Title: Malignant melanoma arising from a perianal fistula and harbouring a BRAF gene mutation: a case report

Transcription

1 Author's response to reviews Title: Malignant melanoma arising from a perianal fistula and harbouring a BRAF gene mutation: a case report Authors: Conrado Martinez-Cadenas (martinezconrado@yahoo.es) Nuria Bosch (nubosch@yahoo.es) Lucas Peñas (lucaspeas@yahoo.es) Esther Flores-Couce (cutiflor@hotmail.com) Enrique Ochoa (enrique.ochoa@hospital2000.net) Javier Munárriz (jmunarriz@comcas.es) Juan P Aracil (juanpablo.aracil@gmail.com) Marcos Tajahuerce (mtajahuerce@hotmail.com) Ramón Royo (royolab@comcas.es) Rafael Lozoya (petor@pobladores.com) Enrique Boldó (eboldo@yahoo.com) Version: 3 Date: 31 May 2011 Author's response to reviews: see over

2 Cover Letter Title: Malignant melanoma arising from a perianal fistula and harbouring a BRAF gene mutation: a case report MS: Dear Tonilynn Manibo (on behalf of Prof Bhudev Das), First of all, my co-authors and I are very grateful to the reviewers for their constructive comments about the manuscript. We have considered all of them and have revised the manuscript accordingly. We would like to submit a revised version of the above manuscript to BMC Cancer (changes made are tracked and highlighted in yellow). We have also changed a couple of references (added one and deleted one) as suggested by Reviewer 1. Below is a pointby-point response to the reviewers comments. Also, as required by the BMC Editorial Office, the manuscript has been restructured. The Consent section has been suppressed and its content relocated at the end of the Methods section, a Discussion section has been added and the Conclusion section has been limited to a summary of the main findings of the work. We hope that the changes made are satisfactory, and look forward to hearing from you. Sincerely, Conrado Martinez-Cadenas (on behalf of all authors) Response to reviewers Reviewer: Tae-Gyun Kim A. Mayor Compulsory Revisions 1. In their histopathological pictures, cellular nests are only confined to the upper dermis, not involving overlying epithelial tissues. In the histopathology of melanoma samples, one can easily find the pagetoid spreading or epidermotropism, thus epidermis is frequently involved by malignant cells. Do the authors certainly insist that the dermal nests are malignant melanoma? Is there any possibility that it is an intradermal nevus rather than melanoma? (Also, cellular atypia and hyperchromasia of nuclei are less prominent.) The diagnosis of melanoma in this case was really never in doubt. Our first inkling was that it was a metastasis from a melanoma, but that was discarded as soon as the adjoining atypical lentiginous melanocytic hyperplasia was found in subsequent tissue sections (even so, whole body CT scan and abdominal ultrasound were performed to look for possible metastasis in the patient). The diagnosis of melanoma rested mainly on the following characteristics displayed by the lesion: 1) presence of atypical hyperplastic and anaplastic cells with prominent nucleoli, 2) lack of maturation in the depth of the lesion, 3) mitotic activity in deep cells and 4) presence of both atypical melanocytes and small melanocytic nests inside the epidermis (see pictures, though probably not the most representative examples, and

3 possibly not migrating). These were the four most important features for the diagnosis of melanoma. Besides, two other secondary features were 5) abundant melanin in the depth of the lesion and throughout it and 6) also plenty of melanin in the upper portion of the epidermis, including the stratum corneum (horny layer). Please see accompanying additional photomicrographic images from the case. The main reason put forward by the pathologists to explain the lack of visible pagetoid spreading or epidermotropism was that the area of union between the intradermal melanoma nests and the overlying epidermis is missing from the histological sections. Possible causes suggested were that 1) the site of that union was probably erased, removed or wiped out by the abundant abscesses and inflammatory processes taking place in the region, 2) that it had simply been missed due to the tangential sections performed on the fistula tissue piece (let s keep in mind that this was a chance finding, nothing suggested beforehand that this tissue block was hiding a melanoma), or 3) that it was lost due to technical reasons during the cutting or trimming of the paraffin block.

