Bashar Dabbas

MDS-associated somatic mutations and clonal hematopoiesis are common in idiopathic cytopenias of undetermined significance

Establishing a diagnosis in patients suspected of having a myelodysplastic syndrome (MDS) can be challenging and could be informed by the identification of somatic mutations. We performed a prospective study to examine the frequency and types of mutations encountered in 144 patients with unexplained cytopenias. Based on bone marrow findings, 17% were diagnosed with MDS, 15% with idiopathic cytopenias of undetermined significance (ICUS) and some evidence of dysplasia, and 69% with ICUS and no dysplasia. Bone marrow DNA was sequenced for mutations in 22 frequently mutated myeloid malignancy genes. Somatic mutations were identified in 71% of MDS patients, 62% of patients with ICUS and some dysplasia, and 20% of ICUS patients and no dysplasia. In total, 35% of ICUS patients carried a somatic mutation or chromosomal abnormality indicative of clonal hematopoiesis. We validated these results in a cohort of 91 lower-risk MDS and 249 ICUS cases identified over a 6-month interval. Mutations were found in 79% of those with MDS, in 45% of those with ICUS with dysplasia, and in 17% of those with ICUS without dysplasia. The spectrum of mutated genes was similar with the exception of SF3B1 which was rarely mutated in patients without dysplasia. Variant allele fractions were comparable between clonal ICUS (CCUS) and MDS as were mean age and blood counts. We demonstrate that CCUS is a more frequent diagnosis than MDS in cytopenic patients. Clinical and mutational features are similar in these groups and may have diagnostic utility once outcomes in CCUS patients are better understood.

Deep exploration of the immune infiltrate and outcome prediction in testicular cancer by quantitative multiplexed immunohistochemistry and gene expression profiling

ABSTRACT Platinum-based chemotherapy is usually curative for patients with testicular germ cell tumors (TGCT), but a subset of patients experience disease progression and poor clinical outcomes. Here, we tested whether immune profiling of TGCT could identify novel prognostic markers and therapeutic targets for this patient cohort. We obtained primary and metastatic TGCT samples from one center. We performed immune profiling using multiplexed fluorescence immunohistochemistry (FIHC) for T-cell subsets and immune checkpoints, and targeted gene expression profiling (Nanostring nCounter Immune panel). Publically available data sets were used to validate primary sample analyses. Nearly all samples had some degree of T-cell infiltration and immune checkpoint expression. Seminomas were associated with increased CD3+ T-cell infiltration, decreased Regulatory T-cells, increased PD-L1, and increased PD-1/PD-L1 spatial interaction compared with non-seminomas using FIHC. Gene expression profiling confirmed these findings and also demonstrated increased expression of T-cell markers (e.g., IFNγ, and LAG3) and cancer/testis antigens (e.g., PRAME) in seminomas, whereas non-seminomas demonstrated high neutrophil and macrophage gene signatures. Irrespective of histology, advanced TGCT stage was associated with decreased T-cell and NK-cell signatures, while Treg, neutrophil, mast cell and macrophage signatures increased with advanced stage. Importantly, cancer/testis antigen, neutrophil, and CD8+/regulatory T-cell signatures correlated with recurrence free survival. Thus, deep immune characterization of TGCT using IHC and gene expression profiling identified activated T-cell infiltration which correlated with seminoma histology and good prognosis. These results may provide a rationale for testing of anti-PD-1/PD-L1 agents and suggest prognostic markers.

Abstract 853: Novel quantitative multiplexed PD-1/PD-L1 immunohistochemistry test provides superior prediction of treatment response in melanoma patients

While PD-1/L1 axis-targeted therapies have shown promising clinical responses, their use in combination therapies is associated with both benefits and safety concerns. Response rates for single-agent anti-PD-1 therapies are significantly higher in biomarker positive patients, therefore there is a need to utilize predictive diagnostics to enhance benefit-risk profiles and guide treatment decisions. To address this, we developed a novel quantitative multiplexed immunohistochemistry assay that provides objective quantitation of PD-L1 positive cells, but more importantly assesses interactions with immune cells (PD-1+ or CD8+) in formalin-fixed paraffin embedded (FFPE) clinical specimens. The clinical validity of this assay was verified in a small series of melanoma patients treated with anti-PD1 targeted therapies/agents. FFPE melanoma tissues from patients who received anti-PD-1 therapy were fluorescently stained with a combination of anti-PD-1, PD-L1, and S100 antibodies plus DAPI or a combination of anti-CD8, PD-L1, and S100 antibodies plus DAPI. Each slide was then imaged on Vectra platform and the frequencies of biomarker positive cells (PD-L1, PD-1, and CD8) and their interaction scores were objectively evaluated using proprietary Automated Quantitative Analysis (AQUA®) algorithms. Analytical sensitivity, precision, and accuracy were established using standardized PD-L1 and PD-1 tissue control arrays composed of cell lines and lymphoid organs, while range of biomarker expression was verified on archived melanoma clinical specimens (n = 30), including samples taken from melanoma patients prior to anti-PD-1 therapies (n = 21). Frequencies of PD-L1 positive cells could be accurately quantified within 1% to 100% range in predefined control cell line mixtures. PD-L1 and PD-1 measurements were highly reproducible (R2 = 0.98 and 0.97, respectively). A broad range of PD-L1 and PD-1 expression and interaction scores were observed in archival clinical specimens (n = 53). In a cohort of 21 advanced melanoma patients treated with nivolumab (n = 5) or pembrolizumab (n = 16), the PD-1/L1 interaction score was found to reliably distinguish responders from non-responders (p = 0.03) while PD-L1 alone (p = 0.15) or CD-8 alone (p = 0.23) did not. Additionally, patients exhibiting higher PD-1/L1 interaction scores had superior response rates (78% vs. 17%, p = 0.03). Responders experienced significantly longer median progression-free survival (177 vs. 85 days, p = 0.014), and fewer deaths (22% vs 58%) compared with patients having lower PD-1/L1 interaction scores. In terms of diagnostic utility, the PD-1/L1 multiplex test showed superior predictive power (78% Positive Predictive Value, 83% Negative Predictive Value) compared with PD-L1 expression alone. Additional studies are underway to fully establish diagnostic utility and aid in treatment guidance. Citation Format: Jennifer Bordeaux, Douglas Johnson, Jeffrey Sosman, Ju Young Kim, Christine Vaupel, Bashar Dabbas, Justin Cates, Jeff Hall, Jelveh Lameh, Shabnam Tangri, Naveen Dakappagari. Novel quantitative multiplexed PD-1/PD-L1 immunohistochemistry test provides superior prediction of treatment response in melanoma patients. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 853.

