Molecular and clinical analyses of PHF6 mutant myeloid neoplasia provide their pathogenesis and therapeutic targeting.

PHF6 mutations (PHF6MT) are identified in various myeloid neoplasms (MN). However, little is known about the precise function and consequences of PHF6 in MN. Here we show three main findings in our comprehensive genomic and proteomic study. Firstly, we show a different pattern of genes correlating with PHF6MT in male and female cases. When analyzing male and female cases separately, in only male cases, RUNX1 and U2AF1 are co-mutated with PHF6. In contrast, female cases reveal co-occurrence of ASXL1 mutations and X-chromosome deletions with PHF6MT. Next, proteomics analysis reveals a direct interaction between PHF6 and RUNX1. Both proteins co-localize in active enhancer regions that define the context of lineage differentiation. Finally, we demonstrate a negative prognostic role of PHF6MT, especially in association with RUNX1. The negative effects on survival are additive as PHF6MT cases with RUNX1 mutations have worse outcomes when compared to cases carrying single mutation or wild-type.

Proteomic characterisations of ulcerative colitis endoscopic biopsies associate with clinically relevant histological measurements of disease severity.

AIMS AND METHODS: Accurate protein measurements using formalin-fixed biopsies are needed to improve disease characterisation. This feasibility study used targeted and global mass spectrometry (MS) to interrogate a spectrum of disease severities using 19 ulcerative colitis (UC) biopsies. RESULTS: Targeted assays for CD8, CD19, CD132 (interleukin-2 receptor subunit gamma/common cytokine receptor gamma chain), FOXP3 (forkhead box P3) and IL17RA (interleukin 17 receptor A) were successful; however, assays for IL17A (interleukin 17A), IL23 (p19) (interleukin 23, alpha subunit p19) and IL23R (interleukin 23 receptor) did not permit target detection. Global proteome analysis (4200 total proteins) was performed to identify pathways associated with UC progression. Positive correlation was observed between histological scores indicating active colitis and neutrophil-related measurements (R2=0.42-0.72); inverse relationships were detected with cell junction targets (R2=0.49-0.71) and ß-catenin (R2=0.51-0.55) attributed to crypt disruption. An exploratory accuracy assessment with Geboes Score and Robarts Histopathology Index cut-offs produced sensitivities/specificities of 72.7%/75.0% and 100.0%/81.8%, respectively. CONCLUSIONS: Pathologist-guided MS assessments provide a complementary approach to histological scoring systems. Additional studies are indicated to verify the utility of this novel approach.

Immune Escape Mechanisms in Intravascular Large B-Cell Lymphoma: A Molecular Cytogenetic and Immunohistochemical Study.

OBJECTIVES: Intravascular large B-cell lymphomas (IVLBCLs) are rare extranodal LBCLs in which relapse is relatively frequent. We sought to further characterize potential immune escape mechanisms in IVLBCLs that newer therapies can exploit. METHODS: A series of 33 IVLBCLs were evaluated for programmed cell death ligand 1 (PD-L1) and PD-L2 expression by immunohistochemistry (IHC), chromosomal alterations (CAs) in the PDL1/PDL2 locus by fluorescence in situ hybridization, and loss of major histocompatibility complex (MHC) class I and II expression by IHC. RESULTS: Cases were subclassified as classical (n = 22) or hemophagocytic syndrome (HPS)-associated (n = 11) variants. A total of 12 cases (39%; n = 12/31) expressed PD-L1 and/or PD-L2. CAs were seen in 7 cases (7/29 [24%]) and included gains, amplifications, and rearrangements. CAs in classical variant cases (24%; n = 5/21) included gains (n =1), gains with concurrent rearrangements (n = 2), and amplifications (n = 2). The 2 HPS-associated variant cases with CAs (25%; n = 2/8) both showed amplification, including 1 case with a concurrent rearrangement. A majority of cases with CAs (71%; n = 5/7) were PD-L1/PD-L2 IHC positive. Among PD-L1/PD-L2 IHC-positive cases, 45% harbored a CA. Loss of MHC class I and/or class II was seen in 27% (n = 9/33) of cases. CONCLUSIONS: Altogether, our data show that 65% (n = 20/31) of IVLBCLs may exploit immune evasion strategies through PD-L1/PD-L2 expression or downregulation of MHC proteins.

