Review: Head and neck squamous cell carcinoma in sub-Saharan Africa.

AIM: Review the literature from 1990 to 2013 to determine known anatomic sites, risk factors, treatments, and outcomes of head and neck squamous cell carcinoma (HNSCC) in sub-Saharan Africa. METHODS: Using a systematic search strategy, literature pertaining to HNSCC in sub-Saharan Africa was reviewed and patient demographics, anatomic sites, histology, stage, treatment, and outcomes were abstracted. The contributions of human immunodeficiency virus (HIV), human papillomavirus (HPV) and behavioural risk factors to HNSCC in the region were assessed. RESULTS: Of the 342 papers identified, 46 were utilized for review, including 8611 patients. In sub-Saharan Africa, the oropharyngeal/oral cavity was found to be the most common site, with 7750 cases (90% of all cases). Few papers distinguished oropharyngeal from oral cavity, making identification of possible HPV-associated oropharyngeal squamous cell carcinoma (SCC) difficult. SCC of the nasopharynx, nasal cavity, or paranasal sinuses was identified in 410 patients (4.8% of all cases). Laryngeal SCC was found in 385 patients (4.5% of all cases), and only 66 patients (0.8% of all cases) with hypopharyngeal SCC were identified. In 862 patients with data available, 43% used tobacco and 42% used alcohol, and reported use varied widely and was more common in laryngeal SCC than that of the oropharyngeal/oral cavity. Toombak and kola nut use was reported to be higher in patients with HNSCC. Several papers reported HIV-positive patients with HNSCC, but it was not possible to determine HNSCC prevalence in HIV-positive compared to negative patients. Reports of treatment and outcomes were rare. CONCLUSIONS: The oropharyngeal/oral cavity was by far the most commonly reported site of HNSCC reported in sub-Saharan Africa. The roles of risk factors in HNSCC incidence in sub-Saharan Africa were difficult to delineate from the available studies, but a majority of patients did not use tobacco and alcohol.

Environmental risk factors for oesophageal cancer in Malawi: A case-control study.

AIM: There is a high burden of oesophageal cancer in Malawi with dismal outcomes. It is not known whether environmental factors are associated with oesophageal cancer. Without knowing this critical information, prevention interventions are not possible. The purpose of this analysis was to explore environmental factors associated with oesophageal cancer in the Malawian context. METHODS: A hospital-based case-control study of the association between environmental risk factors and oesophageal cancer was conducted at Kamuzu Central Hospital in Lilongwe, Malawi and Queen Elizabeth Central Hospital in Blantyre, Malawi. Ninety-six persons with squamous cell carcinoma and 180 controls were enrolled and analyzed. These two groups were compared for a range of environmental risk factors, using logistic regression models. Unadjusted and adjusted odds ratios and 95% confidence intervals (CI) were calculated. RESULTS: Firewood cooking, cigarette smoking, and use of white maize flour all had strong associations with squamous cell carcinoma of the oesophagus, with adjusted odds ratios of 12.6 (95% CI: 4.2-37.7), 5.4 (95% CI: 2.0-15.2) and 6.6 (95% CI: 2.3-19.3), respectively. CONCLUSIONS: Several modifiable risk factors were found to be strongly associated with squamous cell carcinoma. Research is needed to confirm these associations and then determine how to intervene on these modifiable risk factors in the Malawian context.

Review: Head and Neck Squamous Cell Carcinoma in Sub-Saharan Africa

Aim.Review the literature from 1990 to 2013 to determine known anatomic sites; risk factors; treatments; and outcomes of head and neck squamous cell carcinoma (HNSCC) in sub-Saharan Africa.Methods.Using a systematic search strategy; literature pertaining to HNSCC in sub-Saharan Africa was reviewed and patient demographics; anatomic sites; histology; stage; treatment; and outcomes were abstracted. The contributions of human immunodeficiency virus (HIV); human papillomavirus (HPV) and behavioural risk factors to HNSCC in the region were assessed. Results of the 342 papers identified; 46 were utilized for review; including 8611 patients. In sub-Saharan Africa; the oropharyngeal/oral cavity was found to be the most common site; with 7750 cases (90% of all cases). Few papers distinguished oropharyngeal from oral cavity; making identification of possible HPV-associated oropharyngeal squamous cell carcinoma (SCC) difficult. SCC of the nasopharynx; nasal cavity; or paranasal sinuses was identified in 410 patients (4.8% of all cases). Laryngeal SCC was found in 385 patients (4.5% of all cases); and only 66 patients (0.8% of all cases) with hypopharyngeal SCC were identified. In 862 patients with data available; 43% used tobacco and 42% used alcohol; and reported use varied widely and was more common in laryngeal SCC than that of the oropharyngeal/oral cavity. Toombak and kola nut use was reported to be higher in patients with HNSCC. Several papers reported HIV-positive patients with HNSCC; but it was not possible to determine HNSCC prevalence in HIV-positive compared to negative patients. Reports of treatment and outcomes were rare.Conclusions The oropharyngeal/oral cavity was by far the most commonly reported site of HNSCC reported in sub-Saharan Africa. The roles of risk factors in HNSCC incidence in sub-Saharan Africa were difficult to delineate from the available studies; but a majority of patients did not use tobacco and alcohol

