T-cell Receptor Gamma Gene Rearrangement Research Articles

Open-Capsule Budesonide for Refractory Celiac Disease.

Refractory celiac disease (RCD) is a rare condition often associated with poor prognosis. Various immunosuppressive medications (IMs) have been used with modest success. We describe outcomes in patients treated with open-capsule budesonide (OB), including those for whom IM treatment failed. We identified RCD patients treated with OB at Mayo Clinic, Rochester, Minnesota from 2003 to 2015. Demographic, serologic, and clinical variables were analyzed. We identified 57 patients who received OB for suspected RCD. Based on clonal T-cell receptor gamma gene rearrangement or aberrant phenotype of intraepithelial lymphocytes (IELs), 13 patients (23%) were classified as having RCD-2 and 43 (75%) as RCD-1. In one patient (2%) TCR gene rearrangement status was unknown. Most patients were women (69%), mean (s.d.) age was 60.5 (3.5) years and body mass index was 28.4 (4.5) kg/m2. The majority had diarrhea (72%), with median of 6 bowel movements per day (range, 4-25). IM treatment (azathioprine, systemic corticosteroids, or regular budesonide) had failed in nearly half. Twenty-four patients (42%) had anemia and 12 (21%) had hypoalbuminemia. All had Marsh 3 lesions on biopsy: 3a (19%), 3b (46%), and 3c (35%). After OB therapy, the majority had clinical (92%) and histologic (89%) improvement. Follow-up biopsy in 7 out of 13 patients with RCD-2 (53%) showed an absence of clonal TCR gamma gene rearrangement/aberrant IEL phenotype previously seen. On follow-up, 2 patients (4%) died of enteropathy-associated T-cell lymphoma. Most patients with RCD show clinical and histopathologic improvement with OB therapy, including those with failure of IMs. OB is a promising therapeutic option for management of RCD.

Systematic STAT3 Mutation Testing Identifies Patients with Unsuspected T-Cell Large Granular Lymphocytic Leukemia

▪The initial clinical presentation of T-cell large granular lymphocytic leukemia (T-LGL) and myelodysplastic syndromes (MDS) can be similar, each characterized by unexplained peripheral cytopenias. However, these diseases are pathobiologically distinct and associated with stark differences in prognosis and therapy. T-LGL is a clonal lymphoid disorder defined by phenotypically abnormal cytotoxic T cells and an indolent clinical course, while MDS is a clonal disorder of hematopoietic stem cells defined by ineffective hematopoiesis, morphologic dysplasia, and an elevated risk of acute leukemia. Despite these differences, distinction between T-LGL and MDS can be challenging and misdiagnosis can significantly delay initiation of appropriate therapy. The recent identification of STAT3 mutations in LGL may facilitate this distinction: activating STAT3 mutations occur in 40-70% of T-LGL cases, primarily within the SH2 domain, but have not been reported in patients with MDS without concomitant T-LGL. STAT3 is included within our clinical next generation sequencing (NGS) panel, which is used to evaluate patients with known or suspected hematologic malignancies, primarily acute myeloid leukemia, MDS and myeloproliferative neoplasms, as well as various lymphocytic leukemias. We report the frequency and type of STAT3 mutations within our patient population and assess the impact of this information on diagnosis.Between 1/1/2015 and 6/30/2016, 3414 samples (primarily peripheral blood (PB) or bone marrow (BM)) from 2530 unique patients evaluated at Brigham and Women's Hospital and/or Dana-Farber Cancer Institute underwent clinical NGS with a custom, 95-gene, amplicon-based panel (PMID: 27339098). Exons 2-17 and 21-23 of the STAT3 gene were analyzed in each sample using reference transcript 1 (NM_139276).We identified 40 patients with 40 candidate STAT3 mutations (Figure 1). Based on domain localization, variant allele fraction, and population allele frequency, we classified these sequence variants as somatic SH2 domain mutations (n = 21), somatic non-SH2 domain mutations of unknown significance (n = 5) or germline variants (n = 14). Of the 21 patients with somatic SH2 domain mutations, 9 carried a prior diagnosis of T-LGL and 8 were concurrently diagnosed with T-LGL by conventional diagnostic criteria (clonal aberrant T cells in the setting of neutropenia, anemia, or lymphocytosis). The final 4 patients with STAT3 SH2 domain mutations were unexpected diagnoses of T-LGL. These 4 patients were initially referred from outside institutions for MDS based on the reported presence of unilineage erythroid dysplasia (n=2), unquantified ring sideroblasts (n=1), or pancytopenia with unspecified marrow findings (n=1). In 3 of these cases, the STAT3 mutation discovery prompted T-cell flow cytometric analysis of peripheral blood, which revealed an aberrant immunophenotype, and T-cell receptor gamma gene rearrangement studies, which were clonal; these tests are pending in the 4th case. BM evaluation was performed in 12 of 21 patients, including the 4 with suspected MDS; in all cases, the findings did not meet diagnostic criteria for MDS by expert hematopathology review and all showed a normal karyotype. Five additional cases demonstrated somatic non-SH2 domain STAT3 mutations of unknown pathobiologic significance: 3 myeloid neoplasms, 1 chronic lymphocytic leukemia, and 1 autoimmune hemolytic anemia. Additional non-STAT3 mutations were also frequently identified in tumors other than T-LGL.Our experience demonstrates that STAT3 sequencing is a critical component of the evaluation of unexplained cytopenias, and identification of a mutation can clarify ambiguous phenotypes thus averting the consequences of misdiagnosis or diagnostic delay. Notably, several series have reported that MDS and T-LGL can infrequently occur concurrently and thus identification of a STAT3 mutation and a clonal T-LGL population does not exclude the possibility of concomitant MDS. In our cohort, however, no T-LGL patient with a STAT3 mutation demonstrated pathologic evidence of MDS and the majority (19 of 21) showed no other myeloid-associated somatic mutations. In addition, 5 cases in our cohort had likely somatic, non-SH2 domain STAT3 mutations in the context of disparate clinical scenarios, suggesting that these mutations may have a pathogenic role in other hematologic malignancies, a subject of future study. [Display omitted] DisclosuresDeAngelo:Ariad: Consultancy; Pfizer: Consultancy; Novartis: Consultancy; Amgen: Consultancy; Baxter: Consultancy; Incyte: Consultancy; Celgene: Consultancy. Stone:Celator: Consultancy; Jansen: Consultancy; ONO: Consultancy; Agios: Consultancy; Novartis: Consultancy; Pfizer: Consultancy; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Juno Therapeutics: Consultancy; Amgen: Consultancy; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Karyopharm: Consultancy; Merck: Consultancy; Roche: Consultancy; Seattle Genetics: Consultancy; Sunesis Pharmaceuticals: Consultancy; Xenetic Biosciences: Consultancy. Lindsley:MedImmune: Research Funding; Takeda Pharmaceuticals: Consultancy.

