A casual role for Human Papilloma virus-16 for the Head and Neck Cancers word wide-and role of HPV vaccine altering the carriage rate of oropharyngeal HPV16?

Dear Editor

Infection with Human Papilloma Virus (HPV) 16 and 18 mostly is well established for development of cervical cancers in pre and post menopausal women through out the world. Professor H. Zur Housen was awarded the Nobel Prize for medicine and physiology for establishing role of HPV in development of cancer in 2008 instead of HSV2. But a pathogenic role for this HPV in non ano- genital cancer has yet been unclear till now. Tobacco and alcohol is considered today as important etiological factor for Head and Neck Squamous cell cancers(HNSCC). Epidemiological and laboratory evidences howevver now suggest this conclusion that in addition to tobacco and alcohol, HPV may be also the causative agent for the head and neck SCC. In our Institute at IPGME&R, Kolkata-20, West Bengal, India, from Pathology department the major bulk of cancer are diagnosed in the male population in any year is HNSCC and more then 95% of these sufferers gives however history of either smoking cigarette or local made Bidi or chewing Pan Parag products for long periods. These people are usually older and middle aged male. What about those young people dignosed with HNSCC? As with cervical cancer HNSCC is today a world wide public health problem with more then 9-10 lacks population per year are diagnosed by the Histopathologists having HNSCC. High risk HPV like HPV16 or HPV18 is probably not necessary for development of HNSCC. In our laboratory, we carried a work for 2 years on Role of HPV 16 and HNSCC mostly in younger people particularly and found HPV type 16 DNA is present in primary[poorly differentiated] and in metastatic cell nucleus in high copy numbers frequently integrated and transcriptionaly active by ISH technique. We are not sure whether these findings is casual association of HPV16 with HNSCC or etiology. Researches over past several years had also shown a string and consistent association between high risk HPV and distinct subset of HNSCC . However in our study these HPV associated HNSCC are characterized clinically by their location within the laryngeal and palatal tonsil of oropharynx. Their poorly differentiated histopatholgy and their frequent occurrence in nonsmoking and young patients then in HNSCC not associated with HPV. The proportion of HNSCC that is associated with HPV may be greater in a nonsmoker. HPV positive cancer may also occur in smokers. However it is unclear whether elevated risk of HNSCC for contamination of HPV infection and tobacco addiction is synergistic or additive with HPV? Thus screening for HPV in oral cavity and development of oral PAP smear might lead to early diagnosis and treatment for HNSCC. The prevalence of oral HPV infection presently a pre-requisite for HPV associated HNSCC was around 7% in centers without cancer in the IARC study [1] Another possibility for prevention of HPV associated HNSCC lies in HPV vaccine as we consider. Systemic Immunization with a protective HPV16 vaccine will be highly effective no doubt in preventing persistence HPV 16 in female genital tract. It is however not known to us whether such a vaccine will also alter the carriage rate of oropharyngial HPV16 and HNSCC.

Professor Pranab kumar Bhattacharya MD(cal), FIC path(Ind.) , Professor of Pathology, In charge of Histopathology Unit, in charge Blood Bank &VCCTC, Cytogenetics. Institute of Post Graduate Medical Education & Research, 244a AJC Bose Road, Kolkta-20, West Bengal, India; **Mr. Ritwik Bhattacharya, B.Com(cal); ***Mr. Rupak Bhattacharya BSc(Cal)MSc(JU) of 7/51 purbapalli, Sodepur Dist 24 parganas(North) Kol- 110, West Bengal, India; ****Mrs Dahlia Mukherjee BA(hons) Swamiji Road, Habra N-24 parganas, W.B, India, *****Miss upasana Bhattacharya of Mahamyatala, Garia kol-86,Daughter of Prof. Bhattacharya**** Dr Debasis Bhattacharya Ms(cal) FRCS(eng). Associate Professor, dept. of Surgery IPGME&R, Dr. Tarun Mandal MBBS(cal) , Dr. Ananya Pal MD(PGT) Pathology Dept. Of Pathology Institute of Post Graduate Medical Education & Research, 244a AJC Bose Road, Kolkta-20, West Bengal, India

Reference
1. Herrero R, Castellasaguex, Pawlita M etal “ Human Paplloma Virus and Oral Cancer, The international gency for Research on Cancer-a multicenter study J. Natl .cancer Institute 2003:95:1272-83

Dear Editor,

We fully support the integrated approach to hematopathological diagnostics, which has been advanced by the WHO 2008 classification and we acknowledge the prominent role of immunophenotyping in this classification (1). The integrated approach is still not fully applied even in the most advanced countries. In some other countries, (hemato)pathologists still struggle, having only bone marrow (BM) tissue core biopsies to work with. In some centers and countries, there may be a partial redundancy in immunophenotyping with both flow cytometry and immunohistochemistry (IHC) methods available.

