Ashok R. Mehta

Oncogene amplification in squamous cell carcinoma of the oral cavity

We have determined the prevalence of amplification of c‐myc, N‐myc, L‐myc, H‐ras, Ki‐ras, and N‐ras oncogenes in 23 cases of squamous cell carcinoma of the oral cavity, using Southern hybridization analysis of DNA extracted from the primary tumor tissues. Nick‐translated oncogene probes and oncogene inserts labeled to high specific activities were used. We observed a 5‐ to 10‐fold amplification of one or more of c‐myc, N‐myc, Ki‐ras and N‐ras oncogenes in 56% of the tumor tissue samples, with these oncogenes not being amplified in the peripheral blood cells of the same patients, L‐myc and H‐ras were not amplified in any of our samples. The oncogene amplifications seemed to be associated with advanced stages of squamous cell carcinomas, with the ras and myc family oncogenes being amplified in stages 3 and 4. Hybridization with N‐myc detected an additional 2.3 kb EcoRI fragment, along with the normal 2.1 kb fragment. Our data also demonstrated amplification of multiple oncogenes in the same tumor tissue sample. About 60% of the samples with amplified oncogenes showed simultaneous amplification of 2 or more oncogenes. The results showing different oncogene amplifications in similar tumors, as well as multiple oncogene amplifications in the same tumor, suggest that these oncogenes may be alternatively or simultaneously activated in oral carcinogenesis.

Detection of HPV-16 genome in human oral cancers and potentially malignant lesions from India.

The presence of high risk human papilloma virus (HPV) 16 and 18 was examined in 100 oral cancer patients of Indian descent, 80 patients with potentially malignant oral lesions and corresponding clinically normal mucosa from 48 of these patients. Additionally, presence of HPV-33, -6 and -11 was also studied in 86 oral cancers, 50 potentially malignant oral lesions and 30 corresponding normal oral mucosa. All the patients with oral cancer and oral lesions, were long term tobacco-chewers, and a majority of the patients were in Advanced Stages III and IV. The DNA samples were amplified by polymerase chain reaction (PCR) using HPV L1 consensus primers. Typing of HPV was performed by Southern hybridization analysis of the PCR products using HPV-16, -18, -33, -6 and -11 type specific oligonucleotide probes. HPV-16 was detected in 15 out of 100 (15%) oral tumours, 27 out of 80 (34%) potentially malignant lesions and 15 out of 48 (31%) of the corresponding normal mucosa in the patients with oral lesions. HPV-18 was not detected in any of the oral cancers, oral lesions and normal mucosa. HPV-33 and the low-risk HPV-6 and -11 were also not detected in the oral cancers, oral lesions and corresponding normal mucosa. A significantly higher prevalence of HPV-16 was observed in oral lesions (27 out of 80, 34%) as compared to oral cancers (15 out of 100, 15%). The observed difference of 19% (95% confidence interval [CI]: 6%, 31%), between these two proportions was statistically significant at the 5% level of significance. Our data indicates that HPV-16 may play a direct role in a certain proportion of oral cancers; whereas in a subpopulation of oral cancers HPV-16 infection may be vital in the early events associated with development of potentially malignant oral lesions, and the presence of the virus not essential in the progression of the oral lesion to frank malignancy.

Complications of the pectoralis major myocutaneous flap in the oral cavity : A prospective evaluation of 220 cases

A prospective study of 220 consecutive pectoralis major myocutaneous flaps used for oral cavity reconstruction from March of 1990 to February of 1991 showed that 89 patients (40.5 percent) developed flap-related complications and 33 patients (15 percent) had complications unrelated to the flap; 92 patients (42 percent) had an uneventful recovery and there were 6 (2.7 percent) postoperative deaths. Sixty patients (27 percent) developed flap necrosis, of whom only 6 (2.7 percent) had total flap loss. Major partial loss occurred in 20 patients (9 percent) and minor flap loss occurred in 34 (15.5 percent). Flap necrosis was significantly lower in the purely myocutaneous flaps (p < 0.00000) vis-à-vis the bipedicled and osteocutaneous flaps. Fistula formation, wound infection, dehiscence at the flap margin, and postoperative hematomas occurred with comparable frequency in both groups. The female gender, primary tongue cancer, subtotal or total glossectomy, bipedicling of flaps, prior chemotherapy, and presence of systemic disease (diabetes) emerged as significant risk factors for flap necrosis on multivariate analysis (p < 0.005).

