Laborde, Rebecca R.

The aim of this retrospective study was to describe the oncologic and functional results of treating oropharyngeal squamous cell carcinoma with transoral robotic surgery and neck dissection as monotherapy. A review was performed, including all patients who underwent transoral robotic surgery and neck dissection as the only means of therapy for oropharyngeal carcinoma from March 2007 to July 2009 at a single tertiary care academic medical center. We reviewed all cases with ≥ 24-month follow-up. Functional outcomes included tracheostomy dependence and oral feeding ability. Oncologic outcomes were stratified by human papillomavirus (HPV) status and tobacco use and included local, regional, and distant disease control, as well as disease-specific and recurrence-free survival. Eighteen patients met study criteria. Ten patients (55.6%) were able to eat orally in the immediate postoperative period, and 8 (44.4%) required a temporary nasogastric tube for a mean duration of 13.6 days (range 3 to 24 days) before returning to an oral diet. No patient required placement of a gastrostomy tube, and all patients are tracheostomy-tube–free. Among the HPV-positive nonsmokers (12/18, 66.7%), Kaplan-Meier estimated 3-year local, regional, and distant control rates were 90.9%, 100%, and 100%, respectively. Kaplan-Meier estimated disease-specific survival and recurrence-free survival were 100% and 90.9%, respectively. No complications occurred.

Abstract Background High frequency mutations in both the colony-stimulating factor 3 receptor (CSF3R) and SET binding protein-1 (SETBP1) have recently been described in World Health Organization (WHO)-defined chronic neutrophilic leukemia (CNL) (NEJM 2013;368:1781; Leukemia. 2013. Prepublished on 2013/04/23). Response to treatment with ruxolitinib (10-15 mg twice-daily) was also described in one patient with CSF3RT618I mutation (NEJM 2013;368:1781). Primary cells from this patient were reportedly sensitive to inhibition by ruxolitinib (IC50, 127 nM). In a report of 12 patients with WHO-defined CNL, CSF3RT618I was seen in 10 (83%) patients, four (40%) of whom also expressed SETBP1 mutations (Leukemia. 2013. Prepublished on 2013/04/23). In the current study, we investigated the clonal distribution of mutant CSF3R and SETBP1in a CNL patient expressing both mutations and describe our observations regarding the effect of JAK inhibitors, both in vitro and in vivo. Methods Under institutional review board approved protocol, peripheral blood and buccal cells were collected and density gradient centrifugation was applied to enrich for mononuclear and granulocyte cell fractions. Antibody-labeled magnetic bead separation was used to obtain CD3+ and CD34+ cell fractions. DNA sequencing was used to screen for CSF3R and SETBP1 mutations. Single colonies were obtained by plating mononuclear cells in duplicate with cytokine enriched methylcellulose with and without the addition of fedratinib (a JAK2 inhibitor) or a commercially available JAK1 inhibitor. On day 11, erythroid and granulocyte individual colonies were counted and collected at each concentration. Single colonies were screened for both SETBP1 and CSF3Rmutations. Results The study patient was a 66-year-old lady with history of radiation therapy after lumpectomy for breast cancer in 1997. In October, 2012 the discovery of three synchronous lesions in the left breast necessitated mastectomy. White blood cell count (WBC) was approximately 13 x 10(9)/L at the time. Subsequently, her WBC gradually increased to 180 x 10(9)/L. Bone marrow examination on March 15, 2013 showed predominantly granulocytic proliferation with over 95% cellularity and no dysplastic features or monocytosis. Cytogenetic studies and mutation screening for JAK2V617F and BCR-ABL1were negative. A diagnosis of CNL was made and the patient was placed on hydroxyurea therapy. i) In vitro studies The patient harbored both CSF3RT618I and SETBP1D868N mutations in granulocytes, mononuclear cells and CD34+ myeloid progenitors; neither mutation was present in buccal or CD3+ cells. Colony forming unit (CFU) assay-derived 30 single colonies (15 erythroid and 15 granulocyte) were analyzed for the presence of CSF3R and SETBP1 mutations and all (100%) harbored heterozygous SETBP1D868N and 27 (90%) heterozygous CSF3RT618I; two granulocyte colonies were wild-type and one homozygous for CSF3RT618I. Fedratinib and the JAK1 inhibitor revealed activity in suppressing colony formation by >50% at a drug concentration of ≥600 