Characterization of Myelodysplastic Syndromes (MDS) with T-Cell Large Granular Lymphocyte Proliferations (LGL)

▪Introduction:MDS include a spectrum of hematopoietic stem cell malignancies characterized by bone marrow failure and dysplastic morphology. LGL is a clonal proliferation of cytotoxic T cells, which manifest as neutropenia, anemia, and thrombocytopenia and is associated with autoimmune disorders. LGL in association with MDS has been previously reported. However, clinicopathological features, prognostic, and predictive factors in those patients diagnosed with both LGL and MDS is not well studied.Methods:We identified patients at Moffitt Cancer Center (MCC) diagnosed with MDS who were previously tested for the presence of LGL clonal populations by peripheral blood flow cytometry at time of first visit. An LGL population was defined by the standard flow cytometry immunophenotype and clonality confirmed by T-cell receptor gamma and beta gene rearrangement.. Next Generation sequencing data was available for 151 patients. Recurrent somatic gene mutations were compared between patients with an LGL clone and those without.Results:Of the 675 patients with MDS tested for LGL in the database, 206 (30.5%) had an LGL clonal population. The mean LGL absolute cell count in the peripheral blood was 335/µL. Table-1 summarizes the baseline characteristics of the two groups.There was no difference in response to azacitidine therapy. Among 50 patients with LGL clone who received azacitidine with available data on response, the rate of hematological improvement or better (HI+) was 38%. The (HI+) was 28% among 105 patients evaluable for response without LGL clone. P .14The median overall survival (OS) was for patients with no LGL clone was 65 months (mo) compared to 46 mo (p .024). The median OS for lower risk MDS patients (low/int-1 by International Prognostic Scoring System [IPSS]) was 68 mo versus 97 mo for those with or without LGL proliferation, respectively (P .005). In higher risk MDS, there was no difference in median OS between those with or without LGL expansion, respectively (20 mo versus 16 mo, p .7). The median OS for patients with very low/ low Revised-Internatinal Prognostic Scoring System (R-IPSS) was 96 mo if LGL proliferation was detected compared to 128 mo if it was not, (p value .016). For intermediate R-IPSS the median OS was 65 mo and 41 mo with or without LGL proliferation (p .16). Finally, for high/very high R-IPSS the median OS was 18 and 16 mo with or without LGL proliferation, (p .84) In cox regression analysis the presence of an LGL clone was independently prognostic for OS after adjusting for age and R-IPSS, Hazard ratio 1.3, p = .05. Somatic gene mutation data were available for 151 patients; there was no statistically significant difference in the distribution of any mutation except IDH-2 (Table-2). The most common somatic mutations observed among patients with LGL clone were SF3B1 19%, TET-2 16%, U2AF1 13%, IDH-2 13%, RUNX-1 13%, and ASXL-1 10%. In patients without an LGL clone the most common somatic mutations were TET-2 26%, ASXL-1 20%, DNMT3A 16%, TP53 13%, SF3B1 12%.Conclusion:An LGL clone is demonstrable in approximately 30% of patients with MDS in association with advancing age. The presence of LGL proliferation was associated with worse OS in lower risk MDS pts. Although the spectrum of somatic gene mutations were similar, the presence of IDH-2 mutation and absence of DNMT3A or TP53 gene mutationscharacterized LGL+ cases. [Display omitted] [Display omitted] DisclosuresRoe:Celgene: Speakers Bureau; Alexion: Speakers Bureau; Seattle Genetics: Speakers Bureau. Sweet:Pfizer: Speakers Bureau; Novartis: Consultancy, Speakers Bureau; Ariad: Consultancy, Speakers Bureau; Incyte Corporation: Research Funding; Karyopharm: Honoraria, Research Funding. Sokol:Seattle Genetics: Consultancy; Spectrum: Consultancy. Komrokji:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Speakers Bureau; Incyte: Consultancy.

Characterization of a PCR-based lymphocyte clonality assay as a complementary tool for the diagnosis of feline lymphoma.

Differentiation between resident mature lymphocyte populations and small cell lymphoma cannot be made by cytological review alone and remains challenging in histopathological review. These cases warrant application of complementary tools like PCR-based immunoglobulin (IG) and T-cell receptor (TCR) clonality testing for confirmation. In this prospective study, diagnostic sensitivity and specificity of different primer sets for routine diagnosis of feline TCR gamma (TCRG) and complete IG heavy chain (IGH) gene rearrangements were assessed. Fine needle aspirates from 20 feline lymphoma cases and lymph node material from 10 cats without hematopoietic neoplasia were subjected to clonality testing. Feline lymphoma cell lines and previously confirmed patient material served as positive control. Detection limits for clonal populations within a polyclonal background was 90% for B-cells and 50% for T-cells. Diagnostic sensitivity and specificity of the clonality assay were 70% and 90%. Overall diagnostic accuracy was 77%, positive predictive value 93% and negative predictive value 60%.

Peripheral T-cell lymphomas of follicular helper T-cell type frequently display an aberrant CD3−/dimCD4+ population by flow cytometry: an important clue to the diagnosis of a Hodgkin lymphoma mimic

AbstractNodal follicular helper T-cell-derived lymphoproliferations (specifically the less common peripheral T-cell lymphomas of follicular type) exhibit a spectrum of histologic features that may mimic reactive hyperplasia or Hodgkin lymphoma. Even though angioimmunoblastic T-cell lymphoma and peripheral T-cell lymphoma of follicular type share a common biologic origin from follicular helper T-cells and their morphology has been well characterized, flow cytometry of peripheral T-cell lymphomas of follicular type has not been widely discussed as a tool for identifying this reactive hyperplasia/Hodgkin lymphoma mimic. We identified 10 peripheral T-cell lymphomas of follicular type with available flow cytometry data from five different institutions, including two cases with peripheral blood evaluation. For comparison, we examined flow cytometry data for 8 classical Hodgkin lymphomas (including 1 lymphocyte-rich classical Hodgkin lymphoma), 15 nodular lymphocyte predominant Hodgkin lymphomas, 15 angioimmunoblastic T-cell lymphomas, and 26 reactive nodes. Lymph node histology and flow cytometry data were reviewed, specifically for the presence of a CD3−/dimCD4+ aberrant T-cell population (described in angioimmunoblastic T-cell lymphomas), besides other T-cell aberrancies. Nine of 10 (90%) peripheral T-cell lymphomas of follicular type showed a CD3−/dimCD4+ T-cell population constituting 29.3% (range 7.9–62%) of all lymphocytes. Five of 10 (50%) had nodular lymphocyte predominant Hodgkin lymphoma or lymphocyte-rich classical Hodgkin lymphoma-like morphology with scattered Hodgkin-like cells that expressed CD20, CD30, CD15, and MUM1. Three cases had a nodular growth pattern and three others exhibited a perifollicular growth pattern without Hodgkin-like cells. Epstein–Barr virus was positive in 1 of 10 cases (10%). PCR analysis showed clonal T-cell receptor gamma gene rearrangement in all 10 peripheral T-cell lymphomas of follicular type. By flow cytometry, 11 of 15 (73.3%) angioimmunoblastic T-cell lymphomas showed the CD3−/dimCD4+ population (mean: 19.5%, range: 3–71.8%). Using a threshold of 3% for CD3−/dimCD4+ T cells, all 15 nodular lymphocyte predominant Hodgkin lymphoma controls and 8 classical Hodgkin lymphomas were negative (Mann–Whitney P=0.01, F-PTCL vs Hodgkin lymphomas), as were 25 of 26 reactive lymph nodes. The high frequency of CD3−/dimCD4+ aberrant T cells is similar in angioimmunoblastic T-cell lymphomas and peripheral T-cell lymphomas of follicular type, and is a useful feature in distinguishing peripheral T-cell lymphomas of follicular type from morphologic mimics such as reactive hyperplasia or Hodgkin lymphoma.

