Lisa Durkin

STAT3 mutations unify the pathogenesis of chronic lymphoproliferative disorders of NK cells and T-cell large granular lymphocyte leukemia

Chronic lymphoproliferative disorders of natural killer cells (CLPD-NKs) and T-cell large granular lymphocytic leukemias (T-LGLs) are clonal lymphoproliferations arising from either natural killer cells or cytotoxic T lymphocytes (CTLs). We have investigated for distribution and functional significance of mutations in 50 CLPD-NKs and 120 T-LGL patients by direct sequencing, allele-specific PCR, and microarray analysis. STAT3 gene mutations are present in both T and NK diseases: approximately one-third of patients with each type of disorder convey these mutations. Mutations were found in exons 21 and 20, encoding the Src homology 2 domain. Patients with mutations are characterized by symptomatic disease (75%), history of multiple treatments, and a specific pattern of STAT3 activation and gene deregulation, including increased expression of genes activated by STAT3. Many of these features are also found in patients with wild-type STAT3, indicating that other mechanisms of STAT3 activation can be operative in these chronic lymphoproliferative disorders. Treatment with STAT3 inhibitors, both in wild-type and mutant cases, resulted in accelerated apoptosis. STAT3 mutations are frequent in large granular lymphocytes suggesting a similar molecular dysregulation in malignant chronic expansions of NK and CTL origin. STAT3 mutations may distinguish truly malignant lymphoproliferations involving T and NK cells from reactive expansions.

Combination of ibrutinib with ABT-199: synergistic effects on proliferation inhibition and apoptosis in mantle cell lymphoma cells through perturbation of BTK, AKT and BCL2 pathways

Mantle cell lymphoma (MCL) is considered incurable with a shorter survival than indolent lymphomas. Current standard therapeutic regimens have high initial response rates, however, even with high dose therapy, drug resistance is common and recurrence is anticipated (Garraway & Janne, 2012). Adaptive resistance often results in progression to more clinically aggressive disease. Most patients eventually relapse with a median overall survival of 4–6 years. Thus, more effective therapies are needed. Abnormal activation of Bruton tyrosine kinase (BTK) mediated B-cell-receptor (BCR) signalling pathway contributes to the pathogenesis of B-cell malignancies (Davis et al, 2010). Signals mediated by BTK trigger cell survival pathways such as NFjB, RAS/RAF/MEK/ERK and PI3K/AKT. BTK is therefore an attractive target for inhibition of B-cell growth. A clinical trial suggested Ibrutinib is a promising agent in patients with relapsed or refractory MCL (Wang et al, 2013). However, considering that genetic mutations cause resistance to ibrutinib in chronic lymphocytic leukaemia (CLL) patients and altered signalling pathways are common mechanisms of resistance to single agents, Ibrutinib monotherapy is not expected to cure MCL (Zucca & Bertoni, 2013). The BCL2 family proteins play a critical role in regulating apoptosis and lymphoid malignancies frequently have high BCL2 expression. The BH3-only mimetic ABT-199 selectively inactivates BCL2 and is a promising drug for treatment of BCL2-dependent cancers. However, acquired mutations could cause resistance to ABT-199 in lymphoma cells (Fresquet et al, 2014). These observations indicate potential clinical challenges of applying ABT-199 as a single agent. Combination of ABT199 with other agents may be a strategy to overcome acquired resistance. The effectiveness of ABT-199 as a single agent or in combination is largely unexplored in MCL. Given the presumed non-overlapping pathways, we hypothesized that ibrutinib/ABT-199 combination may overcome the resistance observed with single agents alone. To test this, a new MCL cell line CCMCL1 (Zhao et al, 2013) was examined for the response to these agents. Synergistic inhibition of proliferation was confirmed in 22 of 24 tested combinations, based on combination index (CI) values <1, in which 18 combinations had CI values <0 7, suggesting strong synergy (Fig 1A). Synergistic apoptosis induction was observed in all combinations (Fig 1B). The same assay with four additional MCL cell lines, Jeko-1, Mino, JVM2 and Rec-1, showed strong synergistic effects, as evidenced by CI values <0 7 with a majority being <0 2 (Fig 1C, D). Testing with primary cells from two cases of recurrent MCL also displayed robust synergy of apoptosis induction (Figure S1). Although these two cases displayed different degrees of response to single agents, synergy was observed for both. Another noteworthy finding of this study is molecular mechanisms underlying the interaction of ibrutinib/ABT-199 in MCL cells. In CCMCL1 cells, ibrutinib caused dephosphorylation of BTK(Y223). ABT-199 had no detectable effect on this target but there was a larger decrease of p-BTK(Y223) upon cells co-treated with ibrutinib/ABT-199. A similar effect was observed on p-AKT(S473) (Fig 2A). Immunoblotting of four other MCL cell lines confirmed that this combination enhanced dephosphorylation of the above signalling molecules (Figure S2), which was associated with survival/proliferation of malignant B-cells. The impact of ibrutinib and/or ABT-199 on BCL2 family proteins (BCL2, MCL1 and BCL2L1) was more cell-line dependent. In CCMCL1 cells, ibrutinib alone downregulated MCL1. Each single agent had little effect on BCL2 and BCL2L1, but the combination down-regulated both of these proteins (Fig 2B). Co-treatment of other MCL cell lines with ibrutinib/ABT-199 resulted in decrease of at least one BCL2 family protein (Figure S3). Ibrutinib/ABT-199 co-treatment also more effectively triggered reduced mitochondrial membrane potential compared to single agent, and more poly (ADP-ribose) polymerase (PARP) cleavage (Fig 2C, D) suggested caspase activation. In summary, we report the therapeutic potential of ibrutinib/ABT-199 combination in MCL cells. This combination displayed strongly synergistic effects in all tested cell lines and primary cells from recurrent MCL patients. These cell lines represent different types of MCL, including aggressive MYCtranslocated CCMCL1 (X. Zhao, S. Shetty, M. R. Smith, J. Bodo, E. D. Hsi unpublished data). Mechanistically, ibrutinib/ ABT-199 interaction caused synergistic effects in MCL cells through perturbation of p-BTK and p-AKT mediated survival signals and of BCL2 family proteins (Fig 2E). Ibrutinib was developed as a BTK inhibitor to block BCR signalling. To date, few studies described alterations of BCR signalling causing any effects on expression or function of BCL2 family proteins. However, BTK is necessary for BCR-induced phosphorylation of cAMP-response elementbinding protein (CREB) (Blois et al, 2004). In mature B cells,

