Suhail K. Mithani

Frequency and phenotypic implications of mitochondrial DNA mutations in human squamous cell cancers of the head and neck.

Mitochondrial genomic mutations are found in a variety of human cancers; however, the frequency of mitochondrial DNA (mtDNA) mutations in coding regions remains poorly defined, and the functional effects of mitochondrial mutations found in primary human cancers are not well described. Using MitoChip, we sequenced the whole mitochondrial genome in 83 head and neck squamous cell carcinomas. Forty-one of 83 (49%) tumors contained mtDNA mutations. Mutations occurred within noncoding (D-loop) and coding regions. A nonrandom distribution of mutations was found throughout the mitochondrial enzyme complex components. Sequencing of margins with dysplasia demonstrated an identical nonconservative mitochondrial mutation (A76T in ND4L) as the tumor, suggesting a role of mtDNA mutation in tumor progression. Analysis of p53 status showed that mtDNA mutations correlated positively with p53 mutations (P < 0.002). To characterize biological function of the mtDNA mutations, we cloned NADH dehydrogenase subunit 2 (ND2) mutants based on primary tumor mutations. Expression of the nuclear-transcribed, mitochondrial-targeted ND2 mutants resulted in increased anchorage-dependent and -independent growth, which was accompanied by increased reactive oxygen species production and an aerobic glycolytic metabolic phenotype with hypoxia-inducible factor (HIF)-1α induction that is reversible by ascorbate. Cancer-specific mitochondrial mutations may contribute to development of a malignant phenotype by direct genotoxic effects from increased reactive oxygen species production as well as induction of aerobic glycolysis and growth promotion.

DNA global hypomethylation in squamous cell head and neck cancer associated with smoking, alcohol consumption and stage

Epigenetic changes have been implicated in the pathogenesis of solid tumors, including head and neck squamous cell carcinoma (HNSCC). Prior efforts have primarily examined regional promoter hypermethylation as a silencer of tumor suppressor gene expression. To analyze the global state of methylation in the HNSCC genome, we utilize pyrosequencing of repetitive elements (LINEs) to compare the state of global methylation in HNSCC to normal aerodigestive mucosa. 137 samples (119 HNSCC tumors and 18 normal mucosal tissues) were digested to extract DNA and subjected to bisulfite treatment. Treated DNA was amplified using PCR primers for the repetitive LINEs sequence and produced a heterogeneous sample of products, from many genomic loci. These products were pyrosequenced to quantitatively evaluate their global genomic methylation status. HNSCC specimens showed global hypomethylation, with a mean level of genomic methylation of 46.8% methylated with a standard deviation of 9.0. Conversely, the normal upper airway mucosa had a global methylation level of 54.0 and a standard deviation of 4.6 (Mann–Whitney p value < 0.001). The tumor specimens also showed an increasing degree of hypomethylation associated with advanced tumor stage (ANOVA p‐value of 0.003). About 67% of HNSCCs are globally hypomethylated when evaluated against the minimum level of methylation in the normal mucosal specimens. Degree of global hypomethylation was associated with smoking history, alcohol use and stage in univariate analysis (p‐value 0.02), however, only HNSCC diagnosis remained significant on multivariate analysis. Despite the presence of regional promoter hypermethylation, HNSCC demonstrates global genomic hypomethylation. The effects of stage, alcohol use and smoking on global hypomethylation were not independently significant.

Coordinated Activation of Candidate Proto-Oncogenes and Cancer Testes Antigens via Promoter Demethylation in Head and Neck Cancer and Lung Cancer

Background Epigenetic alterations have been implicated in the pathogenesis of solid tumors, however, proto-oncogenes activated by promoter demethylation have been sporadically reported. We used an integrative method to analyze expression in primary head and neck squamous cell carcinoma (HNSCC) and pharmacologically demethylated cell lines to identify aberrantly demethylated and expressed candidate proto-oncogenes and cancer testes antigens in HNSCC. Methodology/Principal Findings We noted coordinated promoter demethylation and simultaneous transcriptional upregulation of proto-oncogene candidates with promoter homology, and phylogenetic footprinting of these promoters demonstrated potential recognition sites for the transcription factor BORIS. Aberrant BORIS expression correlated with upregulation of candidate proto-oncogenes in multiple human malignancies including primary non-small cell lung cancers and HNSCC, induced coordinated proto-oncogene specific promoter demethylation and expression in non-tumorigenic cells, and transformed NIH3T3 cells. Conclusions/Significance Coordinated, epigenetic unmasking of multiple genes with growth promoting activity occurs in aerodigestive cancers, and BORIS is implicated in the coordinated promoter demethylation and reactivation of epigenetically silenced genes in human cancers.