Aggressive T-LGL or indolent Hepatosplenic T-cell lymphoma: case with a difficult differential diagnosis

T-cell large granular lymphocytic leukemia (T-LGL) is an indolent lymphoproliferative disorder characterized by persist en t (>6 months ) i nc rease in pe r iphe ra l b lood immunophenotypically distinct, clonal population of large granular lymphocytes, usually between 2–20×10^9/L, without a clearly identifiable cause [1]. Hepatosplenic T-cell lymphoma (HSTL), on the other hand, is a uniformly aggressive disease with a fulminant systemic clinical presentation with massive hepatosplenomegaly, bone marrow involvement, thrombocytopenia and anemia, and B symptoms with survival counted in months (6–16 months) from the time of diagnosis [1]. More indolent variants are not recognized. This is a case with clinical presentation more compatible with T-large granular lymphocytic leukemia (T-LGL) and clinically followed as such, until it manifested clinically aggressively with B symptoms, splenomegaly, lymphadenopathy, and morphologically compatible with HSTL. This case continued to challenge us with the differential of a case of T-LGL with aggressive transformation or a HSTL with indolent presentation. This is a case of a 65-year-old lady, with a 10-year history of Raynaud’s disease, who presented with a mild viral syndrome associated with a slight leucopenia in September of 2007. In 2008, due to persistent cytopenia, a bone marrow biopsy was obtained. Her CBC at that time showed a wbc 1.4 k/uL, with absolute neutropenia, Hg 13gm/dl, and platelets 137 K/uL. There were no B symptoms (fever, weight loss, night sweats). The peripheral smear failed to show an abnormal lymphocyte or large granular lymphocytic population. The bone marrow biopsy showed a small inter-trabecular nodular CD3-positive T-lymphocytic aggregate without significant interstitial T-cell infiltrates. Flow analysis showed an aberrant T-cell phenotype CD3+/CD8dim+/CD7dim+/ CD56dim+/CD2+/CD16+/CD4−/CD5−/CD57− suspicious for a T-cell lymphoproliferative disorder. In 2009, the patient presented with pancytopenia, marked splenomegaly and mild hepatomegaly. The CBC showed wbc 1.7 (Neutrophil 340/ uL), Hg 10.8, and plt 80. The bone marrow was hypercellular marrow (70 %) with decreased granulocytes, increased erythroid precursors, and normal-appearing megakaryocytes. There was an interstitial and moderate intrasinusoidal T-cell infiltrate containing small lymphocytes with round to slight irregular nuclear profiles and ample cytoplasm (HE, CD3+, TIA1+, Granzyme B equivocal negative) Fig. 1 (a–c). Flow analysis showed a similar aberrant phenotype CD3+/ CD8dim+/CD7dim+/ CD56dim+/ CD2+/CD16+/CD4−/ CD5−/CD57− and γδ T-cell receptor-positive. Molecular studies were performed, and TCR beta and gamma gene rearrangements were seen. Cytogenetic studies showed a normal karyotype. FISH studies performed showed no chromosome 7 abnormalities. She was treated with G-CSF for her cytopenias. Unfortunately, this caused a splenic rupture (Fig. 1d). The spleen showed a diffuse red pulp disease with atrophic follicles. In June of 2010, she presented with B symptoms with fevers, night sweats, and a 10-pound weight loss and paraaortic and inguinal lymphadenopathy. There were persistent cytopenias: wbc 2.8, rbc 3.18, plt 204 k. A lymph node biopsy was obtained. The lymph node architecture was effaced by a diffuse population of large transformed lymphocytes (Fig. 1e). By immunostains, they marked as CD3+, CD8+, TIA+ T-cell infiltrate with flow analysis showing a similar phenotype as reported earlier. A peripheral smear reviewed at this time showed numerous large granular lymphocytes (Fig. 1f). She * Prabhjot Kaur prabhjot.kaur@hitchcock.org

T-Prolymphocytic Leukemia Is Sensitive to Polyclonal T Cell-Derived Graft-Versus-Leukemia Effects: Evidence from Minimal Residual Disease Kinetics and TCR Repertoire Diversity Analyses

▪ INTRODUCTIONT-prolymphocytic leukemia (T-PLL) is a rare T cell malignancy with an aggressive clinical course. Preliminary clinical data suggest that allogeneic stem cell transplantation (alloSCT) may provide long-term disease control in a proportion of patients. However, direct evidence that graft-versus-leukemia (GVL) activity is indeed effective in T-PLL is lacking. We sought to investigate GVL in T-PLL by correlating minimal residual disease (MRD) kinetics with immune modulatory interventions (immunosuppression tapering, donor lymphocyte infusions (DLI), chronic graft-versus-host disease (cGvHD)), and T cell receptor (TCR) repertoire diversity alterations after alloSCT. METHODSThe study sample consisted of 10 consecutive patients who received alloSCT for T-PLL at the University of Heidelberg between 2007 and 2015. Quantitative MRD monitoring was performed using clone-specific real-time quantitative PCR (RQ-PCR) of clonal TCR beta (TRB) and/or gamma (TRG) gene rearrangements. Data interpretation followed EuroMRD guidelines. In selected patients, TCR repertoire diversity was analyzed longitudinally by next-generation sequencing (NGS). TRBV-TRBD-TRBJ gene rearrangements were amplified according to BIOMED2 protocol on genomic DNA. PCR products were sequenced on Illumina's MiSeq platform. NGS data were analyzed through a purpose-built bioinformatics immunoprofiler (EuroClonality-NGS consortium). Rearrangements with very similar junctional amino acid sequences and identical TRBV and TRBJ gene usage were defined as clonotypes. RESULTSPatients underwent alloSCT in remission after first-line (8) or salvage (2) alemtuzumab-based therapy. 5 patients were allografted with an unrelated donor, 4 with a related donor, and one received haploidentical alloSCT. Conditioning was fludarabine with cyclophosphamide and/or total body irradiation-based. All patients had a cytological complete response (CR) after alloSCT. 2 patients died early because of acute GvHD, and one had no MRD marker, leaving 7 patients for MRD monitoring. Of these, 3 were MRD- at alloSCT, whereas 5 patients remained or became MRD+ early after alloSCT. In all of these 5 patients, immunosuppression tapering (3) or DLI (2) resulted in significant reduction of MRD levels (range 1-3 log) and was accompanied by cGvHD in 3 patients. However, durable MRD- was obtained in only 2 patients (alive 81+ and 12+ months post transplant), whilst MRD re-increased in 3 patients after 5-28 months despite ongoing cGvHD in one of them. NGS of the TCR repertoire was performed longitudinally in 3 patients with the longest follow-up. A total of 104 samples (blood (n=91) and BM (n=10) plus 3 donor blood samples) were sequenced. The sample at diagnosis showed one or two major clonotypes in all 3 sequenced T-PLLs, predominantly reflecting a mono- or biallelic leukemic TRB gene rearrangement. Kinetics of this leukemic clonotype followed kinetics of RQ-PCR MRD measurement, demonstrating that NGS can be used to quantify MRD in T cell lymphoma. Immediately after transplantation, the TRB repertoire was heavily skewed in all 3 patients, but recovered over time. Also in all 3 patients, MRD responses were reproducibly associated with a shift from a clonal, T-PLL-driven profile to a polyclonal signature. This corresponded to the donor clonotype repertoire and disappeared with increasing MRD levels. In each of the samples after alloSCT, several expanded non-leukemic clonotypes were observed at a maximum frequency from 3% to 54% of total TRB sequences. However, during MRD response, novel dominant clonotypes that could explain a clonal GVL effect did not emerge. Figure 1 shows an example of a patient repeatedly showing MRD response to immune interventions. Currently, 5 patients are alive and in cytological CR (4-81 months after alloSCT), translating into a median relapse-free survival of 40 months. CONCLUSIONSThe MRD responses to immune interventions observed here provide the first direct evidence for the efficacy of GVL in T-PLL. However, the GVL effect in T-PLL appears to be often only limited or transient. It also does not seem to be caused by the emergence of novel dominant T cell clones but is rather relying on a poly-/oligoclonal T cell response. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Composite angioimmunoblastic T-cell and diffuse large B-cell lymphoma

