Nisha J. D'Silva

EGFR, p16, HPV Titer, Bcl-xL and p53, Sex, and Smoking As Indicators of Response to Therapy and Survival in Oropharyngeal Cancer

PURPOSE To prospectively identify markers of response to therapy and outcome in an organ-sparing trial for advanced oropharyngeal cancer. PATIENTS AND METHODS Pretreatment biopsies were examined for expression of epidermal growth factor receptor (EGFR), p16, Bcl-xL, and p53 as well as for p53 mutation. These markers were assessed for association with high-risk human papillomavirus (HPV), response to therapy, and survival. Patient variables included smoking history, sex, age, primary site, tumor stage, and nodal status. RESULTS EGFR expression was inversely associated with response to induction chemotherapy (IC) (P = .01), chemotherapy/radiotherapy (CRT; P = .055), overall survival (OS; P = .001), and disease-specific survival (DSS; P = .002) and was directly associated with current smoking (P = .04), female sex (P = .053), and lower HPV titer (P = .03). HPV titer was significantly associated with p16 expression (P < .0001); p16 was significantly associated with response to IC (P = .008), CRT (P = .009), OS (P = .001), and DSS (P = .003). As combined markers, lower HPV titer and high EGFR expression were associated with worse OS (rho(EGFR) = 0.008; rho(HPV) = 0.03) and DSS (rho(EGFR) = 0.01; rho(HPV) = 0.016). In 36 of 42 biopsies, p53 was wild-type, and only one HPV-positive tumor had mutant p53. The combination of low p53 and high Bcl-xL expression was associated with poor OS (P = .005) and DSS (P = .002). CONCLUSION Low EGFR and high p16 (or higher HPV titer) expression are markers of good response to organ-sparing therapy and outcome, whereas high EGFR expression, combined low p53/high Bcl-xL expression, female sex, and smoking are associated with a poor outcome. Smoking cessation and strategies to target EGFR and Bcl-xL are important adjuncts to the treatment of oropharyngeal cancer.

Chemoselection As a Strategy for Organ Preservation in Advanced Oropharynx Cancer: Response and Survival Positively Associated With HPV16 Copy Number

PURPOSE To test induction chemotherapy (IC) followed by concurrent chemoradiotherapy (CRT) or surgery/radiotherapy (RT) for advanced oropharyngeal cancer and to assess the effect of human papilloma virus (HPV) on response and outcome. PATIENTS AND METHODS Sixty-six patients (51 male; 15 female) with stage III to IV squamous cell carcinoma of the oropharynx (SCCOP) were treated with one cycle of cisplatin (100 mg/m(2)) or carboplatin (AUC 6) and with fluorouracil (1,000 mg/m(2)/d for 5 days) to select candidates for CRT. Those achieving a greater than 50% response at the primary tumor received CRT (70 Gy; 35 fractions with concurrent cisplatin 100 mg/m(2) or carboplatin (AUC 6) every 21 days for three cycles). Adjuvant paclitaxel was given to patients who were complete histologic responders. Patients with a response of 50% or less underwent definitive surgery and postoperative radiation. Pretreatment biopsies from 42 patients were tested for high-risk HPV. RESULTS Fifty-four of 66 patients (81%) had a greater than 50% response after IC. Of these, 53 (98%) received CRT, and 49 (92%) obtained complete histologic response with a 73.4% (47 of 64) rate of organ preservation. The 4-year overall survival (OS) was 70.4%, and the disease-specific survival (DSS) was 75.8% (median follow-up, 64.1 months). HPV16, found in 27 of 42 (64.3%) biopsies, was associated with younger age (median, 55 v 63 years; P = .016), sex (22 of 30 males [73.3%] and five of 12 females [41.7%]; P = .08), and nonsmoking status (P = .037). HPV titer was significantly associated with IC response (P = .001), CRT response (P = .005), OS (P = .007), and DSS (P = .008). CONCLUSION Although the numbers in this study are small, IC followed by CRT is an effective treatment for SCCOP, especially in patients with HPV-positive tumors; however, for patients who do not respond to treatment, alternative treatments must be developed.

Sirtuin-3 (SIRT3), a Novel Potential Therapeutic Target for Oral Cancer

Several sirtuin family members (SIRT1‐7), which are evolutionarily conserved NAD‐dependent deacetylases, play an important role in carcinogenesis. However, their role in oral cancer has not yet been investigated. Therefore, the objective of this study was to investigate whether sirtuins play a role in oral cancer carcinogenesis.