nM. Mutation analysis in post-treatment residual single colonies revealed persistence of both mutations, even under conditions of higher ambient drug concentrations. However, CSF3R unmutated colonies were more likely to emerge than SETBP1unmutated colonies after in vitro drug exposure, which resulted in the appearance of occasional colonies that were negative for both mutations. ii) Treatment with ruxolitinib Ruxolitinib treatment at 10 mg twice-daily was started on 6/10/13, in addition to 500 mg/day of hydroxyurea. At the time, WBC was 86.5 x 10(9)/L, Hgb 11.8 g/dL and platelets 122 x 10(9)/L. On 6/17/13, WBC decreased to 43.9 and hydroxyurea was held. On 6/25/13, WBC increased to 89.6 x 10(9)/L and ruxolitinib was increased to 15 mg twice-daily. On 7/15/13, WBC had further increased to 190 x 10(9)/L and hydroxyurea was added to the treatment regimen at 500 mg/day. On 8/2/13, WBC was recorded at 91.3 x 10(9)/L. Conclusions In a double mutated CNL patient, we found CSF3R and SETBP1 mutations to be myeloid cell-restricted and co-expressed in erythroid and granulocytic cells; the latter antedated the former in order of acquisition. In vitro JAK inhibition had non-selective activity in suppressing myeloid colony formation. Treatment of the study patient with single agent ruxolitinib was ineffective. Disclosures: No relevant conflicts of interest to declare.

Abstract Background JAK inhibitors, including ruxolitinib, are to date incapable of inducing complete (CR) or partial (PR) remissions, reversal of bone marrow (BM) fibrosis or molecular responses in myelofibrosis (MF). This is consistent with the fact that JAK2 mutations are neither specific nor pathogenetically essential for the disease. Other currently available drugs in MF are equally ineffective in terms of disease-modifying activity. Methods In an investigator-sponsored single-center study (ClinicalTrials.gov Identifier: NCT01731951), imetelstat, a lipid-conjugated oligonucleotide inhibitor of human telomerase, was administered to patients with high or intermediate-2 risk MF (JCO 2011). Adverse events were monitored by common terminology criteria (Version 4.03) and responses by the International Working Group criteria (Blood 2013). Eligibility criteria included platelets ≥50 x 10(9)/L. Study drug and funding were provided by Geron Corporation (Menlo Park, CA, USA). Imetelstat was administered by a 2-hour intravenous infusion (9.4 mg/kg) every three weeks (cohort A) or weekly x 3 followed by every three weeks (cohort B). Mutations with prognostic (ASXL1 and SRSF2) or phenotypic (SF3B1 and U2AF1) relevance were screened by DNA sequencing. Quantitative PCR was used to measure JAK2V617F burden (assay sensitivity 0.01%). Laboratory correlative studies included analyses of granulocyte telomere length, mononuclear cell telomerase activity and human telomerase reverse transcriptase (hTERT) isoforms. Results Thirty-three patients were accrued; the first 18 patients enrolled and followed for a minimum of 3 months or discontinued are presented in this abstract: 11 cohort A and 7 cohort B; 44% PMF, 33% post-PV MF and 22% post-ET MF. Median age was 68 years and baseline risk was high in 56% and intermediate-2 in 44%. Seven patients were transfusion-dependent. Median spleen size was 13 cm and 11 patients had constitutional symptoms. Karyotype was abnormal in 7 patients and 89% were JAK2-mutated. Fifteen (83%) patients were previously treated including 7 with a JAK inhibitor and 3 with pomalidomide. i) Toxicity At a median f/u of 3.2 months, 16 (89%) patients remain on treatment; the two discontinuations were from unrelated death and disease progression. In cohort A, there were no grade-4 treatment-related adverse events; grade-3 events were limited to thrombocytopenia in 27% and anemia in 9%. In cohort B, two (29%) patients experienced grade-4 thrombocytopenia; grade-3 events were limited to thrombocytopenia, neutropenia and anemia in one patient each. Dose reduction was necessary in only two (11%) patients because of grade 3 or 4 myelosuppression. ii) Efficacy Overall response rate was 44%. This included five (28%) patients who met the BM and peripheral blood morphologic criteria for CR (n=4) or PR (n=1) and 3 patients with clinical improvement, pending validation of response duration and resolution of drug-induced grade-1 thrombocytopenia. The four (22%) CR patients experienced reversal of BM fibrosis and recovery of normal megakaryocyte morphology. Two CR patients were transfusion-dependent at baseline and