Underwent Gene Expression Profiling Research Articles

Molecular and Histopathological Characterization of Metastatic Cutaneous Squamous Cell Carcinomas: A Case-Control Study.

A subset of patients affected by cutaneous squamous cell carcinoma (cSCC) can exhibit locally invasive or metastatic tumors. Different staging classification systems are currently in use for cSCC. However, precise patient risk stratification has yet to be reached in clinical practice. The study aims to identify specific histological and molecular parameters characterizing metastatic cSCC. Patients affected by metastatic and non-metastatic cSCC (controls) were included in the present study and matched for clinical and histological characteristics. Skin samples from primary tumors were revised for several histological parameters and also underwent gene expression profiling with a commercially available panel testing 770 different genes. In total, 48 subjects were enrolled in the study (24 cases, 24 controls); 67 genes were found to be differentially expressed between metastatic and non-metastatic cSCC. Most such genes were involved in immune regulation, skin integrity, angiogenesis, cell migration and proliferation. The combination of histological and molecular profiles of cSCCs allows the identification of features specific to metastatic cSCC, with potential implications for more precise patient risk stratification.

Influence of TP53 Comutation on the Tumor Immune Microenvironment and Clinical Outcomes With Immune Checkpoint Inhibitors in STK11-Mutant Non-Small-Cell Lung Cancer.

Non-small-cell lung cancer (NSCLC) with STK11mut has inferior outcomes to immune checkpoint inhibitors (ICIs). Using multiomics, we evaluated whether a subtype of STK11mut NSCLC with a uniquely inflamed tumor immune microenvironment (TIME) harboring TP53 comutations could have favorable outcomes to ICIs. NSCLC tumors (N = 16,896) were analyzed by next-generation sequencing (DNA-Seq/592 genes). A subset (n = 5,034) underwent gene expression profiling (RNA-Seq/whole transcriptome). Exome-level neoantigen load for STK11mut NSCLC was obtained from published pan-immune analysis. Tumor immune cell content was obtained from transcriptome profiles using the microenvironment cell population (MCP) counter. ICI data from POPLAR/OAK (n = 34) and the study by Rizvi et al (n = 49) were used to model progression-free survival (PFS), and a separate ICI-treated cohort (n = 53) from Dana-Farber Cancer Institute (DFCI) was used to assess time to treatment failure (TTF) and tumor RECIST response for STK11mutTP53mut versus STK11mutTP53wt NSCLC. Overall, 12.6% of NSCLC tumors had a STK11mut with the proportions of tumor mutational burden (TMB)-high (≥10 mut/Mb), PD-L1 ≥50%, and microsatellite instability-high being 38.3%, 11.8%, and 0.72%, respectively. Unsupervised hierarchical clustering of STK11mut (n = 463) for stimulator of interferon-gamma (STING) pathway genes identified a STING-high cluster, which was significantly enriched in TP53mut NSCLC (P < .01). Compared with STK11mutTP53wt, tumors with STK11mutTP53mut had higher CD8+T cells and natural killer cells (P < .01), higher TMB (P < .001) and neoantigen load (P < .001), and increased expression of MYC and HIF-1A (P < .01), along with higher expression (P < .01) of glycolysis/glutamine metabolism genes. Meta-analysis of data from OAK/POPLAR and the study by Rizvi et al showed a trend toward improved PFS in patients with STK11mutTP53mut. In the DFCI cohort, compared with the STK11mut TP53wt cohort, the STK11mutTP53mut tumors had higher objective response rates (42.9% v 16.7%; P = .04) and also had longer TTF (14.5 v 4.5 months, P adj = .054) with ICI. STK11mut NSCLC with TP53 comutation is a distinct subgroup with an immunologically active TIME and metabolic reprogramming. These properties should be exploited to guide patient selection for novel ICI-based combination approaches.

Integrative transcriptomic and metabolomic analysis reveals alterations in energy metabolism and mitochondrial functionality in broiler chickens with wooden breast

This integrative study of transcriptomics and metabolomics aimed to improve our understanding of Wooden Breast myopathy (WB). Breast muscle samples from 8 WB affected and 8 unaffected male broiler chickens of 47 days of age were harvested for metabolite profiling. Among these 16 samples, 5 affected and 6 unaffected also underwent gene expression profiling. The Joint Pathway Analysis was applied on 119 metabolites and 3444 genes exhibiting differential abundance or expression between WB affected and unaffected chickens. Mitochondrial dysfunctions in WB was suggested by higher levels of monoacylglycerols and down-regulated genes involved in lipid production, fatty acid beta oxidation, and oxidative phosphorylation. Lower levels of carnosine and anserine, along with down-regulated carnosine synthase 1 suggested decreased carnosine synthesis and hence impaired antioxidant capacity in WB. Additionally, Weighted Gene Co-expression Network Analysis results indicated that abundance of inosine monophosphate, significantly lower in WB muscle, was correlated with mRNA expression levels of numerous genes related to focal adhesion, extracellular matrix and intercellular signaling, implying its function in connecting and possibly regulating multiple key biological pathways. Overall, this study showed not only the consistency between transcript and metabolite profiles, but also the potential in gaining further insights from analyzing multi-omics data.

Characterisation of immune checkpoints in Richter syndrome identifies LAG3 as a potential therapeutic target.