Lymphatic mapping and sentinel lymphadenectomy after preoperative therapy for stage II and III breast cancer

AbstractBackground: We evaluated the accuracy of sentinel lymph node dissection (SLND) in patients with stage II and III breast cancer who had received preoperative therapy. Methods: A prospective clinical trial evaluated 122 patients who had SLND followed by axillary lymph node dissection. Thirty-two women had stage II or III breast cancer and received preoperative doxorubicin-based chemotherapy or paclitaxel and radiotherapy. Results: A sentinel lymph node (SLN) was identified in 31 (97%) of 32 patients. The SLN predicted the status of the axillary nodes in 30 (97%) of 31 patients. Eighteen (58%) of 31 had metastases in the SLN. Eighteen of 19 patients with axillary metastases had a tumor-positive SLN (sensitivity, 95%; false-negative rate, 5%). Eight (44%) of 18 women with metastases in the SLN also had metastases in 1 or more nonsentinel nodes. Conclusions: In this relatively small study, the accuracy of SLND in women with stage II or III breast cancer treated with preoperative therapy was similar to that achieved in early-stage breast cancer. If these results are confirmed in a larger cohort, it may be feasible to substitute SLND for routine axillary lymph node dissection in this population. This approach could reduce the morbidity of surgical therapy while preserving the accuracy of axillary staging and maintaining regional control in this high-risk population.

Recurrent abdominal laxity following interpositional human acellular dermal matrix.

Repair of large complex abdominal hernias with significant loss of domain requires component separation in combination with either a synthetic or biologic interpositional material. We previously described an algorithm for complex abdominal hernia repair, which incorporates Alloderm as an interpositional material and selective use of prolene mesh as an overlay. We now report recurrent laxity in a series of patients who were repaired with interpositional Alloderm alone without prolene mesh overlay. We reviewed all patients who underwent repair of massive ventral hernias and identified 7 patients who presented with abdominal wall laxity following component separation with interpositional Alloderm alone. All patients developed laxity within 12 months and required a secondary procedure. At the time of re-exploration, severe attenuation in the Alloderm was noted. The segment was excised, the edges closed primarily, and prolene mesh was placed as an onlay. Although Alloderm has been reported to be an effective biologic material for abdominal hernia reconstruction, we have noted significant laxity requiring secondary intervention.

Predictable Patterns of Intracranial and Cervical Spine Injury in Craniomaxillofacial Trauma: Analysis of 4786 Patients

Background: Patients presenting with traumatic craniomaxillofacial fractures often have occult concomitant injuries. This study was designed to determine whether facial fracture patterns are associated with a particular constellation of concomitant head and neck injuries. Methods: A retrospective review of 4786 consecutive patients diagnosed with maxillofacial fractures at a dedicated urban trauma center from 1998 to 2005 was conducted; maxillofacial fractures and cervical spine injuries were grouped by dividing the craniomaxillofacial skeleton and cervical spine into thirds. Univariate and multivariate logistic regression analyses were used to identify associations between facial fractures and other traumatic injuries. Results: Among all patients with facial fractures, 461 (9.7 percent) also had cervical spine injuries and 2175 (45.5 percent) had associated head injuries. Fractures of the upper face were associated with increased likelihood of mid lower cervical spine injuries, severe intracranial injuries, and increased mortality rates. Unilateral mandible injuries were associated with an increased likelihood of having upper cervical spine injuries, whereas unilateral midface injuries were associated with basilar skull fractures and several intracranial injuries. Finally, bilateral midface injuries were associated with basilar skull fracture and death. Conclusions: Craniomaxillofacial fractures are commonly associated with head and cervical spine injuries that involve predictable patterns of force dispersion from the maxillofacial skeleton and transmission to the cranial vault and cervical spine. These results suggest that concomitant injuries should be investigated closely with distinct types of facial fractures.

Mitochondrial Mutations Are a Late Event in the Progression of Head and Neck Squamous Cell Cancer

Purpose: To determine the timing of mitochondrial mutations in the progression of head and neck squamous cell carcinoma. Experimental Design: Twenty-three mitochondrial mutations were identified in 12 tumors using a high-throughput mitochondrial sequencing array. Areas of adjacent dysplastic and normal epithelium adjacent to tumors were sequenced using conventional methods for the presence of mutations that occurred in the corresponding tumor. Results: Two of 23 (8.7%) tumor mitochondrial mutations (2 of 12 tumors) were present in both the areas of adjacent dysplasia and normal epithelium. Five of 23 (21.7%) tumor mitochondrial mutations (4 of 12 tumors) were present in areas of adjacent dysplasia. Eleven of 12 tumors contained nonsynonymous mutations that resulted in protein coding alterations. A significant difference (P < 0.01, χ2) was found in the incidence of mitochondrial mutation that occurred after development of cancer compared with adjacent areas dysplasia and normal epithelium. Conclusions: The majority of mitochondrial mutations occur during or after the transition of preneoplastic epithelium to cancer in head and neck squamous cell carcinoma, indicating that these are a late event in head and neck carcinogenesis.

