subtype typically unrelated to immunosuppression. This case highlights three novel aspects: (1) ALK + LBCL can arise during immunosuppressive therapy, (2) FCM is valuable for early diagnosis of advanced-stage disease, and (3) alectinib may serve as a potential therapeutic option for patients with poor-prognosis ALK + LBCL and early relapse. Although immune function is usually preserved in ALK + LBCL [ 2 ], the present case represents an unusual occurrence of the disease developing during immunosuppressive therapy for rheumatoid arthritis and systemic lupus erythematosus. A similar case was described in 2007, involving a 33-year-old man with psoriatic arthritis who developed ALK + LBCL after 3 years of methotrexate therapy [ 5 ]. Both cases exhibited complex karyotypes, suggesting a possible link with secondary lymphoproliferative disorders such as methotrexate-associated lymphoproliferative disease. Thus, ALK + LBCL should be considered in the differential diagnosis of lymphoid malignancies occurring in immunosuppressed patients. Given the aggressive nature of ALK + LBCL, early and accurate diagnosis is crucial for improving patient outcomes; however, several diagnostic pitfalls may hinder timely identification. In the present case, initial findings suggested epithelial tumor infiltration of the bone marrow (carcinomatosis) owing to the presence of large, cohesive cell clusters. The adhesive morphology of these tumor cells often leads to evaluation of epithelial markers such as keratins (AE1/AE3), which may result in misdiagnosis as metastatic carcinoma. The differential diagnosis includes epithelioid inflammatory myofibroblastic sarcoma and, among hematologic malignancies, plasmablastic lymphoma. Additionally, ALK expression can be seen in certain lung carcinomas, some of which display plasmacytoid morphology, further complicating distinction from ALK + LBCL. Neuroendocrine tumors may also express ALK and PAX5, necessitating their inclusion in the differential diagnosis. Although exclusion of these entities is essential, reliance on morphology alone often renders diagnosis difficult, representing the first major diagnostic pitfall. The second pitfall concerns FCM detection. Lymphomas typically exhibit tumor populations within the lymphocyte gate on bone marrow FCM; however, this pattern may not be observed in ALK + LBCL. This occurs because ALK + LBCL cells show weak or absent CD45 expression, preventing detection through conventional lymphocyte gating. In the present case, morphologic assessment suggested plasmacytoid features, prompting evaluation for multiple myeloma. However, FCM revealed no strongly CD38-positive clusters, excluding multiple myeloma and initially raising suspicion for epithelial malignancy. Ultimately, identification of a large cell population above the CD45 weakly positive region, together with the surface marker profile CD20⁻CD138 + κ>>λ, supported the diagnosis of ALK + LBCL. Similar FCM findings have been reported previously [ 6 ]. Awareness of this distinctive pattern—and the fact that ALK + LBCL may be missed with standard lymphoma gating—can enhance early recognition. Given that approximately one-quarter of ALK + LBCL cases involve bone marrow infiltration, bone marrow FCM serves as a particularly valuable diagnostic adjunct. At diagnosis, the patient presented with stage IVB disease, an International Prognostic Index score of 5, and a complex karyotype—features associated with extremely poor prognosis and a possible secondary etiology related to immunosuppressive therapy. Early relapse occurred within 1 year after CHOP therapy; however, alectinib achieved 10 months of tumor control and contributed to prolonged survival. The first adult case of ALK + LBCL treated with alectinib was reported in 2022 [ 7 ], followed by three additional reports [ 8 – 10 ]; notably, all involved patients aged 60 years or younger. To our knowledge, this is the first documented case of an elderly patient (> 60 years) with ALK + LBCL successfully treated with alectinib. ALK + LBCL is often diagnosed at an advanced stage and in patients with poor performance status, particularly among the elderly, which frequently limits the feasibility of intensive chemotherapy. Based on clinical experience with crizotinib—another ALK inhibitor—ALK inhibitors appear to have a more favorable toxicity profile than conventional cytotoxic regimens [ 11 , 12 ]. Consistent with this, our patient tolerated alectinib well without significant adverse events. Previous studies have reported that ALK + LBCL in patients younger than 18 years tends to be localized [ 4 ], and some have suggested a bimodal age distribution, with incidence peaks in pediatric and middle-aged populations [ 13 ]. These observations imply that disease biology and clinical behavior may differ across age groups. The favorable response to alectinib in this elderly patient may reflect age-related differences in disease biology. Alternatively, the presence of an underlying methotrexate-associated lymphoproliferative disorder could have influenced treatment sensitivity, warranting further investigation. At present, no standard therapy exists for ALK + LBCL, and intensive chemotherapy regimens are typically used for relapsed or refractory disease. Consequently, treatment options for elderly or chemotherapy-ineligible patients remain extremely limited. The achievement of prolonged survival with single-agent alectinib in this elderly patient with advanced-stage disease—without significant toxicity—represents a noteworthy therapeutic outcome. These findings further support exploration of alectinib-based combination regimens as potential first-line options in future clinical studies. Conclusion We report a rare case of ALK + LBCL diagnosed in an elderly patient receiving immunosuppressive