Rearrangement Studies Research Articles

S3489 The Uncommon Acute Abdomen: Enteropathy-Associated T-Cell Lymphoma Revealed by Jejunal Perforation

Introduction: Long-standing Celiac disease that is untreated or refractory to gluten-free diet can have dire consequences. Among these, Enteropathy-associated T-cell Lymphoma (EATL) is a rare form of non-Hodgkin’s intestinal lymphoma that carries a poor prognosis with high mortality. This neoplasm can manifest as complicated small bowel disease such as obstruction or perforation. We present a patient with Celiac Disease presenting with an acute abdomen found to have jejunal perforation due to EATL. Case Description/Methods: A 51-year-old male with history of biopsy-confirmed Celiac Disease (CD) presented for severe abdominal pain. Vital signs were stable and laboratory findings showed leukocytosis and lactic acidosis. Abdominal CT revealed closed loop obstruction and pneumatosis with hollow viscus perforation. He was taken to the OR for urgent exploratory laparotomy where purulent fluid and a perforation in the mid-jejunum was discovered. Subsequent jejunal resection with primary anastomosis was performed. Post-operative course was complicated by ileus and urinary retention but ultimately made full recovery. Biopsy of the resected jejunum revealed malignant lymphoid infiltrate in a background of mild villous blunting, increased intraepithelial lymphocytes, ulceration, perforation, and acute serositis consistent with EATL. Follow-up endoscopy was consistent with known CD, but otherwise unremarkable. T-cell receptor rearrangement study and bone marrow biopsy were negative. He was initiated on BV-CHP chemotherapy and achieved complete remission after 6 cycles. After induction, he opted for consolidation (BEAM) chemotherapy followed by autologous bone marrow transplant. Unfortunately, this treatment course was complicated by septic shock with multi-organ failure and ultimately decision was made for comfort care. (Figure) Discussion: Strict gluten-free diet (GFD) is the primary prevention of EATL in CD. Non-compliance to GFD has been shown to significantly increase risk of developing EATL. Small bowel perforation is a rare, but well-documented complication of EATL that must be recognized in CD patients presenting with acute abdomen. Current treatment of EATL with chemotherapy and autologous bone marrow transplant has shown promising improvement in survival rates and reduced mortality rate. For CD patients, strict adherence to GFD with close clinical monitoring is of utmost importance to improve quality of life, minimize progression of EATL, and prevent necessity of risky advanced interventions as highlighted in our case.Figure 1.: Axial view of Abdominal CT showing perforation of small bowel.

Pediatric-Type Indolent B-Cell Lymphomas With Overlapping Clinical, Pathologic, and Genetic Features.

Pediatric-type follicular lymphoma (PTFL) and pediatric nodal marginal zone lymphoma (PNMZL) are rare pediatric-type indolent B-cell lymphomas (PedIBCL) that differ clinicopathologically from their adult counterparts. Accurate diagnosis is important to avoid overtreatment but is often challenging. The mutational landscape of PTFL is known and may aid diagnosis, but the genetic features of PNMZL are not well understood. We analyzed 21 cases of PedIBCL according to their clinicopathologic findings and classified them into PTFL (n=11), PNMZL (n=2), and “mixed type” tumors (n=8) showing ambiguous histology. We also analyzed 2 cases of adult B-cell lymphomas showing features of PedIBCL. Targeted sequencing of 121 lymphoma-related genes was performed. The median age of PedIBCL patients was 16 years (range: 3 to 47), and all but 1 PTFL patient were male. All patients presented with limited-stage disease, and only 1 relapsed. There were no significant differences in clinical features among the 3 PedIBCL groups. The most frequently mutated genes were MAP2K1, TNFRSF14, KMT2C, IRF8, and NOTCH2. The genetic features of all groups were similar to the established mutational landscape of PTFL. The 2 adult B-cell lymphomas cases also had MAP2K1, TNFRSF14, and IRF8 mutations, but the clinical features were not typical of PedIBCL. In summary, this study demonstrated that PTFL and PNMZL are similar diseases with overlapping clinical, pathologic, and genetic features; mixed type tumors can also occur. Atypical adult cases with similar histologic features were also observed. Therefore, the disease spectrum of PedIBCL may be much broader than is currently believed.

Tetragonal reconstruction and monoclinic variants rearrangement during heat treatment in zirconia

AbstractAn in situ monoclinic variants selection and rearrangement study on Y2O3–ZrO2 coatings was conducted using electron backscattered diffraction. The sequence growth of variants with the correspondence of (100)m ∼// (010)t, [010]m ∼// [001]t, and [001]m ∼// [100]t (CAB‐OR2) was found initially and converted to the 90°‐rotated sequence growth with (100)m ∼// (100)t, [001]m ∼// [001]t, and [010]m ∼// [010]t (ABC‐OR2) after heat treatment. In another coating, most of the variants first exhibited interleaving growth and changed to typically fourfold growth. This in situ evolution of both variants orientation relationship and arrangement revealed the effects of stress and stress relief on the martensitic transformation. Moreover, the variants selection might be diversified during the cyclic transformation, and the classical phenomenological theory could mainly be proved by the transformation triggered by temperature, instead of external stress. Furthermore, the variants orientation relationship was exactly confirmed by the tetragonal grain reconstructions. In particular, the orientation variation of reconstructed tetragonal grains was also found and could mainly be explained by the behavior of stress relief and dynamic recrystallization.

Subtlety of Granulomatous Mycosis Fungoides: A Retrospective Case Series Study and Proposal of Helpful Multimodal Diagnostic Approach With Literature Review.