became transfusion-independent. Complete molecular responses were documented in 2 CR patients: one had U2AF1Q157P and 10% JAK2V617F and the other SF3B1K666E and 50% JAK2V617F. A third CR patient had a >50% reduction in U2AF1 469_insAGTATG mutation. Among 13 patients with leukocytosis, 10 (77%) normalized their count or had >50% reduction. Eleven (61%) patients had complete or partial resolution of leukoerythroblastosis. iii) Laboratory correlative studies Three (50%) of 6 spliceosome-mutated vs. 1 (8%) of 12 unmutated (p=0.045) achieved CR. Spliceosome-mutated patients were also more likely to experience grade-3/4 myelosuppression (67% vs. 25% ; p=0.09). Treatment was associated with suppression of telomerase activity, shortening of telomere length and alteration of hTERT isoform pattern. Conclusions The current study signifies the potential value of telomerase-based treatment strategies in MF and identifies imetelstat as an active drug in that regard. The observed morphologic and molecular remissions confirm selective anti-clonal activity, which has thus far eluded other drugs in MF, including JAK inhibitors. The association between response and spliceosome mutations suggests a broader application for the drug in myeloid malignancies.</abstract> Disclosures: Stuart: Geron: Consultancy.

Abstract Background The median survival (OS) of patients with primary myelofibrosis (PMF; 6.5y) is significantly shortened compared to reference population (Cervantes et al, JCO 2012; 30:2981). OS is predicted by the four risk categories of IPSS, Dynamic-IPSS and DIPSS-plus score, nevertheless pts heterogeneity persists within these categories, necessitating improved risk stratification. We recently reported that pts harboring mutations in any one of the prognostically relevant ASXL1, EZH2, IDH1/2 and SRSF2 genes constitute a IPSS-and DIPPS-plus independent Molecular High Risk category (MHR+) characterized by significant reduction of OS and leukemia free survival (LFS) (Vannucchi et al, Leukemia 2013). The aim of this work was to analyze the impact of the number of prognostically relevant mutated genes on OS and LFS in PMF. Patients and methods Two independent cohorts are included, a “test cohort” from Europe, analyzed at diagnosis, and a “validation cohort” from Mayo Clinic, analyzed at any time after diagnosis. Mutation analysis was performed in DNA from whole blood or granulocytes using RTQ-PCR, HRM and direct sequencing; all mutations were confirmed at least twice. The prognostic value of the molecular variables with regard to OS and LFS was analyzed by Cox regression. Test cohort It included 490 pts (median age 61y; males = 301) risk stratified by IPSS into high (n=74, 15%), intermediate-2 (n=93, 19% ), intermediate-1 (n=147, 30%) and low (n=176, 36%). The median follow-up was 3.63y (95% CI, 0.06-28.33); 161 pts died (33.0%), of whom 76 (15.6%) had progressed to acute leukemia after a median of 3.4y (0.04-28.3) from diagnosis. One hundred forty-six pts (29.8%) presented at least one of the four aforementioned mutated genes and were classified as MHR+. The OS of MHR+ pts was significantly reduced compared to patients with no mutations (n=344): 80.7 vs 148.9 mo (HR 2.2, CI95% 1.6-3.03). One hundred twelve (22.8%) pts had 1 mutation and 34 (6.9%) had 2 or more mutations. In univariate analysis presence of 2 or more mutated genes was significantly more detrimental for OS (29.5mo; HR 4.12, IC95% 2.6-6.4) than having 1 mutated gene (84.2mo; HR 1.8, IC95% 1.2-2.5) compared with having no mutations (148.9mo). Multivariate analysis results adjusted for IPSS indicated that having two or more mutations was an independent prognostic factor for OS (HR 2.9, 95% CI 1.8-4.5). Notably, the prognostic relevance of harboring two or more mutations involved both lower (low and intermediate-1; HR 1.87, 95% CI 1.3-2.6) and higher (intermediate-2 and high; HR 1.6, 95% CI 1.2-2.1) categories of IPSS. Also LFS resulted significantly shorter (129mo; HR 3.1, 95%CI 1.9-4.8) in MHR+ pts compared with pts with no adverse mutations (323mo). Having 2 or more mutations correlated with greater reduction of LFS (79.6mo; HR 7.02, CI95% 3.9-12.6) than having one mutation only (133.9mo; HR 2.2, IC95% 1.3-3.7) as compared with no mutations (323.2mo). Multivariate analysis showed that IPSS high risk category (HR 4.5; 95% CI 2.3-8.8) and two or more mutated genes (HR 5.3; 95% CI 2.9-9.8) predicted independently for a significant reduction in LFS. The negative impact on LFS of having 2 or more mutated genes