The prognostic significance of B-cell lymphoma 2 (BCL2) expression in patients with diffuse large... more The prognostic significance of B-cell lymphoma 2 (BCL2) expression in patients with diffuse large B-cell lymphoma (DLBCL) is conflicting. We developed an immunohistochemistry (IHC) algorithm and assay to determine BCL2 expression and assessed the prognostic value of BCL2 in newly diagnosed DLBCL cohorts. BCL2 expression was associated with poorer progression-free survival. Findings support use of our BCL2 IHC scoring system and assay to select BCL2-positive patients for future studies. Introduction: The prognostic value of B-cell lymphoma 2 (BCL2) expression in de novo diffuse large B-cell lymphoma (DLBCL) treated with immunochemotherapy is of interest to define a target patient population for clinical development of BCL2 inhibitors. We aimed to develop a reproducible immunohistochemistry algorithm and assay to determine BCL2 protein expression and assess the prognostic value of BCL2 in newly diagnosed DLBCL cohorts. Patients and Methods: The prospectively defined algorithm incorporated BCL2 staining intensity and percentage of BCL2-positive cells. Functionally relevant cutoffs were based on the sensitivity of lymphoma cell lines to venetoclax. This assay was highly reproducible across laboratories. The prognostic impact of BCL2 expression was assessed in DLBCL patients from the phase 3 MAIN (n ¼ 230) and GOYA (n ¼ 366) trials, and a population-based registry (n ¼ 310). Results: Approximately 50% of tumors were BCL2 positive, with a higher frequency in high International Prognostic Index (IPI) and activated Bcellelike DLBCL subgroups. BCL2 expression was associated with poorer progression-free survival in the MAIN study (hazard ratio [HR], 1.66; 95% confidence interval [CI], 0.81-3.40; multivariate Cox regression adjusted for IPI and cell of origin). This trend was confirmed in the GOYA and registry cohorts in adjusted multivariate analyses (GOYA: HR, 1.72; 95% CI, 1.05-2.82; registry: HR, 1.89; 95% CI, 1.29-2.78). Patients with BCL2 immunohistochemistry-positive and IPIhigh disease had the poorest prognosis: 3-year progression-free survival rates were 51% (GOYA) and 37% (registry). Conclusion: Findings support use of our BCL2 immunohistochemistry scoring system and assay to select patients with BCL2-positive tumors for future studies.
Central nervous system (CNS) relapse carries a poor prognosis in diffuse large B-cell lymphoma (D... more Central nervous system (CNS) relapse carries a poor prognosis in diffuse large B-cell lymphoma (DLBCL). Integrating biomarkers into the CNS–International Prognostic Index (CNS-IPI) risk model may improve identification of patients at high risk for developing secondary CNS disease. CNS relapse was analyzed in 1418 DLBCL patients treated with obinutuzumab or rituximab plus cyclophosphamide, doxorubicin, vincristine, prednisone chemotherapy in the phase 3 GOYA study. Cell of origin (COO) was assessed using gene-expression profiling. BCL2 and MYC protein expression was analyzed by immunohistochemistry. The impact of CNS-IPI, COO, and BCL2/MYC dual-expression status on CNS relapse was assessed using a multivariate Cox regression model (data available in n = 1418, n = 933, and n = 688, respectively). High CNS-IPI score (hazard ratio [HR], 4.0; 95% confidence interval [CI], 1.3-12.3; P = .02) and activated B-cell‒like (ABC) (HR, 5.2; 95% CI, 2.1-12.9; P = .0004) or unclassified COO subtype...
Circulating tumor cells offer promise as a surrogate source of cancer cells that can be obtained ... more Circulating tumor cells offer promise as a surrogate source of cancer cells that can be obtained in real time and may provide opportunities to evaluate predictive biomarkers that can guide treatment decisions. In this review, we consider some of the technical hurdles around CTC numbers and suitability of various CTC capture and analysis platforms for biomarker evaluation. In addition, we consider the potential regulatory hurdles to development of CTC-based diagnostics. Finally, we suggest a path for co-development of anticancer therapeutics with CTC-based diagnostics that could enable clinical validation and qualification of CTC-based assays as companion diagnostics.
Proceedings of the National Academy of Sciences, 2011
Deregulation of apoptosis is a common occurrence in cancer, for which emerging oncology therapeut... more Deregulation of apoptosis is a common occurrence in cancer, for which emerging oncology therapeutic agents designed to engage this pathway are undergoing clinical trials. With the aim of uncovering strategies to activate apoptosis in cancer cells, we used a pooled shRNA screen to interrogate death receptor signaling. This screening approach identified 16 genes that modulate the sensitivity to ligand induced apoptosis, with several genes exhibiting frequent overexpression and/or copy number gain in cancer. Interestingly, two of the top hits, EDD1 and GRHL2, are found 50 kb apart on chromosome 8q22, a region that is frequently amplified in many cancers. By using a series of silencing and overexpression studies, we show that EDD1 and GRHL2 suppress death-receptor expression, and that EDD1 expression is elevated in breast, pancreas, and lung cancer cell lines resistant to death receptor-mediated apoptosis. Supporting the relevance of EDD1 and GRHL2 as therapeutic candidates to engage ap...
Apo2L/TRAIL stimulates cancer cell death through the proapoptotic receptors DR4 and DR5, but the ... more Apo2L/TRAIL stimulates cancer cell death through the proapoptotic receptors DR4 and DR5, but the determinants of tumor susceptibility to this ligand are not fully defined. mRNA expression of the peptidyl O-glycosyltransferase GALNT14 correlated with Apo2L/TRAIL sensitivity in pancreatic carcinoma, non-small-cell lung carcinoma and melanoma cell lines, and up to 30% of samples from various human malignancies showed GALNT14 overexpression. RNA interference of GALNT14 reduced cellular Apo2L/TRAIL sensitivity, whereas overexpression increased responsiveness. Biochemical analysis of DR5 identified several ectodomain O-(N-acetyl galactosamine-galactose-sialic acid) structures. Sequence comparison predicted conserved extracellular DR4 and DR5 O-glycosylation sites; progressive mutation of the DR5 sites attenuated apoptotic signaling. O-glycosylation promoted ligand-stimulated clustering of DR4 and DR5, which mediated recruitment and activation of the apoptosis-initiating protease caspase-8. These results uncover a new link between death-receptor O-glycosylation and apoptotic signaling, providing potential predictive biomarkers for Apo2L/TRAIL-based cancer therapy.