Granulomatous mycosis fungoides (GMF) harbors a worse prognosis compared with classic MF and remains a significant diagnostic dilemma. We analyzed clinicopathologic, immunophenotypic, and molecular characteristics of GMF to develop a diagnostic algorithm. Our methodology involved a retrospective case series study of patients with GMF from our database between 2014 and 2020. A total of 8 patients with 9 biopsies of GMF were identified. Skin manifestations had variable clinical phenotype. Histologically, all cases demonstrated atypical CD4 + T-cell infiltrate with scant in 50% (n = 4), focal 37.5% (n = 3), and absent 25% (n = 2) epidermotropism. Granuloma formation was seen in 77.8% biopsies (n = 7) with sarcoid-type granulomas in 57.1% (n = 4) and granuloma annulare-like type in 42.9% (n = 3). In 66.7% of biopsies (n = 6), the CD4:CD8 ratio was >4:1 and 66.6% (n = 6) of biopsies showed ≥50% loss of CD7 expression. T-cell receptor gene rearrangement studies performed on biopsy sections were positive in all biopsies (n = 6), whereas peripheral blood T-cell receptor gene rearrangement studies did not identify clonality. In conclusion, GMF has subtle or absent epidermotropism and variable granulomatous reaction; thus, the diagnosis requires a multimodal approach, and our proposed algorithm provides a framework to approach this diagnostic challenge.

Targeted Therapy for Older Patients with Non-Small Cell Lung Cancer: Systematic Review and Guidelines from the French Society of Geriatric Oncology (SoFOG) and the French-Language Society of Pulmonology (SPLF)/French-Language Oncology Group (GOLF).

Simple SummaryTargeted therapy has become essential in the treatment of non-small cell lung cancer (NSCLC). There are currently no guidelines for older patients who are frailer with regard to this type of treatment. Two learned societies, the French Society of Geriatric Oncology (SoFOG) and the French-language Society of Pulmonology (SPLF)/French-language Oncology Group (GOLF), joined forces to conduct a systematic review of the literature from May 2010 to May 2021 regarding the efficacy, toxicity, and feasibility of targeted therapy in older patients with NSCLC. Guidelines were then drawn up to enable clinicians to adapt the type of targeted therapy proposed according to the oncological and geriatric profile of the older patient with NSCLC.Systematic molecular profiling and targeted therapy (TKI) have changed the face of Non-Small Cell Lung Cancer (NSCLC) treatment. However, there are no specific recommendations to address the prescription of TKI for older patients. A multidisciplinary task force from the French Society of Geriatric Oncology (SoFOG) and the French Society of Pulmonology/Oncology Group (SPLF/GOLF) conducted a systematic review from May 2010 to May 2021. Protocol registered in Prospero under number CRD42021224103. Three key questions were selected for older patients with NSCLC: (1) to whom TKI can be proposed, (2) for whom monotherapy should be favored, and (3) to whom a combination of TKI can be proposed. Among the 534 references isolated, 52 were included for the guidelines. The expert panel analysis concluded: (1) osimertinib 80 mg/day is recommended as a first-line treatment for older patients with the EGFR mutation; (2) full-dose first generation TKI, such as erlotinib or gefitinib, is feasible; (3) ALK and ROS1 rearrangement studies including older patients were too scarce to conclude on any definitive recommendations; and (4) given the actual data, TKI should be prescribed as monotherapy. Malnutrition, functional decline, and the number of comorbidities should be assessed primarily before TKI initiation.

Use of Ultrasensitive RNA In Situ Hybridization for Determining Clonality in Cutaneous B-Cell Lymphomas and Lymphoid Hyperplasia Decreases Subsequent Use of Molecular Testing and Is Cost-effective.

Primary cutaneous B-cell lymphomas (PCBCLs) are diagnostically challenging entities due to significant overlap in clinical and morphologic features with reactive lymphoid proliferations. Traditional methods for evaluating clonality such as immunohistochemistry (IHC) and chromogenic in situ hybridization (CISH) are limited by low sensitivity, which leads to additional costly and time-consuming molecular clonality assays. More recent technology has introduced ultrasensitive bright-field RNA in situ hybridization (BRISH) to the field, which can detect single molecules of light-chain mRNA. The current study evaluated 274 cases of PCBCL in addition to atypical and reactive lymphoid infiltrates, with CISH or BRISH performed on 180 (65.7%). CISH was performed on 105 (58.3%), and BRISH was performed on 75 (41.7%). Significantly fewer immunoglobulin heavy-chain (IGH) rearrangement studies were performed on cases that were evaluated with BRISH as compared with CISH (P=0.02). Subgroup analysis demonstrated that cases with restriction by BRISH were significantly less likely to have subsequent IGH studies performed (P=0.01). The expected costs of cases using CISH versus BRISH were $1053.89 versus $810.32 to the patient and $245.63 versus $225.23 to the laboratory. The use of ultrasensitive BRISH to evaluate clonality in PCBCL reduced the use of IGH rearrangement studies when compared with CISH. In particular, cases with light-chain restriction by BRISH did not result in confirmatory molecular testing. Despite slightly higher costs to the laboratory to perform BRISH, routine use of this methodology can result in cost savings to both the patient and laboratory by decreasing the use of expensive molecular methods.