Analytical Accuracy of RET Fusion Detection by Break-Apart Fluorescence In Situ Hybridization.

CONTEXT.­: RET gene fusions are oncogenic drivers in nonsmall cell lung cancer and nonmedullary thyroid cancer. Selpercatinib (RETEVMO), a targeted inhibitor of RET, was approved by the US Food and Drug Administration for the treatment of RET fusion-positive nonsmall cell lung cancer and nonmedullary thyroid cancer emphasizing the need for rapid and accurate diagnosis of RET fusions. Fluorescence in situ hybridization (FISH) has been used to detect gene rearrangements, but its performance detecting RET rearrangements is understudied. OBJECTIVE.­: To validate and describe the performance of Abbott Molecular RET break-apart FISH probes for detecting RET rearrangements. DESIGN.­: A training set with RET fusion-positive (13) and RET fusion-negative nonsmall cell lung cancer and nonmedullary thyroid cancer samples (12) was used to establish criteria for FISH scoring. The scoring criteria was then applied to a larger validation set of samples (96). RESULTS.­: A cutoff of 19% or more positive nuclei by FISH was established in the training set and determined by the mean ±3 SD. The validation set was tested using Abbott Molecular RET break-apart FISH compared with sequencing. With this cutoff, a sensitivity of 86% (12 of 14) and specificity of 99% (81 of 82) was achieved. Bootstrapping showed sensitivity could be optimized by using a greater than 13% cutoff with indeterminate samples of 13% to 18% abnormal nuclei requiring confirmation by an orthogonal method. Using this 3-tier scoring system sensitivity increased to 100% (14 of 14) and specificity was 96% (79 of 82). CONCLUSIONS.­: Abbott Molecular break-apart FISH probes can be used to detect RET fusions. Laboratories can optimize cutoffs and/or testing algorithms to maximize sensitivity and specificity to ensure appropriate patients receive effective, timely therapy.

Cooperative miRNA-dependent PTEN regulation drives resistance to BTK inhibition in B-cell lymphoid malignancies.

Aberrant microRNA (miR) expression plays an important role in pathogenesis of different types of cancers, including B-cell lymphoid malignancies and in the development of chemo-sensitivity or -resistance in chronic lymphocytic leukemia (CLL) as well as diffuse large B-cell lymphoma (DLBCL). Ibrutinib is a first-in class, oral, covalent Bruton's tyrosine kinase (BTK) inhibitor (BTKi) that has shown impressive clinical activity, yet many ibrutinib-treated patients relapse or develop resistance over time. We have reported that acquired resistance to ibrutinib is associated with downregulation of tumor suppressor protein PTEN and activation of the PI3K/AKT pathway. Yet how PTEN mediates chemoresistance in B-cell malignancies is not clear. We now show that the BTKi ibrutinib and a second-generation compound, acalabrutinib downregulate miRNAs located in the 14q32 miRNA cluster region, including miR-494, miR-495, and miR-543. BTKi-resistant CLL and DLBCL cells had striking overexpression of miR-494, miR-495, miR-543, and reduced PTEN expression, indicating further regulation of the PI3K/AKT/mTOR pathway in acquired BTKi resistance. Additionally, unlike ibrutinib-sensitive CLL patient samples, those with resistance to ibrutinib treatment, demonstrated upregulation of 14q32 cluster miRNAs, including miR-494, miR-495, and miR-543 and decreased pten mRNA expression. Luciferase reporter gene assay showed that miR-494 directly targeted and suppressed PTEN expression by recognizing two conserved binding sites in the PTEN 3'-UTR, and subsequently activated AKTSer473. Importantly, overexpression of a miR-494 mimic abrogated both PTEN mRNA and protein levels, further indicating regulation of apoptosis by PTEN/AKT/mTOR. Conversely, overexpression of a miR-494 inhibitor in BTKi-resistant cells restored PTEN mRNA and protein levels, thereby sensitizing cells to BTKi-induced apoptosis. Inhibition of miR-494 and miR-495 sensitized cells by cooperative targeting of pten, with additional miRNAs in the 14q32 cluster that target pten able to contribute to its regulation. Therefore, targeting 14q32 cluster miRNAs may have therapeutic value in acquired BTK-resistant patients via regulation of the PTEN/AKT/mTOR signaling axis.