Evaluation of cancer biomarkers from blood could significantly enable biomarker assessment by pro... more Evaluation of cancer biomarkers from blood could significantly enable biomarker assessment by providing a relatively non-invasive source of representative tumor material. Circulating Tumor Cells (CTCs) isolated from blood of metastatic cancer patients hold significant promise in this regard.
Purpose: We describe the preclinical pharmacology and antitumor activity of GDC-0068, a novel hig... more Purpose: We describe the preclinical pharmacology and antitumor activity of GDC-0068, a novel highly selective ATP-competitive pan-Akt inhibitor currently in clinical trials for the treatment of human cancers. Experimental Design: The effect of GDC-0068 on Akt signaling was characterized using specific biomarkers of the Akt pathway, and response to GDC-0068 was evaluated in human cancer cell lines and xenograft models with various genetic backgrounds, either as a single agent or in combination with chemotherapeutic agents. Results: GDC-0068 blocked Akt signaling both in cultured human cancer cell lines and in tumor xenograft models as evidenced by dose-dependent decrease in phosphorylation of downstream targets. Inhibition of Akt activity by GDC-0068 resulted in blockade of cell-cycle progression and reduced viability of cancer cell lines. Markers of Akt activation, including high-basal phospho-Akt levels, PTEN loss, and PIK3CA kinase domain mutations, correlate with sensitivity to ...
Purpose: The pathways underlying basal-like breast cancer are poorly understood, and as yet, ther... more Purpose: The pathways underlying basal-like breast cancer are poorly understood, and as yet, there is no approved targeted therapy for this disease. We investigated the role of mitogen-activated protein kinase kinase (MEK) and phosphatidylinositol 3-kinase (PI3K) inhibitors as targeted therapies for basal-like breast cancer. Experimental Design: We used pharmacogenomic analysis of a large panel of breast cancer cell lines with detailed accompanying molecular information to identify molecular predictors of response to a potent and selective inhibitor of MEK and also to define molecular mechanisms underlying combined MEK and PI3K targeting in basal-like breast cancer. Hypotheses were confirmed by testing in multiple tumor xenograft models. Results: We found that basal-like breast cancer models have an activated RAS-like transcriptional program and show greater sensitivity to a selective inhibitor of MEK compared with models representative of other breast cancer subtypes. We also showe...
Purpose: The class I phosphatidylinositol 3′ kinase (PI3K) plays a major role in proliferation an... more Purpose: The class I phosphatidylinositol 3′ kinase (PI3K) plays a major role in proliferation and survival in a wide variety of human cancers. A key factor in successful development of drugs targeting this pathway is likely to be the identification of responsive patient populations with predictive diagnostic biomarkers. This study sought to identify candidate biomarkers of response to the selective PI3K inhibitor GDC-0941. Experimental Design: We used a large panel of breast cancer cell lines and in vivo xenograft models to identify candidate predictive biomarkers for a selective inhibitor of class I PI3K that is currently in clinical development. The approach involved pharmacogenomic profiling as well as analysis of gene expression data sets from cells profiled at baseline or after GDC-0941 treatment. Results: We found that models harboring mutations in PIK3CA, amplification of human epidermal growth factor receptor 2, or dual alterations in two pathway components were exquisitely...
Purpose: Elevated levels or increases in circulating tumor cells (CTC) portend poor prognosis in ... more Purpose: Elevated levels or increases in circulating tumor cells (CTC) portend poor prognosis in patients with epithelial cancers. Less is known about CTCs as surrogate endpoints or their use for predictive biomarker evaluation. This study investigated the utility of CTC enumeration and characterization using the CellSearch platform, as well as mutation detection in circulating tumor DNA (ctDNA), in patients with advanced non–small cell lung cancer (NSCLC). Experimental Design: Forty-one patients were enrolled in a single-arm phase II clinical trial of erlotinib and pertuzumab. Peripheral blood was analyzed for CTC enumeration, EGFR expression in CTCs, and detection of oncogenic mutations in CTCs and ctDNA. Changes in CTC levels were correlated with 2[18F]fluoro-2-deoxy-d-glucose–positron emission tomographic (FDG-PET) and computed tomographic (CT) imaging and survival endpoints. Results: CTCs were detected (≥1 CTC) at baseline in 78% of patients. Greater sensitivity for mutation de...
Venetoclax inhibits BCL2, an antiapoptotic protein that is pathologically overexpressed and that ... more Venetoclax inhibits BCL2, an antiapoptotic protein that is pathologically overexpressed and that is central to the survival of chronic lymphocytic leukemia cells. We evaluated the efficacy of venetoclax in combination with rituximab in patients with relapsed or refractory chronic lymphocytic leukemia. In this randomized, open-label, phase 3 trial, we randomly assigned 389 patients to receive venetoclax for up to 2 years (from day 1 of cycle 1) plus rituximab for the first 6 months (venetoclax-rituximab group) or bendamustine plus rituximab for 6 months (bendamustine-rituximab group). The trial design did not include crossover to venetoclax plus rituximab for patients in the bendamustine-rituximab group in whom progression occurred. The primary end point was investigator-assessed progression-free survival. After a median follow-up period of 23.8 months, the rate of investigator-assessed progression-free survival was significantly higher in the venetoclax-rituximab group (32 events of...