compared to one or no mutations was maintained in the lower (HR 6.4, 95% CI 2.6-15.2) and higher (HR 5.5, 95% CI 2.3-12.8) risk categories. Validation cohort Validation cohort included 262 patients (median age 64 years; males = 167) risk stratified by DIPSS-plus into high (n=89, 34%), intermediate-2 (n=92, 35%), intermediate-1 (n=46, 18%) and low (n=34, 13%). One hundred forty-six pts (56%) displayed none of the four aforementioned mutations, 93 (36%) harbored one mutation whereas 23 (9%) harbored two or more mutations. In univariate analysis, having two or more mutations (HR 3.7; 95% CI 2.2-6.1) was significantly more detrimental for OS than having no mutations, which is more favorable than having one mutation (HR 1.9; 95% CI 1.4-2.7). When adjusted for DIPSS-plus, the presence of two or more mutations retained its significance (HR 2.1; 95% CI 1.3-3.6) and outperformed ASXL1 mutation alone in its prognostic relevance Conclusions Overall, these results show that the number of prognostically relevant mutated genes correlate with OS and LFS in pts with PMF, suggesting that screening for these mutations might help to improve risk stratification. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 1751 Background: JAK inhibitors have significant palliative benefit in myelofibrosis (MF), mainly in the form of improved constitutional symptoms and reduced splenomegaly. Preliminary data suggests that CYT387, a JAK-1/2 inhibitor, also has the ability to produce anemia responses (ASH Annual Meeting, 2011). In general, the mechanism(s) underlying treatment effects of JAK inhibitors remain unclear but likely represent a drug-specific balance between anti-clonal activity and modulation of immuno cellular-cytokine pathways. We conducted a gene expression profiling (GEP) study using primary cells from MF patients undergoing therapy with CYT387 followed by correlation with clinical data. Methods: Study subjects were enrolled in the Phase-1/2 study of CYT387 treatment in patients with primary (PMF), post-polycythemia vera (PPMF) or post-essential thrombocythemia (PTMF) myelofibrosis. Paired research samples were collected; the time points were pre-study and 12 weeks after commencing study treatment. PBMCs were purified from whole blood by Ficoll separation; RNA was isolated from this cell fraction for GEP analysis. Gene expression profiles were generated using Illumnia Human HT-12 v4 microarray. Pair wise analysis was conducted using the Wilcoxon signed-rank test with a p-value cutoff of 0.05 to generate lists of differentially expressed genes between assigned groups. Pathway analysis was conducted to identify relevant pathways enriched for differentially expressed genes. Comprehensive plasma cytokine profiling was performed using Multiplex Bead-Based Luminex technology (Invitrogen, Carlsbad, CA). Results: Seventeen patients were studied based on sample availability; 11 (65%) mere male with median age of 66 years (range 53–85). Twelve (71%) were JAK2V617F mutation positive and the DIPSS-plus risk categorization was 10 (59%) high and 7 (41%) intermediate-2. All patients were evaluable for anemia response; 14 (82%) were red cell transfusion dependent at study start. Nine (53%) patients achieved anemia response by IWG-MRT criteria; of these, 8 patients achieved transfusion independence (minimum non-transfused hemoglobin level of 8 g/dL maintained for at least 12 weeks) and 1 had a sustained >2 g/dL increase in hemoglobin level above baseline. The initial pair wise analysis to identify differential patterns of gene expression compared pre- and post-treatment groups (Figure 1A). This revealed a cluster of significantly (p <0.05) down-regulated genes (minimum 2-fold; median 17-fold) following treatment (displayed in green; upper left quadrant). Pathway enrichment analysis revealed significant associations of these genes with cytokine regulation of immune response, cell proliferation, chemotaxis and cytoskeleton remodeling. We then conducted a pair wise analysis of anemia responders versus non-responders; this revealed a predominance of over expressed gene targets (median 35-fold) in the anemia responder group (Figure 1B) (displayed in red; upper right quadrant). Similar pathway analysis identified enrichment for genes involved in immune system function in this cluster. Conclusions: The current preliminary analysis suggests that genes relevant to immune response-cytokine pathways are significantly over expressed in patients who achieve anemia response following CYT387 therapy. This