Richter syndrome (RS), an aggressive lymphoma occurring in the context of chronic lymphocytic leukaemia/small lymphocytic lymphoma, is associated with poor prognosis when treated with conventional immunochemotherapy, therefore, improved treatments are required. Immune checkpoint blockade has shown efficacy in some B-cell malignancies and modest responses in early clinical trials for RS. We investigated the immune checkpoint profile of RS as a basis to inform rational therapeutic investigations in RS. Formalin-fixed, paraffin-embedded biopsies of RS (n=19), de novo diffuse large B-cell lymphoma (DLBCL; n=58), transformed indolent lymphomas (follicular [tFL], n=16; marginal zone [tMZL], n=24) and non-transformed small lymphocytic lymphoma (SLL; n=15) underwent gene expression profiling using the NanoString Human Immunology panel. Copy number assessment was performed using next-generation sequencing. Immunohistochemistry (IHC) for LAG3 and PD-1 was performed. LAG3 gene expression was higher in RS compared to DLBCL (P=0·0002, log2FC 1·96), tFL (P<0·0001, log2FC 2·61), tMZL (P=0·0004, log2FC 1·79) and SLL (P=0·0057, log2FC 1·45). LAG3 gene expression correlated with the gene expression of human leukocyte antigen Class I and II, and related immune genes and immune checkpoints. IHC revealed LAG3 protein expression on both malignant RS cells and tumour-infiltrating lymphocytes. Our findings support the investigation of LAG3 inhibition to enhance anti-tumour responses in RS.

Generation of Patient-Specific Autologous Liver and Hepatocellular Carcinoma for Development of Cancer Therapies

Introduction: The dire prognosis for patients with advanced Hepatocellular Carcinoma (HCC) is due to paucity of therapeutic options. For optimal patient care, it is critical to develop novel therapies tailored in a patient-specific fashion. This personalized approach requires preclinical models that faithfully recapitulate a patient's cancer and its microenvironment. For accurate evaluation of HCC therapies, we have developed conditions that allow us to grow HCC and non-cancerous hepatocytes in culture in a patient-specific fashion. Hypothesis: Patient-derived proliferating normal hepatocytes and HCC cell lines will retain the genomic landscapes of the patient's liver and tumor, respectively. Methods: Cancer and surrounding non-cancerous tissue from clinical HCC resections were collected. From these tissues, we isolated and expanded cancerous and non-cancerous cells. We developed culture methods to induce proliferation of primary human hepatocytes via generation of small hepatocyte proliferating cells (SHPCs). During cellular expansion, the SHPCs underwent gene expression profiling via RNA-sequencing at passages 0 and 3. Results: We isolated cancerous (Fig.1A) and non-cancerous hepatocytes from HCC resection specimens and cultured these cells to grow HCC and SHPCs. After in vitro expansion, SHPCs maintained expression of hepatocyte specific genes after passaging (Fig.1B). Conclusions: Expansion of human hepatocytes is an innovative technology to generate a proliferating hepatocyte population that maintains its gene expression profile over passages. Our goal is to humanize mouse livers with these non-cancerous hepatocytes followed by transplantation of tumor cells, from the same patient, to recreate HCC and tumor microenvironment to develop personalized therapies.

Distinct Molecular Profiles and Immunotherapy Treatment Outcomes of V600E and V600K BRAF-Mutant Melanoma.

BRAF V600E and V600K melanomas have distinct clinicopathologic features, and V600K appear to be less responsive to BRAFi±MEKi. We investigated mechanisms for this and explored whether genotype affects response to immunotherapy. Pretreatment formalin-fixed paraffin-embedded tumors from patients treated with BRAFi±MEKi underwent gene expression profiling and DNA sequencing. Molecular results were validated using The Cancer Genome Atlas (TCGA) data. An independent cohort of V600E/K patients treated with anti-PD-1 immunotherapy was examined. Baseline tissue and clinical outcome with BRAFi±MEKi were studied in 93 patients (78 V600E, 15 V600K). V600K patients had numerically less tumor regression (median, -31% vs. -52%, P = 0.154) and shorter progression-free survival (PFS; median, 5.7 vs. 7.1 months, P = 0.15) compared with V600E. V600K melanomas had lower expression of the ERK pathway feedback regulator dual-specificity phosphatase 6, confirmed with TCGA data (116 V600E, 17 V600K). Pathway analysis showed V600K had lower expression of ERK and higher expression of PI3K-AKT genes than V600E. Higher mutational load was observed in V600K, with a higher proportion of mutations in PIK3R1 and tumor-suppressor genes. In patients treated with anti-PD-1, V600K (n = 19) had superior outcomes than V600E (n = 84), including response rate (53% vs. 29%, P = 0.059), PFS (median, 19 vs. 2.7 months, P = 0.049), and overall survival (20.4 vs. 11.7 months, P = 0.081). BRAF V600K melanomas appear to benefit less from BRAFi±MEKi than V600E, potentially due to less reliance on ERK pathway activation and greater use of alternative pathways. In contrast, these melanomas have higher mutational load and respond better to immunotherapy.