, 20 pts have been treated. Median age: 63 yrs (range 50-77), 33% del(17p), 43% complex karyotype... more , 20 pts have been treated. Median age: 63 yrs (range 50-77), 33% del(17p), 43% complex karyotype, and 29% and 10% TP53 and NOTCH1 mutation, respectively. Median # prior CLL treatments 2 (range 0-5, prior ibrutinib (n=8), idelalisib (n=2), and ven (n=2); 5 pts no prior treatment). Median # ven + da-R-EPOCH cycles in this ongoing study is 3.5 (range 1-6), including 5 pts who recently enrolled. 4 pts had dose de-escalation of R-EPOCH and 1 pt had dose escalation. ≥Gr 3 heme toxicity included: neutropenia (45%), anemia (35%), thrombocytopenia (25%). ≥Gr 3 non-heme toxicities in ≥15% of pts: febrile neutropenia (20%), hypocalcemia and hypophosphatemia (15% each). No TLS occurred with daily ven ramp-up after 1 cycle of R-EPOCH. Infectious complications: 3 pts with sepsis during C1 of R-EPOCH (prior to starting ven) and 1 pt each with influenza A and norovirus while on combination therapy. 7 pts died, including 4 due to disease progression (2 during C1 before ven), and 1 each due to sepsis, sudden death, and GVHD post-alloHCT. 8 pts are not evaluable for efficacy of the combination (3 are still in C1, 4 had toxicity in C1 and never started ven, 1 pt withdrew in C1). Of the 12 evaluable pts who have started combination therapy, 9 responded (ORR 75%); 8/12 (67%) had CR, all of whom also had undetectable bone marrow MRD for CLL. 5 pts went to alloHCT, with pts still in CR now up to 1.5 yrs post-alloHCT. With a median followup of 3 mo (range 0-17.9), median PFS is 10 mo (Fig 1A), median OS is 16.3 mo (Fig 1B). Conclusions: Our initial data suggest that ven + da-R-EPOCH is a feasible regimen to treat RS. Expected toxicities from intensive chemoimmunotherapy were seen, without significant additional toxicity from ven, including no TLS with daily ven ramp-up. The 67% CR and PFS/OS of 10/16.3 mo are favorable in the context of historical results. Accrual is ongoing, and updated results will be presented.
Background: Expression of PD-L1 in tumor cells and tumor-infiltrating immune cells has been assoc... more Background: Expression of PD-L1 in tumor cells and tumor-infiltrating immune cells has been associated with improved efficacy to anti-PD-1/PD-L1 inhibitors in patients with advanced-stage non-small-cell lung cancer (NSCLC) and emerged as a potential biomarker for the selection of patients to cancer immunotherapies. We investigated the utility of circulating tumor cells (CTCs) and circulating white blood cells (WBCs) as a noninvasive method to evaluate PD-L1 status in advanced NSCLC patients. Patients and methods: CTCs and circulating WBCs were enriched from peripheral blood samples (ISET V R platform; Rarecells) from 106 NSCLC patients. PD-L1 expression on ISET filters and matched-tumor tissue was evaluated by automated immunostaining (SP142 antibody; Ventana), and quantified in tumor cells and WBCs. Results: CTCs were detected in 80 (75%) patients, with levels ranging from 2 to 256 CTCs/4 ml, and median of 60 CTCs/4 ml. Among 71 evaluable samples with matched-tissue and CTCs, 6 patients (8%) showed !1 PD-L1-positive CTCs and 11 patients (15%) showed !1% PD-L1-positive tumor cells in tumor tissue with 93% concordance between tissue and CTCs (sensitivity ¼ 55%; specificity ¼ 100%). From 74 samples with matched-tissue and circulating WBCs, 40 patients (54%) showed !1% PD-L1-positive immune infiltrates in tumor tissue and 39 patients (53%) showed !1% PD-L1 positive in circulating WBCs, with 80% concordance between blood and tissue (sensitivity ¼ 82%; specificity ¼ 79%). We found a trend for worse survival in patients receiving first-line cisplatin-based chemotherapy treatments, whose tumors express PD-L1 in CTCs or immune cells (progression-free and overall survival), similar to the effects of PD-L1 expression in matched-patient tumors. Conclusions: These results demonstrated that PD-L1 status in CTCs and circulating WBCs correlate with PD-L1 status in tumor tissue, revealing the potential of CTCs assessment as a noninvasive real-time biopsy to evaluate PD-L1 expression in patients with advanced-stage NSCLC.
Introduction: Composite scores integrating genes mutation status with the clinical predictor FLIP... more Introduction: Composite scores integrating genes mutation status with the clinical predictor FLIPI have been recently proposed to improve risk stratification for follicular lymphoma (FL) patients. We evaluated the ability of m7-FLIPI and POD24-PI scores to predict progression free survival (PFS) in a large cohort of patients receiving first-line immunochemotherapy, with or without rituximab maintenance. Methods: Tumour biopsies were obtained at FL diagnosis from 252 patients from the PRIMA study, either as FFPE tissues (n = 98) or fresh-frozen tissues (n = 154). After DNA extraction, DNA-targeted sequencing was performed using the Foundation One Heme™ panel. m7-FLIPI and POD24-PI models were applied as originally described. Results: The frequency of non-silent mutations were similar to those previously reported: CREBBP = 75%, EZH2 = 28%, CARD1 = 19%, ARID1A = 19%, EP300 = 16%, FOXO1 = 16% and MEF2B = 12%. We first evaluated the prognostic value of each gene mutation status separately. While some mutations were associated with a longer (MEF2B, EZH2) or shorter (EP300, CREBBP) PFS as previously described, ARID1A or CARD11 mutations were not associated with patients outcome. Moreover, FOXO1 mutations were associated with a good outcome, in opposite to their prognostic weight in the m7-FLIPI. The m7-FLIPI and FLIPI scores classified 28% and 43% of patients as high-risk, respectively. m7-FLIPI correlated with PFS (p = 0•005; OR = 1.74, 95%CI: 1.00-3.02), slightly outperforming the FLIPI 96 ABSTRACT 89% (range 40-100%) in young adults (18-40 yrs) to 74% (range 0-100%, p = 0.01) in the oldest cohort (>70 yrs). No single gene mutation was found to be associated with older age after correction for multiple testing. Conclusions: Our data suggest that older age does not directly impact disease biology and treatment efficacy in FL, and should not be used to guide treatment decisions.
