T-cell Receptor Gamma Gene Rearrangement Research Articles

Molecular and clinicopathological features of granzyme B-negative extranodal NK/T-cell lymphoma

Extranodal NK/T-cell lymphoma (ENKTL) generally expresses cytotoxic molecules, including granzyme B (GZMB), T-cell-restricted intracellular antigen-1 (TIA-1), and perforin; however, the expression of these molecules varies across cases. We performed gene expression profiling and identified unique biological and clinicopathological features of GZMB-negative ENKTL. We reviewed the clinicopathological characteristics of 71 ENKTL samples. Gene expression profiling on nine ENKTLs using multiplexed, direct, and digital mRNA quantification divided ENKTLs into Groups A (n = 7) and B (n = 2) through hierarchical clustering and t-distributed stochastic neighbor embedding. Group B was characterized by downregulation of genes associated with IL6-JAK-STAT3 signaling and inflammatory responses. GZMB mRNA expression was significantly downregulated in Group B. GZMB protein expression was evaluated with immunohistochemistry in all 71 ENKTLs, and expression data of Tyr705-phosphorylated STAT3 (pSTAT3) and MYC from our previous study was utilized. T-cell receptor gamma (TRG) gene rearrangement in the selected samples was also assessed using PCR. GZMB expression was higher in pSTAT3-positive (p = 0.028) and MYC-positive (p = 0.014) ENKTLs. Eighteen percent (13/71) of all ENKTLs were negative for GZMB (defined by positivity <10 %); patients with GZMB-negative ENKTLs were often in a higher clinical stage (p = 0.016). We observed no other correlations with clinical parameters or TRG rearrangement and no significant association between GZMB expression and survival. In conclusion, GZMB expression is highly heterogeneous in ENKTLs and is associated with the activation of the JAK-STAT3 pathway and higher MYC expression. GZMB-negative ENKTLs correlate with an advanced clinical stage, suggesting the potential utility of GZMB immunohistochemistry as a biomarker of ENKTL.

Anaplastic Large Cell Transformation of Mycosis Fungoides: Case Report and Review of the Literature.

We report a 48-year-old man with CD30+ large cell transformation of mycosis fungoides (tMF) with distinctive anaplastic morphology. The patient initially presented with folliculotropic and syringotropic mycosis fungoides (MF) manifested as occipital scalp plaque and trunk and extremities patches. Six years later, he progressed to the tumor stage from his scalp lesion and developed cervical lymphadenopathy. Lymph node and scalp biopsies showed diffuse infiltration of CD30+ anaplastic cells with multinucleated, hallmark-like, Hodgkin-Reed-Sternberg-like, histiocytoid forms, indistinguishable from anaplastic large cell lymphoma (ALCL). T-cell receptor gamma gene (TCRg) rearrangement studies revealed identical clones in the initial MF scalp lesion and nodal anaplastic lesion, confirming the transformation. Ancillary studies showed absence of IRF4/DUSP22 and ALK rearrangements and positive RB1, SMARCA4, SOCS1, and TP53 mutations. The patient achieved partial response with systemic chemotherapy. Our case is an example of tMF presenting as the morphology and phenotype of ALCL. Because clinical behavior and therapeutic options of tMF and primary cutaneous ALCL may be different, it is clinically relevant to differentiate these 2 entities. The proof of clonal relationship may be useful in diagnostically challenging cases with features overlapping between tMF and primary cutaneous ALCL.

Diagnostic utility of the lymphoid screening tube supplemented with TRBC1 for the assessment of T-cell clonality.

Flow cytometric panels for the investigation of lymphoproliferative disorders, such as the EuroFlow Lymphoid Screening Tube (LST), often fail to demonstrate T-cell clonality, as a suitable clonality marker was unavailable until recently. Aim of this study was to evaluate the added value of supplementing TRBC1, a flow cytometric T-cell clonality marker, to the LST. Flow cytometric analysis was performed on 830 routine samples referred to our lab for suspicion of hematological malignancy. T-cells with monotypic TRBC1-expression were additionally characterized with a 12-color T-cell tube and molecular T-cell receptor gamma gene rearrangement (TRG). LST analysis revealed 97 (11.7%) samples with the presence of a monotypic T-cell population according to TRBC1, including 21 (2.5%) "high-count" (≥500 cells/μL blood or ≥15% of lymphocytes) and 76 (9.2%) "low-count" (<500 cells/μL blood or <15% of lymphocytes) populations. Clinical symptoms indicative for T-CLPD could be correlated to 11/21 "high-count" and 17/76 "low-count" monotypic T-cell populations. Molecular TRG analysis demonstrated a monoclonal result in 76% (16/21) of "high-count" samples and in 64% (42/66; 10 samples not tested) of "low-count" samples, but also in 9/20 samples with polytypic TRBC1 results. Analysis of an LST tube supplemented with TRBC1 led to the detection of a high number of monotypic T-cell populations. The detection of numerous small monotypic T-cell populations raises the question of their clinical significance. A possible flowchart for assessment of these populations, based on the available literature, is proposed. Molecular TRG analysis is complementary and cannot be omitted from T-cell clonality assessment.

T-Cell Monoclonality in the Blood and the Skin Correlates With Poor Response to Treatment in Mycosis Fungoides.