Asymptomatic, reddish-brown papules and plaques in an elderly Japanese woman

Asymptomatic, reddish-brown papules and plaques in an elderly Japanese woman

Transformed Mycosis Fungoides: Clinical and Pathologic Characteristics in a Single Center Retrospective Analysis

IntroductionLarge cell transformation (LCT) in mycosis fungoides is defined as 25% large cells and is traditionally associated with a poor prognosis and a median survival of 3-5 years. There is no consensus on the appropriate management for these patients. We aimed to characterize clinical and pathologic features of patients with transformed mycosis fungoides (tMF) at our institution and to describe treatment patterns and patient outcomes.MethodsWe performed a retrospective review of 63 patients with a histopathologic diagnosis of tMF seen at the Winship Cancer Institute of Emory University from 1990-2021. Clinical data collected from the electronic medical record included demographics, baseline laboratory values, disease characteristics, pathologic characteristics, and therapy. Kaplan-Meier curves for OS and PFS were generated for the whole cohort. Descriptive analysis was performed for covariates. The association of baseline variables with OS was modeled by Cox proportional hazards model.ResultsAmong 63 patients with tMF, there was even distribution among male (54%) and female (46%) patients (Figure 1A). The median age at the time of large cell transformation (LCT) was 63 years (range, 20-87). This population included 46% African American (AA), 50.8% White, 1.6% other races (n=1). At the time of diagnosis, the stage was 1A-1B in 23 (36.5%), 2A in 11 (17.5%), 2B in 20 (31.7%), 3A-3B in 3 (4.8%), 4A in 5 (7.9%) and 4B in 1. The ECOG performance status (PS) was 0-1 in 49 (77.8%), 2 in 7 patients (11.1%), 3 in 3 patients, and 4 in one patient. Most patients had patches and plaques at diagnosis, 23 (36.5%) had tumors, and 9 (14.3%) had ulceration. Only 2 patients had hypopigmented MF. The most common therapies prior to LCT were topical steroids (n=34), phototherapy (n=25), topical nitrogen mustard (n=20), oral retinoids (n=14), and oral methotrexate (n=8). Radiation (RT) was received in 22 patients prior to transformation with 16 having localized RT and 9 total skin electron beam therapy (TSEB).The median time from diagnosis to LCT was 2.1 years (range, 0-32 years). Most patients had advanced stage at the time of LCT (n=45, 86%), with stage 2B the most frequent 23 (36.5%). Other stages at LCT were 1A in 1 patient, 2A in 6, 3A-B in 6, 4A in 14 and 4B in 11. Overall, 47 (74.6%) patients experienced progression in their TNMB stage following LCT. LCT occurred in the skin in 51 patients (81%), lymph nodes in 21 (33.3%), and viscera in 5 (7.9%). The percentage of large cells was >50% in 24 patients (38.1%), and < 50% in 13 (20.6%). CD30 percentage was missing in the majority of patients (n=37, 58.7%). T-cell rearrangement in blood, skin, and lymph nodes was not consistently characterized with data missing in nearly half the patients (n=30, 47.6%). In those with TCR checked in at least one location, it was non-clonal in 19, had clonal persistence in 6 (9.5%), clonality in at least one sample but either not rechecked, or not persistent in 6 (9.5%), and oligoclonal in 2 (n=3.2). The most common treated for LCT was total skin electron beam therapy (TSEB, n=16) and chemotherapy (n=14), followed by brentuximab vedotin and localized RT in 9 patients each. Other treatments included topical corticosteroids (N=2), histone deacetylase inhibitors (n=4), and mogamulizumab (n=4). The median overall survival was 2.3 years from LCT (95% CI 1.1-3.5 years), and the median PFS was 2.6 years (95% CI, 1.1-4.1 years) (Figure 1B) . The median time to treatment following LCT was 30 days (range, -1 day to 2.3 years). By cox regression, neither baseline demographic factors, tumor characteristics, treatment, nor time to LCT were associated with progression-free or overall survival. Only response to treatment for LCT (complete response, partial response, or stable disease) was associated with survival (p=0.041, Figure 1C).ConclusionsWe characterized the clinical features and outcomes of a cohort of patients with tMF seen at Emory University over a 30 year period. We found poor outcomes despite many patients receiving novel and targeted therapies in the modern era. Key pathologic and clinical variables such as CD30 percentage and t-cell gene rearrangement studies were not reported for many patients, suggesting standardized practices are needed in the diagnosis and pathologic evaluation of patients with tMF. Additional pathologic correlatives will be updated at the time of the presentation. [Display omitted] DisclosuresAllen: ADC Therapeutics: Consultancy; Kyowa Kirin: Consultancy; Secure Bio: Consultancy.

Myeloid/Lymphoid Neoplasm with Eosinophilia and a Novel RUFY1-Pdgfrb Rearrangement