Background: Akt, a serine/threonine protein kinase, is a key signaling node in the PI3K/Akt/mTOR ... more Background: Akt, a serine/threonine protein kinase, is a key signaling node in the PI3K/Akt/mTOR pathway and plays an essential role in regulating tumor cell proliferation, migration and survival. PI3K/Akt signaling is up-regulated in gastric cancer, mainly due to loss of PTEN, and is associated with poor prognosis and chemoresistance. Chemotherapeutics such as doxorubicin and 5-FU/cisplatin have been shown to upregulate PI3K/Akt signaling in several solid cancers in response to DNA damage. We have previously reported on a potent highly selective ATP-competitive pan-Akt inhibitor, GDC-0068, which inhibits cell cycle progression and viability of cancer cell lines and xenografts driven by Akt signaling, including those with defects in the tumor suppressor PTEN, oncogenic mutations in PIK3CA, and amplification of HER2. Thus, we hypothesized that the combination of FOLFOX and GDC-0068 would induce anti-tumor activity in primary gastric cancer models. Methods: Gastric cancer cell lines (n=11) were treated with increasing concentrations of GDC-0068 and FOLFOX and evaluated for combinational efficacy over single agent via cell viability assay. For in vivo studies, primary gastric cancer xenograft models were implanted in immuno-compromised mice and treated with GDC-0068 and FOLFOX as single agents or in combination. Results: In vitro, the combination of GDC-0068 and FOLFOX results in enhanced inhibition of tumor cell viability in the cell lines with activation of the PI3K pathway compared to either single agent alone. These results were recapitulated in vivo, in the primary xenograft models STO#240 and STO#182, where the combination of GDC-0068 and FOLFOX resulted in increased tumor growth inhibition or regressions compared to single agents alone. All combinations tested were well tolerated in vivo based on minimal changes in body weights. Conclusions: Our preclinical studies demonstrate improved anti-tumor efficacy when GDC-0068 is combined with FOLFOX in gastric cancer models in vitro and in vivo. The data supports the clinical development of GDC-0068 in combination with FOLFOX for the treatment of gastric cancer. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B190. Citation Format: Ellen Ingalla, Rebecca Hong, Heidi Savage, Elizabeth Punnoose, Sandra Rost, Hartmut Koeppen, Deepak Sampath, Michelle A. Nannini. GDC-0068 is a novel and selective Akt inhibitor that enhances the efficacy of FOLFOX in primary gastric cancer models. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B190.
GDC-0068 is a highly selective ATP-competitive small molecule that inhibits all three isoforms of... more GDC-0068 is a highly selective ATP-competitive small molecule that inhibits all three isoforms of Akt with IC50 values of 5 to 30 nM. GDC-0068 selectively inhibits cancer cells with activated Akt signaling (e.g. via PTEN loss or PIK3CA mutations). Patients with advanced solid tumors were treated with GDC-0068 using a 3+3 escalation design. GDC-0068 was dosed PO QD on a 21-day on, 7-day off schedule; endpoints included safety, pharmacokinetics (PK) and determination of pathway knockdown. Pharmacodynamics (PD) endpoints in surrogate tissue [platelet rich plasma (PRP)] were evaluated in all patients. In addition, at least two patients per cohort had pre- and on-treatment (day 15) tumor biopsies at doses ≥100mg QD. The tumor tissue samples were evaluated using reverse phase protein array (RPPA). Thirty patients were enrolled across 7 cohorts (25, 50, 100, 200, 400, 600 and 800 mg QD). GDC-0068 was generally well-tolerated at doses ≤ 600 mg. Preliminary PK analyses show a dose proportional increase in exposure over the dose range tested. In addition, clinical exposures at doses ≥ 200 mg QD met or exceeded GDC-0068 exposures associated with tumor stasis in multiple PTEN null preclinical xenograft models. PK/PD evaluation showed dose-dependent Akt pathway inhibition in the PRP assay, with ≥70% inhibition of pGSK3 in all patients at doses ≥ 200 mg QD. Pre- and on-treatment tumor biopsies showed ≥50% decrease in pPRAS40 and cyclin D1 in multiple patients at doses of 200 mg QD and higher. In addition, evidence of feedback activation of the MAP kinase pathway after treatment with GDC-0068 was observed in biopsy samples. One patient with PTEN low/ PIK3CA H1047R/ KRAS wt CRC had prolonged stable disease and showed Akt pathway suppression by multiple downstream markers. GDC-0068 is well tolerated at doses ≤ 600 mg with a favorable safety profile and dose proportional pharmacokinetics. Treatment with GDC-0068 results in substantial pathway knockdown in both surrogate and tumor tissues at doses ≥ 200 mg QD. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B154.