The prognostic value of skin and blood T-cell receptor clonality in mycosis fungoides is a matter of debate. Our aim was to ascertain the relation between the presence of a monoclonal T-cell population in the blood and in the skin with response to treatment in patients with mycosis fungoides. Clinical features and follow-up data were retrospectively collected and analyzed in 94 patients with mycosis fungoides seen at a cutaneous lymphoma clinic in a single tertiary center. All patients had results of polymerase chain reaction analysis of T-cell receptor gamma gene rearrangement in lesional skin and in peripheral blood at time of diagnosis. Association of response to treatment with clonality in the tissue and in the blood was assessed. T-cell monoclonality was detected in the skin in 30 of 94 patients, in the blood in 12 of 94 cases and the same clone was found in both tissues in 6 of 94 patients. The presence of a polyclonal T-cell population in the circulation was associated with complete response (P=.006). Lack of response to treatment (stable disease or progression of disease) was associated with T-cell clonality in skin (P=.009), in blood (P=.002) and in both tissues (P < .001). A multivariate analysis showed that T-cell monoclonality in the skin is independently associated with lack of response of mycosis fungoides to therapy. Blood and skin should be studied for T-cell clonality as part of the routine initial workup, even in patients with early-stage disease.

A Unusual Case of Classic Hodgkin Lymphoma (cHL) with Aberrant T-Antigens Expression

Abstract Introduction/Objective T-cell-associated antigens (TCA) are rarely expressed by Hodgkin/Reed-Sternberg (HRS) cells of classical Hodgkin lymphoma (cHL) and is associated with uncertain clinical significance. Methods/Case Report We present a very recent case of a 68 year-old female with no significant past medical history presented with a large right groin mass for 1 month. She denied any fever, night sweats, fatigue or weight loss. CT abdomen/pelvis showed inguinal/pelvic lymphadenopathy. Resection showed effaced nodal architecture with a vague nodular appearance and with large, atypical nucleolated cells in a background of small, mature lymphocytes and histiocytes. Eosinophils are conspicuously absent in the background. The large cells were polylobated lymphocyte and histiocyte rich (L &amp; H) like, classic HRS cells as well as multinucleated in appearance. The morphologic features overlapped between Classic Hodgkin Lymphoma, and Nodular Lymphocyte Predominant Hodgkin Lymphoma. Given the immunohistochemical (IHC) features of the neoplastic cells [CD45 negative, PAX5+ (weak), CD30+ (strong), CD15+ (majority) and MUM1 +] the diagnosis was most compatible with CHL. Interestingly, a subset of the Hodgkin cells (10-20%) aberrantly expressed a number of T-cell markers (CD2, CD3, CD4, and CD8). However, the background smaller T-cells did not show cytologic atypia or aberrancies of T-cell antigen markers. The large cells were negative for CD43 and ALK1. TCR gamma gene rearrangement was also negative for clonal T-lymphocytes, thus excluding the possibility of peripheral T-cell lymphoma with Hodgkin-like cells. Based on the morphology and IHC profile, this case proves to be CHL with multiple TCA expression including CD2, CD3, CD4 and CD8. She was planned for chemotherapy. Four months after the initial diagnosis and after completion of 2 cycles of chemotherapy, her PET CT scan showed improvement with right inguinal adenopathy being smaller in size and decreased avidity, indicating a response to treatment. She is due for 2 more cycles. Till todate she is doing well with no further symptoms. Results (if a Case Study enter NA) NA. Conclusion So far reported cases of classic Hodgkin lymphoma (CHL) with aberrant T-cell antigen (TCA) expression were associated with decreased event-free survival and overall survival when compared with TCA-negative CHLs. Our finding contributes to the literature and improves our knowledge of the disease.

Focal Myositis with CD8+T-Cell predominance: an Inflammatory Myositis Mimicking a Soft Tissue Neoplasm

Abstract Introduction/Objective FM is a rare self-limiting T-cell rich lesion arising within muscles of young adults as a solitary lesion and can be confused with a variety of neoplastic/inflammatory conditions or lymphoma. Here we describe a rare T-cell rich variant of FM with CD8 predominance. Methods/Case Report A 51-year-old female presented with two-month history of left trapezius swelling. MRI showed an enhancing tumor within the muscle, suspicious of sarcoma, less likely myeloma. Results (if a Case Study enter NA) Biopsy showed diffuse infiltration of small lymphocytes in a fibrotic background and atrophic skeletal muscle. These lymphocytes stained positive for CD2, CD3, CD7, CD8, CD5 (partial), TIA1 (partial), CD43, CD57 and negative for CD4, CD30, CD56, CD57, granzyme B, perforin, BCL-6, BCL-2, Pax5, TCL1, and CD56. Ki-67 was (&amp;lt;10%) with few background CD20+ B cells. By flow cytometry, the CD8+ T-cells co-express CD2, CD3, CD7, with dim CD5 expression. TCR gene rearrangement study by PCR showed no clonal TCR Gamma gene rearrangement. Conclusion FM is extremely rare with only 22 cases well-described in the literature, all predominantly composed of CD4+ T-cells, clinically concerning for low-grade sarcomas, as inflammatory myofibroblastic tumor, inflammatory leiomyosarcoma, or liposarcoma. Careful analysis is essential for correct diagnosis. Mimics include other causes of myositis, such as polymyositis and inclusion body myositis. Depending on the extent of lymphocytic infiltrate, gene rearrangement studies might be necessary to rule out clonality. Recognition of this rare entity with excellent prognosis is crucial to provide appropriate management and avoid unnecessary, aggressive procedures.