further suggests a dominant immune component that underpins ineffective hematopoiesis in responding patients. On the basis of broad treatment-related changes in gene expression we suggest that an important component of CYT387's treatment effect is down regulation of these dysregulated pathways. Ongoing studies include validation of select gene targets which will be tested prospectively in future treatment protocols, as well as correlation of gene expression with circulating cytokine-chemokine levels. Disclosures: Pardanani: Bristol-Myers Squibb: Clinical trial support, Clinical trial support Other; YM BioSciences: Clinical trial support, Clinical trial support Other; Sanofi-Aventis: Clinical trial support Other. Off Label Use: Data from Phase −1/2 study of CYT387 use in myelofibrosis is mentioned.

Abstract Abstract 430 Background: The Dynamic International Prognostic Scoring System (DIPSS-plus) uses eight risk factors to predict overall survival (OS) in primary myelofibrosis (PMF): unfavorable karyotype, peripheral blood (PB) blast count ≥1%, platelet count <100 × 109/L, white blood cell count (WBC) >25 × 109/L, hemoglobin level <10 g/dL, red blood cell transfusion need, constitutional symptoms, and age >65 years (JCO 2011;29:392). Among these risk factors, karyotype holds a dominant role in predicting both overall (OS) and leukemia-free (LFS) survival (Blood 2011;118:4595). More recently, certain PMF-associated somatic mutations (IDH, EZH2, ASXL1, SRSF2) have been reported (in print or at recent meetings) to carry prognostic relevance for OS or LFS in PMF while other mutations (JAK2, MPL, TET2, SF3B1) did not. The purpose of the current study was to examine the individual and combined prognostic relevance of the former in the context of cytogenetic risk stratification and DIPSS-plus. Methods: An updated Mayo Clinic database of 1008 karyotypically- and DIPSS-plus-annotated patients with PMF was used to identify study patients in whom DNA at time of referral was available for mutation analysis. OS and LFS were calculated from the time of referral, which was also the time of study sample collection. Results: In an ongoing project, a total of 397 PMF patients have so far been studied for IDH1/2 (n=367), SRSF2 (n=355), ASXL1 (n=260) and EZH2 (n=43) mutations. The corresponding mutational frequencies were 5.4% (20/367), 14.4% (51/355), 31.2% (81/260) and 7% (3/43). These mutations were not exclusive of either each other or JAK2 or MPL mutations and a significant clustering was noted between SRSF2 and IDH mutations; IDH mutational frequency was 21% (10/48) in SRSF2-mutated versus 3% (9/279) in unmutated cases (P<0.0001). Mutational hotspots were P95 in exon 1 for SRSF2 and exons 1, 2 and 5 for ASXL1. OS and LFS effect have so far been examined for ASXL1, SRSF2 and IDH 1/2 mutations; in univariate analysis, all three mutations significantly affected OS (ASXL1 RR 1.9, 95% CI 1.4–2.7; SRSF2 RR 1.7, 95% CI 1.2–2.5; IDH 1/2 RR 1.8, 95% CI 1.1–3.1) whereas only IDH 1/2 (RR 5.5, 95% CI 2.5–11.8) and SRSF2 (RR 3.5, 95% CI 1.7–7.0) but not ASXL1 (p=0.29) mutations affected LFS. The observed prognostic relevance for OS was sustained during multivariable analysis that included ASXL1 with either SRSF2 or IDH mutations, as co-variates, whereas both IDH and SRSF2 mutations remained significant for LFS. Based on the above observations, study patients were classified into two groups: i) unmutated for ASXL1, SRSF2 and IDH1/2 (all unmutated; n=146) and ii) mutated for at least one of the three mutations (one or more mutated; n=130); the latter group constituted 62%, 52%, 18% and 27% of DIPSS-plus high, intermediate-2, intermediate-1and low risk patients, respectively (p<0.0001). The corresponding rates for patients with favorable and unfavorable karyotype were 47% and 43% (p=0.7) and for normal vs. abnormal karyotype were 53% and 36% (p=0.008). The “all unmutated group”, compared to the “one or more mutated group” enjoyed a significantly superior OS (RR 0.5, 95% CI 0.4–0.7) and LFS (RR 0.3, 95% CI 0.2–0.7) that was DIPSS-plus-independent for OS (p=0.03) and karyotype-independent for LFS (p=0.004). The prognostic value of distinguishing “all unmutated” from “one or more mutated” patient groups was most evident for DIPSS-plus intermediate-2 or lower risk groups (p=0.002) and its added value for high-risk group was limited (p=0.23) (Figure). Conclusions: A concomitant analysis strategy reveals that SRSF2, ASXL1 and IDH mutations carry inter-independent prognostic relevance for overall or leukemia-free survival in PMF. Any one of these three mutations is more likely to occur in patients with normal karyotype and such an event predicts shortened survival that is not accounted for by DIPSS-plus and a higher risk of leukemic transformation that is independent of karyotype. The current study suggests that such information might be useful in treatment decisions involving intermediate-2 or lower risk patients. Disclosures: No relevant conflicts of interest to declare.