Causes of cervical lymphadenopathy at Kamuzu Central Hospital.

AIM: Description of pathologic causes of cervical lymphadenopathy at Kamuzu Central Hospital. INTRODUCTION: The evaluation of cervical lymphadenopathy is a common diagnostic challenge facing clinicians. Previously at Kamuzu Central Hospital (KCH) tuberculosis (TB) was reported to be the most common cause of cervical lymphadenopathy However, no recent study has assessed this common diagnostic challenge in Malawi, particularly since the beginning of the HIV epidemic and the subsequent scale-up of antiretroviral therapy. METHODS: We conducted a cross-sectional study of all cervical lymph node specimens from the KCH pathology laboratory between 1 July 2011 and 28 February 2013 and describe patient age, gender, and pathologic diagnoses. RESULTS: Our search of the KCH pathology database yielded 179 cases. Of these, 143 (77%) were histologic specimens (open biopsy or core needle samples) while 34 (23%) were cytology specimens. The age range was from 0 to 76 years with a mean of 30 (SD: 19). In adults, the most common diagnosis was malignancy (n=41, 35%), while in children 15 cases each of malignancy and benign masses were diagnosed. Only 6 cases (5%) of TB were diagnosed in adults, and 4 cases (6%) of TB were diagnosed in children. CONCLUSION: Our study shows more malignancy and much less TB than a prior study of cervical lymphadenopathy at KCH. With the successful initiaion of the KCH Pathology Laboratory in 2011, we recommend biopsy or FNA early in the workup of cervical lymphadenopathy to prevent long delays in diagnosis and treatment of curable cancers.

Different cellular p16(INK4a) localisation may signal different survival outcomes in head and neck cancer.

BACKGROUND: Recently, the management of head and neck squamous cell carcinoma (HNSCC) has focused considerable attention on biomarkers, which may influence outcomes. Tests for human papilloma infection, including direct assessment of the virus as well as an associated tumour suppressor gene p16, are considered reproducible. Tumours from familial melanoma syndromes have suggested that nuclear localisation of p16 might have a further role in risk stratification. We hypothesised p16 staining that considered nuclear localisation might be informative for predicting outcomes in a broader set of HNSCC tumours not limited to the oropharynx, human papilloma virus (HPV) status or by smoking status. METHODS: Patients treated for HNSCC from 2002 to 2006 at UNC (University of North Carolina at Chapel Hill) hospitals that had banked tissue available were eligible for this study. Tissue microarrays (TMA) were generated in triplicate. Immunohistochemical (IHC) staining for p16 was performed and scored separately for nuclear and cytoplasmic staining. Human papilloma virus staining was also carried out using monoclonal antibody E6H4. p16 expression, HPV status and other clinical features were correlated with progression-free (PFS) and overall survival (OS). RESULTS: A total of 135 patients had sufficient sample for this analysis. Median age at diagnosis was 57 years (range 20-82), with 68.9% males, 8.9% never smokers and 32.6% never drinkers. Three-year OS rate and PFS rate was 63.0% and 54.1%, respectively. Based on the p16 staining score, patients were divided into three groups: high nuclear, high cytoplasmic staining group (HN), low nuclear, low cytoplasmic staining group (LS) and high cytoplasmic, low nuclear staining group (HC). The HN and the LS groups had significantly better OS than the HC group with hazard ratios of 0.10 and 0.37, respectively, after controlling for other factors, including HPV status. These two groups also had significantly better PFS than the HC staining group. This finding was consistent for sites outside the oropharynx and did not require adjustment for smoking status. CONCLUSION: Different p16 protein localisation suggested different survival outcomes in a manner that does not require limiting the biomarker to the oropharynx and does not require assessment of smoking status.

Management of the carotid artery at the skull base.