Evaluation of noncytotoxic DNMT1-depleting therapy in patients with myelodysplastic syndromes

BACKGROUND Mutational inactivation in cancer of key apoptotic pathway components, such as TP53/p53, undermines cytotoxic therapies that aim to increase apoptosis. Accordingly, TP53 mutations are reproducibly associated with poor treatment outcomes. Moreover, cytotoxic treatments destroy normal stem cells with intact p53 systems, a problem especially for myeloid neoplasms, as these cells reverse the low blood counts that cause morbidity and death. Preclinical studies suggest that noncytotoxic concentrations of the DNA methyltransferase 1 (DNMT1) inhibitor decitabine produce p53-independent cell-cycle exits by reversing aberrant epigenetic repression of proliferation-terminating (MYC-antagonizing) differentiation genes in cancer cells. METHODS In this clinical trial, patients with myelodysplastic syndrome (n=25) received reduced decitabine dosages (0.1-0.2 mg/kg/day compared with the FDA-approved 20-45 mg/m2/day dosage, a 75%-90% reduction) to avoid cytotoxicity. These well-tolerated doses were frequently administered 1-3 days per week, instead of pulse cycled for 3 to 5 days over a 4- to 6-week period, to increase the probability that cancer S-phase entries would coincide with drug exposure, which is required for S-phase-dependent DNMT1 depletion. RESULTS The median subject age was 73 years (range, 46-85 years), 9 subjects had relapsed disease or were refractory to 5-azacytidine and/or lenalidomide, and 3 had received intensive chemoradiation to treat other cancers. Adverse events were related to neutropenia present at baseline: neutropenic fever (13 of 25 subjects) and septic death (1 of 25 subjects). Blood count improvements meeting the International Working Group criteria for response occurred in 11 of 25 (44%) subjects and were highly durable. Treatment-induced freedom from transfusion lasted a median of 1,025 days (range, 186-1,152 days; 3 ongoing), and 20% of subjects were treated for more than 3 years. Mutations and/or deletions of key apoptosis genes were frequent (present in 55% of responders and in 36% of nonresponders). Noncytotoxic DNMT1 depletion was confirmed by serial BM γ-H2AX (DNA repair/damage marker) and DNMT1 analyses. MYC master oncoprotein levels were markedly decreased. CONCLUSION Decitabine regimens can be redesigned to minimize cytotoxicity and increase exposure time for DNMT1 depletion, to safely and effectively circumvent mutational apoptotic defects. TRIAL REGISTRATION Clinicaltrials.gov NCT01165996. FUNDING NIH (R01CA138858, CA043703); Department of Defense (PR081404); Clinical and Translational Science Award (CTSA) (UL1RR024989); and the Leukemia and Lymphoma Society (Translational Research Program).