Overlapping Features of Primary Cutaneous Marginal Zone Lymphoproliferative Disorder and Primary Cutaneous CD4 + Small/Medium T-Cell Lymphoproliferative Disorder : A Diagnostic Challenge Examined by Genomic Analysis.

Primary cutaneous marginal zone lymphoproliferative disorder (PCMZL) and primary cutaneous CD4 + small/medium T-cell lymphoproliferative disorder (CD4 + TLPD) are indolent lymphoproliferative disorders. However, cases with overlapping features can be challenging. We identified 56 CD4 + TLPD and 38 PCMZL cases from our pathology archives. Clinical, morphologic, and immunophenotypic features were reviewed. Polymerase chain reaction for immunoglobulin (IG) and T-cell receptor gamma (TRG) gene rearrangements were analyzed. Next-generation sequencing studies were performed on 26 cases with adequate material, 19 with CD4 + TLPD, and 7 with PCMZL. CD4 + TLPD presented mostly (91%) as solitary lesions, located in the head and neck area (64%), while PCMZL occurred mostly in the upper extremity (47%) and trunk (34%). Lesions were sometimes multiple (40%) and recurrences (67%) were more common. Cases of PCMZL had an increase in reactive CD3 + T cells, with frequent programmed cell death protein 1 expression, whereas cases of CD4 + TLPD often contained abundant reactive B cells. Twenty-five cases were identified as having overlapping features: 6 cases of PCMZL were clonal for both IG and TRG; 11 cases of CD4 + TLPD were clonal for IG and TRG and 6 cases of CD4 + TLPD had light chain-restricted plasma cells. By next-generation sequencing, 23 variants were detected in 15 genes, with PCMZL more likely to show alterations, most commonly affecting TNFAIP3 and FAS, altered in 5 cases. Both entities have an indolent clinical course with response to conservative therapy and management, and warrant interpretation as a lymphoproliferative disorder rather than overt lymphoma.

S3445 Microscopic Enteritis: A Rare Manifestation of T-cell Promyelocytic Leukemia

Introduction: An abnormal infiltration of intraepithelial lymphocytes in the intestinal mucosa is described as microscopic enteritis (ME). ME is a heterogeneous condition that can be found in, but not limited to, celiac disease, autoimmune disorders, food protein intolerance, parasitic infections, and NSAID use. Thus, an obvious cause of ME is difficult to elucidate and requires thorough work up. We report a case of a patient who was found to have ME thought to be secondary to infiltrative T-cell promyelocytic leukemia (T-PLL). Case Description/Methods: A 68-year-old male with a history of type II diabetes and hyperlipidemia presented with intermittent fever, abdominal pain, nausea, and emesis. He was found to have small bowel obstruction with severe enteritis of 3/4 of the small bowel (distal jejunum to the ileocecal valve). He had a small bowel resection with pathology revealing necrotizing lymphocytic enteritis. Bidirectional endoscopy revealed mild colonic, ileal and duodenal edema with biopsies revealing increased intraepithelial lymphocytes. He was empirically treated with intravenous methylprednisolone with resolution of fevers. He was discharged home with a follow up with gastroenterology for ME and with hematology for persistent leukocytosis. He continued to have recurrent fevers with persistent lymphocytic leukocytosis (14,200 WBC/microliter). Infectious (acid fast bacteria, fungi, cytomegalovirus, adenovirus, syphilis) and rheumatologic diagnostics were unrevealing. He underwent bone marrow biopsy and peripheral blood examination with immunostains, flow cytometry, and cytogenetic studies and was found to have clonal T-cell receptor gamma gene rearrangement concerning for T-PLL. He was treated with alemtuzumab; however developed worsening disease and disseminating opportunistic infections (cytomegalovirus and aspergillosis). He passed away shortly after his diagnosis. Discussion: ME is a challenging diagnosis as its etiology can be difficult to elucidate. Prompt, timely evaluation should be prioritized to improve outcomes. T-PLL is a rare leukemia due to abnormal growth of T-lymphocytes and typically presents with leukocytosis, weight loss, and infection; however, gastrointestinal symptoms may be a presenting symptom.

Treatment of indolent cutaneous T-cell lymphoma with hypofractionated radiation therapy in three cats.