Smad3 has a critical role in TGF-β-mediated growth inhibition and apoptosis in colonic epithelial cells

BACKGROUND Smad proteins play a key role in TGF-beta signaling that regulates cell proliferation, differentiation, and apoptosis. Mice deficient in Smad3 develop colonic adenocarcinoma. MATERIALS AND METHODS We developed a Smad3-deficient colonocyte cell line that was used to study TGF-beta-mediated growth inhibition and induction of apoptosis was compared to young adult mouse colonocyte (YAMC) control cells. Growth inhibition was assessed by cell count and ((3)H)-thymidine incorporation assay. Transcriptional response to TGF-beta was measured by transfecting the reporters p3TP-Lux and p(CAGA)(9)-MLP-luc. TGF-beta-induced apoptosis was assessed using ELISA and Hoechst staining. Mediators of cell-cycle arrest and apoptosis were assayed by Western blot. RESULTS Smad3-/- cells were resistant to TGF-beta-mediated growth inhibition compared to control cells. Ninety-eight percent of cell count growth inhibition observed in YAMC cells, while 34% inhibition was observed in Smad3-/- cells after TGF-beta treatment. ((3)H)-thymidine incorporation was inhibited by 61% in YAMC cells, while Smad3-/- cells showed 25% inhibition after TGF-beta treatment. Smad3-/- cells were deficient in luciferase reporter induction by TGF-beta. TGF-beta induced apoptosis 8-fold in YAMC cells, but had no effect on apoptosis in Smad3-/- cells. p21(Cip11) and PAI-1 are induced in YAMC cells by TGF-beta, but unchanged in Smad3-/- cells. TGF-beta decreases cyclin D1 levels in YAMC cells but does not affect levels in Smad3-/- cells. CONCLUSIONS Our findings suggest that the loss of Smad3 contributes to resistance of TGF-beta growth inhibition and apoptosis in colonic epithelium. This may represent a mechanism by which cells are able to escape antiproliferative controls and embark on a pathway toward neoplasia.

Mitochondrial Resequencing Arrays Detect Tumor-Specific Mutations in Salivary Rinses of Patients with Head and Neck Cancer

Purpose: Alterations of the mitochondrial genome have been identified in multiple solid tumors and in many head and neck squamous cell carcinomas (HNSCC). Identification of mitochondrial mutations in the salivary rinses of patients with HNSCC has potential application in disease detection. In this study, we used the MitoChip v2.0 mitochondrial genome resequencing array to detect minor populations of mitochondrial DNA in salivary rinses of patients with HNSCC. Experimental Design: Salivary rinses from 13 patients with HNSCC, whose tumors carried mitochondrial mutations, were collected before surgical resection. DNA isolated from salivary rinses and serial dilutions of DNA derived from HNSCC-derived cell lines with known mitochondrial mutations were sequenced using the MitoChip, and analyzed using a quantitative algorithm which we developed to detect minor populations of mitochondrial DNA from MitoChip probe intensity data. Results: We detected heteroplasmic populations of mitochondrial DNA up to a 1:200 dilution using MitoChip v2.0 and our analysis algorithm. A logarithmic relationship between the magnitude of assay intensity and concentration of minor mitochondrial populations was shown. This technique was able to identify tumor-specific mitochondrial mutations in salivary rinses from 10 of 13 (76.9%) patients with head and neck cancer. Conclusions: Minor populations of mitochondrial DNA and disease-specific mitochondrial mutations in salivary rinses of patients with HNSCC can be successfully identified using the MitoChip resequencing array and the algorithm which we have developed. This technique has potential application in the surveillance of patients after resection and may have applicability in the surveillance of body fluids in other tumor types.

Inactivation of the tumor suppressor genes causing the hereditary syndromes predisposing to head and neck cancer via promoter hypermethylation in sporadic head and neck cancers.

Fanconi anemia (FA) and dyskeratosis congenita (DC) are rare inherited syndromes that cause head and neck squamous cell cancer (HNSCC). Prior studies of inherited forms of cancer have been extremely important in elucidating tumor suppressor genes inactivated in sporadic tumors. Here, we studied whether sporadic tumors have epigenetic silencing of the genes causing the inherited forms of HNSCC. Using bisulfite sequencing, we investigated the incidence of promoter hypermethylation of the 17 Fanconi- and DC-associated genes in sporadic HNSCC. Genes that only showed methylation in the tumor patients were chosen for quantitative methylation-specific PCR (qMSP) in a set of 45 tumor and 16 normal patients. Three gene promoters showed differences in methylation: FancB (FAAP95, FA core complex), FancJ (BRIP1, DNA Helicase/ATPase), and DKC1 (dyskeratin). Bisulfite sequencing revealed that only FancB and DKC1 showed no methylation in normal patients, yet the presence of promoter hypermethylation in tumor patients. On qMSP, 1/16 (6.25%) of the normal mucosal samples from non-cancer patients and 14/45 (31.1%) of the tumor patients demonstrated hypermethylation of the FancB locus (p < 0.05). These results suggest that inactivation of FancB may play a role in the pathogenesis of sporadic HNSCC.