Infiltrating lymphocytes and human papillomavirus-16–associated oropharyngeal cancer†‡§

Human papillomavirus‐16 (HPV‐16)–associated carcinoma of the oropharynx has a favorable prognosis. Such patients have elevated CD8+ T‐lymphocyte levels that correlate with response to chemotherapy and survival. Tumor‐infiltrating lymphocyte (TIL) subpopulations were assessed in pretreatment biopsies from a prospective patient cohort to determine if TIL subsets differed by HPV status, clinical factors, or patient outcome or correlated with peripheral blood T‐cell levels.

Correlation of Cellular Immunity With Human Papillomavirus 16 Status and Outcome in Patients With Advanced Oropharyngeal Cancer

OBJECTIVE to determine whether the favorable outcome associated with human papillomavirus (HPV) 16-positive oropharyngeal cancer is related to a patients adaptive immunity. SETTING academic medical center. PATIENTS forty-seven of 66 previously untreated patients (6 of 20 patients with stage III and 41 of 46 with stage IV cancer) in a prospective clinical trial of chemoradiotherapy. INTERVENTION all patients were treated with a single course of neoadjuvant chemotherapy followed by either surgery (for nonresponders) or chemoradiotherapy. MAIN OUTCOME MEASURES pretreatment levels (percentages and absolute counts) of CD3, CD4, CD8, natural killer, and B cells and overall white blood cell counts were measured by flow cytometry. Correlations of subsets with HPV-16 status, tumor subsite, cancer stage, T class, N class, smoking status, performance status, sex, response to chemoradiotherapy, p53 mutation type, epidermal growth factor receptor expression, and disease-specific and overall survival were determined. RESULTS after a median follow-up of 6.6 years, improved survival was associated with an elevated percentage of CD8 cells (P = .04), a low CD4:CD8 ratio (P = .01), low epidermal growth factor receptor expression (P = .002), and HPV status (P = .02). The percentage of CD8 cells was significantly higher (P = .04) and the CD4:CD8 ratio was significantly lower (P = .02) in HPV-16-positive patients. A higher percentage of CD8 cells was associated with response to induction chemotherapy (P = .02) and complete tumor response after chemoradiotherapy (P = .045). CONCLUSION these findings confirm previous correlations of outcome with circulating CD8 cell levels and support the conjecture that improved adaptive immunity may play a role in the favorable prognosis of patients with HPV-16-positive cancers.

Adenovirus Encoding Human Platelet-Derived Growth Factor-B Delivered to Alveolar Bone Defects Exhibits Safety and Biodistribution Profiles Favorable for Clinical Use

Platelet-derived growth factor (PDGF) gene therapy offers promise for tissue engineering of tooth-supporting alveolar bone defects. To date, limited information exists regarding the safety profile and systemic biodistribution of PDGF gene therapy vectors when delivered locally to periodontal osseous defects. The aim of this preclinical study was to determine the safety profile of adenovirus encoding the PDGF-B gene (AdPDGF-B) delivered in a collagen matrix to periodontal lesions. Standardized alveolar bone defects were created in rats, followed by delivery of matrix alone or containing AdPDGF-B at 5.5 x 10(8) or 5.5 x 10(9) plaque-forming units/ml. The regenerative response was confirmed histologically. Gross clinical observations, hematology, and blood chemistries were monitored to evaluate systemic involvement. Bioluminescence and quantitative polymerase chain reaction were used to assess vector biodistribution. No significant histopathological changes were noted during the investigation. Minor alterations in specific hematological and blood chemistries were seen; however, most parameters were within the normal range for all groups. Bioluminescence analysis revealed vector distribution at the axillary lymph nodes during the first 2 weeks with subsequent return to baseline levels. AdPDGF-B was well contained within the localized osseous defect area without viremia or distant organ involvement. These results indicate that AdPDGF-B delivered in a collagen matrix exhibits acceptable safety profiles for possible use in human clinical studies.

The tumor suppressor gene rap1GAP is silenced by miR-101-mediated EZH2 overexpression in invasive squamous cell carcinoma

Rap1GAP is a critical tumor suppressor gene that is downregulated in multiple aggressive cancers, such as head and neck squamous cell carcinoma, melanoma and pancreatic cancer. However, the mechanistic basis of rap1GAP downregulation in cancers is poorly understood. By employing an integrative approach, we demonstrate polycomb-mediated repression of rap1GAP that involves Enhancer of Zeste Homolog 2 (EZH2), a histone methyltransferase in head and neck cancers. We further demonstrate that the loss of miR-101 expression correlates with EZH2 upregulation, and the concomitant downregulation of rap1GAP in head and neck cancers. EZH2 represses rap1GAP by facilitating the trimethylation of histone 3 at lysine 27, a mark of gene repression, and also hypermethylation of rap1GAP promoter. These results provide a conceptual framework involving a microRNA–oncogene–tumor suppressor axis to understand head and neck cancer progression.