PURPOSE: To determine appropriate management of benign lesions with significant involvement of the carotid artery at the skull base and present an algorithm for safe treatment of these patients. MATERIALS AND METHODS: From 1982 to 1999, 115 patients with significant parapharyngeal space masses were treated at our institution. Of these patients, 43 had lesions involving the carotid artery at the skull base and served as the basis for this study. All patients underwent preoperative computed tomography or magnetic resonance imaging scans to determine carotid involvement, and all had preoperative 4-vessel arteriograms and carotid occlusion tests with continuous electroencephalography or neurologic examination monitoring to predict safety of carotid sacrifice. RESULTS: Of 43 patients, 41 passed carotid occlusion testing and were treated surgically. Of these patients, 33 (81%) underwent resection of their lesions with preservation of the internal carotid artery, 5 (12%) had resection with bypass or reconstruction of the artery, and 3 (7%) had en bloc resections without artery reconstruction. There were no transient or permanent neurologic sequelae in any patient. CONCLUSIONS: When carotid artery encasement occurs in the setting of benign lesions at the skull base, safe resection with vascular preservation is possible in most cases. If carotid artery resection is necessary, vascular bypass or reconstruction is recommended to minimize neurologic morbidity.

Sinogenic subdural empyema and Streptococcus anginosus.

Subdural empyema (SDE) is most commonly caused by sinusitis and, without early diagnosis and neurosurgical intervention, is associated with high mortality. In a patient with sinusitis who presents with mental status changes, the diagnosis of SDE should be suspected on clinical grounds, even in the absence of significant computed tomographic findings. Computed tomography with contrast is a useful aid in the diagnosis of SDE, but findings may be subtle, and contrasted magnetic resonance imaging is superior. The association of Streptococcus anginosus sinusitis and related intracranial sequelae is important owing to the potentially catastrophic complications and should be recognized by otolaryngologists. In view of the rapidly progressing nature of sinogenic SDE, physicians should strongly consider early institution of aggressive therapy consisting of craniotomy with concurrent sinus drainage in patients in whom sinogenic SDE is suspected on clinical grounds, particularly in the presence of S. anginosus-positive sinus cultures.

Three-dimensional xenograft model of dysplastic human laryngeal mucosa.

OBJECTIVE: Development of new therapeutic interventions in head and neck squamous cell carcinoma (HNSCC) will be facilitated by a model system that incorporates the ease of manipulation found in current tissue culture systems while retaining the three dimensional architecture that defines these malignancies. STUDY DESIGN: Original scientific investigation. METHODS: We describe a modification of a normal respiratory mucosa model system which recreates premalignant mucosal histology. Grossly normal appearing human mucosa is harvested from laryngectomy specimens, the mucosal epithelium selectively removed by protease treatment and placed in conventional tissue culture. After 7 days, the cells are seeded into denuded rat tracheas, which are in turn implanted in flank pockets of athymic nu/nu mice. The tracheas are incubated for three weeks, removed and the mucosa examined histologically. RESULTS: As originally described, normal pseudostratified squamous epithelium can be re-established in this system. Using human dysplastic mucosa as a starting material, mucosal histologies of respiratory dysplasia, squamous metaplasia, squamous dysplasia and squamous carcinoma in situ can be established. CONCLUSION: This system will provide a paradigm for future therapeutic interventions to modify the progression of squamous metaplasia to dysplasia, carcinoma in situ and invasive squamous cell carcinoma.

ras mutations and expression in head and neck squamous cell carcinomas.

Mutational activation and overexpression of the family of ras proto-oncogenes have been associated with many human tumors. The role of mutations of H-ras, K-ras, and N-ras, as well as expression of the respective protein products (p21s) in normal mucosa, dysplastic mucosa, and squamous cell carcinomas (SCCs) of the head and neck has not been fully described. In our study, 51 tumors (40 paraffin embedded and 11 fresh frozen) were examined to determine if mutational activation of ras is an important molecular event in head and neck SCC. Analyses of codons 12, 13, and 61 of H-ras, K-ras, and N-ras revealed no mutations, suggesting that mutational activation of ras is not important in the majority of head and neck SCCs. Immunocytochemistry (ICC) was used to define the expression of H-ras, K-ras, and N-ras in normal mucosa, dysplastic mucosa, and SCC of the head and neck and to determine if expression of ras family members correlated with early or late events in the development of SCC. Expression of p21N-ras in nine samples of histologically normal head and neck mucosa revealed moderate staining in the basal proliferative layers with progressively less staining as cells matured. The most superficial layers of normal mucosa failed to express p21N-ras. A low level of p21H-ras was expressed in all layers of normal mucosa while K-ras was not expressed. ICC of SCC tumor sections revealed cytoplasmic expression of N-ras in nine of nine tumors, H-ras in five of nine tumors, and K-ras in one of nine tumors. Expression of H-ras, K-ras, and N-ras in head and neck SCC was not related to histologic differentiation or TNM staging; however, p21N-ras was overexpressed in seven of nine tumors. Furthermore, the pattern of N-ras expression in dysplastic lesions revealed expression in all layers of the mucosa in contrast to normal mucosa, which expresses p21N-ras primarily in the basal proliferative layer. The change in p21N-ras expression pattern in dysplastic mucosa and its overexpression in the majority of tumors suggest that loss of control of N-ras expression may be an early step in carcinogenesis of head and neck SCC.