MYD88 L265P somatic mutation: its usefulness in the differential diagnosis of bone marrow involvement by B-cell lymphoproliferative disorders.

OBJECTIVES To examine the usefulness of the MYD88 L265P somatic mutation in identifying cases of lymphoplasmacytic lymphoma (LPL) from other lymphoplasmacytic neoplasms in bone marrow biopsy specimens. METHODS We studied 64 bone marrow biopsy specimens with involvement by various small B-cell lymphomas or plasma cell myeloma. RESULTS The MYD88 L265P somatic mutation was present in 13/13 cases of LPL, 1/13 cases of hairy cell leukemia, and absent in the other mature B-cell neoplasms tested. A test set of diagnostically challenging bone marrow cases with lymphoplasmacytoid morphology (B-cell lymphoma, not otherwise specified) was selected for additional review and reclassified, without knowledge of the MYD88 L265P status. Of those 16 cases, 7 were positive for MYD88, including 4/4 cases that were reclassified as LPL during the review. CONCLUSIONS Although not entirely specific, MYD88 L265P is a useful adjunct for bone marrow diagnosis in separating LPL from other small B-cell lymphomas and plasma cell myeloma.

Expression of TweakR in breast cancer and preclinical activity of enavatuzumab, a humanized anti-TweakR mAb

BackgroundThe receptor for the cytokine TWEAK (TweakR) is a cell surface member of the tumor necrosis factor receptor superfamily with diverse biological roles. TNFRSF family members are appealing therapeutic targets in oncology due to their aberrant expression and function in tumor cells. The goal of the current study was to examine the potential of TweakR as a therapeutic target in breast cancer.MethodsExpression of TweakR in primary breast cancer tissues and metastases was characterized using immunohistochemistry. To determine the functional relevance of TweakR, breast cancer cell lines were treated in vitro and in vivo with enavatuzumab, a humanized mAb against TweakR.ResultsOverexpression of TweakR was observed in infiltrating tumors compared to normal adjacent breast tissues, and strong staining of TweakR was observed in all subtypes of invasive ductal breast cancer. In addition, a positive correlation of TweakR and HER2 expression and co-localization were observed, irrespective of ER status. TweakR expression was also observed in bone metastasis samples from primary breast cancer but rarely in benign tumors. Enavatuzumab inhibited the in vitro growth of TweakR-expressing breast cancer cell lines, and this activity was augmented by cross-linking the mAb. In addition, enavatuzumab significantly inhibited the in vivo growth of multiple breast cancer xenograft models including a model of metastasis.ConclusionsTweakR is highly expressed in all subtypes of invasive ductal breast cancer, and enavatuzumab administration exhibited a dose-dependent inhibition of primary tumor growth and lung metastasis and enhanced the antitumor activity of several chemotherapy agents currently used to treat breast cancer. These data provide the rationale to evaluate enavatuzumab as a potential therapy for the treatment of breast cancer.

Quantitative In Situ Detection of Phosphoproteins in Fixed Tissues Using Quantum Dot Technology

Detection and quantitation of phosphoproteins (PPs) in fixed tissues will become increasingly important as additional inhibitors of protein kinases enter clinical use and new disease entities are defined by molecular changes affecting PP levels. We characterize fixation conditions suitable for accurate PP quantitation that are achievable in a clinical laboratory and illustrate the utility of in situ quantitation of PPs by quantum dot (QD) nano-crystals in two models: (1) a therapeutic model demonstrating effects of a targeted therapeutic (quantitative reduction of phospho-GSK3β) in xenografts treated with enzas-taurin; and (2) a diagnostic model that identifies elevated levels of nuclear phospho-STAT5 in routine bone marrow biopsies from patients with acute myeloid leukemia based on the presence of the activating FLT3-ITD mutation. Finally, we document production of a well-characterized tissue microarray of widely available cell lines as a multilevel calibrator for validating numerous phosphoprotein assays. QD immunofluorescence is an ideal method for in situ quantitation of PPs in fixed samples, providing valuable cell type—specific and subcellular information about pathway activation in primary tissues. (J Histochem Cytochem 57:701–708, 2009)