Abstract Purpose: Immunotherapeutic strategies to treat patients with renal cell carcinoma (RCC) offer new opportunities for disease management. Further improvements to immunotherapy will require additional understanding of the host response to RCC development. Experimental Design: Using a novel approach to understanding the immune status of cancer patients, we previously showed that patients with a certain immune profile had decreased overall survival. Here, we examine in more detail the phenotypic changes in peripheral blood and the potential consequences of these changes in RCC patients. Results: We found that CD14+HLA-DRlo/neg monocytes were the most predominant phenotypic change in peripheral blood of RCC patients, elevated nearly 5-fold above the average levels measured in healthy volunteers. Intratumoral and peritumoral presence of CD14 cells was an independent prognostic factor for decreased survival in a cohort of 375 RCC patients. The amount of peripheral blood CD14+HLA-DRlo/neg monocytes was found to correlate with the intensity of CD14 staining in tumors, suggesting that the measurement of these cells in blood may be a suitable surrogate for monitoring patient prognosis. The interaction of monocytes and tumor cells triggers changes in both cell types with a loss of HLA-DR expression in monocytes, increases of monocyte survival factors such as GM-CSF in tumors, and increased production of angiogenic factors, including FGF2. Conclusions: Our results suggest a model of mutually beneficial interactions between tumor cells and monocytes that adversely affect patient outcome. Clin Cancer Res; 21(18); 4224–33. ©2015 AACR.

Abstract Chromosome rearrangements that result in gene fusions have important roles in the initial steps of tumorigenesis especially in leukemias and lymphomas, but the biological and clinical impact of gene fusions in the common solid tumor has been less appreciated. We previously generated RNAseq data from 12 oropharyngeal tumors along with matched normal tissue from the same patients. We utilized and compared two bioinformatic pipelines, SnowShoes-FTD and FusionHunter to identify two novel fusion events involving of MGST3-ZMAT5 and MS4A7-DKFZp586P0123. These fusions were validated utilizing. Reverse transcriptase polymerase chain reaction (RT-PCR) and Sanger sequencing validated. In the first translocation the 5′ UTR of MGST3 was fused with exon 1 of ZMAT5, and in the second translocation the exon 2 of MS4A7 was fused with 3′ UTR of DKFZp586P0123. These precise fusions were not present in the matched normal tissue, however, they were only found in the single tumor in which they were identified. Given a growing and powerful set of high-throughput massively parallel sequencing technologies, the future for genome-wide analysis of chromosome rearrangement appears open in many directions. Ultimately, we hope that this integrative methodology will elucidate key aspects of tumor biology as well as generate novel targets for cancer diagnostics and therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3100. doi:1538-7445.AM2012-3100

Abstract There are a number of human genes which span very large chromosomal regions. Forty genes span greater than 1 megabase and 200 are 500 Kb or larger. We and others have demonstrated that many of the largest genes are derived from within regions of profound genomic instability known as common fragile sites (CFSs). We have been studying oropharyngeal cancers which are derived from the base of tongue, tonsil and larynx. While the traditional etiologic factors underlying the development of this aggressive cancer is a history of smoking and drinking there are an increasing number of younger patients developing this cancer without these risk factors. The most likely cause of these cancers is the presence of human papillomavirus which we detect in over 90% of oropharyngeal cancers derived from the Mayo Clinic. In an attempt to better understand the molecular alterations that underlie oropharyngeal cancers arising