The prognostic impact of consensus molecular subtypes (CMS) and its predictive effects for bevacizumab benefit in metastatic colorectal cancer: molecular analysis of the AGITG MAX clinical trial

The consensus molecular subtypes (CMS) is a transcriptome-based classification of colorectal cancer (CRC) initially described in early-stage cohorts, but the associations of CMS with treatment outcomes in the metastatic setting are yet to be established. This study aimed to evaluate the prognostic impact of CMS classification and its predictive effects for bevacizumab benefit in metastatic CRC by correlative analysis of the AGITG MAX trial. The MAX trial previously reported improved progression-free survival (PFS) for the addition of bevacizumab (B) to chemotherapy [capecitabine (C)±mitomycin (M)]. Archival primary tumours from 237 patients (50% of trial population) underwent gene expression profiling and classification into CMS groups. CMS groups were correlated to PFS and overall survival (OS). The interaction of CMS with treatment was assessed by proportional hazards model. The distribution of CMS in MAX were CMS1 18%, CMS2 47%, CMS3 12%, CMS4 23%. CMS1 was the predominant subtype in right-sided primary tumours, while CMS2 was the predominant subtype in left-sided. CMS was prognostic of OS (P = 0.008), with CMS2 associated with the best outcome and CMS1 the worst. CMS remained an independent prognostic factor in a multivariate analysis. There was a significant interaction between CMS and treatment (P-interaction = 0.03), for PFS, with hazard ratios (95% CI) for CB+CBM versus C arms in CMS1, 2, 3 and 4: 0.83 (0.43-1.62), 0.50 (0.33-0.76), 0.31 (0.13-0.75) and 1.24 (0.68-2.25), respectively. This exploratory study found that CMS stratified OS outcomes in metastatic CRC regardless of first-line treatment, with prognostic effects of CMS groups distinct from those previously reported in early-stage cohorts. In CMS associations with treatment, CMS2 and possibly CMS3 tumours may preferentially benefit from the addition of bevacizumab to first-line capecitabine-based chemotherapy, compared with other CMS groups. Validation of these findings in additional cohorts is warranted. This is a molecular sub-study of MAX clinical trial (NCT00294359).

Abstract 3141: Immune gene expression and prognosis in localized clear cell (cc) renal cell carcinoma (RCC)

Abstract Background: We previously reported that increased infiltration of immune cells (lymphocytes, plasma cells, macrophages, neutrophils) in localized ccRCC is associated with recurrence. In these tumors we now study the immune gene expression to identify a signature to predict recurrence. Methods: We identified pts with ccRCC with &amp;gt;T1b tumor who underwent nephrectomy for whom we had annotation for objective tumor recurrence and a minimum follow-up of 2 years. All histologic slides were examined and tumor section with the highest amount of intratumoral immune infiltration were selected from each tumor for gene expression profiling. Total mRNA isolated from the macrodissected FFPE tumor underwent gene expression profiling for 770 genes in the Immune Profile Panel by NanoString Technologies. Digital raw counts of mRNA abundance were normalized using positive controls as well as 16 housekeeping genes. The mean signals were used to calculate fold change in gene expression between recurrers and non-recurrers, and a p-value &amp;lt;0.05 using empirical Bayes methods was considered significant. Cox regression was also conducted to correlate gene expression with time to recurrence. Results: Of 132 patients (pts), 24 (18%) had recurrence and 108 did not. The median age of pts was 59 years and 57 were female. The median time to recurrence was 25.7 months. Fifty pts had low immune infiltration score and 82 pts had high infiltration based on previously conducted morphologic assessment. Only 4 genes (IL8, NCAM1, ARG2, PPBP) had &amp;gt;1.5 times increased mean expression in recurrers vs non-recurrers (p&amp;lt;0.05). Only 5 genes (CX3CL1, HLA-G, VCAM1, IL17RB, CXCL14) had &amp;gt;1.5 times decreased mean expression in recurrers vs non-recurrers (p&amp;lt;0.05). Overexpression of the following immunoregulatory genes was associated with a shorter time to recurrence: TGFB1, STAT3, NFATC4, B7-H3, ADORA2A, OX-40L, IL10, RUNX1, LILRA4, MARCO, CR1, FCER2, IL2RA (HR 1.5-3.6, p&amp;lt;0.05). When compared to pts with low lymphocyte infiltration, pts with high lymphocyte infiltration overexpressed T cell (CD3E, CD5), cytotoxic T cell (CD8B, SLAMF6, IL2RB, GZMK, TCF7, CTSW, EBI3), Th1 (CD38, CXCR3, CXCR6, IL16), Th2 (ITK) and Tregs (IL2RB, IRF4, TIGIT) genes (FDR p&amp;lt;0.01). Conclusions: Specific immune related somatic genes were differentially expressed based on recurrence and time to recurrence in patients with localized ccRCC undergoing surgery. With further validation, these genes warrant functional validation since they may represent therapeutic targets in the perioperative setting. The results also highlight the heterogeneity of immune cell infiltration. Citation Format: Pooja Ghatalia, Karthik Devarajan, Jennifer Gordetsky, Essel Dulaimi, Sejong Bae, Gurudatta Naik, Guru Sonpavde, Elizabeth Plimack. Immune gene expression and prognosis in localized clear cell (cc) renal cell carcinoma (RCC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3141.

