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The majority of diffuse large B-cell lymphoma (DLBCL) tumors contain mutations in histone-modifying enzymes (HMEs), indicating a potential therapeutic benefit of histone deacetylase inhibitors (HDIs), and preclinical data suggest that HDIs augment the effect of rituximab. In this randomized phase 2 study, we evaluated the response rate and toxicity of panobinostat, a pan-HDI administered 30 mg orally 3 times weekly, with or without rituximab, in 40 patients with relapsed or refractory de novo (n = 27) or transformed (n = 13) DLBCL. Candidate genes and whole exomes were sequenced in relapse tumor biopsies to search for molecular correlates, and these data were used to quantify circulating tumor DNA (ctDNA) in serial plasma samples. Eleven of 40 patients (28%) responded to panobinostat (95% confidence interval [CI] 14.6-43.9) and rituximab did not increase responses. The median duration of response was 14.5 months (95% CI 9.4 to “not reached”). At time of data censoring, 6 of 11 patients had not progressed. Of the genes tested for mutations, only those in MEF2B were significantly associated with response. We detected ctDNA in at least 1 plasma sample from 96% of tested patients. A significant increase in ctDNA at day 15 relative to baseline was strongly associated with lack of response (sensitivity 71.4%, specificity 100%). We conclude that panobinostat induces very durable responses in some patients with relapsed DLBCL, and early responses can be predicted by mutations in MEF2B or a significant change in ctDNA level at 15 days after treatment initiation. This clinical trial was registered at www.ClinicalTrials.gov (#{"type":"clinical-trial","attrs":{"text":"NCT01238692","term_id":"NCT01238692"}}NCT01238692).

Purpose: Relapsed or refractory diffuse large B-cell lymphoma (rrDLBCL) is fatal in 90% of patients, and yet little is known about its biology. Experimental Design: Using exome sequencing, we characterized the mutation profiles of 38 rrDLBCL biopsies obtained at the time of progression after immunochemotherapy. To identify genes that may be associated with relapse, we compared the mutation frequency in samples obtained at relapse to an unrelated cohort of 138 diagnostic DLBCLs and separately amplified specific mutations in their matched diagnostic samples to identify clonal expansions. Results: On the basis of a higher frequency at relapse and evidence for clonal selection, TP53, FOXO1, MLL3 (KMT2C), CCND3, NFKBIZ, and STAT6 emerged as top candidate genes implicated in therapeutic resistance. We observed individual examples of clonal expansions affecting genes whose mutations had not been previously associated with DLBCL including two regulators of NF-κB: NFKBIE and NFKBIZ. We detected mutations that may be affect sensitivity to novel therapeutics, such as MYD88 and CD79B mutations, in 31% and 23% of patients with activated B-cell–type of rrDLBCL, respectively. We also identified recurrent STAT6 mutations affecting D419 in 36% of patients with the germinal center B (GCB) cell rrDLBCL. These were associated with activated JAK/STAT signaling, increased phospho-STAT6 protein expression and increased expression of STAT6 target genes. Conclusions: This work improves our understanding of therapeutic resistance in rrDLBCL and has identified novel therapeutic opportunities especially for the high-risk patients with GCB-type rrDLBCL. Clin Cancer Res; 22(9); 2290–300. ©2015 AACR.

Increasingly, targeted therapies are being developed to treat malignancies. To define targets, determine mechanisms of response and resistance, and develop biomarkers for the successful investigation of novel therapeutics, high-quality tumor biospecimens are critical. We have developed standard operating procedures (SOPs) to acquire and process serial blood and tumor biopsies from patients with diffuse large B-cell lymphoma enrolled in multicenter clinical trials. These SOPs allow for collection and processing of materials suitable for multiple downstream applications, including immunohistochemistry, cDNA microarrays, exome sequencing, and metabolomics. By standardizing these methods, we control preanalytic variables that ensure high reproducibility of results and facilitate the integration of datasets from such trials. This will facilitate translational research, better treatment selection, and more rapid and efficient development of new drugs. See all the articles in this CEBP Focus section, “Biomarkers, Biospecimens, and New Technologies in Molecular Epidemiology.” Cancer Epidemiol Biomarkers Prev; 23(12); 2688–93. ©2014 AACR.

Histone deacetylase inhibitors (HDACi) have modest objective response rates ranging from 5.5% to 49% [1–5] as single agents. Furthermore, HDACi exert their anti-tumor effects in different ways, lik...