A58-year-old female smoker with a history of hypertension presented to the emergency department with a recurrent episode of worsening shortness of breath over five days. Her exam was significant for sinus tachycardia, cervical lymphadenopathy, and a firm left supraclavicular node. Laboratory values demonstrated a normal white cell count and differential, normocytic anemia, thrombocytopenia, and a lactate dehydrogenase of 803 IU/L. She was HIV negative. Computerized tomography (CT) scan of the chest/abdomen/pelvis demonstrated extensive bilateral cervical, supraclavicular and axillary adenopathy, mediastinal and hilar lymphadenopathy with infiltrate and consolidation of the right lower and posterior upper lobe and encasement of the right pulmonary artery, as well as splenomegaly with paraaortic and retroperitoneal adenopathy. An echocardiogram revealed a normal left ventricle and a severe pericardial effusion. She underwent a pericardial window and an excisional supraclavicular lymph node biopsy, which revealed T-cells with features of angioimmunoblastic T-cell lymphoma (AITL) and large atypical B-cells consistent with diffuse large Bcell lymphoma (DLBCL) (Figure 1A). Destruction of the lymph node architecture was noted, with many medium-sized atypical lymphocytes with irregular nuclei and moderate amounts of pale cytoplasm. In addition, scattered and focally increased large atypical lymphocytes with prominent nucleoli were seen. Blood vessels were increased. Immunostains demonstrated atypical T-cells expressing CD3 (Figure 1B), CD4 (Figure 1C), CD5, CD2, CD7, CD4, CD10, PD-1 (Figure 1D), as well as clustered large B-cells positive for expression of CD20, Bcl-6 and BOB-1 (Figure 1E). Polymerase chain reaction (PCR) testing revealed a clonal TCR gamma gene rearrangement, as well as a clonal IgH gene rearrangement. Epstein-Barr virus-encoded RNA (EBER) was negative by in situ hybridization (ISH). Bone marrow biopsy and CSF analysis were negative for lymphoid or plasma cell infiltrates. The patient was initiated on R-CHOEP (rituximab, cyclophosphamide, doxorubicin, vincristine, etoposide, prednisone) and completed three cycles of chemotherapy. Her CT scan after three cycles was consistent with a partial response, with significantly diminished bilateral axillary lymphadenopathy, improved mediastinal, perihilar and right lower lobe infiltrates, decreased splenomegaly, and upper abdominal and retroperitoneal adenopathy. The patient eventually had progression of her disease during the course of subsequent R-CHOEP cycles. She then progressed through two cycles of gemcitabine-carboplatin and one course of romidepsin. Her performance status deteriorated, not enabling any further treatment and the patient finally expired, approximately 24 months after initial diagnosis. This case illustrates a rare hematologic malignancy presenting with a dual primary lymphoma population. The treatment of composite lymphoma with simultaneous Band T-cell components is challenging and the less favorable prognosis component determines

A Recurrent T-Cell Receptor γ Rearrangement of the Vγ9-JγP Type Represents the Major Clone of γδ T-Cells in Normal Individuals and Is Highly Suppressed in Lymphoproliferative Disorders

INTRODUCTIONγδ T-cells expressing T-cell receptor (TCR) type Vγ9Vδ2 are well-established mediators of anti-tumor immunity and have demonstrable HLA-independent cytotoxic activity against both B- and T-cell non-Hodgkin lymphoma cells. Initial clinical trials using ex vivo expanded, adoptively transferred γδ T-cells to enhance tumor immunity have shown some promise but overall results have been lackluster. This indicates that wholesale polyclonal expansion prior to transfer is not likely to be effective. Thus more refined approaches are necessary. This requires a better understanding of their TCR repertoire. We recently developed a next generation sequencing (NGS)-based method for evaluating the spectrum T-cell receptor gamma (TRG) gene rearrangements present in clinical samples. In an effort to better understand the TCR repertoire of γδ T-cells we used this strategy to evaluate a series of samples from normal individuals and from individuals with B and T-cell lymphoproliferative disorders (LPDs).METHODSDNA was isolated from samples from 11 normal individuals (all peripheral blood, PB), 11 patients with a T-cell LPD (6 PB, 3 bone marrow; BM, 2 FFPE tissues), and 5 patients with a B-cell LPD (Hairy cell leukemia; HCL, 4 PB, 1 BM). γδ T-cells were sorted from the PB of 4 of the healthy donors by FACS. TRG rearrangements were PCR amplified using consensus primers and NGS libraries were prepared and sequenced on the Ion Torrent PGM platform. The data was analyzed as follows. NGS typically yielded up to 400,000 sequencing reads which were grouped by identical V, J, and CDR3 sequences into unique rearrangements (typically 15-30,000). The prevalence of a particular TRG rearrangement (or CDR3 sequence) was determined by the number of individual NGS reads with this unique sequence per the entire data set (percent of total reads). All rearrangements were then ranked by their prevalence.RESULTSWe sequenced the TRG repertoire of isolated γδ T-cells from the peripheral blood of normal individuals (n=4). We found that a recurrent Vγ9-JγP rearrangement with the CDR3 sequence CALWEVQELGKKIKVF was always (4 of 4 samples) the most prevalent rearrangement in normal γδ T-cells (3.2-11.7-fold more prevalent than the second most common rearrangement; representing 4-11.9% of total reads). Similarly, analysis of a larger set of unsorted normal peripheral blood samples demonstrated high prevalence of the same canonical CDR3 in 5 out of 7 samples, confirming that it is very common in most individuals relative to all TRG rearrangements (among the top 10 most prevalent CDR3s in 4 of 5 samples). We also sequenced the TRG repertoire in 11 samples from patients demonstrating evidence of involvement by a T-cell LPD. Unexpectedly, all Vγ9-JγP type rearrangements were strongly suppressed in these samples including the one with the canonical CDR3. The canonical rearrangement (present in 6 /11 cases), or the most abundant Vγ9-JγP rearrangement when the canonical rearrangement was absent (absent in 5/11 cases), represented on average 0.036 ±0.024 % of all NGS reads in the samples from patients with T-cell LPDs compared to 0.72 ±0.72 % of total NGS reads in the normal controls. This represents on average a 19.9 fold reduction in the T-cell LPD samples. The median rank by abundance of the top Vγ9-JγP rearrangement dropped from 9th (normals) to 291st (T-cell LPD cases) indicating that the suppression was not simply a consequence of the presence of an abundant malignant T-cell clone in the data. The overall distribution of TRG V-segment usage in the normal and neoplastic samples was comparable (Vγ9: 9.9 ±0.55 % and 7.2 ±2.79 %, respectively). In 2 samples from patients with HCL, Vγ9-JγP rearrangements were reduced to a similar extent to that seen in the T-cell LPD cases (9.5-fold reduced; average 0.06% of NGS reads; average rank order 451st). In the other 3 cases of HCL the findings were very similar to those seen in the normal samples.CONCLUSIONSWe identified a recurrent Vγ9-JγP rearrangement by NGS representing the most abundant CDR3 in sorted γδ T-cells from normal individuals. This population, along with other clones with Vγ9 rearrangements, appeared specifically suppressed in all samples from patients with T-cell LPDs and in 2 or 5 samples from patients with B-cell LPDs (HCL), perhaps indicating a role in the disease process. Additional samples from a wider range of T- and B-cell LPDs are being analyzed. DisclosuresNo relevant conflicts of interest to declare.