Feline indolent cutaneous T-cell lymphoma (ICL) is an uncommon neoplastic disease. There is currently no consensus on treatment recommendations for ICL. To report the clinical outcome of three cats with ICL treated with hypofractionated electron-beam radiotherapy (RT). Three privately owned cats with ICL. Medical records and client surveys were reviewed. A diagnosis of probable ICL was based on history, clinical presentation and histopathological findings, and confirmed using CD3 immunohistochemical analysis and PCR for antigen receptor gene rearrangement (PARR). All cats were treated with hypofractionated RT (four fractions of 8Gy). All cats presented with skin lesions characterised by erythema and alopecia that were refractory to previous treatment with systemic glucocorticoids. Before hypofractionated RT treatment, lesions were histologically described as having diffuse infiltration of the dermis with CD3+ T cells. Molecular clonality analysis revealed clonal T-cell receptor gamma gene rearrangement. After RT, two cats showed histological improvement defined by decreased infiltration of lymphocytes, with cellular infiltrate present only in the deeper dermis; one cat had near complete histological resolution of lesions with only minimal residual lymphocytes. One cat was determined to have a complete clinical response while the other showed partial responses. No acute adverse effects of radiation were observed; chronic effects included leukotrichia, partial alopecia and mild fibrosis. All clients reported improvement in quality of life for their cats. Clinical and histological improvement in these cats suggests that hypofractionated RT can be a useful treatment modality for cats with ICL.

TCR γδ Large Granular Lymphocyte Leukemia (LGLL) Displays Features between CD8+ and CD4+ TCR αβ LGLL

BackgroundT-large granular lymphocyte leukemia (T-LGLL) is a rare and heterogeneous lymphoproliferative disorder characterized by the chronic proliferation of clonal Large Granular Lymphocytes (LGLs) with cytotoxic activity. Usually, leukemic cells of this T-cell neoplasia express the αβ T-cell receptor (TCR); only a small subset of cases expresses the γδ TCR denoting the TCRγδ-LGLL. Currently, among the different LGL diseases, TCRγδ-LGLL remains less studied and several clinical and laboratory data already described in TCRαβ-LGLL have not yet been explored in TCRγδ-LGLL. In particular, data are still missing about STAT3 and STAT5b mutations, frequently observed in TCRαβ-LGLL, and about T-LGL clonotypes discovered to be private to TCRαβ-LGLL as compared to healthy controls.AimsThis work was aimed to perform the analysis on: STAT3 and STAT5b mutations, LGL immunophenotype, TCRγ clonotype repertoire and clinical presentations in a group of TCRγδ-LGLL patients. The objective was to reveal common and different features of this rare kind of LGLL with the more frequent TCRαβ-LGLL.MethodsThe cohort of study included 11 patients affected by TCRγδ-LGLL. Sanger sequencing was used for mutational analysis on hot-spot regions of the 2 genes more frequently mutated in LGL disorders, STAT3 and STAT5b . Immunophenotype of LGL clone was defined by flow cytometry analysis. CDR3 repertoire and frequency distribution of TCR gamma gene rearrangements were obtained by Next-Generation Sequencing (NGS).ResultsTCRγδ-LGLL showed a higher occurrence of STAT mutations (82%) as compared to TCRαβ-LGLL (43%). In detail, 9 out of 11 TCRγδ LGLL patients were characterized by STAT3 or STAT5b mutations in a mutually exclusive pattern. Being characterized by both STAT3 and STAT5b mutations, TCRγδ LGLL resembles both CD8+ TCRαβ-LGLL and CD4+ TCRαβ-LGLL, the first being mostly associated to STAT3 mutations and the latter to STAT5b . Similarly, TCRγδ-LGLL showed the same relationship observed between immunophenotype and kind of mutation in TCRαβ-LGLL: TCRγδ-LGLL patients with CD56- LGL immunophenotype were characterized by STAT3 mutations (as in CD8+ TCRαβ-LGLL), while TCRγδ-LGLL patients with CD56+ LGL immunophenotype by STAT5b mutations (as in CD4+ TCRαβ-LGLL). Anyway, in TCRγδ-LGLL no correlation was found between mutations and clinical course as observed in CD8+ TCRαβ-LGLL patients carrying STAT mutations, rather, we observed that patients with γδLGLs positive for Vδ2 showed frequently indolent course, while Vδ1 was associated to a more symptomatic disease (4 out of 5 symptomatic patients were Vδ1+). By NGS we obtained the TCR gamma repertoire of each patient and we revealed that all patients were clonal with the exception of 2 showing a polyclonal pattern. The 2 polyclonal cases were the only 2 patients without STAT mutations. Among the remaining STAT3 mutated patients (n=4), 3 were polyclonal and one biclonal, while STAT5b mutated patients (n=5) were more frequently monoclonal (4/5 monoclonal and 1/5 biclonal). Considering the CDR3 repertoire of the immunodominant clones, as observed in TCRαβ-LGLL, common clonotype sequences were present with low frequency in almost all TCRγδ-LGLL patients. Interestingly, 2 different CDR3 sequences were found shared in different patients at frequency >10% of the total rearrangements. In terms of clonal rearrangements, Vγ3-Jγ1/2, Vγ9-JγP and Vγ8-Jγ1/2 were the combination usages most frequently detected, showing a limited combinatorial diversity of TCR genes similarly to CD4+ and differently from CD8+ TCRαβ-LGLL, this last having larger variability. Analysis about the clonotype repertoire in a 2 years-follow up showed that patients maintain their clonal pattern along the time.ConclusionsOur results highlight common and different characteristics of TCRγδ-LGLL as compared to TCRαβ-LGLL. The data suggest that TCRγδ-LGLL can be considered the intersection of the 2 types of TCRαβ-LGLL, sharing CD8+ or CD4+ T-LGLL mutational and phenotype features. Similarly to TCRαβ-LGLL, also TCRγδ disease showed a decreased diversity of TCR repertoire, with common TCR sequences shared among patients and with limited number of Vγ-Jγ combinations. A distinctive feature of TCRγδ-LGLL is that STAT mutations do not correlate with a symptomatic clinical behavior, rather, Vδ1 expression appears to be indicative for symptomatic disease. DisclosuresGalimberti:Bristol-Myers Squibb: Speakers Bureau; Novartis: Speakers Bureau; Incyte: Speakers Bureau; Pfizer: Speakers Bureau.