Decreased BIM expression in BCL2-negative follicular lymphoma: a potential mechanism for resistance to apoptosis.

Follicular lymphoma (FL) is characterized by t(14; 18)(q32; q21), leading to overexpression of the antiapoptotic molecule BCL2; however, a subset of FLs lack BCL2 rearrangement and BCL2 expression as analyzed by immunohistochemistry (IHC). In this study, we evaluated expression of antiapoptotic (MCL1 and BCL-XL) and proapoptotic proteins (BIM) by IHC in both BCL2(-) and BCL2(+) FLs. FLs diagnosed between 2009 and 2019 were reviewed to identify BCL2(-) cases by IHC (assessed by clone 124). Immunohistochemical analyses for BCL2 (EP36), MCL1, BIM, BCL-XL, and Ki-67 were performed on tissue microarrays or whole slides. BCL2 (EP36) was interpreted as positive (≥10%) or negative (<10%). Ki-67 was interpreted on tumor cells in 10% increments. The remaining immunohistochemical analysis results were scored on tumor cells in 10% increments, and intensity was interpreted as weak, moderate, or strong to derive an H-score. Twenty-four BCL2(-) FLs were initially identified, but on further testing with BCL2(EP36) immunohistochemical staining, 5 of 24 were reclassified as BCL2(+), leaving 19 BCL2(-) FLs. Thirty-three BCL2(+) FLs were selected with sufficient tissue for additional immunohistochemical analyses. There was no significant difference in expression of antiapoptotic BCL-XL or MCL1 between BCL2(-) and BCL2(+) FLs (p = 0.75 and 0.28, respectively). However, proapoptotic BIM expression was significantly lower in BCL2(-) FLs than in BCL2(+) FLs (p = 0.002). In our study, 21% of putative BCL2(-) FLs were BCL2(+) when tested with alternative clones, supporting the practice of having more than one BCL2 clone in immunohistochemical laboratories. Decreased BIM expression in BCL2(-) FLs could have an overall antiapoptotic effect and represent an alternate mechanism for cell survival in BCL2(-) FLs.

Targeting BCL-2 in B-cell malignancies and overcoming therapeutic resistance.

Defects in apoptosis can promote tumorigenesis and impair responses of malignant B cells to chemotherapeutics. Members of the B-cell leukemia/lymphoma-2 (BCL-2) family of proteins are key regulators of the intrinsic, mitochondrial apoptotic pathway. Overexpression of antiapoptotic BCL-2 family proteins is associated with treatment resistance and poor prognosis. Thus, inhibition of BCL-2 family proteins is a rational therapeutic option for malignancies that are dependent on antiapoptotic BCL-2 family proteins. Venetoclax (ABT-199, GDC-0199) is a highly selective BCL-2 inhibitor that represents the first approved agent of this class and is currently widely used in the treatment of chronic lymphocytic leukemia (CLL) as well as acute myeloid leukemia (AML). Despite impressive clinical activity, venetoclax monotherapy for a prolonged duration can lead to drug resistance or loss of dependence on the targeted protein. In this review, we provide an overview of the mechanism of action of BCL-2 inhibition and the role of this approach in the current treatment paradigm of B-cell malignancies. We summarize the drivers of de novo and acquired resistance to venetoclax that are closely associated with complex clonal shifts, interplay of expression and interactions of BCL-2 family members, transcriptional regulators, and metabolic modulators. We also examine how tumors initially resistant to venetoclax become responsive to it following prior therapies. Here, we summarize preclinical data providing a rationale for efficacious combination strategies of venetoclax to overcome therapeutic resistance by a targeted approach directed against alternative antiapoptotic BCL-2 family proteins (MCL-1, BCL-xL), compensatory prosurvival pathways, epigenetic modifiers, and dysregulated cellular metabolism/energetics for durable clinical remissions.

Targeting of CD38 by the Tumor Suppressor miR-26a Serves as a Novel Potential Therapeutic Agent in Multiple Myeloma.