▪Myeloid/lymphoid neoplasms associated with eosinophilia (MLN-Eo) and tyrosine kinase fusion genes include rearrangements of PDGFRB with over 30 different partner genes, most commonly ETV6-PDGFRB. Rearrangements are evident by cytogenetic analysis and fluorescence in situ hybridization (FISH) break-apart probes. Herein, we describe a novel rearrangement partner for PDGFRB.A 25-year-old man with a medical history significant for gout and hypertension presented to medical care after several days of progressive dyspnea, chest pain, fatigue, epistaxis, and rash. Physical exam revealed palpable cervical, axillary, and inguinal lymphadenopathy and massive splenomegaly. By CT scan the spleen measured 25 cm and diffuse lymphadenopathy was identified in the chest, abdomen and pelvis with the largest lymph node mass measuring 8.9 x 2.5 cm. Transthoracic echocardiogram showed normal heart function and no evidence of myocardial injury. The patient subsequently developed respiratory failure requiring intubation and mechanical ventilation, shock requiring vasopressors, and renal failure requiring renal replacement therapy. Initial laboratory evaluation demonstrated a leukocytosis to 83.3 x 10 9/L with neutrophilic predominance with left shift and eosinophilia (10% of WBCs, absolute eosinophil count of 8.2 x 10 9/L); hemoglobin of 8.3 g/dL and hematocrit of 25%; and thrombocytopenia to 50 x 10 9/L. Ferritin was 660 ng/mL, lactate dehydrogenase 384 U/L, and uric acid 10.4 mg/dL. Secondary causes of eosinophilia including infection, immunodeficiency, atopy, medications, and rheumatologic disease were ruled out. The patient was positive for strongyloides IgG antibodies and received ivermectin.Core biopsy of a lymph node was non-diagnostic and could not repeated safely due to progressive severe thrombocytopenia. Skin biopsy demonstrated a perivascular leukocytoclasis, but no abnormal B or T cell population. Bone marrow aspirate and biopsy revealed a hypercellular marrow (90%) with mild trilineage dysplasia, granulocytic hyperplasia, moderate reticulin fibrosis, and slightly increased mast cells. Blasts were not increased and no hemophagocytic histiocytes were identified. Eosinophils accounted for 6.9% of myeloid cells. Flow cytometry did not reveal an abnormal B or T lymphoid, blast or myeloid population.Peripheral blood flow cytometry did not reveal an abnormal B or T-cell population and T-cell rearrangement studies showed an oligoclonal population. No clonal B cell population was identified.FISH, which identified a rearrangement of PDGFRB in 28% of cells, confirmed the diagnosis. Karyotype analysis revealed an abnormal male karyotype with a paracentric inversion of 5q with breakpoints at 5q32 and 5q35. FusionPlex® Solid Tumor Panel (Clinical Genomics Laboratory, University of Washington SOM) identified a novel fusion between RUFY1 and PDGFRB. RUFY1 is expressed in most tissues and encodes a protein that contains a RUN domain and a FYVE-type zinc finger domain and plays a role in early endosomal trafficking (Figure). We are currently demonstrating the fusion is oncogenic and sensitive to tyrosine kinase inhibition in Ba/F3 cells. The patient's remarkable clinical response, however, supports imatinib sensitivity and is in keeping with other published reports of MLN-Eo with PDGFRB rearrangements. Initial treatment included high dose steroids for 7 days and hydroxyurea briefly. Imatinib 400 mg daily was started, then decreased to 200 mg daily due to gastrointestinal toxicity. Follow-up is limited (8 weeks). However, the patient has recovered clinically. Six weeks after starting imatinib he had renal recovery and has achieved a complete hematologic response except for mild anemia due to resolving acute kidney injury. Splenomegaly and peripheral lymphadenopathy have resolved. PDGFRB FISH has declined to 9%.Inversions of chromosome 5 have very rarely been described in association with eosinophilia, but only one report has specifically identified a rearrangement of PDGFRB. This report described a novel rearrangement involving exons 12 to 23 of PDGFRB at 5q32 and the 5′ (UTR) of G3BP1 at 5q33.1. Notably, this specific rearrangement was undetectable by PDGFRB break-apart FISH. These reports and ours highlight the clinical relevance of NGS in identifying novel rearrangement partners. Furthermore, patient-specific ddPCR assays can be designed for sensitive monitoring. [Display omitted] DisclosuresOehler: BMS: Consultancy; OncLive: Honoraria; Pfizer: Research Funding; Takeda: Consultancy; Blueprint Medicines: Consultancy.

PAX5 P80R mutated acute leukemia followed by genetically-related histiocytic proliferation with clonal IGH gene rearrangements

Abstract Introduction/Objective PAX5 is a transcription factor critical for B-cell development and its genetic alterations are common in B lymphoblastic leukemia/lymphoma (B-ALL). We report a case of PAX5 P80R mutated acute leukemia with predominantly monocytic immunophenotype followed by a genetically-related histiocytic proliferation. Methods/Case Report A 27-year-old male presented with pancytopenia, epistaxis, and blurry vision. Bone marrow exam showed 95% blasts with nuclear indentations, occasional prominent nucleoli and basophilic cytoplasm. Blasts were positive for bright CD33, HLA-DR, CD14, bright CD64, partial CD123 and partial CD19. In addition, a minute population of B lymphoblasts positive for CD19, CD20, and dim TdT was seen. The AML FISH panel showed markedly aneuploid population with all probes demonstrating 3 to 5 signals. PAX5 P80R and KRAS G13D mutations were detected by NGS. Post induction bone marrow showed no evidence of acute leukemia with normal cytogenetics and FISH results. Subsequent two bone marrow exams performed due to progressive cytopenias demonstrated a prominent histiocytic proliferation with sheets of mature appearing histiocytes with abundant cytoplasm, oval to folded nuclei and occasional hemophagocytosis. This population was positive for CD163, CD14, CD68R, CD68, cyclin D1 with variable OCT2 expression and low proliferative activity. PAX5 P80R and KRAS G13D were detected. AML FISH panel showed aneuploidy in histiocytic appearing cells and IgH gene rearrangement studies by PCR showed prominent clonal population. The patient remained pancytopenic and died of disseminated fungal infection. Results (if a Case Study enter NA) NA Conclusion PAX5 P80R mutation has been primarily reported in B-ALL and is exceedingly rare in acute myeloid leukemias. This alteration has been linked to downregulation of B-cell lineage genes affecting B-cell maturation, and not surprisingly a proportion of PAX5 P80R mutated B-ALL cases show a switch to a monocytic lineage. The reported case demonstrates diagnostic caveats including unusual features of acute leukemia at the time of initial diagnosis and subsequent genetically-related histiocytic proliferation.