T-Cell Monoclonality As Assessed By TCR-Sequencing: A Potential Biomarker of Response in Patients with MDS with Autoimmune Cytopenias Treated with IVIG

Background: We have previously reported 75% of patients (pts) achieved clinical responses to treatment with intravenous immunoglobulin (IVIG) in pts with myelodysplastic syndromes (MDS) that exhibit autoimmune cytopenias in the context of T-cell clonality. T-cell clonality was previously determined via peripheral blood (PB) gel-based testing of T-cell receptor (TCR) beta (TRB) and/or gamma (TRG) gene rearrangements or flow cytometry. IVIG was shown to be variably effective in normalizing hemolytic parameters and cytopenias (Feld et al, Blood 2019). Since next generation sequencing of TRG and TRB (TCR-seq) offers better resolution as to the identity of T-cell clones, we reassessed T-cell clonality in a subset of our cohort and longitudinally evaluated the persistence of clones with IVIG treatment. Methods: Among 27 pts who were previously treated with IVIG +/- steroids, we processed the PB of 9 pts who had serial samples of leftover DNA available for TCR-Seq analysis (7 had samples available prior to treatment). Clinical responses were per MDS IWG 2006 criteria, whereas hemolytic responses were established by either a complete normalization or a mild response if there was &amp;gt;50% improvement in hemolytic parameters from baseline. TCR-Seq was performed on 100ng DNA using the Lymphotrack® TRG and TRB Assays on Illumina® MiSeq platform according to manufacturer's instructions (Invivoscribe, Inc.). Using the LymphoTrackMiseq2.4.3 software T-cell clonality was interpreted as monoclonal (MC), oligoclonal, small clone(s) of uncertain significance (SCUS) or polyclonal (PC), based on percent total merged clonal reads and polyclonal background. Results: Baseline characteristics of our 9 pt cohort included 8 males and 1 female, a median age of 70 years (range 45-82), and a median duration of treatment with IVIG of 23 months. Six pts received steroids in addition to IVIG. Diagnosis included 8 pts with MDS and one with large granular lymphocytic (LGL) leukemia. Of the MDS pts measurable by IPSS criteria, 6 were Int-1 and one was low risk. Expanded lymphocyte populations were detected in 7 pts by PB flow cytometry and all pts exhibited T-cell clonality by gel-based assessment. All were TRB positive (+) and 7 were TRG+ by PCR. In 3 pts, 4 serial samples were available for TCR-seq; 4 pts had 3 samples available and 2 had only 2 serial samples. Samples were collected over a period of 5 months up to 4-5 years. With the better resolution afforded by TCR-Seq, 3 pts exhibited only PC populations of T-cells for TRG and TRB at all time points, and 1 pt exhibited SCUS in 1 of 3 samplings for TRG. Of the four pts, only one had an erythroid response; importantly, this sample was tested after treatment. One pt had a normalization in their bilirubin, otherwise only mild responses in hemolytic parameters were noted in the remaining 3 PC pts. Median duration of treatment was 1.5 months. Five pts had a MC TRB and/or TRG population by TCR-Seq. Two pts had MC TRG, two had MC TRG &amp; TRB, and one had MC TRB populations. Four of the 5 pts had hematologic improvement (HI): 1 had an erythroid and platelet response, 2 had an erythroid response, and 1 had a platelet response. More significant hemolytic responses were also observed in 3/5 pts, with a mild response in the 2 remaining pts. Only 1 TCR-Seq+ pt had no HI, and this pt had a monoclonal population detected by TCR-Seq after finishing IVIG treatment. Median duration of treatment was 45 months. For all these cases, the same clonal sequences were found across all samples up to 4-5 years. Of 4 evaluable patients, 3 had reductions in their clonal read percentage burden over their treatment period. Conclusions: TCR-Seq of the TRG and TRB genes affords better resolution of T-cell clones and permits tracking of clones over time. On a further subgroup analysis of 9 pts from our cohort of IVIG-treated pts, we found that pts who exhibited monoclonality as established by TCR-Seq were more likely to respond to IVIG, with more significant erythroid and hemolytic responses, associated with a consequential (45+ months) longer treatment duration. Thus, TCR-Seq appears to be a superior option to PCR-based TCR gene rearrangement assays to reliably identify T cell clones in pts with MDS, and may be a useful biomarker to prospectively identify a subset of pts that can respond to IVIG. Further studies are ongoing to test this hypothesis. Disclosures Navada: Onconova Therapeutics Inc: Research Funding. Silverman:Onconova Therapeutics Inc: Patents &amp; Royalties, Research Funding; Medimmune: Research Funding; Celgene: Research Funding.