Multiple myeloma is an incurable refractory hematologic malignancy arising from plasma cells in the bone marrow. Here we investigated miR-26a function in multiple myeloma and tested single-wall carbon nanotube delivery of miR-26a in vitro and in vivo. miR-26a was downregulated in patients with multiple myeloma cells compared with plasma cells from healthy donors. miR-26a overexpression inhibited proliferation and migration and induced apoptosis in multiple myeloma cell lines. To identify the targets of miR-26a, RPMI8226-V-miR-26-GFP and RPMI8226-V-GFP cells were cultured using stable isotope labeling by amino acids in cell culture (SILAC) medium, followed by mass spectrometry analysis. In multiple myeloma cells overexpressing miR-26a, CD38 protein was downregulated and subsequently confirmed to be a direct target of miR-26a. Depletion of CD38 in multiple myeloma cells duplicated the multiple myeloma inhibition observed with exogenous expression of miR-26a, whereas restoration of CD38 overcame the inhibition of miR-26a in multiple myeloma cells. In a human multiple myeloma xenograft mouse model, overexpression of miR-26a inhibited CD38 expression, provoked cell apoptosis, and inhibited cell proliferation. Daratumumab is the first CD38 antibody drug for monotherapy and combination therapy for patients with multiple myeloma, but eventually resistance develops. In multiple myeloma cells, CD38 remained at low level during daratumumab treatment, but a high-quality response is sustained. In daratumumab-resistant multiple myeloma cells, CD38 expression was completely restored but failed to correlate with daratumumab-induced cell death. Therefore, a therapeutic strategy to confer selection pressure to maintain low CD38 expression in multiple myeloma cells may have clinical benefit. SIGNIFICANCE: These results highlight the tumor suppressor function of miR-26a via its targeting of CD38 and suggest the therapeutic potential of miR-26a in patients with multiple myeloma.

Immunohistochemical Expression of Lymphoid Enhancer Binding Factor 1 in CD5-Positive Marginal Zone, Lymphoplasmacytic, and Follicular Lymphomas.

OBJECTIVES: Lymphoid enhancer binding factor 1 (LEF1) is expressed in most cases of chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) and has shown utility in distinguishing CLL/SLL from other small B-cell lymphomas. LEF1 expression has not been systematically studied in CD5-positive marginal zone lymphomas (MZLs), lymphoplasmacytic lymphomas (LPLs), and follicular lymphomas (FLs). We evaluated whether these cases lacked LEF1, helping to distinguish them from CLL/SLL. METHODS: MZLs, LPLs, and FLs expressing CD5 were retrospectively studied for expression of LEF1 by immunohistochemistry. RESULTS: LEF1 was absent in 17 of 18 CD5-positive lymphomas including 13 MZLs (2 nodal, 3 splenic, and 8 mucosa-associated lymphoid tissue lymphomas), 3 LPLs, and 1 of 2 FLs. One grade 3A CD5-positive FL expressed LEF1 in a majority of tumor cells. CONCLUSIONS: LEF1 is not expressed in most CD5-positive MZLs and LPLs; therefore, it is a reliable marker for distinguishing them from CLL/SLL. LEF1 may be expressed in CD5-positive FLs.

Inhibition of cyclin-dependent kinase 9 synergistically enhances venetoclax activity in mantle cell lymphoma.

Mantle cell lymphoma (MCL) is an aggressive and largely incurable subtype of non-Hodgkin's lymphoma. Venetoclax has demonstrated efficacy in MCL patients with relapsed or refractory disease, however response is variable and less durable than CLL. This may be the result of co-expression of other anti-apoptotic proteins such as MCL-1, which is associated with both intrinsic and acquired resistance to venetoclax in B-cell malignancies. One strategy for neutralizing MCL-1 and other short-lived survival factors is to inhibit CDK9, which plays a key role in transcription. Here, we report the response of MCL cell lines and primary patient samples to the combination of venetoclax and novel CDK9 inhibitors. Primary samples represented de novo patients and relapsed disease, including relapse after ibrutinib failure. Despite the diverse responses to each single agent, possibly due to variable expression of the BCL-2 family members, venetoclax plus CDK9 inhibitors synergistically induced apoptosis in MCL cells. The synergistic effect was also confirmed via venetoclax plus a direct MCL-1 inhibitor. Murine xenograft studies demonstrated potent in vivo efficacy of venetoclax plus CDK9 inhibitor that was superior to each agent alone. Together, this study supports clinical investigation of this combination in MCL, including in patients who have progressed on ibrutinib.