Tre-O2-03 - Mogamulizumab-associated rash frequently mimics cutaneous T-cell lymphoma recurrence but heralds a positive response to therapy: an updated single-center case series

Mogamulizumab is a humanized antibody that targets CCR4, a transmembrane chemokine receptor predominantly expressed on effector T helper type 2 and malignant cutaneous T-cell lymphoma (CTCL) cells. This biologic was FDA approved for relapsed/refractory mycosis fungoides (MF)/Sézary syndrome (SS) in 2018. MARs are the most common adverse events reported; however, the clinicopathologic nature of these eruptions remains unclear. To inform future management and recognition of MAR, we report an update on our retrospective case series of MF/SS patients treated with mogamulizumab since its approval as standard of care. Among 24 CTCL (16 SS, 8 MF) patients, 17 developed MAR (68%). MAR development was associated with complete (11/17) or partial (4/17) responses, for an overall response rate (ORR) of 88% to mogamulizumab among patients with MAR. A lower ORR of 28% (2/7) was observed in patients that did not develop MAR. Median MAR time to onset (TTO) was 2.1 mo. (0.7–9.0). Median time to resolution (TTR) was 6.9 mo. (3.5–9.2) in 8/17 patients with MAR resolution. Five distinct but frequently concurrent clinical patterns were identified: lichenoid papules and plaques (9/17), MF-like lesions (9/17), photodistribution (7/17), psoriasiform plaques (7/17), and SS-like erythroderma (3/17). Histopathologic patterns included spongiotic (n=21), lichenoid (n=14), psoriasiform (n=10), and interface (n=8) features across 27 skin biopsies. Eosinophils (n=15), epithelioid granulomas (n=4), and bizarre bi- and multinucleated cells (n=6) that may represent highly activated macrophages were also noted. Although mild, features associated with CTCL were noted in all suspected MAR tissue samples [epidermotropism (n=10), exocytosis (n=12), and lymphoid atypia (n=16)]. When compared to baseline studies, flow cytometry analysis for circulating Sézary cells and TCR rearrangement studies in skin and blood were most often negative during suspected MAR despite the presence of CTCL-associated histological features, emphasizing the importance of ancillary studies in distinguishing MAR from CTCL. In conclusion, MAR may be characterized by distinct clinical, histopathologic, and molecular patterns. Notably, MAR can clinically mimic CTCL lesions and may occur more often in patients with clinical responses, highlighting the need for clinicians to rule out progressive disease vs. drug eruption in treating a condition that historically suffers from poor outcomes and treatment response.

Analysis of clonality in cutaneous B-cell lymphoma and B-cell pseudolymphoma using skin flow cytometry: Comparison of immunophenotyping and gene rearrangement studies.

To identify clonal neoplastic cells in skin affected by B-cell lymphoma using skin flow cytometry (FCM) techniques, we investigated light-chain restriction using skin FCM with clonality assessed by polymerase chain reaction and light-chain restriction by in situ hybridization (ISH). We retrospectively analyzed 16 cases of B-cell lymphoma with cutaneous involvement: primary cutaneous diffuse large B-cell lymphoma, leg type (pcDLBCL-LT) (n=7), DLBCL-not otherwise specified (DLBCL-NOS) (n=6), primary cutaneous follicle center lymphoma (pcFCL) (n=1), and follicular lymphoma (n=2), as well as cutaneous B-cell pseudolymphoma (n=9). Results of skin FCM light-chain restriction analyses were compared with immunoglobulin H (IgH) gene rearrangement and κ/λ ISH findings. Skin FCM detected light-chain restriction in 11 of 14 B-cell lymphoma patients but none of the B-cell pseudolymphoma patients. The sensitivity of skin FCM for distinguishing B-cell lymphoma and B-cell pseudolymphoma was 79%, and the specificity was 100%. Eleven of 13 B-cell lymphoma patients exhibited gene rearrangement (sensitivity 85%), whereas six of seven pseudolymphoma patients were negative (specificity 86%). ISH was positive in three of 16 B-cell lymphoma cases (sensitivity 19%) but none of the B-cell pseudolymphoma cases (specificity 100%). ISH sensitivity was 29% for pcDLBCL-LT, 17% for DLBCL-NOS, and 0% for pcFCL and follicular lymphoma. Skin FCM therefore appears to be more sensitive than ISH in detecting light-chain restriction in DLBCL and follicular lymphoma, and as sensitive as IgH gene rearrangement analysis in detecting clonality. Skin FCM is thus a promising diagnostic tool for identifying monoclonal neoplastic B-cell populations.