In 1998, we reported that a significant proportion of breast and/or ovarian cancer families of French Canadian descent harbor specific germline mutations in BRCA1 or BRCA2 attributed to common founders. Here we report the frequency of previously described mutations (n = 7) and 13 mutations identified in French Canadian families since 1998, in a new group of families (n = 88). Four of the previously described mutations, 4446C>T, 2953delGTAinsC, 8765delAG and 6085C>T, account for 72% and 69% of mutation-positive families in previously (n = 81) and recently ascertained groups, respectively. Only 2 of 13 recently identified mutations were found in more than 1 family: 3875delGTCT (n = 2) and 3398delAAAAG (n = 4). The 2 groups (ascertained pre- and post-gene discovery) did not differ significantly when distribution of mutations based on cancer syndrome phenotype and age of diagnosis or number of breast cancer cases were compared. Five common mutations accounted for a significant proportion (84%) of all mutation-positive families. The age of diagnosis of female breast cancer in mutation-negative families was significantly higher than that of the mutation-positive families (p

Ovarian carcinomas exhibit extensive heterogeneity, and their etiology remains unknown. Histological and genetic evidence has led to the proposal that low grade ovarian serous carcinomas (LGOSC) have a different etiology than high grade carcinomas (HGOSC), arising from serous tumours of low malignant potential (LMP). Common regions of chromosome (chr) 3 loss have been observed in all types of serous ovarian tumours, including benign, suggesting that these regions contain genes important in the development of all ovarian serous carcinomas. A high-density genome-wide genotyping bead array technology, which assayed >600,000 markers, was applied to a panel of serous benign and LMP tumours and a small set of LGOSC, to characterize somatic events associated with the most indolent forms of ovarian disease. The genomic patterns inferred were related to TP53, KRAS and BRAF mutations. An increasing frequency of genomic anomalies was observed with pathology of disease: 3/22 (13.6%) benign cases, 40/53 (75.5%) LMP cases and 10/11 (90.9%) LGOSC cases. Low frequencies of chr3 anomalies occurred in all tumour types. Runs of homozygosity were most commonly observed on chr3, with the 3p12-p11 candidate tumour suppressor region the most frequently homozygous region in the genome. An LMP harboured a homozygous deletion on chr6 which created a GOPC-ROS1 fusion gene, previously reported as oncogenic in other cancer types. Somatic TP53, KRAS and BRAF mutations were not observed in benign tumours. KRAS-mutation positive LMP cases displayed significantly more chromosomal aberrations than BRAF-mutation positive or KRAS and BRAF mutation negative cases. Gain of 12p, which harbours the KRAS gene, was particularly evident. A pathology review reclassified all TP53-mutation positive LGOSC cases, some of which acquired a HGOSC status. Taken together, our results support the view that LGOSC could arise from serous benign and LMP tumours, but does not exclude the possibility that HGOSC may derive from LMP tumours.

INTRODUCTION: Novel therapies which consider the molecular diversity of diffuse large B cell lymphoma (DLBCL) are needed to salvage patients who fail to respond to standard chemoimmunotherapy. The majority of DLBCL contain mutations in histone modifying enzymes (HME) which suggests that histone deacetylase inhibitors (HDI) should be active. Preclinical data suggest that they also augment the effect of rituximab. METHODS: In this randomized phase II study, we evaluated the response rate and toxicity of panobinostat, a pan-HDI, 30 mg orally 3 times a week, with or without rituximab, in 40 patients with relapsed and refractory DLBCL (median 3 prior regimens, range1-9). Tumors were sequenced for mutations known to be associated with DLBCL, and serial biopsies were analyzed for changes in biomarkers. RESULTS: 21 patients were treated with panobinostat alone and 19 with panobinostat and rituximab. Overall, 12/40 patients (30%) responded to panobinostat (95% confidence interval 16.6%-46.5%) the addition of rituximab did not increase responses. The median duration of response was 14.5 months (95% confidence interval 9.4 to not reached) (Figure). At a median follow up of 17.5 months for responders, 6 of 12 patients have not progressed. Patients with dual expression of MYC and BCL2 (6/27) and those with FAS mutations (5/33) did not respond although responses were seen in 5/10 patients with TP53 mutations. Responses were seen in 8/24 patients with mutations in HME genes. Patients with MEF2B mutations had the greatest odds of response (P CONCLUSION: Panobinostat induces durable responses in some patients with relapsed DLBCL, and accompanying biomarker analyses identified DLBCL subgroups likely to respond. Figure 1. Progression free survival overall and for responders, N=40 patients. Figure 1. Progression free survival overall and for responders, N=40 patients. Disclosures Assouline: Novartis: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Honoraria, Research Funding; Janssen: Honoraria; Celgene: Consultancy. Off Label Use: panobinostat for the treatment of diffuse large B cell lymhpoma. Crump:Seattle Genetics: Consultancy; Celgene: Consultancy; Roche Canada: Consultancy. Tosikyan:Novartis: Consultancy.