Abstract 4683: Co-existence of SRSF2 and EZH2 gene mutations in myeloid malignancies

SRSF2 is recurrently mutated in a variety of myeloid malignancies, including chronic myelomonocytic leukemia (CMML), myelodysplastic syndrome (MDS), myeloproliferative neoplasm (MPN), and acute myeloid leukemia (AML); and most alterations involve the codon proline-95 (P95). In vitro studies have shown that the SRSF2-P95H mutation affects splicing of EZH2 and functionally reduces its protein expression. Inactivating mutations of EZH2, located at 7q36.1, have also been reported in a variety of myeloid malignancies. Studies have shown that SRSF2 and EZH2 gene mutations are mutually exclusive events. In this study, we retrospectively analyzed next-generation sequencing results of 16,767 patient cases with a clinical indication of a possible myeloid malignancy. All exons of the EZH2 gene and exon 1 of SRSF2 gene were sequenced. A total of 33 cases (0.2%) had mutations in both SRSF2 and EZH2 genes; 70% were male (age: 58->89 years) and 30% were female (age: 64->89 years). Of the 20 cases with sufficient material for rendering a pathologic diagnosis, 40% were CMML; 10% were MDS/MPN, unclassifiable; 10% were AML with MDS-related changes and monocytic differentiation; 10% were AML with MDS-related changes and no monocytic differentiation; 10% were MDS; 10% were AML, not otherwise specified, with monocytic differentiation; 5% were AML, not otherwise specified, without monocytic differentiation; and 5% were primary myelofibrosis. In total, 60% (12/20) of the cases with co-existing SRSF2 and EZH2 mutations displayed monocytic differentiation. In 97% (32/33) of the cases, SRSF2 mutations occurred in the hot spot codon, proline-95 [P95H (13 cases), P95R (8), P95L (6), P95T (3), P95V (1), and P95 deletion (1)]. In regards to EZH2, a single mutation was detected in 88% (29/33) of the cases. Double mutations were found in four cases with both mutations detected simultaneously in three cases and, acquisition of a second frameshift mutation noted in one case. EZH2 loss-of-function mutations including frameshift, nonsense, and splice-site mutations were detected in 40% (13/33) of the cases. Missense mutations were detected in 48% (16/33) of the cases, and 56% of these mutations were located in the conserved catalytic SET domain of the EZH2 protein. The other mutations included an in-frame insertion involving the SET domain and splice-region mutations. Cytogenetic information was available for 16 cases with 56% showing a normal karyotype. Trisomy 8 was the predominant chromosomal abnormality observed, especially in cases of AML. One case with AML with MDS-related changes showed monosomy 7 and an EZH2-R690H mutation. In conclusion, our data demonstrate that mutations in SRSF2 and EZH2 can infrequently co-exist in different subtypes of myeloid malignancies, and are associated with monocytic differentiation in most cases. Citation Format: Durga P. Cherukuri, Brian Kwok, Jacqueline Lekostaj, Paris Petersen, Peter Bui, Farzad Nooraie, Li Cheng, Francesca Fike, David A. Taylor, Bashar Dabbas, Matthew J. Mcginniss. Co-existence of SRSF2 and EZH2 gene mutations in myeloid malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4683. doi:10.1158/1538-7445.AM2017-4683

CD20-positive T-cell lymphoma involving bone marrow: report of four cases

Lymphomas are classified based on clinical presentation, morphology, immunophenotyping and molecular studies. NonHodgkin lymphomas are broadly divided into B cell and T/NK cell types [1]. Historically, CD20 has been regarded as a specific B-cell marker and has been used to distinguish mature B-cell from T-cell neoplasms [2]. However, several reports have shown CD20 expression in peripheral T-cell lymphomas diagnosed in lymph nodes, skin lesions, mediastinal masses, adrenal masses, gastric lesions, and pancreatic masses [3–7]. In this report, we describe four patients with CD20positive T-cell lymphomas involving the bone marrow. Although little is known about the prognostic significance of CD20 expression in T-cell lymphomas, its identification may cause diagnostic difficulty. Therefore, a combination of morphologic examination, immunophenotypic analysis with a broad panel of antibodies, and molecular studies is necessary for determination of the cell lineage andmaking a correct diagnosis.