Mutations in oncogenes such as EGFR, KRAS and PIK3CA, have been shown to have profound effects on... more Mutations in oncogenes such as EGFR, KRAS and PIK3CA, have been shown to have profound effects on pathway signaling and tumor growth. These genetic aberrations are potentially valuable predictors for patient stratification and therapeutic responses to targeted therapies directed against these signaling pathways. Here we evaluate the utility of circulating tumor DNA as a “liquid biopsy” for predictive diagnostics and real-time monitoring of disease. A fundamental challenge with this approach is that, due to the low frequency of the mutant tumor DNA in circulation, a highly sensitive assay is required to detect the single base pair alterations. We have designed and investigated several assay formats, including TaqMan and scorpion-ARM assays for mutation detection using a digital PCR platform. We also employed LNA/PNA blockers and thermostable restriction digestion in PCR reaction to enrich the mutant DNA. The combination of the biased amplification and digital PCR dramatically increased mutation detection 10-100 fold. To assess clinical utility of the technology, data on PIK3CA mutation screening of matched patient tumor and plasma samples from a phase I clinical trial will also be reported. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3180. doi:10.1158/1538-7445.AM2011-3180
BCL-2 family proteins dictate survival of human multiple myeloma (MM) cells making them attractiv... more BCL-2 family proteins dictate survival of human multiple myeloma (MM) cells making them attractive drug targets. Indeed, MM cells are sensitive to antagonists that selectively target pro-survival proteins such as BCL-2/BCL-X<sub>L</sub> (ABT-737 and ABT-263/navitoclax) or BCL-2 only (ABT-199/GDC-0199/venetoclax). Resistance to these three drugs is mediated by expression of MCL-1. However, given the selectivity profile of venetoclax it is unclear whether co-expression of…
The prognostic significance of B-cell lymphoma 2 (BCL2) expression in patients with diffuse large... more The prognostic significance of B-cell lymphoma 2 (BCL2) expression in patients with diffuse large B-cell lymphoma (DLBCL) is conflicting. We developed an immunohistochemistry (IHC) algorithm and assay to determine BCL2 expression and assessed the prognostic value of BCL2 in newly diagnosed DLBCL cohorts. BCL2 expression was associated with poorer progression-free survival. Findings support use of our BCL2 IHC scoring system and assay to select BCL2-positive patients for future studies. Introduction: The prognostic value of B-cell lymphoma 2 (BCL2) expression in de novo diffuse large B-cell lymphoma (DLBCL) treated with immunochemotherapy is of interest to define a target patient population for clinical development of BCL2 inhibitors. We aimed to develop a reproducible immunohistochemistry algorithm and assay to determine BCL2 protein expression and assess the prognostic value of BCL2 in newly diagnosed DLBCL cohorts. Patients and Methods: The prospectively defined algorithm incorporated BCL2 staining intensity and percentage of BCL2-positive cells. Functionally relevant cutoffs were based on the sensitivity of lymphoma cell lines to venetoclax. This assay was highly reproducible across laboratories. The prognostic impact of BCL2 expression was assessed in DLBCL patients from the phase 3 MAIN (n ¼ 230) and GOYA (n ¼ 366) trials, and a population-based registry (n ¼ 310). Results: Approximately 50% of tumors were BCL2 positive, with a higher frequency in high International Prognostic Index (IPI) and activated Bcellelike DLBCL subgroups. BCL2 expression was associated with poorer progression-free survival in the MAIN study (hazard ratio [HR], 1.66; 95% confidence interval [CI], 0.81-3.40; multivariate Cox regression adjusted for IPI and cell of origin). This trend was confirmed in the GOYA and registry cohorts in adjusted multivariate analyses (GOYA: HR, 1.72; 95% CI, 1.05-2.82; registry: HR, 1.89; 95% CI, 1.29-2.78). Patients with BCL2 immunohistochemistry-positive and IPIhigh disease had the poorest prognosis: 3-year progression-free survival rates were 51% (GOYA) and 37% (registry). Conclusion: Findings support use of our BCL2 immunohistochemistry scoring system and assay to select patients with BCL2-positive tumors for future studies.
Central nervous system (CNS) relapse carries a poor prognosis in diffuse large B-cell lymphoma (D... more Central nervous system (CNS) relapse carries a poor prognosis in diffuse large B-cell lymphoma (DLBCL). Integrating biomarkers into the CNS–International Prognostic Index (CNS-IPI) risk model may improve identification of patients at high risk for developing secondary CNS disease. CNS relapse was analyzed in 1418 DLBCL patients treated with obinutuzumab or rituximab plus cyclophosphamide, doxorubicin, vincristine, prednisone chemotherapy in the phase 3 GOYA study. Cell of origin (COO) was assessed using gene-expression profiling. BCL2 and MYC protein expression was analyzed by immunohistochemistry. The impact of CNS-IPI, COO, and BCL2/MYC dual-expression status on CNS relapse was assessed using a multivariate Cox regression model (data available in n = 1418, n = 933, and n = 688, respectively). High CNS-IPI score (hazard ratio [HR], 4.0; 95% confidence interval [CI], 1.3-12.3; P = .02) and activated B-cell‒like (ABC) (HR, 5.2; 95% CI, 2.1-12.9; P = .0004) or unclassified COO subtype...
Circulating tumor cells offer promise as a surrogate source of cancer cells that can be obtained ... more Circulating tumor cells offer promise as a surrogate source of cancer cells that can be obtained in real time and may provide opportunities to evaluate predictive biomarkers that can guide treatment decisions. In this review, we consider some of the technical hurdles around CTC numbers and suitability of various CTC capture and analysis platforms for biomarker evaluation. In addition, we consider the potential regulatory hurdles to development of CTC-based diagnostics. Finally, we suggest a path for co-development of anticancer therapeutics with CTC-based diagnostics that could enable clinical validation and qualification of CTC-based assays as companion diagnostics.
Proceedings of the National Academy of Sciences, 2011
Deregulation of apoptosis is a common occurrence in cancer, for which emerging oncology therapeut... more Deregulation of apoptosis is a common occurrence in cancer, for which emerging oncology therapeutic agents designed to engage this pathway are undergoing clinical trials. With the aim of uncovering strategies to activate apoptosis in cancer cells, we used a pooled shRNA screen to interrogate death receptor signaling. This screening approach identified 16 genes that modulate the sensitivity to ligand induced apoptosis, with several genes exhibiting frequent overexpression and/or copy number gain in cancer. Interestingly, two of the top hits, EDD1 and GRHL2, are found 50 kb apart on chromosome 8q22, a region that is frequently amplified in many cancers. By using a series of silencing and overexpression studies, we show that EDD1 and GRHL2 suppress death-receptor expression, and that EDD1 expression is elevated in breast, pancreas, and lung cancer cell lines resistant to death receptor-mediated apoptosis. Supporting the relevance of EDD1 and GRHL2 as therapeutic candidates to engage ap...
Apo2L/TRAIL stimulates cancer cell death through the proapoptotic receptors DR4 and DR5, but the ... more Apo2L/TRAIL stimulates cancer cell death through the proapoptotic receptors DR4 and DR5, but the determinants of tumor susceptibility to this ligand are not fully defined. mRNA expression of the peptidyl O-glycosyltransferase GALNT14 correlated with Apo2L/TRAIL sensitivity in pancreatic carcinoma, non-small-cell lung carcinoma and melanoma cell lines, and up to 30% of samples from various human malignancies showed GALNT14 overexpression. RNA interference of GALNT14 reduced cellular Apo2L/TRAIL sensitivity, whereas overexpression increased responsiveness. Biochemical analysis of DR5 identified several ectodomain O-(N-acetyl galactosamine-galactose-sialic acid) structures. Sequence comparison predicted conserved extracellular DR4 and DR5 O-glycosylation sites; progressive mutation of the DR5 sites attenuated apoptotic signaling. O-glycosylation promoted ligand-stimulated clustering of DR4 and DR5, which mediated recruitment and activation of the apoptosis-initiating protease caspase-8. These results uncover a new link between death-receptor O-glycosylation and apoptotic signaling, providing potential predictive biomarkers for Apo2L/TRAIL-based cancer therapy.
