Correspondence *Queensland Institute of Medical Research, Bancroft Centre, 300 Herston Road, Brisbane, Qld 4006, Australia

Email maher.gandhi@qimr.edu.au

The case

A 75-year-old man was referred to a tertiary teaching hospital with a 2-month history of peripheral lymphocytosis and bilateral inguinal nodes. Previous blood counts were normal. He was diagnosed with myasthenia gravis involving the limbs and bulbar muscles 30 months before presentation and was initially treated with intravenous immunoglobulin, prednisolone and pyridostigmine. Methotrexate was added to this regimen at an average weekly dose of 17.5 mg 1 month later. At the time of presentation, he had bilateral inguinal nodes measuring 2 cm but no other palpable lymphadenopathy or organomegaly and was receiving a weekly regimen of pyridostigmine and 25 mg methotrexate. He described no systemic symptoms such as weight loss, fevers or night sweats. There was mild bilateral ptosis and muscle fatigability with normal bulbar function and mild limb weakness graded as 2a on the Myasthenia Gravis Foundation of America Clinical Classification. Eastern Cooperative Oncology Group (ECOG) performance status was 1. Peripheral blood count revealed hemoglobin at 127 g/L (normal value 135–180 g/L), platelets at 105 10⁹/L (normal value 140–400 10⁹/L), leukocytes at 12.6 10⁹/L (normal value 4–11 10⁹/L) and lymphocytes at 9.2 10⁹/L (normal value 1–4 10⁹/L). Lymphocytes were medium sized, with indented nuclei and inconspicuous nucleoli. Serum lactate dehydrogenase was 255 U/L (upper limit of normal 250 U/L). Peripheral blood flow cytometry revealed a -restricted monoclonal B-cell population that was positive for CD5, CD19 and CD20 and negative for CD10 and CD23. A t(11;14) (q13;q32) translocation was noted by fluorescent in situ hybridization (Figure 1). Right inguinal lymph-node excisional biopsy showed an expanded mantle zone of medium-sized lymphocytes and prominent hyalinized blood vessels surrounding atrophied residual germinal centers. Lymphocytes were positive for CD5, CD20 and cyclin D1 (Figure 2), confirming the diagnosis of mantle-cell lymphoma (MCL). Epstein–Barr virus (EBV)-encoded RNA in situ hybridization (EBER-ISH) was negative. CT staging revealed axillary, mediastinal, hilar, retroperitoneal, mesenteric, para-aortic, iliac and inguinal lymphadenopathy measuring up to 36 mm in maximal dimension with no hepatosplenic enlargement. [¹⁸F]-2-fluoro-2-deoxy-D-glucose-PET avidity was present in all enlarged nodal groups. Bone-marrow morphology and flow cytometry showed heavy MCL involvement. The patient was diagnosed as having Ann Arbor clinical stage IVA MCL with a high–intermediate International Prognostic Score.

Figure 1 Fluorescent in situ hybridization of peripheral blood imprints for t(11;14) translocation was performed using Vysis LSI IgH/CCND1 dual color, dual fusion translocation probe.

(A) A normal lymphoid nucleus containing two chromosome 11 (red) and two chromosome 14 signals (green). (B) Mantle-cell lymphoma t(11;14) nucleus containing one red, one green and two red/green fusion signals, indicated by arrows (1000 original magnification).

Full figure and legend (13K)Figures & Tables indexDownload Power Point slide (216K)

Figure 2 Cyclin D1 immunostaining of the patient's right inguinal lymph node.

The cyclin D1 staining reveals intense nuclear positivity (brown; 800 original magnification).