Although molecular genetic profiling of ovarian cancers harboring germline BRCA1/BRCA2 mutations suggest that pathways in common with sporadic cases are involved in the pathogenesis of the disease, overall survival differs between BRCA1, BRCA2 and sporadic disease. To further dissect molecular pathways involved, which could account for differences in pathogenesis of the disease, we have investigated genomic landscapes in the BRCA1 and BRCA2 mutated ovarian cancers. Chromosomal anomalies were assessed in 28 specimens with BRCA1 (n=15), BRCA2 (n=12) or BRCA1 and BRCA2 (n=1) mutations using high-density Illumina SNP arrays. The majority (22/28) are serous adenocarcinomas while the remaining samples were either endometroid (2/28) or mixed adenocarcinomas (4/28). The cases harbor BRCA1/BRCA2 mutations from a French Canadian population, which exhibit strong founder effects. Allelic imbalance, copy number differences, intrachromosomal breaks and homozygous deletions were inferred visually using the Genome Viewer module of the BeadStudio software followed by ASCAT analysis. A statistical analysis was used to directly compare genotypes of BRCA1 versus BRCA2 positive samples. TP53 gene mutation status was also assessed. All samples were found to harbor a somatic TP53 mutation comprised of missense (19/28), nonsense (2/28), frame-shift (4/28) and (3/28) splice mutations. The results were compared to independently derived data generated previously from our group, which was largely comprised of sporadic cases (Wojnarowicz et al 2012), and to the genomic data from the Cancer Genome Atlas project (TCGA). The genomic landscapes of BRCA1 and BRCA2 mutated cancers overlap those reported in previous studies. However, there were significant differences in the genomic landscapes involving chromosome 6q between BRCA1 and BRCA2 mutation-positive cancers. This genomic distinction between BRCA1 and BRCA2 ovarian cancer samples may point to a region containing genes important in the etiology or progression of hereditary cancer. Citation Format: Eman AlShehri, Moria Belanger, Suzanna Arcand, Kathleen Klein Oros, Celia Greenwood, Patricia N. Tonin. Characterization of genomic landscapes of BRCA1 and BRCA2 implicated ovarian cancer specimens from a founder french canadian population [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-TECH-1101.

The BRCAPRO, Couch, Myriad I and II, Ontario Family History Assessment Tool (FHAT), and Manchester models have been used to predict BRCA1 or BRCA2 mutation carrier status of women at high risk for developing the heritable form of breast and ovarian cancers. We have evaluated these models for their accuracy in classifying 224 French Canadian families with at least three cases of breast cancer (diagnosed before the age of 65 years), ovarian cancer, or male breast cancer where mutation status was known for an index affected case used to assess the model. This series includes 44 BRCA1 and 52 BRCA2 mutation-positive families. Using receiver operator characteristics analyses, the C-statistics were found to be 0.81, 0.80, 0.79, and 0.74 for the BRCAPRO, FHAT, Manchester, and Myriad II models, respectively, when incorporating both BRCA1 and BRCA2 mutation carrier predictions. For the BRCAPRO model, 75% scored greater than a 0.43 probability in the mutation-positive group and 75% scored less than 0.50 in the mutation-negative group. Only 38 of 128 (30%) mutation-negative group had a probability greater than 0.43 with the BRCAPRO model. While all models were highly predictive of carrier status, the BRCAPRO model was the most accurate where a cut-off of 10% would have eliminated 60 of 128 (47%) mutation-negative families for genetic testing and only miss 10 of 96 (10%) mutation-positive families. A review of the cancer phenotypes with high BRCAPRO probabilities showed that significantly more metachronous bilateral breast cancer cases occurred in BRCA1/2 mutation carrier families in comparison to mutation-negative families, a feature which is not discriminated in the BRCAPRO model.

High-grade ovarian serous carcinomas (HGSC) are characterized by TP53 mutations and non-random patterns of chromosomal anomalies, where the nature of the TP53 mutation may correlate with clinical outcome. However, the frequency of common somatic genomic events occurring in HGSCs from demographically defined populations has not been explored. Whole genome SNP array, and TP53 mutation, gene and protein expression analyses were assessed in 87 confirmed HGSC samples with clinical correlates from French Canadians, a population exhibiting strong founder effects, and results were compared with independent reports describing similar analyses from unselected populations. TP53 mutations were identified in 91% of HGSCs. Anomalies observed in more than 50% of TP53 mutation-positive HGSCs involved gains of 3q, 8q and 20q, and losses of 4q, 5q, 6q, 8p, 13q, 16q, 17p, 17q, 22q and Xp. Nearly 400 regions of non-overlapping amplification or deletion were identified, where 178 amplifications and 98 deletions involved known genes. The subgroup expressing mutant p53 protein exhibited significantly prolonged overall and disease-free survival as compared with the p53 protein null subgroup. Interestingly, a comparative analysis of genomic landscapes revealed a significant enrichment of gains involving 1q, 8q, and 12p intervals in the subgroup expressing mutant p53 protein as compared with the p53 protein null subgroup. Although the findings show that the frequency of TP53 mutations and the genomic landscapes observed in French Canadian samples were similar to those reported for samples from unselected populations, there were differences in the magnitude of global gains/losses of specific chromosomal arms and in the spectrum of amplifications and deletions involving focal regions in individual samples. The findings from our comparative genomic analyses also support the notion that there may be biological differences between HGSCs that could be related to the nature of the TP53 mutation.