Mycosis fungoides with CD20 expression: report of two cases and review of the literature

CD20 expression is exceedingly rare in T-cell lymphomas. Most published cases have been diagnosed as peripheral T-cell lymphomas, not otherwise specified. Only 18 cases of CD20-positive mycosis fungoides (MF) have been previously reported. Here, we describe two cases of CD20-positive MF. Patient 1 was an 84-year-old woman who presented with a 5-year history of multiple pruritic erythematous papules coalescing into thin plaques over 80% of her body surface area. She expired after developing tumors and large cell transformation. Patient 2 was a 67-year-old woman with a long-standing history of tumor stage MF with large cell transformation. She developed a nodular plaque while receiving topical and systemic therapy. In both cases, the neoplastic T-cells demonstrated a CD4-positive immunophenotype with loss of pan-T-cell markers and a monoclonal T-cell receptor gamma gene rearrangement. CD20 was expressed by a significant population of the neoplastic T-cells, but these T-cells lacked expression of other B-cell markers, including CD79a, CD19 and PAX5. This report adds to and summarizes the small body of literature describing CD20-positive MF, and discusses diagnostic and clinical implications.

Aggressive Subcutaneous Panniculitis-Like CD30+ Peripheral T-Cell Lymphoma with Diffuse EBER Expression.

T-cell lineage lymphoma with an intense membranous and paranuclear CD30 expression in the absence of ALK1 raises a differential diagnosis of peripheral T-cell lymphoma (PTCL), NOS and anaplastic large cell lymphoma (ALCL), ALK negative. However, Epstein-Barr virus is consistently negative in ALCL and is not considered an implicating factor in its pathogenesis. We describe a case of T-cell lymphoma showing anaplastic large cell morphology with scattered hallmark cells and a uniform CD30 and Epstein-Barr virus encoded early RNA (EBER) expression that primarily involved the subcutaneous tissue at presentation. On incisional biopsy, the neoplastic cells were positive for CD3, CD2, and CD30 while negative for LCA, CD20, PAX5, CD56, ALK1, and cytotoxic granules. Molecular analysis identified a positive T-cell receptor (beta and gamma) gene rearrangement by PCR. Proliferation index approached 100% and the patient had a rapidly progressive course; the subcutaneous lesions more than doubled in size within couple of weeks with new evidence for widespread systemic involvement. This case emphasizes a rare EBV association with a CD30 positive T-cell lymphoma where the morphologic and immunophenotypic findings are otherwise nondiscriminatory between PTCL, NOS and ALCL, ALK negative.

Genomic Characterization Of Histiocytic Lesions Following Pediatric T-Cell Acute Lymphoblastic Leukemia

IntroductionThe development of histiocytic lesions after acute lymphoblastic leukemia (ALL) is rare, occurring in 6 of 971 patients enrolled on BFM treatment regimens for T-ALL (Trebo 2005). Few cases of histiocytosis arising after a history of T-ALL have been characterized at the molecular level for genomic alterations. One case of fatal Langerhans cell histiocytosis (LCH) following treatment of T-ALL revealed activating mutations in NOTCH1; in contrast, no NOTCH1 mutations were identified in 24 other cases of LCH or Rosai-Dorfman disease without a previous history of T-ALL (Rodig 2008). In patients without prior leukemia, BRAF V600 mutations have been identified in a significant proportion of patients with LCH or Erdheim-Chester Disease but not in other histiocytoses (Haroche 2012). Methodology and Principal FindingsNext generation focused exomic sequencing of 236 genes and 47 Introns was conducted on samples of histiocytic lesions from two patients with a previous history of T-ALL. Case 1A 2 year-old male presented with marked adenopathy, mediastinal mass and white blood cell count 67,000 cells/uL with 30% blasts. The blasts expressed CD45, CD2, CD3 (surface/cyto), CD5, CD7, CD38, CD45 (bright), TCR gamma-delta, but were negative for CD4, CD8, and TdT. Karyotype was 46,XY,t(8;14)(q24;q11.2),der(12)t(12;20)(q11;q13.3),der(20)t(12;20)(q21;q13.3) with rearrangement of MYC (8q24) confirmed by FISH. He was treated for T-ALL per Children's Oncology Group (COG) protocol AALL0434 and had 5% residual bone marrow blasts at day 29 of induction. MRD-negative remission was ultimately achieved with high-dose cytarabine and methotrexate followed by consolidation with nelarabine. He underwent matched unrelated cord blood transplant following conditioning with cyclophosphamide and total body irradiation with cranial boost, and engrafted at day +14. Surveillance bone marrow at day +110 revealed systemic juvenile xanthogranuloma (JXG) without T-ALL. PET/CT revealed FDG-uptake in the diffusely enlarged spleen and throughout the skeleton. Due to progressive cytopenias, therapy was initiated with vinblastine and prednisone as per LCHIII. However, refractory cytopenias exacerbated by splenic sequestration developed following induction, and he was then treated with thalidomide and splenectomy. The spleen weighed 404 grams (expected 40 grams) and was diffusely infiltrated with JXG. The cytopenias dramatically improved and he continued thalidomide for 2 months until PET scan demonstrated progression. Genomic analysis of the JXG lesion revealed NRAS G13D mutation, and FISH demonstrated MYC rearrangement identical to the initial T-ALL sample. Case 2A 12-year-old male presented with WBC 142,700 cells/uL and CNS leukemia. Flow cytometry showed T-ALL with CD2, surface CD3, CD4, CD5, CD7, CD8, CD24 (subset), CD71, HLA-DR (subset) and TdT (partial). There was a clonal TCR gamma gene rearrangement and a biallelic CDKN2A (p16) deletion by FISH. He was enrolled on COG AALL0434 and had a rapid response with remission in both CNS and marrow at induction day 29. Following completion of high-dose methotrexate interim maintenance he developed hepatosplenomegaly, pancytopenia and elevated serum bilirubin, ferritin, and triglycerides. Bone marrow aspirate showed rare hemophagocytosis but no evidence of T-ALL. He was treated with dexamethasone and etoposide with no response. Follow-up bone marrow revealed brisk hemophagocytosis and a diffuse histiocytic neoplasm. Karyotype was 48,XY,+7,+11[2] /49,idem,+18[3] /46,XY[14]. PET/CT showed hepatosplenomegaly with FDG uptake in anterior mediastinum, hepatic nodules, spleen, and bone marrow. He was treated with Campath and then with intensive chemotherapy with fludarabine, cytarabine, and liposomal daunorubicin with no response and ultimately succumbed to disease. Genomic analysis of the clonal histiocytic infiltrate revealed KRAS G12C, BRAF G469V, NOTCH1 Q2440, and CCND2 G268R mutations, and FISH positive for biallelic CDKN2A (p16) deletion similar to original T-ALL. ConclusionsOur extensive genomic characterization suggests a unique molecular pathogenesis for histiocytic disorders arising after T-cell ALL and identified RAS signaling pathway and NOTCH1 mutations. Furthermore, these findings strongly indicate a potential derivation or trans differentiation from the malignant leukemic stem cell clone. Disclosures:No relevant conflicts of interest to declare.