Phospho-ERKTHR202/Tyr214 is overexpressed in hairy cell leukemia and is a useful diagnostic marker in bone marrow trephine sections

BRAF V600E mutations are present in virtually all cases of hairy cell leukemia (HCL). We hypothesized that detection of phospho-ERK (pERK) in tissue sections may be a useful marker for diagnosis of HCL. pERK/CD20 double immunostaining was performed on 90 formalin-fixed bone marrow trephine samples affected with small B-cell lymphoproliferative disorders, including 28 cases of HCL. pERK staining was observed in all 28 cases of HCL and in 1 of 62 cases of non-HCL B-cell lymphoproliferative disorders. By allele-specific polymerase chain reaction, all 11 cases of HCL with available DNA were positive for BRAF V600E, as was the 1 pERK+ non-HCL case. The remaining 31 non-HCL cases tested were negative for BRAF V600E. The sensitivity and specificity of pERK for diagnosis of HCL was 100% and 98%, respectively. We conclude that the presence of pERK as detected by immunohistochemical staining is a useful surrogate marker for BRAF V600E in the diagnosis of HCL.

Molecular Subtype Classification of Formalin-Fixed, Paraffin-Embedded Diffuse Large B-Cell Lymphoma Samples on the ICEPlex® System

Microarray gene expression profiling (GEP) has been used to identify molecular subtypes of diffuse large B-cell lymphoma (DLBCL) based on the similarity of GEP to a putative “cell of origin” (COO) and defines two molecular subtypes: activated B-cell-like (ABC) DLBCL and germinal centre B-cell-like (GCB) DLBCL (Alizadeh, et al 2000). This dichotomization has prognostic and biological significance, with the ABC subtype having a worse outcome and distinct pathobiology that includes activation of the B-cell receptor and nuclear factor (NF)-κB pathways (Lenz, et al 2008). Attempts to reduce this subclassification to practice using immunohistochemistry are fraught with technical and interpretive issues such that a need for practical quantitative molecular assays exists (Coutinho, et al 2013, de Jong, et al 2007, de Jong, et al 2009, Salles, et al 2011). Indeed, studies have refined this COO concept using limited gene sets, including a 14-gene model to assign ABC and GCB DLBCL subtype developed by Wright et al (2003) as well as a recently-described DLBCL subtyping assay based on parsimonious digital gene expression (Nanostring) technology (Scott, et al 2014). In order to support clinical trials for therapies targeting populations enriched in ABC DLBCL and to offer prognostic information for DLBCL patients as part of our clinical service, we developed a novel multiplex, single-tube, gene expression assay on the ICEPlex® system (PrimeraDx, Mansfield, MA), which allows differentiation between GCB and ABC DLBCL subtypes in formalin-fixed paraffin-embedded (FFPE) specimens using a Food and Drug Administration-cleared platform.

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.

Dual expression of MYC and BCL2 proteins predicts worse outcomes in diffuse large B-cell lymphoma

Abstract Recent studies suggested that MYC and BCL2 protein co-expression is an independent indicator of poor prognosis in diffuse large B-cell lymphoma. However, the immunohistochemistry protocols for dual-expression staining and the scoring cut-offs vary by study. Sixty-nine cases of diffuse large B-cell lymphoma were evaluated for MYC and BCL2 protein expression using various cut-offs that have been recommended in prior studies. Independent of the International Prognostic Index risk group, cases with dual protein expression of BCL2 and MYC using ≥50%/40% cut-offs and ≥70%/40% had significantly shorter overall survival than cases without. It was verified in this patient population that the use of BCL2 and MYC immunohistochemistry, performed with available in vitro diagnostic-cleared antibodies, provides rapid prognostic information in patients with de novo diffuse large B-cell lymphoma. This study has practical implications for diagnostic laboratories and serves as a guide for implementation in the setting of future clinical trials.