Sarangi, Vivekananda

Abstract We previously identified five single nucleotide polymorphisms (SNPs) at four susceptibility loci for diffuse large B-cell lymphoma (DLBCL) in individuals of European ancestry through a large genome-wide association study (GWAS). To further elucidate genetic susceptibility to DLBCL, we sought to validate two loci at 3q13.33 and 3p24.1 that were suggestive in the original GWAS with additional genotyping. In the meta-analysis (5662 cases and 9237 controls) of the four original GWAS discovery scans and three replication studies, the 3q13.33 locus (rs9831894; minor allele frequency [MAF] = 0.40) was associated with DLBCL risk [odds ratio (OR) = 0.83, P = 3.62 × 10−13]. rs9831894 is in linkage disequilibrium (LD) with additional variants that are part of a super-enhancer that physically interacts with promoters of CD86 and ILDR1. In the meta-analysis (5510 cases and 12 817 controls) of the four GWAS discovery scans and four replication studies, the 3p24.1 locus (rs6773363; MAF = 0.45) was also associated with DLBCL risk (OR = 1.20, P = 2.31 × 10−12). This SNP is 29 426-bp upstream of the nearest gene EOMES and in LD with additional SNPs that are part of a highly lineage-specific and tumor-acquired super-enhancer that shows long-range interaction with AZI2 promoter. These loci provide additional evidence for the role of immune function in the etiology of DLBCL, the most common lymphoma subtype.

Background:Diffuse Large B Cell Lymphoma (DLBCL) is characterized by both biologic and genetic heterogeneity. While significant therapeutic advances have been made, nearly 30% of cases fail frontline treatment. Recent data suggest that 5–10% of DLBCL can be categorized as double/triple hit lymphoma (DH/TH), defined as having concurrent MYC, BCL2 and/or BCL6 gene rearrangements, identified by fluorescence in‐situ hybridization (FISH) probes. According to the 2017 revision of the World Health Organization classification of lymphoid neoplasms, DH/TH DLBCL is now considered a high grade B‐cell lymphoma (HGBL). While gene rearrangements represent significant genetic events in cancer, copy number alterations (CNAs) also play an important role, often through genomic amplification of oncogenes or loss of tumor suppressors. Despite their similarities, the contributions of CNAs in MYC, BCL2, and BCL6, alone, or in combination with gene rearrangements, are largely unknown in DLBCL and could identify a group of previously uncharacterized patients that may have high expression of MYC, BCL2 and/or BCL6 driven by alternative mechanisms.Aims:The goal of the study is to define the landscape and prognostic significance of gene rearrangements and copy gains in MYC, BCL2, and BCL6 in a cohort of newly diagnosed DLBCL.Methods:A total of 176 cases of DLBCL with both FISH and CNA data were available for the study. FISH was performed on formalin‐fixed paraffin‐embedded sections using break‐apart probes to MYC, BCL2, and BCL6 in the Mayo Clinic Department of Lab Medicine and Pathology. Somatic copy number alterations were identified using whole exome sequencing and the Affymetrix OncoScan® FFPE assay on patient tumor FFPE tissue, and were interrogated using Nexus Copy Number 9.0 software. All patients were treated with immunochemotherapy and prospectively followed for event‐free and overall survival.Results:FISH identified 3 groups of previously described patients, DH/TH or HGBL (n = 11), translocation positive (single MYC, BCL2, BLC6, or double BCL2/BLC6, n = 86), and translocation negative (n = 79). Expanding upon the translocation positive subgroup, copy number analysis identified patients (19 of 86, 22%) with copy number gains in a second or third gene resulting in genomic hits at both MYC and BCL2/6; we classified these as alternative‐double/triple‐hit (A‐DH/TH). An example of this would be a MYC translocation plus a BCL2 and/or BCL6 gain (n = 5), or a BCL2 and/or BCL6 translocation plus a copy gain in MYC (n = 14). Similar interrogation of a publically available dataset (n = 250), revealed similar proportions of these patient groups in an independent cohort of DLBCL, validating our findings. Cell of origin classification of the A‐DH/TH cases revealed that they were 53% GCB. A‐DH/TH patients, in comparison to the translocation‐positive/copy‐gain‐negative cases, were at greater risk of an event (Cox HR = 1.5, 95% CI = 0.71–3.0) and the cases trended towards a worse overall survival (log‐rank P = 0.065; Cox HR = 2.0, 95% CI = 0.94–4.3). Patients with copy‐gains alone had similar outcomes as those with no copy‐gain or translocations, suggesting a central role for at least one translocation event in predicting outcome.Summary/Conclusion:Our data suggest that use of CNA data in combination with traditional FISH can identify a unique subset of DLBCL, A‐DH/TH, that are missed by standard clinical practice. More importantly, we find that these patients do poorly and may benefit from more aggressive treatment, although we do acknowledge that our population size was small and further validation is needed.image