Lymphomatoid papulosis with associated cerebellar lesion of similar histology and T-cell clonality

A 9-year old boy presented with a 4-month history of a truncal monomorphic eruption with self-healing papulonecrotic lesions. A skin biopsy revealed a dermal infiltrate of CD4, CD8 and CD30-positive T-cells, consistent with lymphomatoid papulosis. He responded to 4 months of treatment with narrowband UVB phototherapy (311 nm) which stabilised his disease. Five years later he presented with an acute onset of nausea and vomiting, dizziness, headache and ataxia. Magnetic resonance imaging of the brain revealed a lesion in the cerebellum and stereotactic resection was undertaken. Histology showed CD4, CD8 and CD30-positive T-cells similar to his skin lesions, with a monoclonal T-cell receptor (TCR) gamma gene rearrangement. Subsequent analysis of the skin detected a monoclonal band of the same size as the cerebellar lesion. Treatment was initiated for a primary central nervous system (CNS) lymphoma but ceased after one course of high-dose methotrexate. Opinion on the pathology was divided as to whether the cerebellar lesion represented an atypical reactive T-cell lymphoproliferative response or a T-cell lymphoma. On follow-up 2 years later, the patient remains clinically and radiologically clear, making CNS lymphoma unlikely.

Long-Term Follow-Up of Refractory Mycosis Fungoides Which Achieved Remission with the Addision of Isotretinoin to Methotrexate and Psoralen Plus Ultraviolat A Therapy

Dear Editor: Mycosis fungoides (MF) is the most common cutaneous T cell lymphoma. Many therapies are considered based on the patient's individual presentation1. Early stage MF is known to be easily responsive to conventional therapy. However, there is some early stage MF refractory to conventional therapy and thus need other treatment modalities2. A 38-year-old man was presented with a 3-year history of asymptomatic multiple erythematous to dusky red edematous patches over his trunk, legs, neck and face (Fig. 1A). The skin involvement measured about 45% of total body surface. Skin biopsy specimens from the leg showed clustered epidermotrophic atypical lymphocytes in the epidermis with a band-like dense lymphocytic infiltrate in the dermis (Fig. 2A). In immunohistochemical staining, lymphocytes were positive for CD3 and CD4. A T-cell receptor-gamma gene rearrangement revealed the monoclonality in the polymerase chain reaction (PCR) analysis (Fig. 2B). The biopsy finding on the left palpable inguinal lymph node was dermatopathic lymphadenitis. According to TMNB staging, the patient was diagnosed as MF stage IIA (T2N1M0B0). Initially, he was treated with low-dose methotrexate (7.5 mg/wk) and psoralen plus ultraviolet A (PUVA) twice a week, with maximum doses of 1.3 J/m2. However, the disease showed no response during the 5 weeks of therapy. As first generation retinoids, such as isotretinoin, have been shown to be effective in early stage MF for a long period of time1, isotretinoin (20 mg/day) was newly added to his treatment regimen for the following 6 weeks; consequently, the lesions showed remission (Fig. 1B). According to a recent study, many conventional treatments become ineffective when the skin involvement is over 10%, even in early stage MF2. Likewise, although the patient was diagnosed as an early stage MF in this case, the skin lesions were refractory to the initial treatment. Eventually, after the addition of isotretinoin, the skin lesions showed a dramatic improvement without relapse for 2 years. Retinoids are the so-called biological response modifiers with different mechanisms of action than traditional cytotoxic chemotherapies. The biological effects of retinoids in MF are related to its boosting immune function by inducing anti-tumor responses and leading to the apoptosis of tumor cells3. When the retinoid is combined with methotrexate or PUVA, it strengthens the effectiveness of treatment by exhibiting combined actions of each agent and decreasing the side effects by lowering the doses of total UVA irradiation4. Nowadays, many studies have focused on retinoid X receptor-selective rexinoid, bexarotene. However, the efficacy of bexarotene compared to traditional retinoids, such as isotretinoin, has yet to be determined3. Thomsen et al.5 reported the effectiveness of isotretinoin on MF. However, no study has yet reported about the effectiveness of isotretinoin as a component of this combination therapy for refractory early stage MF. As we observed the dramatic therapeutic effect with the addition of isotretinoin for refractory MF, we suggest that isotretinoin could still be a type of effectual therapeutic option for combination therapy of early stage MF, particularly when the skin involvement is over 10% or refractory to the conventional treatment. Fig. 1 (A) Edematous dusky red to erythematous patches on the back and the left leg before treatment. (B) Remission on the back and left leg with addition of isotretinoin to methotrexate and psoralen plus ultraviolet A therapy. Fig. 2 (A) Epidermotrophism with clustered atypical lymphocytes in the epidermis and dense lymphocytic infiltrate with hyperchromatic nuclei in the dermis (HE inset: H&E, ×200). (B) The result of T-cell receptor-gamma gene ...