Leukemia Cutis in a Patient with Rare Indolent T-Prolymphocytic Leukemia - A Case Report and Review of the Literature

Abstract Introduction/Objective T-cell prolymphocytic leukemia (T-PLL) is rare, comprising 2% of mature lymphocytic leukemias in adults. Clinical course is usually aggressive, however, 20-30% patients can have an indolent early phase. Most patients present with extensive disease with peripheral blood lymphocytosis, hepatosplenomegaly, and lymphadenopathy. T-PLL has a higher incidence in patients with ataxia-telangiectasia. Methods We present a case of a 67-year old man with history of somatic mutation of ataxia-telangiectasia gene (ATM) and multiple malignancies, including melanoma, skin and laryngeal squamous cell carcinomas, adrenal adenoma and spindle cell lung tumor (diagnosed on biopsy), who developed leukemia cutis during an indolent phase of T-PLL. Results Initial diagnosis of T-PLL was on routine complete blood count when patient had mild atypical lymphocytosis (5.5 x 109/L). Flow-cytometry immunophenotyping detected CD4 + 8 positive mature T-cells with no aberrant loss of T- cell antigens, and molecular studies confirmed clonal T-cell receptor beta and gamma gene rearrangements. T-PLL associated leukemia cutis was diagnosed on a leg skin nodule biopsy 10 months later. Subsequent bone marrow biopsy demonstrated low level involvement by T-PLL. The patient’s T-PLL followed an indolent trajectory for three years and treatment was withheld until symptomatic presentation. Rapid disease progression was heralded with emergence of widespread T-PLL leukemia cutis, rapidly increasing lymphocytosis, extensive lymphadenopathy, splenomegaly and increasing LDH levels. Conclusion Skin involvement is reportedly identified in 20% to 50% of T-PLL cases in some series. Skin manifestations can be heterogenous with a broad clinical differential. T-PLL associated leukemia cutis may not be recognized owing to the overall low incidence and in skin lesions appearing during an undiagnosed indolent early phase of T-PLL.We highlight the importance of accurate evaluation of non-specific skin lesions in early detection of T- PLL. Being aware of this diagnosis and early engagement in an interdisciplinary approach may allow for better outcomes of this aggressive malignancy.

T-Cell Mediated Cytopenias in Patients with Myelodysplastic Syndromes Respond to Intravenous Immunoglobulin

Background: Patients with Myelodysplastic Syndrome (MDS) with cytopenias have limited treatment options to improve blood counts apart from immune-modulatory drugs (IMIDS), hypomethylating agents (HMA), cytokines and transfusional support. Immune dysregulation is common in MDS and occurs in up to 35% of pts (LR Silverman, Cancer Med, 2017). Expanded T-cell clonal populations in the peripheral blood and bone marrow in the presence of MDS may play a role in aggravating MDS related cytopenias, increasing the need for transfusions or exacerbating symptoms. Intravenous immunoglobulin (IVIG) may interact with Fc receptors on T cells or have suppressive effects on the concurrent large granular lymphocytic leukemia (LGL) clone. Targeted therapy with IVIG could be a novel strategy to mitigate the cytopenias in MDS and other bone marrow failure pts. Methods: We reviewed in a single-center series 27 consecutive pts who had either MDS, aplastic anemia (AA), or a myeloproliferative neoplasm (MPN) along with an LGL clone seen on TCR-rearrangement studies and/or peripheral blood flow cytometry in the blood and/or bone marrow. Of the 27 pts, 22 had MDS, 2 had MPN, and 1 each with AA, LGL, or Multiple Myeloma. IVIG was initially used as a single agent administered at a dose of 500mg/kg/day over 4 hours once weekly x4 weeks. After "induction", administration was changed to every other week, and then in responding pts, the interval was extended to every 3-4 weeks. In pts with a stable response, IVIG was discontinued and pts observed. After the initial 4 pts, pts were treated concurrently with prednisone 20 mg/d with a taper over 4 weeks, except in pts in which prednisone was a relative contra-indication. Overall hematologic improvement was determined by MDS IWG 2006 criteria, while hemolytic response was determined to be either a complete normalization (CR) or a partial normalization (PR) if there was &gt;50% improvement in deviation from normal LDH, reticulocytes, indirect bilirubin, or haptoglobin values from baseline. Results: All pts received IVIG administered at 500mg/kg, once weekly, to be spaced out maximally as tolerated. 74% (20/27) of pts received steroids. Baseline characteristics of the pt population: median age of 70 years (range 39-87), representing 21 males and 6 females. Of the pts with MDS evaluable for risk stratification by IPSS-R criteria [n=18], 3 were very low, 5 low, 6 intermediate, 2 high, and 2 very high risk categories. By WHO criteria, the greatest representation was 11/20 RCMD, followed by 3/20 with RAEB2. Baseline cytogenetics [n=21] included 3 pts with very poor, 2 poor, 3 intermediate, 11 good, and 2 very good risk profiles. Pts had a range of 0-6 mutations, most common were SETBP1, ASXL1, TP53 (n=4) and SRSF2, U2AF1, PDGFRB (n=3). 24/27 pts had positive TCR beta and 13/27 had positive TCR gamma gene rearrangements. ORR for any cytopenia was 77.8% (21/27), and for any hemolytic response was 76.2% (16/21). ORR for individual cell lines included: 59.2% (16/27) erythroid, 48.1% (13/27) neutrophil, and 33.3% (9/27) platelet responses. For hemolytic responses, CR/PR rate were measured by LDH (29.6%/11.1%), bilirubin (22.2%/14.8%), retic (33.3%/14.8%), and haptoglobin (22.2%/11.1%). For the 14 MDS pts with very low, low, and intermediate IPSS-R risk, ORR was: 64.3% (9/14) any cytopenia, 35.7% (5/14) erythroid, 50.0% (7/14) neutrophil, 14.3% (2/14) platelets. Combined CR+PR rates for hemolysis in the lower risk categories were 42.9% LDH, 21.4% bili, 42.9% retic, 28.6% haptoglobin responses. For the 5 non-MDS pts, ORR for any cytopenia was 100%, and 60% for any hemolytic response. There were no reported significant adverse events with IVIG administration. Conclusions: IVIG can be used as a first line treatment for pts with autoimmune cytopenias associated with LGL clones and MDS (along with other hematological malignancies). Treatment with IVIG yields an ORR of 77.8% in the entire cohort and 64.3% in pts with lower risk MDS, with a reduction in hemolysis as well. This report expands on a previous analysis and demonstrates further evidence of efficacy for the use of IVIG in treating cytopenias exacerbated by LGL T-cell clones. IVIG therapy can permit a delay in the start of an HMA or IMID treatment, lowering transfusion burden, and increasing ANC to help prevent infectious complications. Further evaluation in a larger cohort of pts is warranted. Studies exploring the mechanism of action are underway. Disclosures Navada: Onconova Therapeutics Inc: Research Funding. Silverman:Onconova Therapeutics Inc: Patents &amp; Royalties, Research Funding; Celgene: Research Funding; Medimmune: Research Funding.