Generation and Application of T-Cell Lymphoma Patient Derived Tumor Xenograft Models

Introduction:Although pivotal “driver mutations” have been described in T-cell lymphomas (T-NHL), targetable dependences are lacking for many entities. As result, T-NHL patients experience dismal therapeutic responses and have a poor survival. Therapeutic improvements have been hindered by the lack of informative models. Patient-Derived-Tumor-Xenografts (PDTX), recapitulating many of the biological features of primary cancers, represent a novel and powerful platform. Here, we constructed a library of T-NHL PDTX to foster drug discovery programs and new patient-tailored approaches.Methods:Fresh and/or viable cryopreserved samples from primary T-NHL were obtained from Turin, WCM, and MSKCC hospitals. For the initial tumorgraft implants, lymphoma fragments, PBMC or bone marrow cells were implanted in NSG-B2m mice. Seeds from arising tumors were then serially transplanted. MRI was used to follow tumor growth and dissemination. The lymphoma phenotype was assessed by multi-channel flow cytometry and immunohistochemistry. We used TCR gene rearrangement analyses, whole exome sequencing (WES), total RNAseq, and CHIP-Seq (STAT3, methylation marks) to annotate primary and PDTX samples. For the in vitro studies, PDTX were digested and stromal cells were excluded by panning. Enriched Patient-Derived Tumor Cells (PDTC) were cultured in either RPMI/20% FBS or StemSpan/10% KSR media. High-throughput drug screening was performed using a library of 432 compounds (1 µM for 72hrs). Cell titer glo/blue, trypan blue cell count, and annexin-V/7AAD were used to assess cell viability, apoptosis, and proliferation.Results:We implanted 123 fresh T-NHL samples, resulting in the generation of 41 PDTX lines (Fig.1). In addition, 14 implants led to the growth of lymphoblastoid EBV+ populations. All PDTX lines were successfully propagated (5-26 serial passages) with the exception of angioimmunoblastic T-cell lymphoma (AITL) PDTX, which displayed a delay in engraftment over time, and eventually ceased growing (passage 3-7). Serially propagated PDTX showed distinct immunophenotypic profiles, which closely reflected their corresponding primary samples. TCR gene rearrangement studies confirmed the faithful correspondence between donor and PDTX over different generations. WES and total RNA-Seq on primary and serially propagated PDTX (at least T1-T5) yielded a rich landscape of mutations shared by primary tumors and PDTX derivatives. On going mutations were also defined along propagations. CHIP-Seq assay defined a STAT3 classifier in STAT+ PDTC.In vitro, all PDTC were shortly maintained (<7days), with some surviving for 6-8 weeks. To overcome their limited growth in vitro, we tested whether stromal cells (i.e. E4-Endothelial cells, E4-EC) could enhance their survival and/or growth. Indeed, co-culturing of PDTC with E4-ECs rescued cell death induced by ex vivo long-term propagation (peripheral T-cell lymphoma not otherwise specified and NK/T-Cell Lymphoma, PTCL-NOS and NK) or starvation (anaplastic large cell lymphoma, ALCL).PDTC were also used to perform a drug screening targeting major signaling pathways (>3x replicates of 8 ALCL, 1 NK, and 2 PTCL-NOS), demonstrating that each PDTX exhibited a unique drug sensitivity profile, mostly dictated by its genomic landscape. This was epitomized by two PDTC (IL-2 and IL-19) derived from the same PTCL-NOS patient bearing an activating V658F JAK1 somatic mutation. These two PDTC, once exposed to JAK inhibitors, displayed down-regulation of pSTAT3 (24hr) and massive cell death (T3-T6, >95% at 72hrs). Similar results were seen with ALK inhibitors in ALK+ ALCL PDTC (n=5). Moreover, specific drugs were identified to bypass refractory phenotypes in selective PDTC (i.e. proteasome and topoisomerase inhibitors).Conclusions:We built up a large and representative PDTX library from major T-NHL entities. Immunophenotyping, genomics and functional studies showed a close correspondence between primary samples and correspondent PDTX derivatives. Our in vitro models provide a starting point for elucidating stromal:tumor cell interactions that contribute to lymphoma growth and survival in vivo, and evidence that a drug screening can be successfully executed on PDTCs. We predict that this cohort may be a valuable tool for testing novel therapeutic compounds and fostering personalized therapeutic approaches in T-NHL patients. [Display omitted] DisclosuresThompson:Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Other: Founder; Merck: Membership on an entity's Board of Directors or advisory committees; Charles River Laboratories: Membership on an entity's Board of Directors or advisory committees. Aster:Eli Lilly and Company: Membership on an entity's Board of Directors or advisory committees. Ruan:Cell Medica: Research Funding; Pharmacyclics: Research Funding; Seattle Genetics: Consultancy, Research Funding; Celgene: Consultancy, Research Funding. Dogan:Seattle Genetics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Peer Review Institute: Consultancy; Celgene: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Roche Pharmaceuticals: Consultancy. Cerchietti:Celgene: Research Funding; Lymphoma Research Foundation: Research Funding; Leukemia and Lymphoma Society: Research Funding; Weill Cornell Medicine - New York Presbyterian Hospital: Employment. Horwitz:Celgene: Consultancy, Research Funding; Kyowa-Hakka-Kirin: Consultancy, Research Funding; Infinity/Verastem: Consultancy, Research Funding; Seattle Genetics: Consultancy, Research Funding; BMS: Consultancy; HUYA: Consultancy; Aileron Therapeutics: Research Funding; ADCT Therapeutics: Research Funding; Millenium/Takeda: Consultancy, Research Funding; Mundipharma: Consultancy; Forty-Seven: Consultancy, Research Funding.