Biomarker Quality Assurance (QA) Findings From the Comprehensive Oncology Measures for Peripheral T-Cell Lymphoma Treatment (COMPLETE) Registry

AbstractAbstract 4263 Background:COMPLETE is a registry of peripheral T-cell lymphoma (PTCL) patients from academic and community settings in the US. For the registry, selected biomarkers are recorded to determine which markers may have informed the diagnosis of PTCL. Research staff at participating sites enter data from pathology reports into an electronic data capture (EDC) system; however, pathology reports can be difficult to interpret, particularly for those who have not received specialized training. To ensure that biomarker data in COMPLETE accurately reflects pathology reports, a biomarker QA initiative was undertaken. A similar initiative was pursued by the International T-Cell Project and is the subject of a separate, parallel abstract. Methods:The COMPLETE biomarker QA initiative includes review of on-site data entered for 26 biomarkers commonly assessed in the diagnostic work-up of PTCL. External review by a board-certified pathologist is triggered once a site has entered the biomarker data into the EDC system for enrolled patients and uploaded the de-identified pathology reports. All patients enrolled at a site within 30 days of the first patient were reviewed. A single mismatch between the site-entered data and the reviewer's findings counts as an error. Findings of the review are entered into the EDC system to facilitate analysis and queries are sent to the site in cases where corrections are needed. Results:Data for 119 patients from 43 centers comprising a sum of 3570 biomarker entries were reviewed by an external hematopathologist. The mean number of phenotypic markers assessed per patient was 12 (range: 4–20), and the mean number of gene rearrangement tests performed was 0.2 (range: 0–3). Of the 119 patients, 13 (11%) had no EDC entry errors, 33 (28%) had 1–2 errors, 34 (29%) had 3–5 errors, 31 (26%) had 6–10 errors and the remaining 8 cases (7%) had 11–15 errors. The reviewer agreed with 74% of entries where the site noted a finding was positive; of the remaining entries where the site indicated the findings were positive, the reviewer noted they were negative (12%), not assessed (10%) or indeterminate (5%). For entries where the site indicated the finding was negative, the reviewer was in agreement 79% of the time; for the remaining 21% of negative-reported entries by the site, the reviewer indicated the marker was not assessed (12%), indeterminate (6%) or positive (3%). The reviewer was in agreement with the site in 92% of the entries where the site indicated the marker was not assessed; otherwise, the reviewer found the marker was negative (4%), positive (3%) or indeterminate (1%). The markers accounting for the most errors (each marker accounting for 5% or more of errors) were CD8, CD20, CD30, Epstein-Bar Virus (EBV) and T-cell receptor gamma gene rearrangement (TCR-g). The types of errors for these markers are noted in the table below.Marker, N (%) Errors OverallType of Error Noted by the Reviewer for Markers with the Most Errors, n (%)Finding is recorded in non-neoplastic cellsPathology report unclearTest performed, result not in pathology reportMarker is positive or negative; site notes oppositeMarker is not assessed or indeterminate; site notes positive or negative resultMarker is positive or negative; site notes marker was not assessedOtherCD8, 40 (8%)1 (3%)12 (30%)2 (5%)11 (28%)7 (18%)6 (15%)1 (3%)CD20, 47 (9%)24 (51%)6 (13%)0 (0%)5 (11%)6 (13%)6 (13%)0 (0%)CD30, 25 (5%)5 (20%)3 (12%)1 (4%)3 (12%)6 (24%)7 (28%)0 (0%)EBV, 27 (5%)1 (4%)2 (7%)0 (0%)0 (0%)0 (0%)23 (85%)1 (4%)TCR-g, 24 (5%)0 (0%)4 (17%)0 (0%)0 (0%)5 (21%)12 (50%)3 (13%)Sum may exceed 100% due to rounding.Recording the type of EBV test performed [in situ hybridization (ISH) and immunohistochemistry (IHC)] presented further challenges, accounting for 8% of errors. Common errors included: 1) the site indicated the test was not performed when it was performed per report and 2) the site indicated a particular test (ISH, IHC) was performed but the method used was unclear in the report. Conclusions:Results of the COMPLETE biomarker QA analysis indicate that errors in interpretation or recording of biomarker data from pathology reports are common. For registries and other observational studies, periodic reviews by an external pathologist may guide site training and improve the accuracy of data collection. For interventional clinical trials in PTCL, central review of all biomarker data by expert hematopathologists should be considered. Disclosures:Hsi:Allos: Research Funding; Eli Lilly: Research Funding; Abbott: Research Funding; Cellerant Therapeutics: Research Funding; BD Biosciences: Research Funding; Millenium: Research Funding. Gruver:MedNet Solutions: Honoraria. Foss:Allos: Consultancy; Celgene: Consultancy; Seattle Genetics: Consultancy; Eisai: Consultancy; Celgene: Study Grant, Study Grant Other; Merck: Study Grant, Study Grant Other. Carson:Allos: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau. Pinter-Brown:Allos: Consultancy, Membership on an entity's Board of Directors or advisory committees. Horwitz:Allos: Consultancy, Research Funding. Rosen:Allos: Consultancy, Honoraria. Pro:Celgene: Honoraria, Research Funding; Spectrum : Honoraria; Allos: Honoraria; Seattle Genetics : Research Funding. Gisselbrecht:Allos: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Cytogenetic abnormalities precede morphological abnormalities in developing malignant conditions: Report of 2 cases

We previously hypothesized that cytogenetic abnormalities precede morphological abnormalities in developing malignant conditions. In this context we evaluated additional cases to further confirm that hypothesis. We report on 2 additional cases in which clonal cytogenetic abnormalities were observed in otherwise morphologically normal samples. Case 1 is a bone marrow from a 73year old male with transformed follicular lymphoma (FL), while case 2 is a lymph node from a 53-year-old with lymphadenopathy, both referred to the cytogenetics laboratory for evaluation. A 73-year-old male presented with an enlarging left inguinal mass surrounding and obliterating the left iliac vein. A tissue core biopsy of the mass revealed recurrent high grade FL with diffuse large B-cell lymphoma (DLBCL). Examination of a random bone marrow biopsy of the adjacent left posterior iliac crest showed only mild hypercellularity (50%) and no evidence of malignancy, and the results were confirmed by flow cytometry. Cytogenetic evaluation revealed an interstitial deletion, del (9)(q13q32). In case 2, morphologically the lymph node showed extensive paracortical hyperplasia with numerous eosinophils and no clear indication of a neoplastic process with no abnormal lymphoid population observed by flow. PCR studies for TCR gamma and IgH gene rearrangements were negative for clonality. Chromosome analysis demonstrated 47,XY,+add(1)(p22),t(3;14)(q27;q11.2)[13]/46,XY[7]. FISH studies showed a BCL6 gene rearrangement but no TCRAD rearrangement. A subsequent inguinal lymph node biopsy showed DLBCL. These cases along with the other cases in the literature provide further evidence of genetic abnormalities preceding morphological abnormalities in developing malignant conditions.

Erythema multiforme and epitheliotropic T‐cell lymphoma in the oral cavity of dogs: 1989 to 2009

To describe the clinical signs, major laboratory findings, diagnostic procedures and outcome in dogs with erythema multiforme or epitheliotropic T-cell lymphoma of the oral cavity. Retrospective analysis identified 14 client-owned dogs with erythema multiforme or epitheliotropic T-cell lymphoma of the oral cavity. Histological changes were combined with immunohistochemistry and clonality testing data in selected cases, and a clinical follow-up was acquired. Ulcerative stomatitis with no significant or only minor abnormalities on haematology and serum biochemistry panels was common. Histological features were diagnostic in seven cases. The use of immunohistochemistry distinguished the two diseases in an additional three cases. In four cases, the diagnosis remained questionable, and clonality testing for T-cell receptor gamma gene rearrangement helped confirm erythema multiforme in one case. Clinical follow-up revealed erythema multiforme in two and epitheliotropic T-cell lymphoma in one of the remaining questionable cases. Erythema multiforme and epitheliotropic T-cell lymphoma may affect, but are rarely limited to the oral cavity of dogs, and they usually present as stomatitis. Histological features alone are not always diagnostic. Immunohistochemistry and clonality testing may assist in the differentiation between the two, but in ambiguous cases, repeated biopsy and clinical follow-up are essential.