Resistance to BTK inhibition by ibrutinib can be overcome by preventing FOXO3a nuclear export and PI3K/AKT activation in B-cell lymphoid malignancies.

Chronic activation of the Bruton's tyrosine kinase (BTK)-mediated B-cell receptor (BCR) signaling is a hallmark of many B-cell lymphoid malignancies, including chronic lymphocytic leukemia (CLL) and diffuse large B-cell lymphoma (DLBCL). Ibrutinib, an FDA approved, orally administered BTK inhibitor, has demonstrated high response rates, however, complete responses are infrequent and acquired resistance to BTK inhibition can emerge. In this study, we generated ibrutinib-resistant (IB-R) cell lines by chronic exposure of CLL and activated B-cell (ABC)-DLBCL cells to ibrutinib in order to investigate the mechanism of acquired resistance to ibrutinib. IB-R cell lines demonstrated downregulation of FOXO3a and PTEN levels and activation of AKT, with their levels being low in the nuclei of resistant cells in comparison to the sensitive counterparts. Inhibition of PI3K and AKT using idelalisib and MK2206, respectively increased ibrutinib-induced apoptosis in IB-R cells by downregulation of pAKT473 and restoring FOXO3a levels, demonstrating the importance of these cell survival factors for ibrutinib-resistance. Notably, the exportin 1 inhibitor, selinexor synergized with ibrutinib in IB-R cells and restored nuclear abundance of FOXO3a and PTEN, suggesting that nuclear accumulation of FOXO3a and PTEN facilitates increase in ibrutinib-induced apoptosis in IB-R cells. These data demonstrate that reactivation of FOXO3a nuclear function enhances the efficacy of ibrutinib and overcomes acquired resistance to ibrutinib. Together, these findings reveal a novel mechanism that confers ibrutinib resistance via aberrant nuclear/cytoplasmic subcellular localization of FOXO3a and could be exploited by rational therapeutic combination regimens for effectively treating lymphoid malignancies.

Evaluation of PD1/PDL1 Expression and Their Clinicopathologic Association in EBV-associated Lymphoproliferative Disorders in Nonimmunosuppressed Patients.

Epstein-Barr virus (EBV)-associated lymphoproliferative disorders (LPD)/lymphomas in nonimmunosuppressed patients represent a unique entity and have been proposed to be related to immune senescence. Engagement of programmed cell death 1 (PD1) by its ligand programmed death ligand 1 (PDL1) inhibits T-cell activation, and leads to T-cell exhaustion. In clinical trials, therapeutic antibodies that block the PD1-PDL1 axis have shown promising therapeutic activity in certain types of lymphomas. Although PD1/PDL1 has been extensively studied in variety of lymphomas, there are few reports characterizing their expression in EBV-positive LPD. As these group of patients are presumed to be associated with immunosenescence/immune dysregulation, we hypothesize that the immune checkpoint pathway might be relevant in this entity. We explored the expression of PD1, PDL1 and its clinicopathologic association in 6 patients with a total of 8 independent specimens of EBV-positive LPD/lymphomas. We also applied proximity assay, a novel technique, which can identify intermolecular interaction, to evaluate physical interaction or in situ engagement of PD1 and PDL1. We found that the malignant cells in the EBV-positive LPDs express PDL1. PD1-positive tumor-infiltrating lymphocytes can be seen in these tumors. Proximity assay suggests there is active engagement between PD1 and PDL1. To our knowledge, this is the first report on the utility of proximity assay to test the active engagement between PD1 and PDL1 in lymphomas. As some EBV-positive LPDs were positive for PDL1, this subgroup of EBV-positive LPDs might be suitable for PD1/PDL1 antibody therapies.

T-Cell Large Granular Lymphocytic Leukemia and Coexisting B-Cell Lymphomas: A Study From the Bone Marrow Pathology Group.