Elevated protein tyrosine phosphorylation in the optic tract of the chick embryo.

Antibodies specific for protein phosphotyrosyl residues were used to localize sites of protein tyrosine kinase activity in the optic tract of the developing chick by immunoperoxidase staining. In the stage 34 (day 8) chick embryo, phosphotyrosine-modified proteins were abundant within outgrowing neuronal processes in the optic nerve head and nerve fiber layer of the retina, and in the developing stratum opticum at the surface of the optic tectum. These sites corresponded to regions where migrating growth cones and fasciculating bundles of some, but not all, retinal ganglion cell axons were located. Phosphotyrosine-modified proteins were also abundant in and highly restricted to the process-rich layers of the embryonic optic tectum. Phosphotyrosine immunoreactivity decreased dramatically in the corresponding regions of the optic tract of the adult chicken, indicating that protein tyrosine phosphorylation occurred principally in developing, rather than mature, neuronal processes. These findings are in accord with the idea that protein tyrosine phosphorylation may be important in cell-cell or cell-substratum interactions of ganglion cell axons.

D2 dopamine receptors in the human retina: cloning of cDNA and localization of mRNA.

1. We have obtained a cDNA clone encoding a human retinal D2 dopamine receptor. 2. The longest open reading frame (1242 bp) of this clone encodes a protein of 414 amino acids having a predicted molecular weight of 47,000 and a transmembrane topology similar to that of other G protein-coupled receptors. 3. Transient transfection of COS-7 cells with an expression vector containing the clone resulted in expression of a protein possessing a pharmacological profile similar to that of the D2 dopamine receptor found in striatum and retina. 4. Northern blot analysis indicated that, in rat brain and retina, the mRNA for this receptor was 2.9 kb in size. 5. In situ hybridization was performed to examine the distribution of the mRNA for this receptor in human retina. Specific hybridization was detected in both the inner and the outer nuclear layers. 6. These findings are consistent with prior physiological and autoradiographic studies describing the localization of D2 dopamine receptors in vertebrate retinas. Our observations suggest that photoreceptors as well as cells in the inner nuclear layer of human retinas may express the mRNA for this D2 dopamine receptor.

Localization of the normal cellular src protein to the growth cone of differentiating neurons in brain and retina.

The protooncogene c-src is implicated in the development of the vertebrate nervous system. Its product pp60c-src is a tyrosine-specific protein kinase that is expressed in two phases of neural development. An activated form of the pp60c-src is highly enriched in the membrane of nerve growth cones and in the proximal neuritic shaft of differentiating neurons, as shown in brain and retina. A possible role for pp60c-src in neuronal process extension is suggested that may involve cell-substratum adhesion or motility.

Tyrosine phosphorylated proteins accumulate in junctional regions of the developing chick neural retina.

Antibodies specific for protein phosphotyrosyl residues were used to localize sites of action of tyrosine-specific protein kinases in developing chick neural retina by immunoperoxidase staining. Phosphotyrosine-modified proteins became prominent in growth cone- and process-rich regions of embryonic retina during neuronal differentiation. Maximal levels accumulated in the synaptic layers and limiting membranes of the adult retina, where numerous junctional complexes reside. Two major phosphotyrosine-modified proteins in adult retina (80, 42 kDal) increased markedly during maturation. In contrast, the synaptic layers of optic tectum and other brain regions exhibited low protein phosphotyrosine levels. These results suggest a specific role for protein tyrosine phosphorylation in the retina at sites of synapses and other intercellular junctions.

c-src gene product in developing rat brain is enriched in nerve growth cone membranes.