Evaluation of cancer biomarkers from blood could significantly enable biomarker assessment by pro... more Evaluation of cancer biomarkers from blood could significantly enable biomarker assessment by providing a relatively non-invasive source of representative tumor material. Circulating Tumor Cells (CTCs) isolated from blood of metastatic cancer patients hold significant promise in this regard.
Purpose: We describe the preclinical pharmacology and antitumor activity of GDC-0068, a novel hig... more Purpose: We describe the preclinical pharmacology and antitumor activity of GDC-0068, a novel highly selective ATP-competitive pan-Akt inhibitor currently in clinical trials for the treatment of human cancers. Experimental Design: The effect of GDC-0068 on Akt signaling was characterized using specific biomarkers of the Akt pathway, and response to GDC-0068 was evaluated in human cancer cell lines and xenograft models with various genetic backgrounds, either as a single agent or in combination with chemotherapeutic agents. Results: GDC-0068 blocked Akt signaling both in cultured human cancer cell lines and in tumor xenograft models as evidenced by dose-dependent decrease in phosphorylation of downstream targets. Inhibition of Akt activity by GDC-0068 resulted in blockade of cell-cycle progression and reduced viability of cancer cell lines. Markers of Akt activation, including high-basal phospho-Akt levels, PTEN loss, and PIK3CA kinase domain mutations, correlate with sensitivity to ...
Purpose: The pathways underlying basal-like breast cancer are poorly understood, and as yet, ther... more Purpose: The pathways underlying basal-like breast cancer are poorly understood, and as yet, there is no approved targeted therapy for this disease. We investigated the role of mitogen-activated protein kinase kinase (MEK) and phosphatidylinositol 3-kinase (PI3K) inhibitors as targeted therapies for basal-like breast cancer. Experimental Design: We used pharmacogenomic analysis of a large panel of breast cancer cell lines with detailed accompanying molecular information to identify molecular predictors of response to a potent and selective inhibitor of MEK and also to define molecular mechanisms underlying combined MEK and PI3K targeting in basal-like breast cancer. Hypotheses were confirmed by testing in multiple tumor xenograft models. Results: We found that basal-like breast cancer models have an activated RAS-like transcriptional program and show greater sensitivity to a selective inhibitor of MEK compared with models representative of other breast cancer subtypes. We also showe...
Purpose: The class I phosphatidylinositol 3′ kinase (PI3K) plays a major role in proliferation an... more Purpose: The class I phosphatidylinositol 3′ kinase (PI3K) plays a major role in proliferation and survival in a wide variety of human cancers. A key factor in successful development of drugs targeting this pathway is likely to be the identification of responsive patient populations with predictive diagnostic biomarkers. This study sought to identify candidate biomarkers of response to the selective PI3K inhibitor GDC-0941. Experimental Design: We used a large panel of breast cancer cell lines and in vivo xenograft models to identify candidate predictive biomarkers for a selective inhibitor of class I PI3K that is currently in clinical development. The approach involved pharmacogenomic profiling as well as analysis of gene expression data sets from cells profiled at baseline or after GDC-0941 treatment. Results: We found that models harboring mutations in PIK3CA, amplification of human epidermal growth factor receptor 2, or dual alterations in two pathway components were exquisitely...
Purpose: Elevated levels or increases in circulating tumor cells (CTC) portend poor prognosis in ... more Purpose: Elevated levels or increases in circulating tumor cells (CTC) portend poor prognosis in patients with epithelial cancers. Less is known about CTCs as surrogate endpoints or their use for predictive biomarker evaluation. This study investigated the utility of CTC enumeration and characterization using the CellSearch platform, as well as mutation detection in circulating tumor DNA (ctDNA), in patients with advanced non–small cell lung cancer (NSCLC). Experimental Design: Forty-one patients were enrolled in a single-arm phase II clinical trial of erlotinib and pertuzumab. Peripheral blood was analyzed for CTC enumeration, EGFR expression in CTCs, and detection of oncogenic mutations in CTCs and ctDNA. Changes in CTC levels were correlated with 2[18F]fluoro-2-deoxy-d-glucose–positron emission tomographic (FDG-PET) and computed tomographic (CT) imaging and survival endpoints. Results: CTCs were detected (≥1 CTC) at baseline in 78% of patients. Greater sensitivity for mutation de...
Venetoclax inhibits BCL2, an antiapoptotic protein that is pathologically overexpressed and that ... more Venetoclax inhibits BCL2, an antiapoptotic protein that is pathologically overexpressed and that is central to the survival of chronic lymphocytic leukemia cells. We evaluated the efficacy of venetoclax in combination with rituximab in patients with relapsed or refractory chronic lymphocytic leukemia. In this randomized, open-label, phase 3 trial, we randomly assigned 389 patients to receive venetoclax for up to 2 years (from day 1 of cycle 1) plus rituximab for the first 6 months (venetoclax-rituximab group) or bendamustine plus rituximab for 6 months (bendamustine-rituximab group). The trial design did not include crossover to venetoclax plus rituximab for patients in the bendamustine-rituximab group in whom progression occurred. The primary end point was investigator-assessed progression-free survival. After a median follow-up period of 23.8 months, the rate of investigator-assessed progression-free survival was significantly higher in the venetoclax-rituximab group (32 events of...