p53 inactivation in chewing tobacco-induced oral cancers and leukoplakias from India

The inactivation of p53 tumour suppressor gene vis-á-vis point mutation, overexpression and degradation due to Human Papilloma virus (HPV) 16/18 infection, was examined in chewing tobacco-associated oral cancers and oral leukoplakias from India. The analysis of mutations was assessed by polymerase chain reaction (PCR) with single strand conformation polymorphism (PCR-SSCP) of exons 5-9 on DNA from 83 oral cancer cases, and the mutations confirmed by direct nucleotide sequencing of the PCR products. p53 protein expression was evaluated by immunohistochemical analysis on paraffin-embedded sections of 62 representative oral cancer biopsies and 22 leukoplakias, using p53-specific monoclonal antibody DO-7. The presence of HPV16/18 was detected in the 83 oral cancer cases by PCR analysis using HPV L1 consensus sequences, followed by Southern hybridization with type-specific oligonucleotide probes. Forty-six per cent (38/83) of oral cancer tumours showed p53 alterations, with 17% (14/83) showing point mutations, 37% (23/62) with overexpression and 25% (21/83) with presence of HPV16 wherein the E6 HPV16 protein degrades p53. HPV18 was not detected in any of the samples. Ninety-two per cent concordance was observed between missense point mutations and overexpression of p53 protein. A significant correlation was not observed between p53 alterations in oral cancer and clinico-pathological profile of the patients. Twenty-seven per cent (6/22) of oral leukoplakias showed p53 overexpression. The overall p53 alterations in oral cancer tissues and oral lesions are comparable to data from the oral cancers reported in the Western countries with smoking and alcohol-associated oral cancers, and suggest a critical role for p53 gene in a significant proportion of oral cancers from India. The overexpression of p53 protein in leukoplakias may serve as a valuable biomarker for identifying individuals at high risk of transformation to malignant phenotype.

Amplification and Overexpression of Epidermal Growth Factor Receptor Gene in Human Oropharyngeal Cancer

The presence of epidermal growth factor receptor (EGF-R) gene was determined in 84 patients with squamous cell carcinoma (SCC) of the oropharyngeal region--a highly prevalent, chewing-tobacco associated malignancy in India, using Southern hybridisation analysis of DNA extracted from primary tumor tissues. We observed a 3- to 8-fold amplification of EGF-R gene in 19/66 (29%) of the SCCs of the oral cavity, and about 30-fold EGF-R amplification in 2/18 (11%) hypopharyngeal cancers. Dot blot analysis of total RNA from several tumour tissues, revealed overexpression of the EGF-R gene in the examined patients, with the EGF-R gene amplified. 4 patients with single copy EGF-R gene, did not exhibit overexpression of the gene. Within our sample set, no correlation was evident between EGF-R gene amplification and clinico-pathological parameters of the malignancy. The amplification and overexpression of the EGF-R gene observed in the primary tumour tissues of 25% (21/84) of the human oropharyngeal cancers, indicate possible involvement of the gene in the pathogenesis of these cancers.

Loss of allelic heterozygosity at the harveyras locus in human oral carcinomas

SummaryThe Harveyras locus was examined for restriction fragment polymorphism and loss of allelic heterozygosity in 62 oral cancer patients. Southern blot analysis onBamHI digests of the tumour tissue DNA, revealed 23 patients with H-ras-1 heterozygosity. The probes used to study the polymorphism were theBamHI 6.6-kb fragment encoding the complete H-ras-1 sequence plus the variable tandem repeat (VTR) region, and the 1-kbMspI fragment encoding the VTR region. The allelic heterozygosity was better resolved byPvuII and further confirmed byTaqI. In addition,TaqI digestion demonstrated a unique VTR rearrangement indicated by 2.1-kb, 0.9-kb and 0.6-kb fragments, implying additionalTaqI sites, in three of the patients. Further analysis of matched tumor tissue and peripheral blood cell DNA from the same patient demonstrated tumor-associated loss of one of the allelic fragments in 7/23 (30%) of the patients with H-ras-1 heterozygosity. However, the loss was not significantly correlated to clinicopathological parameters staging the disease. Thus, our data showing loss of H-ras-1 alleles and VTR rearrangement, with relatively high incidence (9/23; 39%) in the oral cancer patients at various stages of the disease, implies H-ras-1 involvement as an early event in the process of oral carcinogenesis.