Background:Recent large next generation sequencing studies have redefined the genomic landscape of diffuse large B‐cell lymphoma (DLBCL). While many of the more common genetic variants, primarily mutations, have been biologically characterized, little is known about newly emerging genetic aberrations. In particular, somatic copy number alterations remain poorly studied due to the large genomic regions they cover and the number of genes involved. This, in combination with lack of remission or early relapse in a portion of DLBCL, opens new opportunities to explore yet unidentified genetic contributions to lymphomagenesis.Aims:The goal of this study was to identify variants associated with failure to achieve event free survival at 24 months (EFS24) and define their biologic significance in DLBCL.Methods:Newly diagnosed DLBCL treated with standard immunochemotherapy were included in this study (n = 246). Copy number data was generated by using the molecular inversion probe OncoScan™ FFPE Assay Kit (Affymetrix, Santa Clara, CA, USA). OncoScan OSCHP files were analyzed using Nexus Copy Number 9.0 software (Biodiscovery, El Segundo, USA). Data interpretation and copy number calling was done using the human reference genome GRCh37/hg19. Files were analyzed using the Nexus FASST2‐Segmentation algorithm, which is based on a Hidden Markov Model approach for calling genetic events. For functional analysis, SOCS6 was overexpressed and studied in GCB‐DLBCL cell lines.Results:Upon review of genomic copy number data from DLBCL cases (n = 246), loss of 18q22.1‐q23 was a significant event in our cohort (p = 0.018), is found in 11% (n = 27) of DLBCL, and is enriched in germinal center DLBCL (GCB‐DLBCL) patients (18/107, 17%, p < 0.05) compared to activated B‐cell (ABC)/non‐GCB DLBCL patients (1/93, 1%). Furthermore, GCB‐DLBCL patients with a loss of 18q22.1‐q23 were significantly more likely to fail EFS24 (50%, p < 0.05) compared to GCB‐DLBCL patients without the loss (29%). Genes located at 18q22.1‐q23 include, TMX3, DOK6 and SOCS6. SOCS family proteins have been shown to play an important role in tumor biology, although little is known about SOCS6. SOCS6 gene expression is downregulated in a variety of solid tumors and is hypermethylated in some hematological malignancies. RNASeq data from DLBCL suggest variable expression across tumors. Using a panel of GCB cell lines we found that SOCS6 expression was low to negative, and publically available cell line data suggests this region is hypermethylated. Expression of SOCS6 was higher in a panel of mantle cell lymphoma and ABC‐DLBCL cell lines and this again correlated with their cell line methylation data, where no hypermethylation was found in those cell lines. Together, this data suggests that downregulation of SOCS6 through genomic loss or hypermethylation may have biologic importance. To explore the role of SOCS6, we restored expression in GCB cell lines and immuno‐fluorescence staining suggest localization to the nucleus and cytoplasm, as previously reported in other cell types. Studies to explore the role of SOCS6 in cytokine signaling and STAT family protein regulation are ongoing.Summary/Conclusion:Using genome copy number data from 246 DLBCL, we have found that loss of 18q22.1‐q23 (p = 0.018) is associated with GCB‐DLBCL and failure to achieve EFS24. Furthermore, we have identified that SOCS6 may have clinical and biologic significance in GCB‐DLBCL.