Abstract A071: AG-120 (ivosidenib), a first-in-class mutant IDH1 inhibitor, promotes morphologic changes and upregulates liver-specific genes in IDH1 mutant cholangiocarcinoma

Abstract Background: Somatic mutations in the isocitrate dehydrogenase 1 (IDH1) gene have been reported in ~25% of patients with intrahepatic cholangiocarcinoma (CC). The mutant IDH1 (mIDH1) enzyme has a gain-of-function activity, catalyzing the reduction of alpha-ketoglutarate to D-2-hydroxyglutarate (2-HG). Accumulation of 2-HG leads to epigenetic dysregulation, and potential silencing of genes that control cellular differentiation. In a preclinical model of liver development, IDH1 mutation resulted in HNF4A silencing and impaired hepatocyte differentiation. AG-120 (ivosidenib) is a first-in-class, oral, selective inhibitor of mIDH1 that has been shown to reduce 2-HG levels in plasma and tumor tissue in preclinical and clinical studies, and to induce differentiation of leukemic blasts in patients with acute myeloid leukemia. In solid tumors, the consequences of IDH1 mutations and the effects of inhibition are less understood. In AG120-C-002 (ClinicalTrials.gov NCT02073994), a phase 1 clinical trial in mIDH1 advanced solid tumors, 73 patients with CC were treated with AG-120 at doses ranging from 100 mg twice daily to 1200 mg once daily. Among the phase 1 CC subjects who underwent on-study biopsies, we assessed morphologic and gene expression profile changes to understand the relationship between these parameters. Methods: Biopsy samples were collected from patients with CC enrolled in AG120-C-002 at screening, Cycle 3 Day 1, and in some cases Cycle 7 Day 1, disease progression, and end of treatment. Fourteen pre- and post-dose formalin-fixed paraffin-embedded (FFPE) tumor sample pairs were evaluable for morphology assessment (from 27 FFPE pre- and post-dose pairs collected), and 19 pre- and post-dose fresh frozen tumor sample pairs underwent gene expression profiling (from 38 pre- and post-dose pairs collected). Both morphology and gene expression data were assessed in eight patients to elucidate their relationship. Hematoxylin and eosin stained sections were evaluated for a cholangiocellular growth pattern and cytoplasmic alterations by two gastrointestinal pathologists from independent institutions. Gene expression profiles were generated by RNA sequencing to relate to morphologic changes. Results: Morphologic assessment of 14 pre- and post-dose sample pairs showed that upon 8-24 weeks of AG-120 treatment, a subset of CCs exhibited change in two phenotypes: (a) an increase of 20% or more in the cholangiocellular growth pattern (n=5), and (b) a reduction in the volume of cytoplasm (n=8). Changes in both phenotypes (a) and (b) were seen in five cases. RNA sequence profiling in samples with increased cholangiolar histology also showed a trend toward increased liver-specific gene expression. Conclusions: This is the first demonstration that AG-120 treatment may induce morphologic and molecular changes in a subset of mIDH1 CCs. Correlation of changes in morphology and gene expression with specific clinical outcomes will be described. Further studies, such as methylation changes, protein change by immunohistochemistry, and preclinical studies, are warranted to understand the biology of these morphologic and gene expression changes. Citation Format: Yuko Ishii, Carlie Sigel, Maeve A. Lowery, Lipika Goyal, Camelia Gliser, Liewen Jiang, Susan Pandya, Bin Wu, Sung Choe, Vikram Deshpande. AG-120 (ivosidenib), a first-in-class mutant IDH1 inhibitor, promotes morphologic changes and upregulates liver-specific genes in IDH1 mutant cholangiocarcinoma [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A071.

Ph-like acute lymphoblastic leukemia: a high-risk subtype in adults

AbstractPhiladelphia chromosome (Ph)-like acute lymphoblastic leukemia (ALL) is a high-risk subtype of ALL in children. There are conflicting data on the incidence and prognosis of Ph-like ALL in adults. Patients with newly diagnosed B-cell ALL (B-ALL) who received frontline chemotherapy at MD Anderson Cancer Center underwent gene expression profiling of leukemic cells. Of 148 patients, 33.1% had Ph-like, 31.1% had Ph+, and 35.8% had other B-ALL subtypes (B-other). Within the Ph-like ALL cohort, 61% had cytokine receptor-like factor 2 (CRLF2) overexpression. Patients with Ph-like ALL had significantly worse overall survival (OS), and event-free survival compared with B-other with a 5-year survival of 23% (vs 59% for B-other, P = .006). Sixty-eight percent of patients with Ph-like ALL were of Hispanic ethnicity. The following were associated with inferior OS on multivariable analysis: age (hazard ratio [HR], 3.299; P < .001), white blood cell count (HR, 1.910; P = .017), platelet count (HR, 7.437; P = .005), and Ph-like ALL (HR, 1.818; P = .03). Next-generation sequencing of the CRLF2+ group identified mutations in the JAK-STAT and Ras pathway in 85% of patients, and 20% had a CRLF2 mutation. Within the CRLF2+ group, JAK2 mutation was associated with inferior outcomes. Our findings show high frequency of Ph-like ALL in adults, an increased frequency of Ph-like ALL in adults of Hispanic ethnicity, significantly inferior outcomes of adult patients with Ph-like ALL, and significantly worse outcomes in the CRLF2+ subset of Ph-like ALL. Novel strategies are needed to improve the outcome of these patients.