4 Due to the unusual location and the overall rarity and peculiarity of this melanoma, this case was consulted and discussed with two other independent Pathology Services from different Hospitals. All pathologists independently agreed that it was indeed a melanoma, mainly because of the accumulation of numerous signs, indicators and hints (summarized in the six points above) that, overall, pointed towards a clear diagnosis of melanoma. A summary of these signs has also been added to the Case Presentation section of the manuscript. 2. From the previous study, acal or mucosal melanoma show a low frequency of BRAF mutation, instead, these types of melanoma show aberrations in other genes, such as KIT or CCND1. Such a difference of genetic alterations indicates distinct genetic pathways in the development of melanoma depending on the anatomical site of the primary lesion. The authors should discuss the above fact in their conclusion section because their melanoma case arose in sun-protected area rather than in sun-exposed area. A paragraph in the Discussion section has been added regarding genetic alterations in genes such as KIT or CCND1 in acral and mucosal melanomas. Briefly, we have to take into account that this melanoma was neither a mucosal nor an acral melanoma. It was a melanoma that appeared inside a fistula located in the perianal region. This melanoma did not arise in a mucosal epithelium, but inside a fistulous tract that had been already epithelialized. Even so, had we not found a mutation in the BRAF gene, we would have tried to find a mutation in the KIT gene, since around 39% of mucosal melanomas have showed alterations in that gene [1]. Since we found a BRAF mutation, there was no point in searching for mutations in the KIT or CCND1 genes, because concomitant mutations in BRAF and in genes such as KIT or CCND1 are non-existent or extremely rare [1]. This melanoma was simply an extremely rare occurrence, possibly a unique finding, discovered just by chance. Possibly, it does not belong to any of the melanoma types currently recognized in the field: mucosal, acral, chronic sun-damaged skin and non-chronic sun-damaged skin melanoma [2]. B. Minor Essential Revisions 1. Pseudoepitheliomatous hyperplasia is a hitopathologic reaction pattern which can be seen among diverse dermatologic conditions. It's not an etiologic factor for skin cancers. Please omit the section for PEH from the manuscript. We entirely agree with the reviewer here. We have completely omitted the paragraph and reference regarding pseudoepitheliomatous hyperplasia from the manuscript. C. Discretionary Revisions 1. Did the patient run whole body CT scan or whole body PET CT scan? Because CT scan can't catch a small tumor lesions compared to PET CT scan. The patient went trough a whole body CT scan (at that moment, a PET CT scan was not available at our hospital) and an abdominal ultrasound examination. In any case, as mentioned in the manuscript text, it is almost impossible that this melanoma was a metastasis, since the patient is perfectly healthy nine years after diagnosis, and the melanoma was located next to a lesion showing atypical lentiginous melanocytic hyperplasia, suggesting that the melanoma had a local origin.

5 2. 'Conclusion' is too lengthy. The text of the manuscript has been restructured to avoid such a lengthy Conclusion section. As recommended by the BMC Editorial Office, a Discussion section has been added, as well as a short Conclusion section summarizing the major findings of the study. 3. page9, 'hence the prognosis and good progress after WLE.' please revise this phrase. This sentence has finally been eliminated from the manuscript, since the authors felt it was redundant. Reviewer: Fiona Henriquez This article is well presented and describes a very interesting case. The authors also present an interesting hypothesis as a conclusion in that oxidative stress may cause the BRAF mutation. I feel that this may need more clarification as oxidative stress is a common situation during inflammatory responses, which may mean that the BRAF gene may be subject to mutating very easily and this is not the case. Would it be possible to explicitly describe the example that they reference in citation 12? I feel that this would give more substantial evidence in the absence of performing in vitro experiments to elucidate the effect of oxidative stress on the BRAF gene. Although the mechanism that results in BRAF mutations still remains unknown, this mechanism must not depend only on DNA damage by direct UV exposure or through its photoproducts, since some non-uv-exposed melanomas and many other cancer types (thyroid, colorectal, pancreas, ovarian, etc.) also harbor BRAF mutations. One of the proposed mechanisms is that oxidative damage brought about by UV-light or, more important in this case, by chronic inflammatory processes may cause BRAF mutations (although the means by which oxidation causes BRAF mutagenesis have not been elucidated yet). In citation reference No. 12, the authors studied the BRAF gene in 13 mucosal melanomas and they did not find any BRAF mutations. In their discussion section, they argue that some mucosal melanomas [1] and other cancer types might generate BRAF mutations due to chronic inflammatory processes happening in the tissue where they originated. These BRAF mutations might be caused by an as yet unidentified oxidative lesion generated by chronic inflammation. Besides being an extremely rare case presentation, the fistula melanoma reported in this study carried the most common BRAF mutation. Thus, this case supports the view that inflammation (ubiquitous and omnipresent in fistulas) might have a say in BRAF mutagenesis. We are not concluding that all inflammation must cause BRAF mutations, but that persistent or chronic inflammatory processes in the absence of UV exposure (such as chronic pancreatitis in pancreatic cancer [3], thyroiditis in thyroid cancer [4], chronic bowel inflammation in colon cancer [5] and, in our case, a longstanding fistula in a melanoma), can make the BRAF gene more susceptible to genetic mutations.

6 REFERENCES 1. Curtin JA, Busam K, Pinkel D, Bastian BC: Somatic activation of KIT in distinct subtypes of melanoma. J Clin Oncol 2006, 24: Curtin JA, Fridlyand J, Kageshita T, Patel HN, Busam KJ, Kutzner H, Cho KH, Aiba S, Bröcker EB, LeBoit PE, Pinkel D, Bastian BC: Distinct sets of genetic alterations in melanoma. N Engl J Med 2005, 353: Schönleben F, Qiu W, Bruckman KC, Ciau NT, Li X, Lauerman MH, Frucht H, Chabot JA, Allendorf JD, Remotti HE, Su GH: BRAF and KRAS gene mutations in intraductal papillary mucinous neoplasm/carcinoma (IPMN/IPMC) of the pancreas. Cancer Lett 2007, 249: Kimura ET, Nikiforova MN, Zhu Z, Knauf JA, Nikiforov YE, Fagin JA: High prevalence of BRAF mutations in thyroid cancer: genetic evidence for constitutive activation of the RET/PTC-RAS-BRAF signaling pathway in papillary thyroid carcinoma. Cancer Res 2003, 63: Aust DE, Haase M, Dobryden L, Markwarth A, Löhrs U, Wittekind C, Baretton GB, Tannapfel A: Mutations of the BRAF gene in ulcerative colitis-related colorectal carcinoma. Int J Cancer 2005, 115:673-7