therapy. This case highlights the need to consider ALK + LBCL in the differential diagnosis of methotrexate-associated lymphoproliferative disorders. Because most ALK + LBCL cases are identified at advanced stages, FCM may serve as a valuable adjunct for early detection and timely initiation of treatment. Furthermore, the durable disease control achieved with alectinib in this patient suggests that ALK inhibition could represent a promising therapeutic option for elderly or chemotherapy-ineligible individuals with this aggressive lymphoma. Declarations Declarations Competing Interests: The authors declare that they have no competing interests. Ethics Approval: The study was approved by the Ethics Committee of Tokyo Metropolitan Tama Medical Center. Informed Consent: The patient provided written informed consent for publication. Funding: None. Author Contribution MT conceptualized the study. YH collected the data, interpreted the results, and drafted the manuscript. MM, DS, KI, KK, and YK contributed to data acquisition and clinical interpretation. HO provided pathological evaluation and expert advice from a histopathological perspective. All authors (MT, YH, MM, DS, KI, KK, HO, and YK) critically revised the article and approved the final version of the manuscript. Acknowledgments: Not applicable. References Delsol G, Lamant L, Mariamé B, Pulford K, Dastugue N, Brousset P, Rigal-Huguet F, Al Saati T, Cerretti DP, Morris SW, Mason DY (1997) A new subtype of large B-cell lymphoma expressing the ALK kinase and lacking the 2; 5 translocation. Blood 89:1483–1490 Castillo JJ, Beltran BE, Malpica L, Marques-Piubelli ML, Miranda RN (2021) Anaplastic lymphoma kinase-positive large B-cell lymphoma (ALK + LBCL): a systematic review of clinicopathological features and management. Leuk Lymphoma 62:2845–2853. https://doi.org/10.1080/10428194.2021.1941929 Li S (2009) Anaplastic lymphoma kinase-positive large B-cell lymphoma: a distinct clinicopathological entity. Int J Clin Exp Pathol 2:508–518 Laurent C, Do C, Gascoyne RD, Lamant L, Ysebaert L, Laurent G, Delsol G, Brousset P (2009) Anaplastic lymphoma kinase-positive diffuse large B-cell lymphoma: a rare clinicopathologic entity with poor prognosis. J Clin Oncol 27:4211–4216. https://doi.org/10.1200/jco.2008.21.5020 Stachurski D, Miron PM, Al-Homsi S, Hutchinson L, Harris NL, Woda B, Wang SA (2007) Anaplastic lymphoma kinase-positive diffuse large B-cell lymphoma with a complex karyotype and cryptic 3' ALK gene insertion to chromosome 4 q22-24. Hum Pathol 38:940–945. https://doi.org/10.1016/j.humpath.2006.12.019 Reichard KK, McKenna RW, Kroft SH (2007) ALK-positive diffuse large B-cell lymphoma: report of four cases and review of the literature. Mod Pathol 20:310–319. https://doi.org/10.1038/modpathol.3800742 Soumerai JD, Rosenthal A, Harkins S et al (2022) Next-generation ALK inhibitors are highly active in ALK-positive large B-cell lymphoma. Blood 140:1822–1826. https://doi.org/10.1182/blood.2022015443 Wirk B, Malysz J, Choi E, Songdej N (2023) lorlatinib induces rapid and durable response in refractory anaplastic lymphoma kinase-positive large B-cell lymphoma. JCO Precis Oncol 7:e2200536. https://doi.org/10.1200/po.22.00536 Takiar R, Phillips TJ (2023) Durable responses with ALK inhibitors for primary refractory anaplastic lymphoma Kinase-positive large B-cell lymphoma. Blood Adv 7:2912–2916. https://doi.org/10.1182/bloodadvances.2022007537 Wang H, Bai J, Zhang B, Pei Z, Zhao Y, Gu J, Fang Z, Liu M, Liang X, Song Q (2024) Alectinib combined with VRCD and BV monoclonal antibody for the treatment of ALK-positive large B-cell lymphoma: a case report and literature review. https://doi.org/10.3389/fonc.2024.1480880 . Front Oncol14:1480880 Gambacorti-Passerini C, Messa C, Pogliani EM (2011) Crizotinib in anaplastic large-cell lymphoma. N Engl J Med 364:775–776. https://doi.org/10.1056/NEJMc1013224 Gambacorti Passerini C, Farina F, Stasia A et al (2014) Crizotinib in advanced, chemoresistant anaplastic lymphoma kinase-positive lymphoma patients. J Natl Cancer Inst 106:djt378. https://doi.org/10.1093/jnci/djt378 Yin WH, Guo N, Tian XY, Li Y, Li Z (2012) Pediatric anaplastic lymphoma kinase-positive large B-cell lymphoma: a case report and review of the literature. Pediatr Dev Pathol 15:318–323. https://doi.org/10.2350/11-11-1118-cr.1 Additional Declarations No competing interests reported. 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. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8042084","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Case Report","associatedPublications":[],"authors":[{"id":554617754,"identity":"022bf419-0ec0-49d7-82de-426889d6cb1d","order_by":0,"name":"Yudai Hayashi","email":"","orcid":"","institution":"Tokyo Metropolitan Tama Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Yudai","middleName":"","lastName":"Hayashi","suffix":""},{"id":554617755,"identity":"93a46f30-f446-4e6d-8f73-a15f4fc648f0","order_by":1,"name":"Masao Tsukada","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6ElEQVRIiWNgGAWjYFADHgYGCYYKBgYDBggmBAygWs4YkKqFsc2AsHLd9uMPP3xg+GNvcObwwxs/5/2JNpduYCguwKPF7EyOseQMBoPEDWfbjC17txnk7pxzgMF4Bj4tB3IYpHkYDBIMzjOYSfACtWy4kcBgzINPy/nnj38DtdgbnGf/Jvl3DjFabiSYgWxh3HC2x0yat4EoLW/MLGcwGCfOPHOm2FrmmHHuhjsHG/D75Xz64xsfGOTs+c6kb7z5pkYud8Pt5mPG+EIMDBj/IfOAsWNMSAcakGBgfkyillEwCkbBKBjeAABJtU/dYg1F2AAAAABJRU5ErkJggg==","orcid":"","institution":"Tokyo Metropolitan Tama Medical Center","correspondingAuthor":true,"prefix":"","firstName":"Masao","middleName":"","lastName":"Tsukada","suffix":""},{"id":554617756,"identity":"145f765f-4236-4959-a176-67c4947e9c39","order_by":2,"name":"Marina Matsui","email":"","orcid":"","institution":"Tokyo Metropolitan Tama Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Marina","middleName":"","lastName":"Matsui","suffix":""},{"id":554617757,"identity":"abba266d-ed98-421e-8844-ad10694936b1","order_by":3,"name":"Daisuke