Mesenchymal Stem Cells Preconditioned with Myeloma Cells from High-Risk Patients Support the Growth of Myeloma Cells from Low-Risk Patients

Introduction:Each disease stage in myeloma (MM) is associated with parallel changes in both the MM clone and the bone marrow (BM) microenvironment. Mesenchymal cell lineages derived from mesenchymal stem cells (MSCs), including osteoblasts, adipocytes and pericytes play an important role in MM cell growth mediated by the modification of the MM niche in the BM.The overall goal of the study was to test and identify changes induced in MSCs by high-risk (HR) MM cells that impact MSC function and promote oncogenic pathways capable of supporting low-risk (LR) MM cells.Methods:Normal MSCs were either cultured alone ("unconditioned") or co-cultured with MM cells for 5 days. The cultured and co-cultured cells were trypsinized, replated for 40 min, followed by serial washing to remove MM cells from the adherent MSCs. More than 95% of the remaining adherent cells after co-culture were MSCs ("preconditioned"). The unconditioned and preconditioned MSCs or their 24 hrs conditioned media (CM; 50%) were tested for their ability to support the 5-days growth of CD138+ MM cells from LR (n=4) and HR (n=3) patients.To identify factors altered in MSCs by HR MM cells, the unconditioned and preconditioned MSCs and their serum-free conditioned media (n=4) underwent gene expression profiling and proteomic analysis. Whole bone biopsy gene expression profiles from newly diagnosed patients with MM enrolled in Total Therapy clinical trials were used to correlate the altered expression of factors in preconditioned MSCs with their expression in clinical samples.Results:Growth of all MM cells tested was increased by inclusion of MSCs preconditioned with HR MM cells by 2.2± 0.2 (p<0.0004) and by CM from these MSCs by 9.6±2.0 (p<0.006), compared to culture of MM cells in fresh media. In contrast, CM from unconditioned MSCs increased growth of HR MM cells by 2.6±0.6 (p<0.01) fold but had minor effect on growth of LR MM cells. CM from MSCs preconditioned with HR MM cells increased growth of LR and HR MM cells by 5.7±0.1 (p<0.0002) and 2.6±1.2 (p<0.04), compared to culture of MM cells in CM from unconditioned MSCs, respectively. Growth of LR MM cells was higher by 2.9±0.3 fold using CM from MSCs preconditioned with HR MM cells than by using CM from MSCs preconditioned with LR MM cells (p<0.005).To determine the role of cell-cell contact, we compared the effect of the preconditioned MSCs and their CM on growth of LR and HR MM cells. Growth of LR MM cells (p< 0.003) and HR MM cells (p< 0.005) was higher when cultured in CM than in co-culture with MSCs. These data imply that soluble factors from preconditioned MSCs support MM cell proliferation and that adhesion of MM cells to MSCs may restrain proliferation.Genes overexpressed in preconditioned MSCs included growth factors (e.g. IL6) and receptors (e.g. EDNRA); genes underexpressed include factors associated with activity of osteoblasts (e.g. ITGBL1) and adipocytes (IGFBP2).A proteomic analysis showed a reduced level of the secreted factors IGFBP2 and ITGBL1 and increase level of IL6 in CM from MSCs preconditioned with HR MM cells compared to CM from unconditioned MSCs. IGFBP2 mediates local bioavailability of IGF1 and IGF2 and is also involved in bone formation and angiogenesis independently of the IGF axis. ITGBL1 is involved in osteoblastogenesis whereas EDNRA is known to be expressed by pericytes.Global gene expression profiles from patient material showed that EDNRA and IGFBP2 are not expressed in MM cells but are highly expressed in cultured MSCs compared to hematopoietic cells in buffy coat BM samples. EDNRA is overexpressed (p<0.005) whereas IGFBP2 is underexpressed (p<0.005) in whole BM biopsy samples from MM patients with HR disease compared to patients with LR disease (p<0.005) and in Focal Lesion compared to random BM biopsies taken from the same patients (p<0.0000006 for EDNRA and p<0.02 for IGFBP2).IHC staining of patients’ bone biopsies showed higher numbers of EDNRA+ mesenchymal-like cells in MM (n=10) than MGUS/AMM (n=10, p<0.0003) and in HR MM BM than LR MM BM (p<0.03). IHC analysis also revealed that IGFBP2 is highly expressed by immature adipocytes and that its expression in these cells is reduced in HR MM BM.Conclusion:Preconditioning of MSCs is essential for promoting growth of MM cells from LR patients. Factors altered in MSCs by HR MM cells are linked to signaling pathways known to directly stimulate MM cell growth and markers associated with distinct MSC lineages changed in HR MM niche. DisclosuresDavies:Janssen: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Celgene: Consultancy, Honoraria. Barlogie:Signal Genetics: Patents & Royalties. Morgan:Janssen: Research Funding; Univ of AR for Medical Sciences: Employment; Celgene: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria; Bristol Meyers: Consultancy, Honoraria.

High-Risk Subtype of Ph-like Acute Lymphoblastic Leukemia (ALL) in Adults: Dismal Outcomes of CRLF2+ ALL Patients Treated with Intensive Chemotherapy