Assessment of immunoglobulin heavy chain, immunoglobulin light chain, and T-cell receptor clonality testing in the diagnosis of feline lymphoid neoplasia.

Differentiation between neoplastic and reactive lymphocytic proliferations can be challenging in cats. PCR for antigen receptor rearrangements (PARR) testing is a useful diagnostic tool to assess clonality of a lymphoid population. Previous feline PARR studies evaluated clonality of complete immunoglobulin heavy chain V-D-J (IGH-VDJ) and T-cell receptor gamma (TRG) gene rearrangements. We aimed to evaluate the sensitivity and specificity of feline PARR primers targeting complete IGH-VDJ and TRG rearrangements, as well as incomplete IGH-DJ, kappa deleting element (Kde), and immunoglobulin lambda light chain (IGL) gene rearrangements in defined feline neoplasms and nonneoplastic controls. Fluorescently labeled PCR primers were designed to amplify complete IGH-VDJ, incomplete IGH-DJ, Kde, IGL, and TRG gene rearrangements in two multiplexed PCR reactions, and PCR products were analyzed by fragment analysis. Fresh tissue samples from 12 flow cytometrically confirmed B-cell lymphomas, 26 cytologically confirmed gastric and renal lymphomas of presumed B-cell origin, 30 flow cytometrically confirmed T-cell leukemias, and 11 negative control cats were tested. Using four immunoglobulin primer sets (IGH-VDJ, IGH-DJ, Kde, and IGL), clonal immunoglobulin rearrangements were detected in 87% (33/38) of the presumed B-cell neoplasms. The IGH-VDJ reaction alone only detected clonality in 50% (19/38) of these cases. TRG rearrangements were clonal in 97% (29/30) of the T-cell leukemia cases. All negative control samples had polyclonal immunoglobulin and TRG rearrangements. The PARR assay developed in this study is useful for assessing clonality in feline lymphoid neoplasms. Clonality assessment of incomplete IGH-DJ, Kde, and IGL rearrangements helped identify clonal B-cell neoplasms not detected with complete IGH-VDJ PARR alone.

PCR GeneScan and Heteroduplex Analysis of Rearranged Immunoglobulin or T-Cell Receptor Genes for Clonality Diagnostics in Suspect Lymphoproliferations.

Assessment of the presence of clonal lymphoproliferations via polymerase chain reaction (PCR)-based analysis of rearranged immunoglobulin (IG) or T-cell receptor (TR) genes is a valuable method in the diagnosis of suspect lymphoproliferative disorders. Additionally, this methodology can be used for evaluating dissemination of lymphoma cells and for studying the clonal relationship between multiple (different locations) or consecutive (over time) lymphomas. Here we describe an integrated approach to assess clonality via analysis of Ig heavy chain (IGH), Ig kappa (IGK), TCR beta (TRB), and TCR gamma (TRG) gene rearrangements, based on the standardized multiplex PCRs as originally developed by the European BIOMED-2 consortium. The described protocol covers the pre-analytical phase of DNA isolation (from formalin-fixed paraffin-embedded and fresh tissues, body fluids, peripheral blood, and bone marrow), the analytical phase of PCR GeneScan and heteroduplex analysis, and the post-analytical interpretation of the obtained profiles, following established guidelines.