Differentiating rat neurons express high levels of the protooncogene product pp60c-src, a 60-kDa tyrosine kinase of unknown function encoded by c-src. pp60c-src was found to be concentrated at least 9-fold in membranes from a subcellular fraction of nerve growth cones, the motile tips of outgrowing neuronal processes. Indirect immunofluorescence staining of cultured chick retinal explants showed pp60c-src in neuronal growth cones and processes, with the antigen particularly concentrated in growth cones of long neurites. pp60c-src in growth cone membranes was an active tyrosine-specific protein kinase with elevated tyrosine-specific protein kinase activity and reduced electrophoretic mobility characteristic of the form of pp60c-src in central nervous system neurons. pp60c-src was present at lower levels in subcellular fractions from mature rat brain but synaptosomal membranes were not enriched. Preferential localization of an active form of pp60c-src in nerve growth cone membranes and persistence of pp60c-src in mature neurons suggest that this tyrosine kinase is important in growth cone-mediated neurite extension and synaptic plasticity.

Band 3 tyrosine kinase in avian erythrocyte plasma membrane is immunologically related to pp60c-src.

We have identified in the plasma membrane of the chicken erythrocyte a 60-kDa tyrosine-specific protein kinase immunologically related to the transforming protein pp60v-src of Rous sarcoma virus. The erythrocyte protein kinase phosphorylated heavy chains of tumor-bearing rabbit (TBR) antibodies reactive with pp60c-src at tyrosine in immune complex protein kinase assays. The kinase was identified as a 60-kDa protein by [35S]methionine labeling of erythrocytes and by autophosphorylation in immune complexes. The kinase migrated on two-dimensional gel electrophoresis with an apparent pI and molecular mass similar to pp60c-src. A plasma membrane-enriched fraction isolated from chicken red cells contained the majority of the kinase activity. The kinase was solubilized from the plasma membrane by the detergents 0.5% (wt/vol) Na-deoxycholate and 1% (vol/vol) Nonidet P-40. One molar NaCl was much less effective, indicating a strong association of the kinase with the plasma membrane. Incubation of the plasma membrane fraction with [32P]ATP resulted in tyrosine phosphorylation of the anion transport protein band 3. Band 3 phosphorylation was blocked by TBR antibodies, indicating that the kinase recognized by pp60c-src antibodies was responsible for band 3 phosphorylation. These results demonstrate that the avian erythrocyte plasma membrane contains a tightly bound tyrosine-specific protein kinase identical or closely related to pp60c-src and that this kinase is responsible for band 3 phosphorylation in vitro.

A tyrosine kinase related to pp60c-src is associated with membranes of Electrophorus electricus electric organ.

A tyrosine-specific protein kinase immunologically related to pp60c-src, the cellular homolog of the Rous sarcoma virus-transforming protein, was expressed at elevated levels in the electric organ of the electric eel Electrophorus electricus. The electric organ kinase phosphorylated antibodies reactive with pp60c-src at tyrosine residues in immune complex protein kinase assays and was associated with electric organ membranes enriched in acetylcholine receptors. The protein recognized by anti-pp60c-src antibodies was phosphorylated in endogenous membrane phosphorylation reactions and was shown to have a relative molecular mass of 57 kDa by two-dimensional gel electrophoresis. In immune complex protein kinase assays the 57-kDa protein was phosphorylated at threonine by a distinct threonine kinase from the electric organ. The tyrosine kinase was purified 844-fold from electric organ membranes by chromatography on omega-aminohexyl agarose, phosphocellulose, and casein-Sepharose. Threonine kinase activity in immunoprecipitates was not observed in the tyrosine kinase fractions after the first step. Incubation of the casein Sepharose fraction with [gamma-32P]ATP-Mn2+ in solution resulted in phosphorylation of only the 57-kDa protein. Phosphorylation occurred solely at tyrosine, suggesting that the kinase is capable of autophosphorylation. The structural and functional properties of the 57-kDa electric organ kinase indicate that the 57-kDa electric organ protein is a member of the src subfamily of tyrosine kinases and is closely related to pp60c-src.

RecA protein rapidly crystallizes in the presence of spermidine: a valuable step in its purification and physical characterization.

The RecA protein of Escherichia coli, whether pure or in a crude cell lysate, will rapidly form small crystals (microcrystals) in the presence of low concentrations of spermidine. We describe the conditions of time, pH, and polyamine concentration over which crystallization occurs. Microcrystal formation is inhibited by concentrations of chloride over 25 mM and concentrations of phosphate or sulfate ions as low as 2 mM. Crystallization is not inhibited by high concentrations of other proteins, and the RecA protein microcrystals are easily collected by brief centrifugation. This provides a powerful purification step with high yield. Using this novel property, we prepared over 200 mg of RecA protein at least 95% pure with a single-strand DNA-dependent ATPase activity of 98% from 65 g of cells in 2-3 days. Spermidine was easily removed from the RecA protein by dialysis.