, 20 pts have been treated. Median age: 63 yrs (range 50-77), 33% del(17p), 43% complex karyotype... more , 20 pts have been treated. Median age: 63 yrs (range 50-77), 33% del(17p), 43% complex karyotype, and 29% and 10% TP53 and NOTCH1 mutation, respectively. Median # prior CLL treatments 2 (range 0-5, prior ibrutinib (n=8), idelalisib (n=2), and ven (n=2); 5 pts no prior treatment). Median # ven + da-R-EPOCH cycles in this ongoing study is 3.5 (range 1-6), including 5 pts who recently enrolled. 4 pts had dose de-escalation of R-EPOCH and 1 pt had dose escalation. ≥Gr 3 heme toxicity included: neutropenia (45%), anemia (35%), thrombocytopenia (25%). ≥Gr 3 non-heme toxicities in ≥15% of pts: febrile neutropenia (20%), hypocalcemia and hypophosphatemia (15% each). No TLS occurred with daily ven ramp-up after 1 cycle of R-EPOCH. Infectious complications: 3 pts with sepsis during C1 of R-EPOCH (prior to starting ven) and 1 pt each with influenza A and norovirus while on combination therapy. 7 pts died, including 4 due to disease progression (2 during C1 before ven), and 1 each due to sepsis, sudden death, and GVHD post-alloHCT. 8 pts are not evaluable for efficacy of the combination (3 are still in C1, 4 had toxicity in C1 and never started ven, 1 pt withdrew in C1). Of the 12 evaluable pts who have started combination therapy, 9 responded (ORR 75%); 8/12 (67%) had CR, all of whom also had undetectable bone marrow MRD for CLL. 5 pts went to alloHCT, with pts still in CR now up to 1.5 yrs post-alloHCT. With a median followup of 3 mo (range 0-17.9), median PFS is 10 mo (Fig 1A), median OS is 16.3 mo (Fig 1B). Conclusions: Our initial data suggest that ven + da-R-EPOCH is a feasible regimen to treat RS. Expected toxicities from intensive chemoimmunotherapy were seen, without significant additional toxicity from ven, including no TLS with daily ven ramp-up. The 67% CR and PFS/OS of 10/16.3 mo are favorable in the context of historical results. Accrual is ongoing, and updated results will be presented.
Background: Expression of PD-L1 in tumor cells and tumor-infiltrating immune cells has been assoc... more Background: Expression of PD-L1 in tumor cells and tumor-infiltrating immune cells has been associated with improved efficacy to anti-PD-1/PD-L1 inhibitors in patients with advanced-stage non-small-cell lung cancer (NSCLC) and emerged as a potential biomarker for the selection of patients to cancer immunotherapies. We investigated the utility of circulating tumor cells (CTCs) and circulating white blood cells (WBCs) as a noninvasive method to evaluate PD-L1 status in advanced NSCLC patients. Patients and methods: CTCs and circulating WBCs were enriched from peripheral blood samples (ISET V R platform; Rarecells) from 106 NSCLC patients. PD-L1 expression on ISET filters and matched-tumor tissue was evaluated by automated immunostaining (SP142 antibody; Ventana), and quantified in tumor cells and WBCs. Results: CTCs were detected in 80 (75%) patients, with levels ranging from 2 to 256 CTCs/4 ml, and median of 60 CTCs/4 ml. Among 71 evaluable samples with matched-tissue and CTCs, 6 patients (8%) showed !1 PD-L1-positive CTCs and 11 patients (15%) showed !1% PD-L1-positive tumor cells in tumor tissue with 93% concordance between tissue and CTCs (sensitivity ¼ 55%; specificity ¼ 100%). From 74 samples with matched-tissue and circulating WBCs, 40 patients (54%) showed !1% PD-L1-positive immune infiltrates in tumor tissue and 39 patients (53%) showed !1% PD-L1 positive in circulating WBCs, with 80% concordance between blood and tissue (sensitivity ¼ 82%; specificity ¼ 79%). We found a trend for worse survival in patients receiving first-line cisplatin-based chemotherapy treatments, whose tumors express PD-L1 in CTCs or immune cells (progression-free and overall survival), similar to the effects of PD-L1 expression in matched-patient tumors. Conclusions: These results demonstrated that PD-L1 status in CTCs and circulating WBCs correlate with PD-L1 status in tumor tissue, revealing the potential of CTCs assessment as a noninvasive real-time biopsy to evaluate PD-L1 expression in patients with advanced-stage NSCLC.
Introduction: Composite scores integrating genes mutation status with the clinical predictor FLIP... more Introduction: Composite scores integrating genes mutation status with the clinical predictor FLIPI have been recently proposed to improve risk stratification for follicular lymphoma (FL) patients. We evaluated the ability of m7-FLIPI and POD24-PI scores to predict progression free survival (PFS) in a large cohort of patients receiving first-line immunochemotherapy, with or without rituximab maintenance. Methods: Tumour biopsies were obtained at FL diagnosis from 252 patients from the PRIMA study, either as FFPE tissues (n = 98) or fresh-frozen tissues (n = 154). After DNA extraction, DNA-targeted sequencing was performed using the Foundation One Heme™ panel. m7-FLIPI and POD24-PI models were applied as originally described. Results: The frequency of non-silent mutations were similar to those previously reported: CREBBP = 75%, EZH2 = 28%, CARD1 = 19%, ARID1A = 19%, EP300 = 16%, FOXO1 = 16% and MEF2B = 12%. We first evaluated the prognostic value of each gene mutation status separately. While some mutations were associated with a longer (MEF2B, EZH2) or shorter (EP300, CREBBP) PFS as previously described, ARID1A or CARD11 mutations were not associated with patients outcome. Moreover, FOXO1 mutations were associated with a good outcome, in opposite to their prognostic weight in the m7-FLIPI. The m7-FLIPI and FLIPI scores classified 28% and 43% of patients as high-risk, respectively. m7-FLIPI correlated with PFS (p = 0•005; OR = 1.74, 95%CI: 1.00-3.02), slightly outperforming the FLIPI 96 ABSTRACT 89% (range 40-100%) in young adults (18-40 yrs) to 74% (range 0-100%, p = 0.01) in the oldest cohort (>70 yrs). No single gene mutation was found to be associated with older age after correction for multiple testing. Conclusions: Our data suggest that older age does not directly impact disease biology and treatment efficacy in FL, and should not be used to guide treatment decisions.