from different risk factors we utilized the power of Next Generation sequencing on the Illumina GA IIx to compare the transcriptomes of 12 oropharyngeal cancers to patient matched normal tissue. We first observe a division of patients into two groups of oropharyngeal cancers: those caused by mutations and other alterations induced by a history of smoking and drinking and those associated with HPV infection caused by viral-induced genomic instability. We then examined the expression of the 200 largest human genes in the oropharyngeal tumors and find that each tumor has inactivated the expression of at least 20 different very large genes. There is a distinct pattern of inactivation of these genes in different oropharyngeal cancers depending upon the underlying etiology. Some of the genes most frequently inactivated include Dlg2 (the human homolog of the large discs tumor suppressor gene from drosophila and a mutation target in a number of other cancers); ERBB4 (Her4 and a member of the EGFR family), BAI3 (a brain specific angiogenesis inhibitor that is also mutated in multiple cancers), and ANK2 (mutated in long QT patients). Each of these genes may be functioning as tumor suppressors as has already been demonstrated for the two large CFS genes, FHIT and WWOX. The inactivation of so many of these genes in each oropharyngeal tumor may have a profound effect upon the phenotype of the resulting tumor. When we examine the expression of the large genes most frequently inactivated in each of the two groups of oropharyngeal cancers in panels of breast or lung cancer samples we find that we do not detect significant loss of expression of these genes. We believe that genomic instability promotes alterations in the unstable CFS regions but that there is selective inactivation of different large CFS genes in different cancers. The signature of which specific genes are inactivated may have phenotypic and clinical significance which we are just beginning to investigate. 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 3885. doi:10.1158/1538-7445.AM2011-3885

Abstract There is considerable clinical interest in the role that human papillomavirus (HPV) plays in the development of oropharyngeal cancers. A variety of assays are currently available to stratify patients based on HPV status prior to treatment. The most frequently utilized are PCR and In Situ hybridization (ISH) to detect the presence of HPV DNA sequences. The increasing clinical interest in determining HPV patient status relates to compelling evidence that a subset of patients with HPV positive tumors have better clinical outcomes. Evidence also suggests that patients with HPV positive tumors should be further divided into groupings based on levels of viral activity and that this may represent additional clinical information necessary to develop appropriate treatment plans. The most frequently applied assay to identify HPV activity in a patient sample is to detect the over-expression of the surrogate protein marker p16 by Immunohistochemisty (IHC), which has been shown to increase expression in response to HPV active infections in oropharyngeal cancer. An alternative technique to quantitatively detect the presence and level of transcriptional activity of HPV in tissue samples is to measure the expression of oncogenes E6 and E7. We have analyzed the fresh frozen tissue samples corresponding to matched tumor, normal and metastatic patient tissues using a quantitative PCR (qPCR) approach to determine the expression of HPV oncogenes and p16 gene expression. We observed that there are three groups of oropharyngeal tumors when grouped by HPV16 E6 and E7 gene expression. The first are the most rare and are tumors that contain no HPV sequences or expression of the E6 and E7 ocogenes. The second are tumors that contain HPV DNA sequences but very low expression of E6 or E7 and the third are tumors that contain HPV DNA sequence and robust expression of E6 and E7. Additionally, we found variable correlation between HPV16 E6 and E7 expression and p16 gene expression, suggesting that factors other than HPV oncogene expression may influence the expression of p16. Our results would indicate that the best assays for quantifying the activity of an HPV infection in oropharyngeal cancers would be those optimized to accurately measure either E6 and E7 transcripts or their protein products directly out of paraffin-embedded tissues. 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 3859. doi:10.1158/1538-7445.AM2011-3859