Shinoda","email":"","orcid":"","institution":"Tokyo Metropolitan Tama Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Daisuke","middleName":"","lastName":"Shinoda","suffix":""},{"id":554617758,"identity":"f0332386-d978-4e83-901b-e2e212f3ebd7","order_by":4,"name":"Kanichi Iwama","email":"","orcid":"","institution":"Tokyo Metropolitan Tama Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Kanichi","middleName":"","lastName":"Iwama","suffix":""},{"id":554617759,"identity":"a383a054-7a62-4be1-9561-e32ae0197a95","order_by":5,"name":"Koichi Kajiwara","email":"","orcid":"","institution":"Tokyo Metropolitan Tama Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Koichi","middleName":"","lastName":"Kajiwara","suffix":""},{"id":554617760,"identity":"7f1a6496-dde8-4d59-b770-82a7de4cd8c0","order_by":6,"name":"Haruka Okada","email":"","orcid":"","institution":"Tokyo Metropolitan Tama Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Haruka","middleName":"","lastName":"Okada","suffix":""},{"id":554617761,"identity":"284707fd-11f9-4ca6-96df-eac0fe7b793a","order_by":7,"name":"Yasuji Kozai","email":"","orcid":"","institution":"Tokyo Metropolitan Tama Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Yasuji","middleName":"","lastName":"Kozai","suffix":""}],"badges":[],"createdAt":"2025-11-05 23:08:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8042084/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8042084/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":97672904,"identity":"74684092-833f-49e5-9505-84721a32f551","added_by":"auto","created_at":"2025-12-08 09:39:04","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":5917312,"visible":true,"origin":"","legend":"","description":"","filename":"ManuscriptID1e7b2da6451b40109555c7efcc659579v1.0.docx","url":"https://assets-eu.researchsquare.com/files/rs-8042084/v1/1ed254ee47d4b20140bd0e4d.docx"},{"id":97659010,"identity":"0fab1af1-48a1-4b37-9a4a-73c1fd0947c5","added_by":"auto","created_at":"2025-12-08 07:35:07","extension":"json","order_by":1,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":8559,"visible":true,"origin":"","legend":"","description":"","filename":"1e7b2da6451b40109555c7efcc659579.json","url":"https://assets-eu.researchsquare.com/files/rs-8042084/v1/4306de098df3e1fb31e434e7.json"},{"id":97674663,"identity":"d7708707-8bd5-4937-acb2-bd226a6cfe3b","added_by":"auto","created_at":"2025-12-08 09:43:48","extension":"xml","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":50559,"visible":true,"origin":"","legend":"","description":"","filename":"1e7b2da6451b40109555c7efcc6595791enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-8042084/v1/01a203409beeb9f752a3bb4c.xml"},{"id":97674895,"identity":"7bc1d520-8997-4fd6-87de-0c46db29b3e4","added_by":"auto","created_at":"2025-12-08 09:44:39","extension":"png","order_by":3,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":3838481,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8042084/v1/403faceeb03eeff967d077d4.png"},{"id":97659018,"identity":"9446bbfe-3349-41ed-9ae3-df426eb5bee1","added_by":"auto","created_at":"2025-12-08 07:35:07","extension":"png","order_by":4,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":281311,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8042084/v1/58bc00b771c21a667e70994f.png"},{"id":97659014,"identity":"81788f44-4bc2-402f-bd8b-9670805fefd8","added_by":"auto","created_at":"2025-12-08 07:35:07","extension":"png","order_by":5,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":235082,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8042084/v1/ef78ddce4eb907a17faf8497.png"},{"id":97672639,"identity":"55b26c5b-fe1a-48b9-9c01-3058d3d247ef","added_by":"auto","created_at":"2025-12-08 09:38:31","extension":"jpeg","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":567321,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8042084/v1/315b1027ca287dc88be48b9e.jpeg"},{"id":97659020,"identity":"d581a4ef-a571-415b-a6a8-2759d8166f1b","added_by":"auto","created_at":"2025-12-08 07:35:07","extension":"png","order_by":7,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":169844,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8042084/v1/e73df8c5908e0998c1ceaf91.png"},{"id":97674915,"identity":"88131a4d-d132-4d70-8d89-b778acf9a75a","added_by":"auto","created_at":"2025-12-08 09:44:43","extension":"png","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":464804,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8042084/v1/c29a015b90fe026977829ec4.png"},{"id":97659019,"identity":"4d9573a5-c5d6-416d-8df2-d94e2ca2ab76","added_by":"auto","created_at":"2025-12-08 07:35:07","extension":"png","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":60223,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8042084/v1/f1b19e5a52f13741c99f6c2d.png"},{"id":97659024,"identity":"3bcce115-7fe2-4d20-8921-55e3c1bf5bce","added_by":"auto","created_at":"2025-12-08 07:35:07","extension":"png","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":51037,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8042084/v1/feaeaaf1c0420190783b02dc.png"},{"id":97659022,"identity":"b4e03523-991d-4f02-b077-8bc607dfea71","added_by":"auto","created_at":"2025-12-08 07:35:07","extension":"png","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":370773,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8042084/v1/d0fc5c93ab00d45c7d71face.png"},{"id":97659026,"identity":"f30890b0-203c-434e-9c70-de248b279c6a","added_by":"auto","created_at":"2025-12-08 07:35:07","extension":"png","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":168787,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8042084/v1/cb8fc0571f5816829add51e3.png"},{"id":97674899,"identity":"29f6858a-4dd5-4883-89a7-7a5e9f57cb25","added_by":"auto","created_at":"2025-12-08 