This is not universally the case and one must rely much more on one or the other source of information. If the histopathologists are restricted to histology, they need to be assured that the applied IHC methodology is accurate. Quality assurance (QA), quality control (QC), proficiency testing, reagent documentation and validation are standard parts of everyday practice in clinical laboratories (2). IHC is one area of laboratory practice where such standardization and quality assurance procedures have been addressed relatively late (2,3). The complexity of IHC presents challenging issues within pre-analytical, analytical, and post-analytical components, which still make true standardization difficult. However, optimization in this area has become possible due to rapidly advancing technologies. Also, we have increased our understanding of each of these components, which makes it possible to start the standardization process (3,4).

Tissue processing has been recognized as an essential pre-analytical component that greatly determines the outcome of IHC stains (5,6). When very few IHC markers were employed in the evaluation of BM biopsies, there was no emphasis on QA/QC issues. Currently we face a challenge in the light of increasing number of applied markers and the variation in tissue processing of the BM biopsies. The results of IHC testing in BM biopsies are not exempt from the principles that apply for any other tissue biopsy. This means that none of the components of the IHC testing can be neglected. Even in the situation where one type of fixative is used and no decalcification is applied, pre-analytical components must be subjected to the standardization process. Analytic variables such as the antigen retrieval protocol, the detection system, and the immunostaining platform play an important role in the quality of IHC stains.

Our pilot study included only a fraction of European Bone Marrow Working Group laboratories. However, it disclosed that even in this limited number of laboratories, a large variety of tissue processing and staining methods were in use. It clearly shows that a currently used approach to external QA/proficiency testing is not possible for BM biopsies. At the same time our study shows that there is a need of such external QA/QC to assure reproducibility of the results. BM biopsy remains excluded from the good practices of QC/QA, which are already established in surgical pathology. This is important in the light of the increase in number of tests applied to BM biopsy, which is happening despite advances in other, possibly more sophisticated methods of tissue analysis. Moreover, introducing class II tests to this type of tissue, a new landmark in BM tissue biopsy IHC is approaching.

The priority must be given to stand-alone IHC tests. However, we disagree with Dr.Orazi that standardization and external QA/QC should be limited to class II tests. Many of the class I tests are equally complex to perform and may have critical impact for the final diagnosis (5). If any clinical test deserves to be done, it also deserves to be performed optimally and interpreted correctly. The integrated approach of WHO 2008, at least in our minds, was never constructed to support integration of either false-negative or false-positive IHC results or to obscure deficiencies of technically suboptimal results.

Sincerely yours,

Emina Emilia Torlakovic, MD, PhD, Kikkeri Naresh, M.B.B.S.; D.C.P.; M.D.;F.R.C.Path., Marcus Kremer, MD, Jon van der Walt, M.D., F.R.C.Path., Elizabeth Hyjek, MD, PhD Anna Porwit, MD, PhD,

References:

1. Swerdlow SH, Campo E, Harris NL, Jaffe ES, Pileri SA, Stein H, Thiele J, Vardiman JW. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. WHO Press, Geneva, 2008.

2. Taylor CR. Editorial--a personal perspective. Appl Immunohistochem Mol Morphol. 2007;15:121-3.

3. Taylor CR. Quality assurance and standardization in immunohistochemistry. A proposal for the annual meeting of the Biological Stain Commission, June, 1991. Biotech Histochem. 1992;67:110-7.

4. Bogen SA, Vani K, McGraw B, Federico V, Habib I, Zeheb R, Luther E, Tristram C, Sompuram SR. Experimental validation of peptide immunohistochemistry controls. Appl Immunohistochem Mol Morphol. 2009;17:239-46.

5. Goldstein NS, Hewitt SM, Taylor CR, Yaziji H, Hicks DG; Members of Ad- Hoc Committee On Immunohistochemistry Standardization. Recommendations for improved standardization of immunohistochemistry. Appl Immunohistochem Mol Morphol. 2007;15:124-33.

6. Taylor CR, Shi S-R, Barr N, Wu N. Techniques of Immunohistochenmistry: Principles, pitfalls, and standardization. In: Dabbs D. Diagnostic Immunohistochemistry. Churchill Livingstone, Philadelphia, PA, USA, 2nd Ed. 2006:17-19.