We previously reported an extremely rare case of follicular dendritic cell sarcoma (FDCS) presented as a thyroid mass. Given the rarity of this disease, there are no personalized and molecularly targeted treatment options due to the lack of knowledge in the genomic makeup of the tumor. A 44-year-old white woman was diagnosed with an extranodal FDCS in thyroid. The patient underwent a total thyroidectomy, central compartment dissection, parathyroid re-implantation, and adjuvant radiation therapy. Tumor DNA sequencing of 236 genes by FoundationOne panel found truncating mutations in PTEN and missense mutations in RET and TP53. However, patient-matched germline DNA was not sequenced which is critical for identification of true somatic mutations. Furthermore, the FoundationOne panel doesn't measure genomic rearrangements which have been shown to be abundant in sarcomas and are associated with sarcoma tumorigenesis and progression. In the current study, we carried out comprehensive genomic sequencing of the tumor, adjacent normal tissues, and patient-matched blood, in an effort to understand the genomic makeup of this rare extranodal FDCS and to identify potential therapeutic targets. Eighty-one somatic point mutations were identified in tumor but not in adjacent normal tissues or blood. A clonal truncating mutation in the CLTCL1 gene, which stabilizes the mitotic spindle, was likely a driver mutation of tumorigenesis and could explain the extensive copy number aberrations (CNAs) and genomic rearrangements in the tumor including a chr15/chr17 local chromothripsis resulted in 6 expressed fusion genes. The fusion gene HDGFRP3→SHC4 led to a 200-fold increase in the expression of oncogene SHC4 which is a potential target of the commercial drug Dasatinib. Missense mutations in ATM and splice-site mutation in VEGFR1 were also detected in addition to the TP53 missense mutation reported by FoundationOne.

Abstract Background: The paucity of effective therapeutic agents for hepatocellular cancer (HCC) underscores the critical need for more effective therapeutic strategies. Recent studies indicate lipid biosynthesis and desaturation is required for HCC survival. Targeting these may prove beneficial because such changes contribute to therapeutic resistance. Stearoyl CoA desaturase (SCD1), a key mediator of fatty acid (FA) biosynthesis and rate-limiting in conversion of saturated fatty acids (SFAs) to mono-unsaturated fatty acids (MUFAs), is upregulated in HCC and many other cancers. As such, we therapeutically targeted a novel lipogenic tumor survival mechanism mediated by SCD1 as a means to combat the chemoresistance associated with HCC. In so doing, we evaluated a novel lead SCD1 inhibitor in HCC. Methods: Paraffin embedded patient HCC tissues were examined for SCD1 expression. Using combined computational and synthetic chemistry approaches, we synthesized four novel specific SCD1 inhibitors with SSI-4 being the lead SCD1 inhibitor. HCC cell lines were examined using proliferation assays for response to SSI-4. IC50 concentrations for blocking SCD1 enzyme activity was determined. Blood half-life and bioavailability of single dose SSI-4 was determined. Mechanisms of action of SCD1 were examined that included Endoplasmic reticulum (ER) stress. In vivo, antitumor activity was determined using HCC patient derived xenograft (PDX) mouse models. Results: We identified elevated SCD1 mRNA and protein in HCCs tissues. SSI-4 dose-dependently inhibits cell proliferation in HCC cell lines with specificity demonstrated by oleic acid (MUFA) co-culture. Single dose oral gavage SSI-4 demonstrated a half-life of ~4 hours and excellent oral bioavailability. SSI-4 was well tolerated with long-term daily dosing. SSI-4 treatment of HCC cells and tumors led to endoplasmic reticulum (ER) stress followed by apoptotic cell death. Single agent SSI-4 demonstrated antitumor activity in HCC PDX mouse models with suppression of ER stress regulated proteins. Conclusions: Targeting a novel lipid metabolic pathway in HCC may provide effective therapy for aggressive HCC. Citation Format: John Alton Copland, Laura A. Marlow, Ilah Bok, James L. Miller, Matsuda Akiko, Yan W. Asmann, Vivekananda Sarangi, Steven R. Alberts, Kabir Mody, Lewis R. Roberts, Mark J. Truty, Tushar C. Patel. Targeting stearoyl CoA desaturase 1 (SCD1) in hepatobilliary carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 192. doi:10.1158/1538-7445.AM2017-192

Clinical studies have identified specific genetic variants in dihydropyrimidine dehydrogenase (DPD; DPYD gene) as predictors of severe adverse toxicity to the commonly used chemotherapeutic 5‐fluorouracil (5‐FU); however, these studies have focused on European and European‐American populations. Our laboratory recently demonstrated that additional variants in non‐European haplotypes are predictive of 5‐FU toxicity. The objective of this study was to identify potential risk variants in an understudied East African population relevant to our institution's catchment area. The DPYD protein‐coding region was sequenced in 588 individuals of Somali or Kenyan ancestry living in central/southeast Minnesota. Twelve novel nonsynonymous variants were identified, seven of which significantly decreased DPD activity in vitro. The commonly reported toxicity‐associated variants, *2A, D949V, and I560S, were not detected in any individuals. Overall, this study demonstrates a critical limitation in our knowledge of pharmacogenetic predictors of 5‐FU toxicity, which has been based on clinical studies conducted in populations of limited diversity.