09:44:39","extension":"xml","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":50991,"visible":true,"origin":"","legend":"","description":"","filename":"1e7b2da6451b40109555c7efcc6595791structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8042084/v1/9857c693fcfc140c42120593.xml"},{"id":97659027,"identity":"1838b6aa-c4ac-441c-8e19-e0a79aa8a005","added_by":"auto","created_at":"2025-12-08 07:35:07","extension":"html","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":56790,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8042084/v1/6149b8643f36a9a3871f5a9b.html"},{"id":97659011,"identity":"b4ebb9d0-b865-40be-b644-ade2b994b293","added_by":"auto","created_at":"2025-12-08 07:35:07","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":510662,"visible":true,"origin":"","legend":"\u003cp\u003eBone marrow aspiration. Clusters of large, atypical tumor cells were observed. Atypical cells were present in 70% of the sample.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8042084/v1/6f17c3b5049bce3592a749cb.png"},{"id":97659012,"identity":"eca9ffaf-5181-4f0d-a2db-781a6f5cf24a","added_by":"auto","created_at":"2025-12-08 07:35:07","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":245317,"visible":true,"origin":"","legend":"\u003cp\u003eBlue indicates the CD19⁻CD138⁺ cell population, while red represents the CD19⁻CD138⁻ cell population. A) This is a cell surface marker. The CD19⁻CD138⁺ cell population accounted for 44.5% of the total, and all cells in this subset were CD19⁻CD20⁻. B) This is a cytoplasmic marker. The CD19⁻CD138⁺ cell population showed κ light chain restriction, with 64.6% expressing κ.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8042084/v1/fe2f00dce737f13c54dfa9b3.png"},{"id":97659017,"identity":"245fafe6-e5b2-49d4-b1d0-bbb2ab93679e","added_by":"auto","created_at":"2025-12-08 07:35:07","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":488791,"visible":true,"origin":"","legend":"\u003cp\u003eRight supraclavicular lymph node biopsy specimen. The scale bar represents 50 μm. Hematoxylin and eosin staining was performed (a, b). Immunohistochemical staining shows CD3⁻CD20⁻CD30⁻CD138+BoB1+IgA+κ+λ⁻ (c–k).\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8042084/v1/48ab72d08bd54a125b99391a.jpg"},{"id":97659013,"identity":"777d2332-e663-4361-a5fb-2ad5897bc5e6","added_by":"auto","created_at":"2025-12-08 07:35:07","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":184103,"visible":true,"origin":"","legend":"\u003cp\u003eRight deep cervical to supraclavicular lymph nodes showed enlargement with fluorodeoxyglucose (FDG) uptake (SUVmax 10.60). Upper to middle mediastinal lymph nodes demonstrated enlargement with FDG uptake (SUVmax 12.64–17.33). FDG uptake was observed in lymph nodes around the celiac trunk to hepatic artery (SUVmax 10.86). High FDG uptake was noted in axial bones including the spine, thoracic cage, and pelvis, as well as bilateral proximal femurs (SUVmax 22.67).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8042084/v1/3fd02152f831e6bffc164251.png"},{"id":99711755,"identity":"e6fbadf2-1c4b-4d0f-b315-1ce42636a834","added_by":"auto","created_at":"2026-01-07 13:39:50","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1841423,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8042084/v1/1041f1a2-21a4-4b9f-b85f-744d801f7e73.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Flow Cytometry-Aided Early Diagnosis and Alectinib Response in Advanced ALK-Positive Large B-Cell Lymphoma: A Case Report","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAnaplastic lymphoma kinase (ALK)-positive large B-cell lymphoma (LBCL) is an exceptionally rare subtype of B-cell lymphoma first described by Delsol et al. in 1997 [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. It affects a wide range, from young to elderly patients, and typically occurs independent of immunosuppression [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The disease follows an aggressive clinical course, with a median overall survival (OS) of 1.8 years (95% confidence interval [CI], 1.2\u0026ndash;2.2) and a 5-year survival rate of 28% (95% CI, 17\u0026ndash;40%). Patients with stage IV disease have particularly poor outcomes, with a median OS of approximately 1 year (95% CI, 0.8\u0026ndash;1.5) and a 5-year survival rate of 5% (95% CI, 0\u0026ndash;18%) [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Morphologically, ALK\u0026thinsp;+\u0026thinsp;LBCL consists of large, cohesive cells that can closely resemble epithelial malignancies, such as lung carcinomas, and poorly differentiated variants may display plasmacytoid features. Immunohistochemically, tumor cells are typically negative for B-cell-associated antigens (CD19, CD20, CD79a, and PAX5) and T-cell-associated markers, while exhibiting aberrant ALK expression. Because ALK expression is also seen in certain lung carcinomas and neuroendocrine tumors, accurate diagnosis can be difficult. Flow cytometry (FCM) further complicates the diagnostic process, as these tumors often lack both B- and T-cell antigens and show variable cell size, weak to negative CD45 expression, and frequent CD38 negativity [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Consequently, most cases are diagnosed at an advanced stage (III\u0026ndash;IV) [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], reflecting both the biological aggressiveness of the disease and the diagnostic challenges it presents. Consequently, diagnosis is often delayed, and many patients present with advanced disease and poor performance status that preclude intensive therapy. To date, no standard treatment has been established for ALK\u0026thinsp;+\u0026thinsp;LBCL. Conventional combination chemotherapy with cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) or CHOP-like regimens has shown limited efficacy, particularly in advanced-stage disease. Although long-term survival has occasionally been reported in transplant-eligible pediatric patients, therapeutic options remain extremely limited for relapsed disease, elderly patients, or those unfit for chemotherapy.