Dear Editor

The authors use sweeping generalizations to make their case. This is just one example: "while the role of external quality assurance programs is well established as a valuable tool for QA/QC in diagnostic immunohistochemistry in surgical pathology, such approaches are notably lacking in bone marrow biopsy pathology". However, in reality, no published data support a need for an external QA/QC program for bone marrow biopsies, as proposed by the authors. A long list of unpublished results from the NordiQC cannot be considered as equivalent to published peer-reviewed evidence and should not have been included among the references. Moreover, the authors themselves do not provide any data which may suggest a need for external quality assurance for class I tests, the only tests that they have evaluated in this study. The markers that the authors have tested are and they should most definitely remain class I tests. If the authors’ intent was to address the issue of analyzing potential class II markers which can be used on bone marrow biopsy, those are the ones they should have studied. In regard to a need for class II markers in bone marrow biopsy evaluation, it is unclear, at least to this reader, why immunohistology should be entitled to such a prominent place within a complex diagnostic algorithm. Most of the neoplastic conditions in which immunophenotyping can be useful, greatly benefit from a balanced integrated diagnostic approach. Such an approach is the one mandated by WHO 2008 classification. As they stand now, the conclusions that are provide by this paper are subjective ones and most definitely not based on evidence.

Attilo Orazi

Dear Editor,

In HL-60 cells lovastatin reduces the intracellular pH in dose- dependent manner and the fall in pH correlates with the extent of DNA degradation (1). More importantly alkalinization suppresses lovostatin-induced apoptosis. A fall in pH is an indication of reductive stress, a stimulus for the expression of HIF, VEGF and NOS. A normal or elevated pH may also be necessary for protein synthesis and cell renewal(2,3). A normal or elevated pH together with the expression of HIF (4) might, therefore, be a sine qua non for neoplastic development, growth and spread. If so how might a normal and particularly an elevated pH be induced and apoptosis inhibited in a tissue at risk of becoming neoplastic?

The ability of adjacent tissues to support the delivery of substrate to newly vascularized tissues is clearly important in maintaining a normal pH but what if it is insufficient to maintain the adequacy of ATP resynthesis by anaerobic glycolysis, the dominant means of ATP resynthesis in tumours? One possibility is that the pH is maintained at normal or elevated levels by the release of ammonia by cellular autophagy, apoptosis and/or necrosis. If so the elevation in pH can be expected to be restricted to a region, possibly the growing edge, interposed between that where there is adequate substrate delivery and that in which there is protein degradation occurring in association with autophagy, apoptosis and/or necrosis. In which case the ability of contiguous tissues to sustain substrate delivery to a growth center might be the critical variable.

Hence the question about the predictive value of VEGF-A and i-NOS expression in adjacent tissues. Might VEGF-A and i-NOS expression in unresected tissue be a better predictor of outcome than that within the tumour, the assumption being that it is the environment, rather than the tumour per se, that determines carcinogenesis and dictates long-term survival? This possibility would have to be examined in a prospective study conducted with surgical cooperation. Which is the cart and which the horse?

Richard G Fiddian-Green

References

1. Perez-Sala D, Collado-Escobar D, Mollinedo F. Intracellular alkalinization suppresses lovastatin-induced apoptosis in HL -60 cells through the inactivation of a pH-dependent endonuclease. J. Biol. Chem., 270:6235-6242, 1995.

2. P E Ballmer, M A McNurlan, H N Hulter, S E Anderson, P J Garlick, and R Krapf. Chronic metabolic acidosis decreases albumin synthesis and induces negative nitrogen balance in humans. J Clin Invest. 1995 January; 95(1): 39?45.

3. E Movilli, R Zani, O Carli, L Sangalli, A Pola, C Camerini, G Cancarini, F Scolari, P Feller and R Maiorca. Correction of metabolic acidosis increases serum albumin concentrations and decreases kinetically evaluated protein intake in haemodialysis patients: a prospective study. Nephrology Dialysis Transplantation, Vol 13, Issue 7 1719-1722, 1998.

4. P. H. Maxwell, G. U. Dachs, J. M. Gleadle, L. G. Nicholls, A. L. Harris, I. J. Stratford, O. Hankinson, C. W. Pugh, and P. J. Ratcliffe. Hypoxia-inducible factor-1 modulates gene expression in solid tumors and influences both angiogenesis and tumor growth. PNAS July 22, 1997 vol. 94 no. 15 8104-8109.