Background: Akt, a serine/threonine protein kinase, is a key signaling node in the PI3K/Akt/mTOR ... more Background: Akt, a serine/threonine protein kinase, is a key signaling node in the PI3K/Akt/mTOR pathway and plays an essential role in regulating tumor cell proliferation, migration and survival. PI3K/Akt signaling is up-regulated in gastric cancer, mainly due to loss of PTEN, and is associated with poor prognosis and chemoresistance. Chemotherapeutics such as doxorubicin and 5-FU/cisplatin have been shown to upregulate PI3K/Akt signaling in several solid cancers in response to DNA damage. We have previously reported on a potent highly selective ATP-competitive pan-Akt inhibitor, GDC-0068, which inhibits cell cycle progression and viability of cancer cell lines and xenografts driven by Akt signaling, including those with defects in the tumor suppressor PTEN, oncogenic mutations in PIK3CA, and amplification of HER2. Thus, we hypothesized that the combination of FOLFOX and GDC-0068 would induce anti-tumor activity in primary gastric cancer models. Methods: Gastric cancer cell lines (n=11) were treated with increasing concentrations of GDC-0068 and FOLFOX and evaluated for combinational efficacy over single agent via cell viability assay. For in vivo studies, primary gastric cancer xenograft models were implanted in immuno-compromised mice and treated with GDC-0068 and FOLFOX as single agents or in combination. Results: In vitro, the combination of GDC-0068 and FOLFOX results in enhanced inhibition of tumor cell viability in the cell lines with activation of the PI3K pathway compared to either single agent alone. These results were recapitulated in vivo, in the primary xenograft models STO#240 and STO#182, where the combination of GDC-0068 and FOLFOX resulted in increased tumor growth inhibition or regressions compared to single agents alone. All combinations tested were well tolerated in vivo based on minimal changes in body weights. Conclusions: Our preclinical studies demonstrate improved anti-tumor efficacy when GDC-0068 is combined with FOLFOX in gastric cancer models in vitro and in vivo. The data supports the clinical development of GDC-0068 in combination with FOLFOX for the treatment of gastric cancer. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B190. Citation Format: Ellen Ingalla, Rebecca Hong, Heidi Savage, Elizabeth Punnoose, Sandra Rost, Hartmut Koeppen, Deepak Sampath, Michelle A. Nannini. GDC-0068 is a novel and selective Akt inhibitor that enhances the efficacy of FOLFOX in primary gastric cancer models. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B190.
GDC-0068 is a highly selective ATP-competitive small molecule that inhibits all three isoforms of... more GDC-0068 is a highly selective ATP-competitive small molecule that inhibits all three isoforms of Akt with IC50 values of 5 to 30 nM. GDC-0068 selectively inhibits cancer cells with activated Akt signaling (e.g. via PTEN loss or PIK3CA mutations). Patients with advanced solid tumors were treated with GDC-0068 using a 3+3 escalation design. GDC-0068 was dosed PO QD on a 21-day on, 7-day off schedule; endpoints included safety, pharmacokinetics (PK) and determination of pathway knockdown. Pharmacodynamics (PD) endpoints in surrogate tissue [platelet rich plasma (PRP)] were evaluated in all patients. In addition, at least two patients per cohort had pre- and on-treatment (day 15) tumor biopsies at doses ≥100mg QD. The tumor tissue samples were evaluated using reverse phase protein array (RPPA). Thirty patients were enrolled across 7 cohorts (25, 50, 100, 200, 400, 600 and 800 mg QD). GDC-0068 was generally well-tolerated at doses ≤ 600 mg. Preliminary PK analyses show a dose proportional increase in exposure over the dose range tested. In addition, clinical exposures at doses ≥ 200 mg QD met or exceeded GDC-0068 exposures associated with tumor stasis in multiple PTEN null preclinical xenograft models. PK/PD evaluation showed dose-dependent Akt pathway inhibition in the PRP assay, with ≥70% inhibition of pGSK3 in all patients at doses ≥ 200 mg QD. Pre- and on-treatment tumor biopsies showed ≥50% decrease in pPRAS40 and cyclin D1 in multiple patients at doses of 200 mg QD and higher. In addition, evidence of feedback activation of the MAP kinase pathway after treatment with GDC-0068 was observed in biopsy samples. One patient with PTEN low/ PIK3CA H1047R/ KRAS wt CRC had prolonged stable disease and showed Akt pathway suppression by multiple downstream markers. GDC-0068 is well tolerated at doses ≤ 600 mg with a favorable safety profile and dose proportional pharmacokinetics. Treatment with GDC-0068 results in substantial pathway knockdown in both surrogate and tumor tissues at doses ≥ 200 mg QD. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B154.
Mutations in oncogenes such as EGFR, KRAS and PIK3CA, have been shown to have profound effects on... more Mutations in oncogenes such as EGFR, KRAS and PIK3CA, have been shown to have profound effects on pathway signaling and tumor growth. These genetic aberrations are potentially valuable predictors for patient stratification and therapeutic responses to targeted therapies directed against these signaling pathways. Here we evaluate the utility of circulating tumor DNA as a “liquid biopsy” for predictive diagnostics and real-time monitoring of disease. A fundamental challenge with this approach is that, due to the low frequency of the mutant tumor DNA in circulation, a highly sensitive assay is required to detect the single base pair alterations. We have designed and investigated several assay formats, including TaqMan and scorpion-ARM assays for mutation detection using a digital PCR platform. We also employed LNA/PNA blockers and thermostable restriction digestion in PCR reaction to enrich the mutant DNA. The combination of the biased amplification and digital PCR dramatically increased mutation detection 10-100 fold. To assess clinical utility of the technology, data on PIK3CA mutation screening of matched patient tumor and plasma samples from a phase I clinical trial will also be reported. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3180. doi:10.1158/1538-7445.AM2011-3180
BCL-2 family proteins dictate survival of human multiple myeloma (MM) cells making them attractiv... more BCL-2 family proteins dictate survival of human multiple myeloma (MM) cells making them attractive drug targets. Indeed, MM cells are sensitive to antagonists that selectively target pro-survival proteins such as BCL-2/BCL-X<sub>L</sub> (ABT-737 and ABT-263/navitoclax) or BCL-2 only (ABT-199/GDC-0199/venetoclax). Resistance to these three drugs is mediated by expression of MCL-1. However, given the selectivity profile of venetoclax it is unclear whether co-expression of…
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Papers by E. Punnoose