\u003c/p\u003e\u003cp\u003eWe describe a case of advanced-stage ALK\u0026thinsp;+\u0026thinsp;LBCL that developed in an elderly patient receiving immunosuppressive therapy for rheumatoid arthritis and systemic lupus erythematosus. FCM successfully characterized the tumor immunophenotype, facilitating early diagnosis and prompt treatment. Upon relapse, the patient was treated with the ALK inhibitor alectinib and achieved durable disease control for 10 months, leading to prolonged survival. To our knowledge, this is the first reported case of an elderly patient (\u0026gt;\u0026thinsp;60 years) with ALK\u0026thinsp;+\u0026thinsp;LBCL treated with alectinib. This case highlights alectinib as a potential therapeutic option for refractory ALK\u0026thinsp;+\u0026thinsp;LBCL and underscores the importance of early diagnostic application of FCM in this rare entity.\u003c/p\u003e"},{"header":"Case Presentation","content":"\u003cp\u003eAn Asian male was diagnosed with elderly-onset rheumatoid arthritis at age 70, presenting with polyarthritis, high rheumatoid factor titers, and elevated C-reactive protein levels. His symptoms were well controlled with methotrexate (4 mg weekly), prednisolone (PSL; 3 mg daily), and iguratimod (50 mg daily). One year later, he developed worsening pericardial effusion and pleuritis. Laboratory findings revealed high antinuclear antibody titers (1:1,280, homogeneous/speckled pattern) and positive anti-double-stranded DNA antibodies, along with arthralgia, leading to a diagnosis of systemic lupus erythematosus. PSL was increased to 30 mg daily and gradually tapered as his condition stabilized with maintenance PSL (4 mg daily) and hydroxychloroquine.\u003c/p\u003e\u003cp\u003eTwo years after the diagnosis of systemic lupus erythematosus, the patient developed lateral chest and back pain accompanied by a 3-kg weight loss and night sweats. Physical examination revealed right supraclavicular lymphadenopathy. Laboratory tests showed the following: white blood cell count, 8.9 \u0026times; 10\u0026sup3;/\u0026micro;L; red blood cell count, 5.04 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e/\u0026micro;L; hemoglobin, 15.2 g/dL; platelet count, 323 \u0026times; 10\u0026sup3;/\u0026micro;L; lactate dehydrogenase, 1,279 U/mL; alkaline phosphatase, 1,062 U/mL; creatinine, 1.34 mg/dL; uric acid, 8.5 mg/dL; and C-reactive protein, 24.49 mg/dL. Immunoglobulin levels were IgG 1,043 mg/dL, IgA 441 mg/dL, and IgM 78 mg/dL. Free κ and λ light chain concentrations were 12.2 mg/L and 13.6 mg/L, respectively, with a κ/λ ratio of 0.90. Serum immunoelectrophoresis detected an IgA-κ monoclonal protein, whereas urine immunoelectrophoresis was negative for Bence Jones protein. HIV testing was negative. Tumor marker levels were within normal limits: carcinoembryonic antigen, 2.2 ng/mL (\u0026lt;\u0026thinsp;5.0); cytokeratin-19 fragment, 1.8 ng/mL (\u0026lt;\u0026thinsp;3.5); squamous cell carcinoma antigen, 0.8 ng/mL (\u0026lt;\u0026thinsp;2.5); and pro-gastrin-releasing peptide, 78.6 pg/mL (\u0026lt;\u0026thinsp;81.0). The soluble interleukin-2 receptor level was markedly elevated at 5,965 U/mL. Computed tomography (CT) demonstrated right supraclavicular and mediastinal lymphadenopathy. A biopsy of the right supraclavicular lymph node revealed atypical large cells with aberrant surface markers on FCM lymphocyte gating. Histopathological examination with hematoxylin and eosin staining suggested plasmacytoma based on morphology; however, immunohistochemistry for epithelial, B-cell, and plasma cell markers was unremarkable, precluding a definitive diagnosis. Bone marrow examination revealed 70.4% large atypical tumor cells, with a nucleated cell count of 3.9 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e/\u0026micro;L, megakaryocyte count of 9.0/\u0026micro;L, and a myeloid-to-erythroid ratio of 3.8 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). FCM using CD45 blast gating did not detect atypical cells within the lymphocyte gate. However, a large population of cells was identified above the weakly CD45-positive region and was characterized as CD3⁻CD19⁻CD20⁻CD79a^(dull)\u0026thinsp;+\u0026thinsp;CD138\u0026thinsp;+\u0026thinsp;κ\u0026gt;\u0026gt;λ (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eImmunohistochemical analysis showed the following profile: CD20⁻CD79a⁻CD138\u0026thinsp;+\u0026thinsp;AE1/AE3⁻CAM5.2⁻TTF-1⁻p63⁻CD4⁻CD8⁻CD30⁻CD34⁻TdT⁻S-100⁻desmin⁻p40⁻PAX5⁻EBER⁻MUM1\u0026thinsp;+\u0026thinsp;Oct2\u0026thinsp;+\u0026thinsp;BoB1\u0026thinsp;+\u0026thinsp;IgA\u0026thinsp;+\u0026thinsp;κ\u0026thinsp;+\u0026thinsp;λ⁻ALK+ (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Fluorescence in situ hybridization demonstrated a 10% split signal pattern, confirming the diagnosis of ALK\u0026thinsp;+\u0026thinsp;LBCL. Positron emission tomography/CT (PET/CT) revealed fluorodeoxyglucose (FDG) uptake in both supra- and infradiaphragmatic lymph nodes and in multiple skeletal sites, including the spine, thoracic cage, pelvis, and bilateral proximal femurs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The patient was staged as IVB with a high-risk International Prognostic Index score of 5. Cytogenetic analysis identified a complex karyotype: 46,XY,add(2)(p13),add(8)(p11.2),add(16)(p13.1). After a 3-day course of PSL (1 mg/kg daily), CHOP chemotherapy\u0026mdash;cyclophosphamide 750 mg/m\u0026sup2; (day 1), doxorubicin 50 mg/m\u0026sup2; (day 1), vincristine 1.4 mg/m\u0026sup2; (day 1), and prednisolone 100 mg (days 1\u0026ndash;5)\u0026mdash;was initiated. Following six cycles of CHOP, PET/CT performed demonstrated complete metabolic response.