T-cell large granular lymphocytic leukemia: treatment experience with fludarabine

OBJECTIVES:The aim of this retrospective study was to investigate the results of T-cell large granular lymphocytic leukemia treatment with fludarabine by assessing the complete hematologic response, the complete molecular response, progression-free survival, and overall survival.METHODS:We evaluated the records of six patients with T-cell large granular lymphocytic leukemia who were treated with fludarabine as a first-, second-, or third-line therapy, at a dose of 40 mg/m2, for three to five days per month and 6 to 8 cycles.RESULTS:Of the six patients investigated with T-cell large granular lymphocytic leukemia who were treated with fludarabine, five (83.3%) were female, and their median age was 36.5 years (range 18 to 73). The median lymphocyte level was 3.4×109/L (0.5 to 8.9). All patients exhibited a monoclonal T-cell receptor gamma gene rearrangement at diagnosis. Two (33.3%) patients received fludarabine as first-line treatment, two (33.3%) for refractory disease, one (16.6%) for relapsed disease after the suspension of methotrexate treatment due to liver toxicity, and one (16.6%) due to dyspesia. A complete hematologic response was achieved in all cases, and a complete molecular response was achieved in five out six cases (83.3%). During a mean follow-up period of 12 months, both the progression-free survival and overall survival rates were 100%.CONCLUSION:T-cell large granular lymphocytic leukemia demonstrated a high rate of complete hematologic and molecular response to fludarabine, with excellent compliance and tolerability rates. To confirm our results in this rare disease, we believe that fludarabine should be tested in clinical trials as a first-line treatment for T-cell large granular lymphocytic leukemia.

Large granular lymphocytosis with severe neutropenia following ipilimumab therapy for metastatic melanoma

To the editor Cytotoxic T-lymphocyte antigen-4 (CTLA-4), a molecule present on activated T cells, is a homologue of CD28 that inhibits B7 co-stimulatory molecules expressed on mature antigen presenting cells. Ipilimumab is a fully humanized monoclonal antibody against CTLA-4 [1]. Ipilimumab is FDA approved in the United States for treatment of unresectable or metastatic melanoma [1,2]. We report the first case of large granular lymphocytosis with severe neutropenia following ipilimumab therapy. A 74 year old female presented with relapsed melanoma in the left lower extremity in Sept 2009. She was treated with temozolomide. In July 2010, she developed metastatic melanoma. She was enrolled onto a clinical trial and treated with ipilimumab on Sept 16, 2010 and October 7, 2010 at 3 mg/kg (232 mg per treatment) q3wks. Her baseline CBC was normal. At the start of the third dose, there was a significant decline in the patient's WBC count. Therefore ipilimumab was stopped. The patient was admitted on November 2, 2010 with fever, malaise and generalized weakness. She was noted to have Hgb 6.2 G/dL, WBC 1.3 × 106 /L with no neutrophils, and platelet count of 158 × 106 /L. The patient had a bone marrow evaluation which showed mildly hypercellular cellular marrow with marked myeloid hypoplasia/aplasia, relative erythroid hyperplasia and mild megakaryocytic atypia. Karyotype analysis demonstrated 46 XX, but FISH analysis showed a 5q31 deletion with EGR1 gene deletion in 14% of the cells. Flow cytometry study showed 46% T-cells with 20% large granular lymphocytes and 12% NK cells. A T-cell receptor gene study by Genzyme showed clonal T-cell receptor gamma gene rearrangement. These findings were consistent with myelodysplasia and large granular lymphocytosis with severe neutropenia. In addition to broad-spectrum antibiotics and G-CSF, the patient was given IVIG without significant response. The CBC on Nov 17 was WBC 0.8, Hgb 8.9, and Platelets 271. CT scans revealed bilateral loculated pleural effusion for which she had thoracentesis. The patient was started on treatment on Nov 18, 2010 with IV methylprednisolone 1 mg/kg q12h, and equine ATG at 15 mg/kg daily × 4 days, as well as cyclosporine 2.5 mg/kg twice daily. G-CSF and antibiotics were continued. The WBC rose to 2.6 the day after ATG was completed (Table ​(Table1).1). Two days later, her WBC was 15.3. G-CSF was stopped. Her general condition improved gradually. She was discharged 11 days later. She remained on cyclosporine and low dose prednisone. Three months later, she remained only on low dose prednisone. Her CBC showed WBC 12.1, Hgb 11.9, and Platelets 259 at last follow-up visit on Feb 23, 2011. Table 1 Complete Blood Counts of the Patient The most common adverse events associated with ipilimumab are immune-related, including enterocolitis, hepatitis, dermatitis and hypophysitis. Severe hematological toxicity is rare. One case of severe autoimmune-related neutropenia was reported and the patient responded rapidly to IVIG infusion, but not to steroids [3]. This case of large granular lymphocytosis with severe neutropenia did not respond to IVIG and steroids, but had a rapid response to ATG, steroids, and cyclosporine immunosuppressive therapy. It is unclear whether the myelodysplasia findings on the bone marrow biopsy were related to the ipilimumab therapy or to previous chemotherapy with temozolomide.

Supratentorial metastatic enteropathy-associated T-cell lymphoma: A case report and literature review

Background:We are describing a rare case of supratentorial metastatic enteropathy-associated T-cell lymphoma (EATL). While these lesions are a rare complication of EATL, the implications are grave and they must be evaluated as a diagnostic possibility when a patient with known celiac disease presents with acute neurological deterioration. In addition, multidisciplinary care teams are recommended by the authors as critical to providing the most comprehensive patient care.Case Description:A 65-year-old female presented to the emergency room with uncontrolled abdominal pain, nausea, and vomiting. Initial abdominal computed tomography (CT) scan indicated a small bowel obstruction with a transition point at the jejunal area. Differential diagnosis included small bowel neoplasm, adhesions, or a reactive intestinal inflammatory process. Shortly after presentation, the patient's clinical condition worsened, requiring emergency small bowel resection. Histological analysis of the resected bowel segments demonstrated medium-sized infiltrating lymphocytes with characteristic pleomorphic nuclei and prominent nucleoli. Immunohistochemical stains revealed tumor cells positive for CD-3. Immunohistochemical analysis for Ki-67 showed a markedly increased proliferative index, with 90% of lymphocytes staining positive. Polymerase chain reaction analysis for T-cell receptor-gamma gene rearrangement was positive, demonstrating the presence of a clonal population of T-cells. The combined morphological and immunophenotypic features of this lesion were consistent with jejunal EATL.Five weeks post-diagnosis, she developed new onset neurological symptoms consisting of changes in her mental status and left facio-brachial weakness. Brain magnetic resonance imaging (MRI) demonstrated a single ill-defined, irregular, right fronto-parietal enhancing lesion surrounded by vasogenic edema. Surgical resection and histopathologic evaluation of the biopsied lesion confirmed the diagnosis of metastatic EATL involving the brain.Conclusion:Intracranial metastasis is a rare but grave complication of EATL and must be evaluated as a diagnostic possibility when a patient with known celiac disease presents with acute neurological deterioration. Although the prognosis of these patients is dismal, aggressive oncology management is mandatory.