Assessing the Utility of Immunoglobulin and T Cell Receptor Gene Rearrangement Studies for Cerebrospinal Fluid Specimens

INTRODUCTION : Immunoglobulin (IGH and IGK) and T cell receptor (TCRG) rearrangements (-R) by polymerase chain reaction (PCR) have emerged as important ancillary tools for assessing clonality of B-cell and T-cell processes, respectively, in specimens such as lymph node, bone marrow and peripheral blood. However, the utility of these gene rearrangement studies in cerebrospinal fluid (CSF) samples has not yet been systematically examined. Cytomorphology and flow cytometry (FC) are currently the standard tools for the assessment of the CSF for leptomeningeal involvement by hematological malignancies. There are inherent limitations to both of these diagnostic methods; thus, it is important to establish the role of IGH/IGK- R and TCRG- R studies in the examination of CSF.METHODS : Pathology reports of 87 consecutive CSF specimens from 61 patients submitted for IGH, IGK and/or TCRG -R studies in the past three years (2015-2017) were retrospectively reviewed. PCR for IGH (using FR3 and JH primers), IGK and TCRG rearrangements was performed using the BIOMED2 assays. The PCR products for all three reactions were analyzed via polyacrylamide gel or capillary electrophoresis. The interpretation was rendered by an attending molecular pathologist. These results were assessed in the context of the corresponding flow cytometry report.RESULTS : Majority of the patients (82% of patients) had a prior diagnosis of a hematologic malignancy (Table 1), including diffuse large B-cell lymphoma (31% of specimens) and acute leukemias (17%), while the remaining patients presented with CNS symptoms with/without a brain mass. Nearly equal numbers of cases were requested by the clinicians concurrently with flow cytometry (46 cases; 53%) or by the hematopathologist evaluating the CSF FC results (41 cases; 47%). In the latter group, PCR was requested due to an atypical finding based on CSF flow cytometry, which most commonly was an abnormal population with an immunophenotype was different from the diagnostic specimen or a suspicious population that was too small to be diagnostic.Of the 61 IGH- R and 35 IGK -R tests requested, the majority of the specimens (74% and 63%, respectively) demonstrated a pseudoclonal pattern or poor amplification because of the paucity of B cells. Of the cases with sufficient cellular material, a monoclonal population was detected by IGH- R and IGK -R in more than half of the cases (56% and 54%, respectively). In contrast, in 75% (24/32) of the cases where TCRG-R was requested, there was an adequate number of T-cells for analysis; of these 24 informative cases, 9 (38%) were monoclonal and 15 (62%) were polyclonal.Overall, 37 of the 87 cases (43%) yielded an informative result for one or more of IGH, IGK, and/or TCRG -R. 38% (14/37) of these cases showed a result that clarified or supported an atypical/suspicious finding by FC, whether submitted by the pathologist (70%) or treating clinician (30%). Eight of the 37 cases (22%) were submitted by the pathologist to confirm the FC findings. In 7 of these 8 cases, the PCR findings corroborated the FC results; there was one discordant case in which a polyclonal T-cell population was seen in a positive FC case of T-lymphoblastic lymphoma/leukemia. Remainder of the cases (14/37, 38%) were submitted by the treating clinicians; if they were submitted together with a concurrent FC analysis (12 of 14 cases), the PCR results confirmed unambiguous FC results (whether negative or positive). In 6 of the 14 cases in which a IGH -R and/or IGK -R identified a monoclonal population, the atypical or neoplastic population constituted <5% of the analyzed cells by FC.CONCLUSIONS : The paucicellular nature of the CSF specimens provides a challenge not only for cytomorphology and FC but also for PCR for IGH, IGK, and TCRG rearrangements. Nonetheless, an informative clonality result was observed in 43% of the cases submitted for PCR analysis. The results of 60% of these cases (26% of the total submitted cases) were helpful for clarifying and/or supporting an FC finding. Thus, PCR for IGH, IGK and TCRG -R of CSF specimens is a useful ancillary diagnostic aid and can help in the interpretation and confirmation of flow cytometry results, particularly when the neoplastic lymphoid cells are of low abundance or exhibit an immunophenotype that is distinct from the diagnostic tumor. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Self-regressing oral CD30-positive, EBV-negative, T-cell lymphoproliferative lesions. A poorly understood process highlighted by ominous clinicopathologic features and indolent behavior

Intraoral, primary, CD30-positive (CD30+) T-cell lymphoproliferative disorders (TLPDs) are uncommon, and their clinicopathologic presentation and management can vary and may be challenging. Herein, we present a retrospective study of 4 examples of self-regressing primary CD30+ TLPD affecting the gingiva. Archived files were retrospectively reviewed for oral CD30+ TLPDs featuring (1) proper immunohistochemical documentation, (2) Epstein-Barr virus negativity, (3) adequate follow-up information corroborating regression, and (4) no history of hematopoietic malignancy or related-mucocutaneous disease. Three women and 1 man (age range, 55-82 years; mean, 68.3 years) presented with rapidly growing gingival ulcers. Microscopic evaluation revealed diffuse infiltration by sheets of large, atypical cells admixed with lymphocytes and eosinophils, showing angiocentric distribution, focal neurotropism, and muscle infiltration. The lesional cells consistently stained for CD3 and CD30 and were variably immunoreactive against CD2, CD4, CD5, CD7, and CD8, but were negative for ALK1 and EBV-encoded small RNA. TCR-γ gene rearrangement studies revealed a monoclonal T-cell population in 1 case. All lesions showed complete regression 2 to 8 weeks postoperatively (mean follow-up, 4.5 weeks). Notwithstanding their alarming clinicopathologic appearance, there are CD30+ TLPDs confined to the oral cavity that have an indolent course. However, clinical staging is essential to exclude aggressive systemic malignancy.