Background:Ph-like acute lymphoblastic leukemia (ALL) is a high-risk subtype of ALL in children. There are limited and conflicted data on the incidence and prognosis of Ph-like ALL in adults.Methods:Patients with newly-diagnosed B-ALL who received frontline chemotherapy at MD Anderson Cancer Center underwent gene expression profiling of leukemic cells to identify Ph-like ALL. Gene expression profiling was performed on 148 RNA samples using either U133 Plus 2.0 microarrays, or a customized Taqman low density array (LDA) card to identify patients with the Ph-like ALL gene signature (Roberts et al. NEJM 2014). An additional 7 previously untreated patients were found to have CRLF2 overexpression by multicolor flow-cytometry (MFC), and received induction chemotherapy at MDACC were included in the outcome analysis (but not for subtype frequency calculation). We performed targeted sequencing of 303 recurrently mutated genes (L300 panel, MDACC) in 40 patients with CRFL2 rearrangements (15 with matched germline control). Minimal residual disease (MRD) was assessed by MFC, with a sensitivity of 0.01%.Results:Of 148 patients, 49 (33.1%) were Ph-like, 46 patients (31.1%) were Ph+, and 53 patients (35.8%) were of other B-ALL subtypes (B-other). The median age of Ph-like cohort was 33.5 years (range, 15-71), Ph+ cohort was 49 years (range, 22-84), and B-other was 38 years (range, 15-79). Within the Ph-like ALL cohort, 61% had overexpression of CRLF2. Patients received hyper-CVAD (80%) or an augmented-BFM regimen (20%). The rate of CR/CRp was similar in the 3 disease subgroups (Ph-like ALL 89%, Ph+ ALL 93%, B-other 94%, p = 0.57). However, patients with Ph-like ALL were significantly less likely to achieve MRD-negative remission (30% vs. 56% for Ph+ ALL vs. 87% for B-other, p <0.001). Patients with Ph-like ALL had significantly worse overall survival (OS) and event-free survival (EFS) compared to B-other with a 5-year survival of 23% (vs. 59% for B-other, p=0.006) (Figure 1A). The poor outcomes of Ph-like ALL were also observed when only hyper-CVAD treated patients were considered. Interestingly, 68% of the patients with Ph-like ALL (78% among the CRLF2+ cohort) were of Hispanic ethnicity. This was significantly higher compared to Ph+ ALL (35%) and B-other (30%), p <0.001. Patients with CRLF2 overexpression had significantly inferior OS, EFS, and remission duration when compared to other genomic subgroups, including the Ph-like non-CRLF2 group (Figure 1B). Notably, 5-year survival in the CRLF2+ group was <20%. The following were independently associated with inferior OS on multivariable analysis: age (hazard ratio [HR] 2.474, p<0.001); WBC count (HR 1.183, p=0.007); platelet count (HR 4.283, p<0.001) and Ph-like ALL (HR 1.579, p=0.04) (Table 1).The most common mutations by L300 sequencing of 40 patients with CRLF2 were JAK2 (n=19, 47.5%), KRAS (n=10, 25%), CRLF2 (n=7, 17.5%), NRAS (n=5, 12.5%), PAX5 (n=5, 12.5%), JAK1 (n=4, 10%) (Figure 2). The CRLF2 F232C mutation, noted in 7 (17.5%) patients in this study, appears more frequent than in pediatric patients (3/134, 2.2%, Chen et al. Blood 2012), and in range with a smaller adult series (3/14, 21.4%, Yoda et al. PNAS 2010). CRLF2 F232C mutations were mutually exclusive with JAK2/JAK1 mutations (except in one patient).Conclusions:Our findings show a high frequency of Ph-like ALL in adults; an increased frequency of Ph-like ALL in adults with Hispanic ethnicity; significantly inferior outcomes of adult patients with Ph-like ALL; and significantly worse outcomes in Ph-like ALL patients with CRLF2 overexpression. The frequency of CRLF2 F232C mutation appears to be higher in adult patients with B-ALL than in the children. Ph-like ALL represents a high-risk disease subtype of adult B-ALL. Novel strategies are needed to improve the outcome of these patients. [Display omitted] [Display omitted] [Display omitted] DisclosuresJain:Pharmacyclics: Consultancy, Honoraria, Research Funding; Genentech: Research Funding; Incyte: Research Funding; BMS: Research Funding; Abbvie: Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Celgene: Research Funding; ADC Therapeutics: Consultancy, Honoraria, Research Funding; Seattle Genetics: Research Funding; Servier: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Infinity: Research Funding; Novimmune: Consultancy, Honoraria. Jabbour:ARIAD: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Novartis: Research Funding; BMS: Consultancy. Cortes:ARIAD: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Teva: Research Funding. O'Brien:Pharmacyclics, LLC, an AbbVie Company: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria. Mullighan:Incyte: Membership on an entity's Board of Directors or advisory committees; Amgen: Speakers Bureau; Loxo Oncology: Research Funding. Konopleva:Reata Pharmaceuticals: Equity Ownership; Abbvie: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; Stemline: Consultancy, Research Funding; Eli Lilly: Research Funding; Cellectis: Research Funding; Calithera: Research Funding.

Feasibility of the AML profiler (Skyline™ Array) for patient risk stratification in a multicentre trial: a preliminary comparison with the conventional approach.