OBJECTIVE: T-cell large granular lymphocytic (T-LGL) leukemia is associated with B-cell lymphomas (BCLs), especially small BCLs. We aimed to explore and expand upon its association with BCLs. METHODS: We retrospectively studied clinicopathologic features of T-LGL leukemia patients with coexisting BCL from January 2001 to December 2016. RESULTS: Among 432 patients with T-LGL leukemia, 22 (5.1%) had an associated B-cell non-Hodgkin lymphoma. Thirteen (59%) patients had large and nine (41%) had small BCL. T-LGL leukemia occurred synchronously with BCL in five, preceded BCL in three, and followed BCL in 14 patients. Anemia was the most common cytopenia (68%). Only one patient had a history of rheumatoid arthritis. CONCLUSION: To our knowledge, this is the first multicenter study looking at the spectrum and incidence of BCLs in patients with T-LGL leukemia and highlights its association with large BCLs (3% of T-LGL leukemias).

pSTAT3/pSTAT5 Signaling Patterns in Molecularly Defined Subsets of Myeloproliferative Neoplasms.

BCR/ABL1-negative myeloproliferative neoplasms (MPNs) are characterized by recurrent mutations in JAK2, CALR, and MPL, each of which has been reported to alter JAK/STAT signaling pathways. This report characterizes JAK/STAT signaling patterns in molecularly defined subsets of MPN utilizing immunohistochemistry for pSTAT3 and pSTAT5. Analysis of 30 BCR/ABL1-negative, nonpolycythemia vera MPN identified 15 (50%) with JAK2 V617F, 2 with MPL mutations (7%), and 8 with CALR mutations (27%). All mutations were mutually exclusive, except for 1 case with concurrent JAK2 V617F and CALR mutations. pSTAT3 staining in megakaryocyte nuclei was found in 4 cases (13%) and was not significantly associated with mutation status. pSTAT5 staining in megakaryocyte nuclei was found in 16 cases (53%), as was significantly associated with JAK2 V617F versus CALR mutation (P=0.009). Erythroid staining for pSTAT5 was seen exclusively in "triple-negative (TN)" cases lacking JAK2 V617F, MPL, and CALR mutations (P=0.006, TN vs. other genotypes), and pSTAT5 staining in megakaryocyte nuclei was seen in 2 TN cases. pSTAT5 staining in TN MPN suggests that other unknown abnormalities in this pathway may contribute to the pathogenesis of these cases. Furthermore, the demonstration of distinct STAT staining patterns in molecularly defined MPN suggests that these mutations result in divergent signaling events that may contribute to the biological and prognostic differences in these molecular subsets of MPN.

GATA4 loss of function in liver cancer impedes precursor to hepatocyte transition.

The most frequent chromosomal structural loss in hepatocellular carcinoma (HCC) is of the short arm of chromosome 8 (8p). Genes on the remaining homologous chromosome, however, are not recurrently mutated, and the identity of key 8p tumor-suppressor genes (TSG) is unknown. In this work, analysis of minimal commonly deleted 8p segments to identify candidate TSG implicated GATA4, a master transcription factor driver of hepatocyte epithelial lineage fate. In a murine model, liver-conditional deletion of 1 Gata4 allele to model the haploinsufficiency seen in HCC produced enlarged livers with a gene expression profile of persistent precursor proliferation and failed hepatocyte epithelial differentiation. HCC mimicked this gene expression profile, even in cases that were morphologically classified as well differentiated. HCC with intact chromosome 8p also featured GATA4 loss of function via GATA4 germline mutations that abrogated GATA4 interactions with a coactivator, MED12, or by inactivating mutations directly in GATA4 coactivators, including ARID1A. GATA4 reintroduction into GATA4-haploinsufficient HCC cells or ARID1A reintroduction into ARID1A-mutant/GATA4-intact HCC cells activated hundreds of hepatocyte genes and quenched the proliferative precursor program. Thus, disruption of GATA4-mediated transactivation in HCC suppresses hepatocyte epithelial differentiation to sustain replicative precursor phenotype.

Performance of a Commercially Available MAL Antibody in the Diagnosis of Primary Mediastinal Large B-Cell Lymphoma.