The Indian experience with immediate tracheoesophageal puncture for voice restoration

A 3-year prospective study on primary tracheoesophageal puncture was carried out at Nanavati Hospital and Tata Memorial Hospital, Bombay, to assess its feasibility in a developing country and its success in vocal rehabilitation. We report our experience with primary tracheoesophageal puncture in 64 patients (57 males and 7 females) following surgical treatment for carcinoma of the pyriform fossa (45 cases) and endolarynx (19 cases). All patients underwent total laryngectomies with or without partial pharyngectomy and primary pharyngeal mucosal closure. Fifty-one patients underwent primary surgery followed by postoperative radiotherapy. At 3 months successful tracheoesophageal speech was achieved in 84% patients, although this percentage decreased to 67% at 9 months. There were no major complications. Successful vocal rehabilitation was related more to “patient factors” such as delays in seeking medical attention when prosthetic valves dislodged, improper use and care of the valves and inability to learn speech with the prosthesis. Overall, primary tracheoesophageal puncture was found to be feasible and should be used more extensively for vocal rehabilitation following laryngectomy in developing countries.

Osteochondroma of the skull base.

Osteochondroma, or osteocartilaginous exostosis, is the most common skeletal neoplasm. The cartilagecapped subperiosteal bone projection accounts for 20% to 50% of benign bone tumors and 10% to 15% of all bone tumors. The tumor is a benign, well-demarcated, sessile or stalked subperiosteal bone projection that is partially or totally topped by a bluish-white hyaline cartilage cap. It is histologically and behaviorally distinct from other cartilaginous tumors such as enchondromas, osteosarcomas, and chondrosarcomas. The most common symptom of an osteochondroma is a painless bump in the proximity of a joint. However, more serious presentations include symptoms related to compression of nerves, blood vessels, or tendinous insertions, which require surgical excision rather than periodic screening. A 38-year-old man presented to Tata Memorial Hospital, Mumbai, India, with a 9-month history of discomfort in the region of his hard palate, tinnitus, and persistent right-ear fullness. The patient denied any epistaxis, oral bleeding, dysphagia, or trismus. Otologic examination revealed a right serous middle ear effusion. Physical examination of his oral cavity revealed a submucosal bulging mass located at the junction of the hard and soft palates just posterior to the right upper alveolar margin. The remaining head and neck examination including cranial nerves was noncontributory. Coronal and axial CT scans of the head and neck revealed a 6 5 cm bony lesion in the right parapharyngeal space (Figs 1 and 2). There was distortion of the nasopharynx with scalloping of the medial aspect of the middle cranial fossa floor and erosion of the right lateral pterygoid plate. However, there was no intracranial invasion. The patient underwent a mandibular swing for access to the right parapharyngeal space. The tumor was then isolated and found to have its bony attachment on the skull base. The mandibular condyle and coronoid were completely free of tumor. The tumor was removed en bloc (Fig 3), and histological analysis was completed.

Moderate-Dose Methotrexate in Head and Neck Cancer

200 mg/m2 methotrexate given intravenously in a running drip for 6 h has been used as an initial adjuvant therapy in 38 patients with advanced head and neck cancer. The response rate is as high as 80%, with 21% achieving complete remission. Histologically, specimens were tumor free in 3 patients. Toxicity in 38 patients included leukopenia (4), mucositis (6) and diarrhea (1). This particular dose of methotrexate appears to be safe and usually does not need leucovorin rescue. Also, when given as initial treatment, it is effective in reduction of tumor bulk. A prolonged randomized trial is essential to determine its role in improving long-term survival.