Deoxyribonucleic acid microarrays allow researchers to measure mRNA levels of thousands of genes in a single experiment and could be useful for diagnostic purposes in patients with acute myeloid leukaemia (AML). We assessed the feasibility of the AML profiler (Skyline™ Array) in genetic stratification of patients with de novo AML and compared the results with those obtained using the standard cytogenetic and molecular approach. Diagnostic bone marrow from 31 consecutive de novo AML cases was used to test MLL-PTD, FLT3-ITD and TKD, NPM1 and CEBPAdm mutations. Purified RNA was used to assess RUNX1-RUNX1T1, PML-RARα and CBFβ-MYH11 rearrangements. RNA remnants underwent gene expression profiling analysis using the AML profiler, which detects chromosomal aberrations: t(8;21), t(15;17), inv(16), mutations (CEBPAdm, ABD-NPM1) and BAALC and EVI1 expression. Thirty cases were successfully analysed with both methods. Five cases had FLT3-ITD. In one case, a t(8;21) was correctly detected by both methods. Four cases had inv(16); in one, the RNA quality was unsatisfactory and it was not hybridized, and in the other three, the AML profiler detected the genetic lesion - this being a rare type I translocation in one case. Two cases with acute promyelocytic leukaemia were diagnosed by both methods. Results for NPM1 mutations were concordant in all but two cases (2/11, non-ABD mutations). Analysis of costs and turnaround times showed that the AML profiler was no more expensive than the conventional molecular approach. These results suggest that the AML profiler could be useful in multicentre trials to rapidly identify patients with AML with a good prognosis. Copyright © 2016 John Wiley & Sons, Ltd.

AKT expression is associated with degree of pathologic response in adenocarcinoma of the esophagus treated with neoadjuvant therapy.

Neoadjuvant chemoradiation (NCRT) has become standard in the treatment of locally advanced esophageal adenocarcinoma (EAC) with survival correlated to degree of pathologic response. The phosphatidyl inositol 3 kinase (PI3K)/protein kinase B (AKT)/mTOR pathway plays an important role in tumorgenesis and resistance. We sought to elucidate the role of this pathway in patients with EAC who received NCRT. After IRB approval, a prospective trial was initiated in which patients with EAC underwent endoscopic biopsies of normal and tumor tissue prior to instituting NCRT. Patients then proceeded to esophagectomy. The pre-treatment tissues underwent gene expression profiling. SAM method was used to analyze expression of AKT within normal and tumor tissue. Expression was then correlated to degree of pathologic response. One-hundred patients were consented for the study, of which 67 met final eligibility. Nineteen patient's tumors ultimately underwent gene expression profiling via microarray. The differential expression of all AKT isoforms in tumor tissue was markedly overexpressed compared to normal tissue (P=6×10(-5)). There were 3 patients designated as pNR, 6 as pPR, and 10 as pCR. Partial and non-responders had higher expressions of AKT compared to pCR with the non-responders consistently illustrated the highest expression of AKT (P=0.02). There was a significant correlation between individual isoforms of AKT-1, AKT-2, and AKT-3 and degree of pathologic response (P=0.002, 0.04, and 0.04 respectively). AKT is overexpressed in patients with AC of the esophagus. Moreover, pathologic response to NCRT may be correlated with degree of AKT expression. Additional data is needed to clarify this relationship to potentially add targeted therapies to the neoadjuvant regimen.

Surrogate molecular markers for IGHV mutational status in chronic lymphocytic leukemia for predicting time to first treatment

ZAP-70 is a marker of clinical outcome in chronic lymphocytic leukemia (CLL), however its assessment suffers from a lack of standardization consensus. To identify novel markers able to surrogate IGHV mutational status, CD19(+)CD5(+)-B-lymphocytes from 216 patients enrolled in a prospective study (ClinicalTrial.gov Identifier:NCT00917540), underwent gene expression profiling. Samples were split into CLL-Training (n=102) and CLL-Validation (n=114) sets, and an independent supervised analysis for IGHV mutational status was performed considering all genes with gene expression equal or above that of ZAP-70. Thirty-one genes (23 up- and 8 down-regulated) and 23 genes (18 up- and 5 down-regulated) satisfied these criteria in the CLL-Training and CLL-Validation sets, respectively, and 20 common genes (15 up and 5 down) were found to be differentially regulated in both sets. Two (SNORA70F, NRIP1) of the down-regulated and 6 (SEPT10, ZNF667, TGFBR3, MBOAT1, LPL, CRY1) of the up-regulated genes were significantly associated with a reduced risk of disease progression in both sets. Forcing the afore-mentioned genes in a Cox multivariate model together with IGHV mutational status, only CRY1 (HR=2.3, 95% CI: 1.1–4.9, P=.027) and MBOAT1 (HR=2.1, 95% CI: 1.1–3.7, P=.018) retained their independent prognostic impact, supporting the hypothesis that these genes may potentially act as surrogates for predicting IGHV mutational status.

Abstract P6-01-10: Prognostic significance of breast cancer index (BCI) in node-positive hormone receptor positive early breast cancer: NCIC CTG MA.14