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eApproximately one year after achieving complete remission, the patient developed abdominal pain accompanied by elevated lactate dehydrogenase and soluble interleukin-2 receptor levels. Bone marrow examination confirmed relapsed ALK\u0026thinsp;+\u0026thinsp;LBCL. The patient received two cycles of gemcitabine (1,000 mg/m\u0026sup2; on days 1 and 8), cisplatin (25 mg/m\u0026sup2; on days 1\u0026ndash;3), and dexamethasone (39.6 mg daily on days 1\u0026ndash;3) (GDP regimen). However, PET/CT demonstrated new FDG uptake in the sternum and pelvis, indicating refractory disease. Alectinib was initiated at 300 mg twice daily, resulting in a progressive decline in soluble interleukin-2 receptor levels. At ten months after the initiation of alectinib, PET/CT performed demonstrated a partial metabolic response. The patient received three additional cycles of GDP, but PET/CT revealed progressive metabolic disease. One cycle of CHOP was administered, after which palliative care was initiated at the patient\u0026rsquo;s request. The patient died, achieving an OS of 3 years and 7 months.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eALK\u0026thinsp;+\u0026thinsp;LBCL accounts for \u0026lt;\u0026thinsp;1% of diffuse LBCL and is recognized as a rare, aggressive subtype typically unrelated to immunosuppression. This case highlights three novel aspects: (1) ALK\u0026thinsp;+\u0026thinsp;LBCL can arise during immunosuppressive therapy, (2) FCM is valuable for early diagnosis of advanced-stage disease, and (3) alectinib may serve as a potential therapeutic option for patients with poor-prognosis ALK\u0026thinsp;+\u0026thinsp;LBCL and early relapse.\u003c/p\u003e\u003cp\u003eAlthough immune function is usually preserved in ALK\u0026thinsp;+\u0026thinsp;LBCL [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], the present case represents an unusual occurrence of the disease developing during immunosuppressive therapy for rheumatoid arthritis and systemic lupus erythematosus. A similar case was described in 2007, involving a 33-year-old man with psoriatic arthritis who developed ALK\u0026thinsp;+\u0026thinsp;LBCL after 3 years of methotrexate therapy [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Both cases exhibited complex karyotypes, suggesting a possible link with secondary lymphoproliferative disorders such as methotrexate-associated lymphoproliferative disease. Thus, ALK\u0026thinsp;+\u0026thinsp;LBCL should be considered in the differential diagnosis of lymphoid malignancies occurring in immunosuppressed patients.\u003c/p\u003e\u003cp\u003eGiven the aggressive nature of ALK\u0026thinsp;+\u0026thinsp;LBCL, early and accurate diagnosis is crucial for improving patient outcomes; however, several diagnostic pitfalls may hinder timely identification. In the present case, initial findings suggested epithelial tumor infiltration of the bone marrow (carcinomatosis) owing to the presence of large, cohesive cell clusters. The adhesive morphology of these tumor cells often leads to evaluation of epithelial markers such as keratins (AE1/AE3), which may result in misdiagnosis as metastatic carcinoma. The differential diagnosis includes epithelioid inflammatory myofibroblastic sarcoma and, among hematologic malignancies, plasmablastic lymphoma. Additionally, ALK expression can be seen in certain lung carcinomas, some of which display plasmacytoid morphology, further complicating distinction from ALK\u0026thinsp;+\u0026thinsp;LBCL. Neuroendocrine tumors may also express ALK and PAX5, necessitating their inclusion in the differential diagnosis. Although exclusion of these entities is essential, reliance on morphology alone often renders diagnosis difficult, representing the first major diagnostic pitfall. The second pitfall concerns FCM detection. Lymphomas typically exhibit tumor populations within the lymphocyte gate on bone marrow FCM; however, this pattern may not be observed in ALK\u0026thinsp;+\u0026thinsp;LBCL. This occurs because ALK\u0026thinsp;+\u0026thinsp;LBCL cells show weak or absent CD45 expression, preventing detection through conventional lymphocyte gating. In the present case, morphologic assessment suggested plasmacytoid features, prompting evaluation for multiple myeloma. However, FCM revealed no strongly CD38-positive clusters, excluding multiple myeloma and initially raising suspicion for epithelial malignancy. Ultimately, identification of a large cell population above the CD45 weakly positive region, together with the surface marker profile CD20⁻CD138\u0026thinsp;+\u0026thinsp;κ\u0026gt;\u0026gt;λ, supported the diagnosis of ALK\u0026thinsp;+\u0026thinsp;LBCL. Similar FCM findings have been reported previously [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Awareness of this distinctive pattern\u0026mdash;and the fact that ALK\u0026thinsp;+\u0026thinsp;LBCL may be missed with standard lymphoma gating\u0026mdash;can enhance early recognition. Given that approximately one-quarter of ALK\u0026thinsp;+\u0026thinsp;LBCL cases involve bone marrow infiltration, bone marrow FCM serves as a particularly valuable diagnostic adjunct.