Myelin and lymphocyte (MAL) protein has been previously reported as a highly specific marker for distinguishing primary mediastinal large B-cell lymphoma (PMBL) from diffuse large B-cell lymphoma, not otherwise specified (DLBCL, NOS). However, there has not been a commercially available MAL antibody for immunohistochemistry. We identified a commercially available MAL monoclonal antibody and evaluated it by immunohistochemistry on 43 cases of PMBL and 63 cases of DLBCL, NOS. We also compared this with a CD200 antibody that was previously reported useful in distinguishing PMBL and DLBCL, NOS. A threshold of 10% positive tumor cells was used to determine positive protein expression. MAL was expressed in 72% cases of PMBL and 0% of cases of DLBCL, NOS (sensitivity=72%, specificity=100%). CD200 was expressed in 81% of PMBL cases and 13% of DLBCL, NOS cases (sensitivity=81%, specificity=87%). To our knowledge, this is the first report on the utility of a commercially available MAL monoclonal antibody in the diagnosis of PMBL. There is a high specificity with good sensitivity in distinguishing PMBL from DLBCL, NOS, similar to previous studies with a noncommercial source. This antibody will likely prove useful in identifying cases of PMBL in routine practice.

CAL2 Immunohistochemical Staining Accurately Identifies CALR Mutations in Myeloproliferative Neoplasms.

OBJECTIVES: Mutations in CALR (calreticulin) have been discovered in 50% to 80% of JAK2 (Janus kinase 2) and MPL (myeloproliferative leukemia protein) wild-type patients with Philadelphia-negative myeloproliferative neoplasm (MPNs). We evaluate the performance of a monoclonal antibody for immunohistochemical detection of CALR mutations. METHODS: A computerized archival search was performed for cases of non-chronic myeloid leukemia (CML) MPNs with available CALR and JAK2 V617F mutational analysis data. Bone marrow biopsy specimens were stained with monoclonal antibody CAL2, and the percentage of stained megakaryocytes was calculated. In select cases, double immunofluorescence staining was done with CAL2 and each of the following: CD61, myeloperoxidase, CD34, and glycophorin A. RESULTS: We studied 38 bone marrow biopsy specimens of non-CML MPNs (primary myelofibrosis, n = 21; essential thrombocythemia, n = 15; and n = 2 post-polycythemia vera myelofibrosis) from 31 patients. All eight bone marrow biopsy specimens from patients with mutant CALR showed strong cytoplasmic staining of the megakaryocytes (83.5%; range, 50%-98%; median, 87%) with the CAL2 antibody. Double immunofluorescence staining of the small mononuclear cells seen in CALR mutant cases revealed them to be myeloid blasts. CONCLUSIONS: Immunohistochemistry in routinely processed bone marrow biopsy specimens for mutated CALR is feasible and accurately identifies mutated cases, including rare cases with additional driver mutations.

Acquired resistance to venetoclax (ABT-199) in t(14;18) positive lymphoma cells.

The chromosomal translocation t(14;18) in follicular lymphoma (FL) is a primary oncogenic event resulting in BCL-2 over-expression. This study investigates activity of the BH3 mimetic venetoclax (ABT-199), which targets BCL-2, and mechanisms of acquired resistance in FL.The sensitivity of FL cells to venetoclax treatment correlated with BCL-2/BIM ratio. Cells with similar expression of anti-apoptotic proteins, but with higher levels of BIM were more sensitive to the treatment. Venetoclax induced dissociation of BCL-2/ BIM complex and a decrease in mitochondrial potential. Interestingly the population of cells that survived venetoclax treatment showed increased p-ERK1/2 and p-BIM (S69), as well as a decrease in total BIM levels. Venetoclax resistant cells initially showed elevated levels of p-AKT and p-Foxo1/3a, a dissociation of BIM/BCL-2/BECLIN1 complex, and a decrease in SQSTM1/p62 level (indicating increased autophagy) together with a slight decline in BIM expression. After stable resistant cell lines were established, a significant reduction of BCL-2 levels and almost total absence of BIM was observed.The acquisition of these resistance phenotypes could be prevented via selective ERK/AKT inhibition or anti-CD20 antibody treatment, thus highlighting possible combination therapies for FL patients.