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Methotrexate treatment was ceased and the patient received eight cycles of fortnightly cyclophosphamide, doxorubicin, vincristine and prednisolone (CHOP) chemotherapy¹ with pegylated granulocyte colony-stimulating factor and 375 mg/m² rituximab at each cycle. Empirically, vincristine was omitted to prevent neuropathic damage. The patient tolerated chemotherapy well. Restaging after three cycles showed partial response, with complete remission after six cycles, and ongoing complete remission at completion.² Repeat marrow examination and flow cytometry were negative and peripheral lymphocyte count was normal. The patient remains in complete remission 12 months later and receives bimonthly rituximab at 700 mg, which will be continued for another year. Despite the fact that pyridostigmine is one-third the dose at MCL diagnosis, the patient's myasthenia gravis is substantially better controlled than before the antilymphoma therapy (graded as 1 on the Myasthenia Gravis Foundation of America Clinical Classification), with increased muscle strength on testing for fatigability and an anti-acetylcholine-receptor antibody titer of 1.84 (>8 at diagnosis).

Discussion of diagnosis

MCL accounts for 5% of all non-Hodgkin's lymphomas (NHL).³ The median age at diagnosis is 63 years and patients usually present in advanced stages. Extranodal involvement occurs in up to 30% of patients with MCL. Poor prognostic factors include age, poor performance status, splenomegaly, nodal (versus non-nodal leukemic) disease and anemia. Adverse biological parameters include blastoid morphology, cyclin D1 and c-myc overexpression.³ Gene-expression profiling is of prognostic value, but was not performed in this case because freshly frozen biopsy material was not obtained.

The diagnosis of MCL is based on histologic, immunophenotypic, cytogenetic and molecular criteria. The t(11;14) (q13;q32) translocation is pathognomonic, occurring in virtually all cases.³ The juxtaposition of the cyclin D1 gene with the immunoglobulin heavy chain variable gene (IgVH) leads to increased production of cyclin D1, which promotes cell-cycle progression from G1 through to S phase. MCL is generally regarded as aggressive and incurable, with one of the poorest long-term outcomes among all B-cell lymphomas; however, case series suggest that a more indolent leukemic disorder may exist within a proportion of patients.⁴ This subtype has the same genotypic features as MCL except for the presence of mutated IgVH genes, which indicate a post-germinal-center origin of the malignant B cells.⁴ These data await confirmation in appropriately designed prospective studies. Of note, this patient had unmutated IgVH genes.

Strictly, this case fits the criteria for a methotrexate-associated lymphoproliferative disorder (LPD; Box 1), which, although uncommon, is a recognized entity in the WHO classification.⁵ Most cases are observed in patients undergoing immunosuppression as treatment for rheumatoid arthritis.⁶ Approximately 50% of the cases are EBV-associated; however, there are conflicting epidemiological data as to whether methotrexate increases the risk of lymphoma per se. Studies have been insufficient in demonstrating an excessive risk of a rare event such as lymphoma after methotrexate treatment, and the lymphoma risk in patients with rheumatoid arthritis in the absence of methotrexate has been estimated as 2–20-fold greater than in the normal population. A prospective study found a significant increase in Hodgkin's lymphoma (HL) but not in NHL in patients with rheumatoid arthritis treated with methotrexate.⁷ There are case reports of partial regression of lymphoma after methotrexate withdrawal in approximately 60% of cases, but reporting bias may lead to overestimation.⁸ Intriguingly, most regressing cases are EBV-positive.⁹

To our knowledge, there are no reported cases of MCL in methotrexate-associated LPD. There is no definitive laboratory test to distinguish between lymphomas in which methotrexate is causally implicated and those in which the development of lymphomas after methotrexate administration is more probably coincidental. This patient exhibited a number of features that would imply the latter. Firstly, at the time of myasthenia gravis diagnosis, the patient was noted to have several small retroperitoneal, mesenteric and mediastinal nodes. None met pathological size criteria, but their multiplicity and clustering raise the possibility of indolent MCL before the development of lymphocytosis. Secondly, the diagnostic specimen was EBER-ISH-negative. It is plausible that cases of EBER-ISH-positive methotrexate-associated LPD represent the principal subset in which methotrexate is a contributory etiological factor. Methotrexate is able to activate EBV viral replication of EBV-immortalized B cells in vitro, and rheumatoid arthritis patients treated with methotrexate have higher circulating viral loads than do similar patients treated with other immunosuppressive regimens.¹⁰ Interestingly, this patient had a modestly elevated EBV DNA of 303 copies/10⁶ peripheral blood mononuclear cells (typically 0–50 copies/10⁶ peripheral blood mononuclear cells in healthy EBV carriers).