Abstract Background: The continuous linear Breast Cancer Index (BCI) risk index combines the ratio of genes HOXB13 to IL17BR (H/I) and the molecular grade index (MGI) (Zhang et al, Clinical Cancer Research, 2013). The BCI signature was developed for node-negative breast cancer patients treated with tamoxifen. We examine here whether linear BCI is prognostic for node-positive hormone-receptor positive tamoxifen-treated patients. Methods: MA.14 randomly assigned 667 hormone positive (HR+), postmenopausal women to 5 years of tamoxifen (TAM) +/- 2 years of octreotide LAR (TAM-OCT). A representative subgroup of 299 patients underwent gene expression profiling by RT-PCR for linear BCI. We performed exploratory analyses restricted to node positive patients. The primary objective was to assess the prognostic effect of BCI on relapse-free survival (RFS). RFS was defined as the time from randomization to the time of recurrence of the primary disease alone, including local and ipsilateral nodal recurrence and metastatic disease, and censoring at longest follow-up or death from another cause. With a median 9.8 years follow-up, the association of BCI with RFS was assessed by multivariate Cox regression including treatment, stratification factors (other than nodal status), and baseline patient and tumor characteristics. Patients were defined to be low risk based on BCI if the adjusted Cox survival was &amp;gt;95%, where adjustment was by trial treatment, stratification factors, and baseline patient and tumor characteristics, including IGF-1, IGFBP-3, and C-peptide. Results: 292 of 299 patient samples passed internal analytical quality control; 116 node positive ER+ve patients had 34 (29.3%) relapses, with adjusted Cox survival at 9.6 years of 87.8%. Fifty-two of the 116 patients (45%) did not receive adjuvant chemotherapy, and experienced 11 (21%) RFS events. In the 116 patients, higher continuous BCI value was associated with shorter RFS (p=0.002): hazard ratio (HR) 1.49 (95% CI 1.16-1.91). Smaller pathologic T had significantly (p=0.03) better RFS HR=0.39, (95%CI 0.17-0.90). With MA.14 patient mean BCI of 5.09532, Cox survival at 4.1 years was 95.2%; 17/34 (50%) who recurred had failed by this time. Discussion: In this subgroup analysis, we found that BCI and tumor size were significant prognostic factors for node-positive hormone-receptor positive patients who were treated with tamoxifen. Citation Format: Dennis Sgroi, Paul Goss, Judy-Anne Chapman, Elizabeth Richardson, Shemeica Binns, Yi Zhang, Cathy Schnabel, Mark Erlander, Kathy Pritchard, Lei Han, Lois Sheperd, Michael Pollack. Prognostic significance of breast cancer index (BCI) in node-positive hormone receptor positive early breast cancer: NCIC CTG MA.14 [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-01-10.

Association of AKT expression with degree of pathologic response in patients with esophageal cancer undergoing neoadjuvant therapy.

89 Background: Neoadjuvant therapy (NCRT) has become standard in the treatment of locally advanced esophageal adenocarcinoma (EAC) with survival correlated to degree of pathologic response. The activation of the PI3K/Akt/mTOR pathway plays an important role in tumorgenesis and resistance to anticancer drugs. The aim of this study was to elucidate the role of AKT in chemoresistance in patients with esophageal adenocarcinoma (AC) who received NCRT. Methods: After IRB approval, a prospective trial was initiated in which patients with EAC requiring NCRT were consented for endoscopic biopsies of normal and tumor tissue prior to instituting therapy. After completion of NCRT patients then proceeded to esophagectomy. The tissues obtained preoperatively underwent gene expression profiling using the Affymetrix 133 Plus 2.0 Gene chip. SAM method was used to analyze significant differentially expression of AKT within normal and tumor tissue. Expression was then correlated to degree of pathologic response. Results: One-hundred patients were consented for the study, of which 67 met final eligibility. Nineteen patient’s tumors ultimately underwent gene expression profiling via microarray. The differential expression of all AKT probesets in tumor tissue was markedly overexpressed compared to normal tissue (p=6x10-5). There were 3 patients designated as pNR, 5 as pPR, and 11 as pCR. Investigating the relationship between the degree of expression of AKT and pathologic response demonstrated a linear correlation between over-expression of AKT and degree of pathologic response. Partial and non-responders had higher expressions of AKT compared to pCR with the non-responders consistently illustrated the highest expression of AKT (p=0.02). There was a significant correlation between individual probesets AKT1, AKT2, and AKT3 and degree of pathologic response (p=0.002, 0.04, and 0.04 respectively). Conclusions: AKT is overexpressed in patients with AC of the esophagus. Moreover, pathologic response to NCRT may be correlated with degree of AKT expression. Additional data is needed to clarify this relationship further and add potential targeted therapies.

Pulsatile exposure to simulated reflux leads to changes in gene expression in a 3D model of oesophageal mucosa

Oesophageal exposure to duodenogastroesophageal refluxate is implicated in the development of Barrett's metaplasia (BM), with increased risk of progression to oesophageal adenocarcinoma. The literature proposes that reflux exposure activates NF-κB, driving the aberrant expression of intestine-specific caudal-related homeobox (CDX) genes. However, early events in the pathogenesis of BM from normal epithelium are poorly understood. To investigate this, our study subjected a 3D model of the normal human oesophageal mucosa to repeated, pulsatile exposure to specific bile components and examined changes in gene expression. Initial 2D experiments with a range of bile salts observed that taurochenodeoxycholate (TCDC) impacted upon NF-κB activation without causing cell death. Informed by this, the 3D oesophageal model was repeatedly exposed to TCDC in the presence and absence of acid, and the epithelial cells underwent gene expression profiling. We identified ~300 differentially expressed genes following each treatment, with a large and significant overlap between treatments. Enrichment analysis (Broad GSEA, DAVID and Metacore™; GeneGo Inc) identified multiple gene sets related to cell signalling, inflammation, proliferation, differentiation and cell adhesion. Specifically NF-κB activation, Wnt signalling, cell adhesion and targets for the transcription factors PTF1A and HNF4α were highlighted. Our data suggest that HNF4α isoform switching may be an early event in Barrett's pathogenesis. CDX1/2 targets were, however, not enriched, suggesting that although CDX1/2 activation reportedly plays a role in BM development, it may not be an initial event. Our findings highlight new areas for investigation in the earliest stages of BM pathogenesis of oesophageal diseases and new potential therapeutic targets.