Citations

The current paradigms of known peripheral immune abnormalities in B-Chronic Lymphocytic Leukemia (CLL) are not able to consistently explain patient complications and it is difficult to correct a given CLL patient's immune status. Here, we expand on our initial report demonstrating bone marrow (BM) hematopoietic dysfunction in untreated CLL patients (Manso et al., Leukemia volume 33, pages 638-652, 2019). CLL patient BM had significantly reduced frequencies and short-term functional capacity of hematopoietic stem and progenitor cells (HSPCs). Additionally, the remaining progenitors exhibited increased protein levels of the key hematopoietic transcriptional regulators GATA-2 and PU.1. We further evaluated the frequency and function of myeloid stem cells from controls and untreated CLL patients by limiting dilution analysis in long-term culture-initiating cell (LTC-IC) assays. Over the 5 week duration of the assay, we observed delayed and partial recovery of myelopoiesis from CLL-derived HSPCs (Figure 1A). These data suggest that removal of HSPCs from the CLL leukemic microenvironment partially recovers their ability to sustain myelopoiesis. A known inflammatory mediator and hematopoiesis-modulating cytokine that is constitutively produced by CLL cells, TNF-α, induced increased expression of GATA-2 and PU.1 in specific HSPC subsets and reduced formation of short-term colony forming units in vitro. Addition of TNF-α to LTC-IC assays resulted in a striking ablation of myelopoiesis in a dose-dependent manner, partially reproducing the ex vivo results (Figure 1B). To further assess the direct impact of CLL cells on HSPC biology, isolated HSPCs from controls were exposed in vitro to leukemic CLL cells. The co-culture induced overexpression of GATA-2 and PU.1 in distinct HSPC populations, recapitulating our ex vivo findings (Figure 1C-D). When cell-cell contact was inhibited by use of Transwell inserts, an intermediate increase in GATA-2 and PU.1 was observed, highlighting the contributions of both soluble mediators and cell-cell contact to HSPC alterations. In both direct and Transwell co-culture conditions, overexpression of GATA-2 and PU.1 was reversed when TNF-α was neutralized (Figure 1E-F). Taken together, these findings indicate a significant role for CLL-derived TNF-α in HSPC modulation and expand our previous observations of BM dysfunction in untreated CLL patients. This data offers new molecular insight into the contribution of the leukemic microenvironment to altered hematopoiesis, contributing to immunodeficiency in CLL, and identifies TNF-α as a potential therapeutic target for correction of hematopoiesis in CLL disease. Disclosures Ding: Merck: Research Funding; DTRM Biopharma: Research Funding. Parikh:AstraZeneca: Honoraria, Research Funding; MorphoSys: Research Funding; AbbVie: Honoraria, Research Funding; Acerta Pharma: Research Funding; Pharmacyclics: Honoraria, Research Funding; Janssen: Research Funding; Ascentage Pharma: Research Funding; Genentech: Honoraria. Novak:Celgene Coorperation: Research Funding. Kay:Agios: Other: DSMB; Celgene: Other: Data Safety Monitoring Board; Infinity Pharmaceuticals: Other: DSMB; MorphoSys: Other: Data Safety Monitoring Board.

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.

6010 Background: An evolutionary action scoring algorithm (EAp53) based on phylogenetic sequence variations and speciation stratifies head and neck squamous cell carcinoma (HNSCC) patients bearing TP53 missense mutations as high-risk (high, EAp53≥75), associated with poor outcomes, or low-risk (low), with similar outcomes as TP53 wild-type (wt), and has been validated as a reliable prognostic marker. This study is designed to further validate prior findings that EAp53 is a prognostic marker for locally advanced HNSCC patients, and assess its predictive value for treatment outcomes to adjuvant bio-chemoradiotherapy. Methods: Eighty one resection specimens from patients treated surgically for stage III or IV human papillomavirus-negative (HPV(-)) HNSCC with high-risk pathologic features, who received either Arm 1) radiotherapy(RT)+cetuximab(CTX)+cisplatin or Arm 2) RT+CTX+docetaxel, as adjuvant treatment in a phase II randomized clinical trial (RTOG 0234) underwent TP53 targeted sequencing, and EAp53 scoring. The EAp53 scores were correlated with clinical outcomes. Due to limited sample sizes, patients were combined into 2 EAp53 groups: wt/low and high/other. Results: At median follow-up of 10 years, there was a significant interaction between treatment and EAp53 group for overall survival (OS) (p = 0.008), disease-free survival (DFS) (p = 0.05) and distant metastasis (DM) (p = 0.004). Within arm 2, high/other showed worse OS [HR 4.69 (1.52-14.50)], DFS [HR 2.69 (1.16-6.21)], and had higher DM [HR 11.71 (1.50-91.68)] than wt/low. Within arm 1, there was no significant difference by EAp53 in OS, DFS and DM. Within the wt/low group, arm 2 had better OS [HR 0.11 (0.03-0.36)], DFS [HR 0.24 (0.09-0.61)], and DM [HR 0.04 (0.01-0.31)] than arm 1 but this was not found in high/other. Conclusions: High/other EAp53 scores were associated with worse survival for patients in arm 2. Arm 2 is associated with better survival than arm 1 for patients with wt/low EAp53. This benefit appears to be largely driven by a reduction in DM. Further validation is required to determine whether EAp53 can be used for personalized post-operative treatment decisions in HPV(-) HNSCC.

Abstract Targeting the B-cell receptor and phosphatidylinositol 3-kinase/mTOR signaling pathways has shown meaningful, but incomplete, antitumor activity in lymphoma. Glycogen synthase kinase 3 (GSK3) α and β are 2 homologous and functionally overlapping serine/threonine kinases that phosphorylate multiple protein substrates in several key signaling pathways. To date, no agent targeting GSK3 has been approved for lymphoma therapy. We show that lymphoma cells abundantly express GSK3α and GSK3β compared with normal B and T lymphocytes at the messenger RNA and protein levels. Utilizing a new GSK3 inhibitor 9-ING-41 and by genetic deletion of GSK3α and GSK3β genes using CRISPR/CAS9 knockout, GSK3 was demonstrated to be functionally important to lymphoma cell growth and proliferation. GSK3β binds to centrosomes and microtubules, and lymphoma cells treated with 9-ING-41 become arrested in mitotic prophase, supporting the notion that GSK3β is necessary for the progression of mitosis. By analyzing recently published RNA sequencing data on 234 diffuse large B-cell lymphoma patients, we found that higher expression of GSK3α or GSK3β correlates well with shorter overall survival. These data provide rationale for testing GSK3 inhibitors in lymphoma patient trials.

The NCCN Guidelines for Hepatobiliary Cancers provide treatment recommendations for cancers of the liver, gallbladder, and bile ducts. The NCCN Hepatobiliary Cancers Panel meets at least annually to review comments from reviewers within their institutions, examine relevant new data from publications and abstracts, and reevaluate and update their recommendations. These NCCN Guidelines Insights summarize the panel’s discussion and updated recommendations regarding systemic therapy for first-line and subsequent-line treatment of patients with hepatocellular carcinoma.