Differential diagnosis

Generalized low-volume lymphadenopathy is usually caused by a malignant LPD encompassing various subtypes of HL and NHL; however, benign and other malignant causes must also be excluded.¹¹ The principal infective causes include HIV and mononucleosis illness caused by EBV, cytomegalovirus, toxoplasma and bartonella. Infections are usually suspected clinically and are diagnosed by culture and/or antibody tests. In this patient, all infection tests were negative except for those in relation to past exposure to EBV and cytomegalovirus. Other benign causes include sarcoidosis, amyloidosis and storage disorders such as Gaucher's disease, although the latter would be highly atypical in this age group.¹² Autoimmune syndromes might rarely cause generalized lymphadenopathy, although this would be an unlikely feature of myasthenia gravis. Patients receiving medications such as phenytoin and carbamazepine can also present with nodal masses. Advanced solid tumors might cause widespread lymphadenopathy, but additional features would be expected. In most cases, generalized lymphadenopathy is diagnosed as a malignant lymphoid disorder following histopathological examination of an excisional biopsy.¹²

The Lambert–Eaton syndrome (LES) is an autoimmune myasthenia affecting the trunk and proximal muscles because of deficient release of acetylcholine at the neuromuscular junction. In contrast to myasthenia gravis, power is initially increased with exercise. LES is classically associated with small-cell carcinoma of the bronchus; however, cases secondary to lymphoma are well recognized. In this case, both the neurological features and anti-acetylcholine-receptor antibodies rule out LES in association with MCL.¹³

Treatment and management

MCL remains an incurable lymphoma, with no plateau seen in survival curves. There is no consensus regarding the optimum standard of care.¹⁴ Anthracycline-based strategies such as CHOP are generally used, although unproven by randomized controlled trials, and provide a median overall survival of approximately 3 years.¹⁴ Dose-intense regimens have their advocates, but are generally not suitable for older patients. The addition of de novo rituximab to the CHOP regimen (R-CHOP) increases response but not survival rates.¹⁵ R-CHOP has, however, been shown to prolong survival in patients with relapsed/refractory disease.¹⁶ A nonsignificant trend towards increased event-free survival has been noted with rituximab maintenance therapy, and factors predictive of prolonged event-free survival with rituximab include Fc gamma receptor IIIA position 158-valine homozygosity.¹⁷ In this case, the patient was heterozygous (valine/phenylalanine) at position 158. Investigational approaches include high-dose chemotherapy with stem-cell support, allogeneic stem-cell transplantation, the proteosome inhibitor bortezomib, the cyclin-dependent kinase modulator flavopiridol and radioimmunotherapy.¹⁴

Cessation of methotrexate is mandatory in patients with methotrexate-associated LPD, but could have resulted in exacerbation of myasthenia, particularly following recovery from the immunosuppressive effects of chemotherapy. The fact that myasthenia gravis is better controlled off methotrexate may be attributable to the B-cell-depleting antibody rituximab, which has been beneficial in cases of refractory myasthenia gravis.¹⁸ Its mechanism is postulated to involve reduction in anti-acetylcholine-receptor antibodies, which inflict damage at the neuromuscular junction.

Conclusions

To our knowledge, this is the first reported case of methotrexate-associated LPD with MCL histology. We wish to highlight the difficulties in ascribing this label to individual patients, and the need for further research to identify those cases in which methotrexate is genuinely causally implicated. Given the rarity of methotrexate-associated LPD, trials can only be conducted in the multicenter clinical setting. Hemato-oncologists treating these patients should publish their findings for the benefit of future patients.

Acknowledgments

We would like to thank Dr RA Seaton for critically reading the manuscript. Written informed consent for release of clinical information was obtained from the patient.

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Subject areas under which this article appears: Hematology