Lennert Lymphoma (lymphoepithelioid lymphoma) with Extensive Necrosis: A Case Report

Lennert lymphoma (lymphoepitheloid lymphoma) is an extremely rare variant of peripheral T-cell lymphoma, not otherwise specified. Here we report a case of Lennert lymphoma diagnosed in a 57-year-old woman. She had a three-year history of waxing and waning lymphadenopathy with a rapid increase in size in the past four months before presentation. A needle biopsy and a fine needle aspiration were non-diagnostic due to extensive necrosis. The patient underwent a right neck lymph node excisional biopsy which showed the lymph node architecture was effaced by numerous and sometimes confluent clusters of epithelioid histiocytes and infiltration of small lymphocytes. Extensive necrosis was present. Immunohistochemical stains revealed a mixed population of B- and T-cells with the T-cells showing diminished T-cell markers CD3, CD5, and CD7. Flow cytometric analysis detected a small population (7% of total lymphocytes) of CD4-positive T-lymphocytes with loss of CD3, CD5, and CD7 expressions. PCR-based T-cell receptor gene rearrangement studies showed positive results (clonal peaks) in both gamma and beta genes. Stains for microorganisms were negative. The overall findings indicate Lennert lymphoma. To our knowledge, this is the first reported case of Lennert lymphoma with extensive necrosis. The patient is undergoing chemotherapy. The diagnosis of Lennert lymphoma can be challenging, particularly in cases with extensive necrosis. Our case highlights that adequate sampling is important in the investigation of patients with suspected Lennert lymphoma. A careful pathologic examination with ancillary studies including flow cytometry, immunohistochmistry, and cytogenetic and molecular studies leads to the accurate diagnosis.

Primary cutaneous T-cell lymphomas in childhood and adolescence.

Primary cutaneous lymphomas are extranodal non-Hodgkin lymphomas of T- or B- cell origin, that predominantly affect older patients but have been reported in all age groups and as early as in the first years of life. Diagnosis of cutaneous lymphomas is challenging and requires high clinical suspicion and close collaboration between dermatologists, pediatric oncologists and pathologists. Skin involvement of non-Hodgkin lymphomas in children or adolescents can either be primary cutaneous or secondary due to an underlying nodal lymphoma. The most common primary cutaneous lymphomas encountered in children are of T-cell origin, with mycosis fungoides being the most prevalent cutaneous T-cell lymphoma, followed by CD30+ lymphoproliferative disorders. While cutaneous lymphomas share clinicopathologic characteristics between juvenile and adult forms, there are important differences in terms of clinical presentation, diagnosis and treatment. The hypopigmented variant of mycosis fungoides seems to be overrepresented in the pediatric age group. Prognosis and treatment of mycosis fungoides are stage dependent. The majority of children present with early-stage disease and respond well to topical corticosteroids and phototherapy.

Cell-blocks and hematolymphoid lesions.

Cell-blocks are an important component for evaluation for hematolymphoid lesions. They are especially critical for immunocharacterization of the lymphoid population especially when flow cytometry is not available or cannot be performed. In addition, cell-blocks allow various molecular pathology tests including gene rearrangement studies and FISH, proteomics analysis, and microbiology/histochemical special stains. Fine-needle aspiration (FNA) for mass lesions, lymphadenopathy, and effusion fluids are common cytopathology specimens which are frequently cell-blocked. The differential diagnosis of enlarged lymph nodes (LNs) and mass lesions is broad and includes reactive processes, granulomatous lesions and malignancies including solid tumor metastases and various types of hematological malignancies, of which lymphoma would be most common. Depending on the patient population, most lymphomas may be diagnosed with immunocharacterization on cell-block or/and flow cytometry in concert with excellent cytomorphology in Diff-Quik stained FNA aspirate smears. However, a proportion of lymphoma cases (up to 12-30%) may still require an excisional LN biopsy to evaluate architectural parameters. Similarly, various effusion fluids suspicious for lymphoma can be immunocharacterized by immunostaining of cell-block sections (or/and by flow cytometry). Availability of quantitatively and qualitatively optimum cell-blocks of specimens to be evaluated for hematolymphoid processes is critical for immunohistochemistry, polymerase chain reaction, in situ hybridization (FISH), and gene expression profiling studies.

Sulfur-Mediated Reactions through Sulfonium Salts and Ylides

AbstractThis Account discusses several new reaction methods developed in our group that utilize sulfur-mediated reactions through sulfonium salts and ylides, highlighting the interplay of rational design and serendipity. Our initial goal was to convert aliphatic C–H bonds into C–C bonds site-selectively, and without the use of transition-metal catalysts. While a proof-of-concept has been achieved, this target is far from being ideally realized. The unexpected discovery of an anti-Markovnikov rearrangement and subsequent studies on difunctionalization of alkynes were much more straightforward, and eventually led to the new possibility of asymmetric N–H insertion of sulfonium ylides through Brønsted acid catalysis.1 Introduction2 Allylic/Propargylic C–H Functionalization3 Anti-Markovnikov Rearrangement4 Difunctionalization of Alkynes5 Asymmetric N–H Insertion of Sulfonium Ylides6 Conclusion

Utility of clonal T-cell rearrangement study in the diagnosis of early mycosis fungoides

To the Editor: The diagnosis of early mycosis fungoides (MF) is challenging, as clinical and histopathologic presentation can mimic inflammatory dermatoses. In 2005, the International Society for Cutaneous Lymphoma (ISCL) established a clinicopathologic algorithm for the diagnosis of early MF based on clinical, histopathologic, molecular, and immunopathologic criteria, requiring a total of 4 points (Table I).1 One criterion, the clonal T-cell receptor (TCR) gene rearrangement analysis, represents a significant cost in the workup of early MF and is often unavailable in-house.