\u003c/p\u003e\u003cp\u003eAt diagnosis, the patient presented with stage IVB disease, an International Prognostic Index score of 5, and a complex karyotype\u0026mdash;features associated with extremely poor prognosis and a possible secondary etiology related to immunosuppressive therapy. Early relapse occurred within 1 year after CHOP therapy; however, alectinib achieved 10 months of tumor control and contributed to prolonged survival. The first adult case of ALK\u0026thinsp;+\u0026thinsp;LBCL treated with alectinib was reported in 2022 [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], followed by three additional reports [\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]; notably, all involved patients aged 60 years or younger. To our knowledge, this is the first documented case of an elderly patient (\u0026gt;\u0026thinsp;60 years) with ALK\u0026thinsp;+\u0026thinsp;LBCL successfully treated with alectinib. ALK\u0026thinsp;+\u0026thinsp;LBCL is often diagnosed at an advanced stage and in patients with poor performance status, particularly among the elderly, which frequently limits the feasibility of intensive chemotherapy. Based on clinical experience with crizotinib\u0026mdash;another ALK inhibitor\u0026mdash;ALK inhibitors appear to have a more favorable toxicity profile than conventional cytotoxic regimens [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Consistent with this, our patient tolerated alectinib well without significant adverse events. Previous studies have reported that ALK\u0026thinsp;+\u0026thinsp;LBCL in patients younger than 18 years tends to be localized [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], and some have suggested a bimodal age distribution, with incidence peaks in pediatric and middle-aged populations [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. These observations imply that disease biology and clinical behavior may differ across age groups. The favorable response to alectinib in this elderly patient may reflect age-related differences in disease biology. Alternatively, the presence of an underlying methotrexate-associated lymphoproliferative disorder could have influenced treatment sensitivity, warranting further investigation. At present, no standard therapy exists for ALK\u0026thinsp;+\u0026thinsp;LBCL, and intensive chemotherapy regimens are typically used for relapsed or refractory disease. Consequently, treatment options for elderly or chemotherapy-ineligible patients remain extremely limited. The achievement of prolonged survival with single-agent alectinib in this elderly patient with advanced-stage disease\u0026mdash;without significant toxicity\u0026mdash;represents a noteworthy therapeutic outcome. These findings further support exploration of alectinib-based combination regimens as potential first-line options in future clinical studies.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eWe report a rare case of ALK\u0026thinsp;+\u0026thinsp;LBCL diagnosed in an elderly patient receiving immunosuppressive therapy. This case highlights the need to consider ALK\u0026thinsp;+\u0026thinsp;LBCL in the differential diagnosis of methotrexate-associated lymphoproliferative disorders. Because most ALK\u0026thinsp;+\u0026thinsp;LBCL cases are identified at advanced stages, FCM may serve as a valuable adjunct for early detection and timely initiation of treatment. Furthermore, the durable disease control achieved with alectinib in this patient suggests that ALK inhibition could represent a promising therapeutic option for elderly or chemotherapy-ineligible individuals with this aggressive lymphoma.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eDeclarations\u003c/h2\u003e\u003cp\u003e\u003ch2\u003eCompeting Interests:\u003c/h2\u003e\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eEthics Approval:\u003c/strong\u003e\u003cp\u003eThe study was approved by the Ethics Committee of Tokyo Metropolitan Tama Medical Center.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003ch2\u003eInformed Consent:\u003c/h2\u003e\u003cp\u003eThe patient provided written informed consent for publication.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding:\u003c/h2\u003e\u003cp\u003eNone.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eMT conceptualized the study. YH collected the data, interpreted the results, and drafted the manuscript. MM, DS, KI, KK, and YK contributed to data acquisition and clinical interpretation. HO provided pathological evaluation and expert advice from a histopathological perspective. All authors (MT, YH, MM, DS, KI, KK, HO, and YK) critically revised the article and approved the final version of the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgments:\u003c/h2\u003e\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eDelsol G, Lamant L, Mariam\u0026eacute; B, Pulford K, Dastugue N, Brousset P, Rigal-Huguet F, Al Saati T, Cerretti DP, Morris SW, Mason DY (1997) A new subtype of large B-cell lymphoma expressing the ALK kinase and lacking the 2; 5 translocation. Blood 89:1483\u0026ndash;1490\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCastillo JJ, Beltran BE, Malpica L, Marques-Piubelli ML, Miranda RN (2021) Anaplastic lymphoma kinase-positive large B-cell lymphoma (ALK\u0026thinsp;+\u0026thinsp;LBCL): a systematic review of clinicopathological features and management. Leuk Lymphoma 62:2845\u0026ndash;2853. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1080/10428194.2021.1941929\u003c/span\u003e\u003cspan address=\"10.1080/10428194.2021.1941929\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLi S (2009) Anaplastic lymphoma kinase-positive large B-cell lymphoma: a distinct clinicopathological entity. Int J Clin Exp Pathol 2:508\u0026ndash;518\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLaurent C, Do C, Gascoyne RD, Lamant L, Ysebaert L, Laurent G, Delsol G, Brousset P (2009) Anaplastic lymphoma kinase-positive diffuse large B-cell lymphoma: a rare clinicopathologic entity with poor prognosis. J Clin Oncol 27:4211\u0026ndash;4216. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1200/jco.2008.21.5020\u003c/span\u003e\u003cspan address=\"10.1200/jco.2008.21.5020\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eStachurski D, Miron PM, Al-Homsi S, Hutchinson L, Harris NL, Woda B, Wang SA (2007) Anaplastic lymphoma kinase-positive diffuse large B-cell lymphoma with a complex karyotype and cryptic 3' ALK gene insertion to chromosome 4 q22-24. Hum Pathol 38:940\u0026ndash;945. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.humpath.2006.12.019\u003c/span\u003e\u003cspan address=\"10.1016/j.humpath.2006.12.019\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eReichard KK, McKenna RW, Kroft SH (2007) ALK-positive diffuse large B-cell lymphoma: report of four cases and review of the literature. Mod Pathol 20:310\u0026ndash;319. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/modpathol.3800742\u003c/span\u003e\u003cspan address=\"10.1038/modpathol.3800742\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSoumerai JD, Rosenthal A, Harkins S et al (2022) Next-generation ALK inhibitors are highly active in ALK-positive large B-cell lymphoma. Blood 140:1822\u0026ndash;1826. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1182/blood.2022015443\u003c/span\u003e\u003cspan address=\"10.1182/blood.2022015443\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWirk B, Malysz J, Choi E, Songdej N (2023) lorlatinib induces rapid and durable response in refractory anaplastic lymphoma kinase-positive large B-cell lymphoma. JCO Precis Oncol 7:e2200536. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1200/po.22.00536\u003c/span\u003e\u003cspan address=\"10.1200/po.22.00536\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTakiar R, Phillips TJ (2023) Durable responses with ALK inhibitors for primary refractory anaplastic lymphoma Kinase-positive large B-cell lymphoma. Blood Adv 7:2912\u0026ndash;2916. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1182/bloodadvances.2022007537\u003c/span\u003e\u003cspan address=\"10.1182/bloodadvances.2022007537\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWang H, Bai J, Zhang B, Pei Z, Zhao Y, Gu J, Fang Z, Liu M, Liang X, Song Q (2024) Alectinib combined with VRCD and BV monoclonal antibody for the treatment of ALK-positive large B-cell lymphoma: a case report and literature review. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fonc.2024.1480880\u003c/span\u003e\u003cspan address=\"10.3389/fonc.2024.1480880\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. Front Oncol14:1480880\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGambacorti-Passerini C, Messa C, Pogliani EM (2011) Crizotinib in anaplastic large-cell lymphoma. N Engl J Med 364:775\u0026ndash;776. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1056/NEJMc1013224\u003c/span\u003e\u003cspan address=\"10.1056/NEJMc1013224\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGambacorti Passerini C, Farina F, Stasia A et al (2014) Crizotinib in advanced, chemoresistant anaplastic lymphoma kinase-positive lymphoma patients. J Natl Cancer Inst 106:djt378. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/jnci/djt378\u003c/span\u003e\u003cspan address=\"10.1093/jnci/djt378\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYin WH, Guo N, Tian XY, Li Y, Li Z (2012) Pediatric anaplastic lymphoma kinase-positive large B-cell lymphoma: a case report and review of the literature. Pediatr Dev Pathol 15:318\u0026ndash;323. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.2350/11-11-1118-cr.1\u003c/span\u003e\u003cspan address=\"10.2350/11-11-1118-cr.1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\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":"B-cell lymphoma, anaplastic lymphoma kinase, alectinib, flow cytometry, immunosuppression","lastPublishedDoi":"10.21203/rs.3.rs-8042084/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8042084/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAnaplastic lymphoma kinase (ALK)-positive large B-cell lymphoma (LBCL) is an exceptionally rare subtype of B-cell lymphoma that typically arises independent of immunosuppression. Patients with stage IV disease have a particularly poor prognosis, with a median overall survival of approximately 1 year (95% confidence interval [CI], 0.8\u0026ndash;1.5) and a 5-year survival rate of 5% (95% CI, 0\u0026ndash;18%). Early diagnosis is often difficult because its morphological features may mimic those of solid tumors. In addition, no standard therapeutic regimen has been established, and many patients present with advanced disease and poor performance status that limit treatment options.\u003c/p\u003e\u003cp\u003eWe describe an elderly patient with ALK\u0026thinsp;+\u0026thinsp;LBCL who developed the disease while receiving immunosuppressive therapy for rheumatoid arthritis and systemic lupus erythematosus. Flow cytometry-aided early diagnosis by characterizing the distinct immunophenotype of the tumor. To our knowledge, this is the first reported case of an elderly patient (\u0026gt;\u0026thinsp;60 years) with ALK\u0026thinsp;+\u0026thinsp;LBCL treated with alectinib. Despite relapsed/refractory disease, the patient achieved tumor control for 10 months, suggesting that alectinib may represent a promising therapeutic option for this rare lymphoma.\u003c/p\u003e","manuscriptTitle":"Flow Cytometry-Aided Early Diagnosis and Alectinib Response in Advanced ALK-Positive Large B-Cell Lymphoma: A Case Report","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-08 07:35:02","doi":"10.21203/rs.3.rs-8042084/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":"a3701986-4825-4782-a990-9adcbdd92fbf","owner":[],"postedDate":"December 8th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-01-07T13:39:35+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-08 07:35:02","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8042084","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8042084","identity":"rs-8042084","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}