Identification of a Putative Tumor Suppressor Gene Rap1GAP in Pancreatic Cancer

Human chromosome 1p35-p36 has long been suspected to harbor a tumor suppressor gene in pancreatic cancer and other tumors. We found that expression of rap1GAP, a gene located in this chromosomal region, is significantly down-regulated in pancreatic cancer. Only a small percentage of preneoplastic pancreatic intraductal neoplasia lesions lost rap1GAP expression, whereas loss of rap1GAP expression occurred in 60% of invasive pancreatic cancers, suggesting that rap1GAP contributes to pancreatic cancer progression. In vitro and in vivo studies showed that loss of rap1GAP promotes pancreatic cancer growth, survival, and invasion, and may function through modulation of integrin activity. Furthermore, we showed a high frequency of loss of heterozygosity of rap1GAP in pancreatic cancer. Collectively, our data identify rap1GAP as a putative tumor suppressor gene in pancreatic cancer.

Rap1A and rap1B ras-family proteins are prominently expressed in the nucleus of squamous carcinomas: nuclear translocation of GTP-bound active form

We recently showed that rap1 regulates growth and proliferation in normal keratinocytes, which provoked us to investigate its expression and regulation in malignant cells. Rap1 is variably expressed in whole cell lysates of squamous cell carcinoma (SCC) cell lines. Immunoblot analysis of nuclear and cytosolic fractions and immunohistochemistry revealed that in addition to cytoplasmic expression, SCC cells also exhibit prominent punctate rap1 expression in the nucleus. This unexpected nuclear distribution was confirmed by the evaluation of human oral cancer specimens by immunohistochemistry, which showed both nuclear and cytoplasmic localization. Cytoplasmic rap1 expression was observed mostly in large differentiated cells, whereas nuclear localization was found in morphologically less differentiated cells. Quantitative reverse transcriptase polymerase chain reaction and Northern blot analysis showed that both rap1A and rap1B are expressed in SCC cell lines although rap1B signals are more prominent. Transfection with enhanced GFP-tagged constitutively active and inactive forms of rap1B demonstrated that the active GTP-bound form translocates to the nucleus whereas inactive rap1BGDP is retained in the cytoplasm, much of which is in a perinuclear distribution. Furthermore, growth factors induce nuclear translocation of rap1 in oral cancer cells. This novel discovery that active, GTP-bound rap1 translocates to the nucleus makes it only the second of over 100 small GTP-binding proteins to be identified in the nucleus, and the striking prominence of rap1 expression in the nucleus of SCC cells suggests that activated rap1 plays a role in the malignant process.

A p38α Selective Mitogen-Activated Protein Kinase Inhibitor Prevents Periodontal Bone Loss

In the oral microbial environment, Gram-negative bacterial derived lipopolysaccharide (LPS) can initiate inflammatory bone loss as seen in periodontal diseases. p38 Mitogen-activated protein kinase (MAPK) signaling is critical to inflammatory cytokine and LPS-induced cytokine expression, which may contribute toward periodontal bone loss. The purpose of this proof-of-principle study was to evaluate the ability of an orally active p38α MAPK inhibitor (SD-282) to reduce periopathogenic LPS-induced alveolar bone loss in an experimental rat model. Five groups of Sprague-Dawley rats received one of the following treatments: LPS injected to the palatal gingiva adjacent to the maxillary molars three times per week for 8 weeks, LPS plus two doses of SD-282 (15 or 45 mg/kg) twice daily by oral gavage, or control groups given drug vehicle (1% polyethylene glycol) or SD-282 (45 mg/kg) only. Baseline and 8-week alveolar bone loss was assessed by microcomputed tomography (μCT) and histological examination. LPS induced severe bone loss over this time period, whereas control groups were unchanged from baseline measurements. Both doses of SD-282 showed significant protection from LPS-induced bone loss. Bone area and volumetric analysis of maxillas by μCT indicated significant loss of bone volume with LPS treatment, which was blocked with the p38 inhibitor. Histological examination indicated significantly fewer tartate-resistant acid phosphatase-positive osteoclasts and a significant decrease in interleukin (IL)-6, IL-1β, and tumor necrosis factor α expression in p38 inhibitor-treated groups compared with LPS groups by immunostaining. Results from this in vivo study suggest that orally active p38 MAPK inhibitors can reduce LPS-induced inflammatory cytokine production and osteoclast formation and protect against LPS-stimulated alveolar bone loss.