Rare case of low-grade extranodal NK/T-cell lymphoma, nasal type, arising in the setting of chronic rhinosinusitis and harboring a novel N-terminal KIT mutation

BackgroundExtranodal NK/T-cell lymphoma, nasal type (ENKTCL-NT), is a rare aggressive subtype of non-Hodgkin lymphoma characterized by angioinvasion, angiodestruction, necrosis and strong association with Epstein-Barr virus (EBV). ENKTCL-NT occurs worldwide and is more prevalent in Asian and the Native American populations of Mexico, Central and South America. It represents approximately 10% of all peripheral T-cell lymphomas worldwide. The aim of this report is to present a rare case of ENKTCL-NT with an unusually indolent clinical course and low-grade histopathologic features.Case presentationA 71-year-old Asian woman with a long-standing history of seasonal rhinosinusitis presented with persistent nasal congestion, cough, and fever unresponsive to antihistamines and antibiotics. Histopathological evaluation of a polypoid nasal mass revealed an atypical infiltrate with predominantly small lymphoid cells that were CD2+, surface CD3-, cytoplasmic CD3+, CD5(dim)+, CD7(dim)+, cytotoxic markers (granzyme B and perforin)+, EBER+ and CD56-. The Ki-67 proliferative index was very low (< 1%). T-cell receptor gamma gene rearrangement studies were positive for a monoclonal rearrangement, and sequencing studies identified a novel KIT mutation (p. K167 M, c. 500 A > T). A diagnosis of low-grade ENKTCL-NT was rendered.ConclusionsOur case of ENKTCL-NT is unusual due to (1) an indolent clinical course (2) low-grade histopathologic features including a low proliferative index (3) lack of CD56 expression and (4) a novel KIT mutation. This case raises awareness of the existence of a subset of cases of ENKTCL-NT that can potentially be misdiagnosed as a reactive process, particularly in patients with recurrent chronic rhinosinusitis.

Subcutaneous Panniculitis-Like T-cell Lymphoma in Dogs: Morphologic and Immunohistochemical Classification.

Canine nonepitheliotropic cutaneous T-cell lymphomas (NECTCL) are poorly characterized. In humans, a number of distinct subtypes of NECTCL have been recognized, including subcutaneous panniculitis-like T-cell lymphoma (SPTCL). Five dogs with subcutaneous T-cell lymphomas histologically similar to SPTCL in humans are herein described. The mean age was 8.5 years (5.5 to 12 years). No breed or sex predilection was identified in this small cohort. Two dogs presented with an acute onset of multiple skin masses and 3 dogs had solitary masses with subsequent development of multiple smaller masses within 0.5 to 2 months post-diagnosis without treatment. Locations, when specified, included shoulder, neck, and ventral abdomen. Two dogs were euthanized following diagnosis and one dog treated with chemotherapy (CCNU) survived 7 months post-diagnosis. Histologically, all cases were characterized by proliferations of either small to intermediate or large sized, CD3-positive T cells that infiltrated the subcutis in a lace-like pattern and frequently rimmed adipocytes. No epitheliotropism was observed, neoplastic cells were often karyorrhectic, and there were regions of extensive necrosis. Heavy infiltrates of histiocytes with prominent phagocytosis masked the lymphoid neoplastic cell population in some sections. A clonal T-cell receptor gamma gene rearrangement was found in 4 of the 5 cases. While SPTCLs typically have a less aggressive clinical course in humans, their biological behavior in dogs remains to be determined. In summary, SPTCL may represent a distinct entity in dogs and needs to be accurately diagnosed to better determine clinical behavior.

An unusual case of hepatosplenic T cell lymphoma in a 59-year-old woman

Hepatosplenic T cell lymphoma (HSTL) is a rare aggressive T cell lymphoma which can be diagnostically challenging. We report a case of a 59-year-old woman presenting with sepsis, hepatosplenomegaly, ascites and multi-organ failure. The liver biopsy revealed active hepatitis and steatosis, additionally, with small numbers of atypical sinusoidal lymphoid cells, occasionally forming short chains, but without sinusoidal expansion, difficult to identify and partly masked by inflammatory changes. The cells showed CD3+, CD2+, CD5–, CD7–, CD4–/CD8–, CD56+, perforin+, TIA1+ and CD30+, suggesting an NK/T cell lymphoma, possibly HSTL, with peripheral T cell lymphoma, NOS, also considered. This prompted a subsequent bone marrow trephine, which contained a morphologically and immunophenotypically similar infiltrate of atypical lymphoid cells. Monoclonal TCR beta and gamma gene rearrangements were identified. Despite chemotherapy with CHOEP, the patient deteriorated rapidly and three months after initial diagnosis, she developed progressive CNS disease and died two weeks later. The liver and bone marrow findings, taken in conjunction with the clinical presentation including hepatosplenomegaly, absence of lymphadenopathy or atypical lymphoid cells in the blood, were considered most in keeping with HSTL. This case highlights the difficulty in diagnosis and appreciation of the neoplastic infiltrate of HSTL on haematoxylin-eosin stained slides, as the neoplastic infiltrate can be inconspicuous and masked by inflammatory changes. A high index of suspicion and low threshold for performing immunohistochemistry is prudent in such cases.

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.