Supplementary Figure 1 from KIT Pathway Alterations in Mucosal Melanomas of the Vulva and Other Sites

Sinonasal malignant melanoma (SNMM) comprises less than 1% of all melanomas and is located in the nasal cavity and the paranasal sinuses. The majority of SNMMs have unknown underlying oncogenic driver mutations. The recent identification of a high frequency of driver mutations in the promoter of the telomerase reverse transcriptase (TERT) gene in cutaneous melanoma led us to investigate whether these mutations also occur in SNMM. Our aim was to determine the TERT promoter mutation frequencies in primary SNMMs. Laser capture microdissection and manual dissection were used to isolate tumour cells from 49 formalin-fixed paraffin-embedded tissues. The tumours were screened for TERT promoter mutations by direct Sanger sequencing. Information on NRAS, BRAF and KIT mutation was available from an earlier study. Overall, 8% (4/49) of SNMMs harboured TERT promoter mutations. One of these mutated tumours had a coexistent NRAS mutation and one had a BRAF mutation. Our findings show that TERT promoter mutations are present in a moderate proportion of SNMM. No conclusion can be drawn on their potential influence on the clinical outcome or tumour progression.

BACKGROUND/AIM Mucosal melanomas arise in non UV-light exposed areas and causative factors are yet unknown. Human polyomaviruses (HPyVs) are rapidly increasing in numbers and are potentially oncogenic, as has been established for MCPyV in Merkel cell carcinoma, an unusual skin cancer type. The aim of the present study was to investigate the association between TSPyV, MWPyV, HPyV6, 7 and 9 and mucosal melanoma. MATERIALS AND METHODS Fifty-five mucosal melanomas, were analyzed by a Luminex assay, for the presence of 10 HPyVs (BKPyV, JCPyV, KIPyV, WUPyV, TSPyV, MWPyV, HPyV6, 7 and 9) and two primate viruses (SV40 and LPyV). RESULTS In 37 samples the DNA quality was satisfactory for analysis. However, none of the samples analyzed were positive for any of the examined viruses. CONCLUSION None of the above-analyzed HPyVs were detected in mucosal melanoma samples, and they are for this reason unlikely to play a major role in the development of this tumor type.

OBJECTIVETo establish population-based trends for sinonasal mucosal melanoma (SNMM) in Sweden.METHODSWe identified 186 patients from the Swedish National Cancer Registry diagnosed with primary melanomas arising from the nasal cavity, paranasal sinuses, or both, during the period 1960 through 2000. Incidence, gender and age, primary anatomical sites, geographic distribution, treatment and survival were investigated.RESULTSThe age-standardized incidence of SNMM increased significantly during the 41-year-period, with a higher overall incidence for females than males, but with a more rapid increase for males than for females. The incidence increased with age, peaking after the eightieth year in both genders. About 70 % of the cases were clinically amelanotic. The most common primary treatment was surgery. Five-year, disease-specific survival rates were poor for all these patients, but women had a significantly better survival time than men. For both genders the survival rate lengthened during the study period, irrespective of therapeutic strategy.CONCLUSIONSNMM is a rare disease, but the incidence in Sweden has increased significantly from 1960 through 2000, although not at the same pace as that of cutaneous malignant melanoma. Both the incidence and the survival were significantly higher in females than in males, but the reason for these gender differences is unknown.

Abstract Purpose: A significant proportion of mucosal melanomas contain alterations in KIT. The aim of this study was to characterize the pattern of KIT, NRAS, and BRAF mutations in mucosal melanomas at specific sites and to assess activation of the KIT downstream RAF/MEK/extracellular signal-regulated kinase (ERK) and phosphoinositide 3-kinase (PI3K)/AKT pathways in mucosal melanoma specimens. Experimental Design: Seventy-one primary mucosal melanomas from various sites were studied. Mutation analysis was done by DNA sequencing. Expression of KIT, phosphorylated (p)-ERK, and p-AKT was evaluated by immunohistochemistry. Results: KIT mutations were detected in 35% (8 of 23) of vulvar, 9% (2 of 22) of anorectal, 7% (1 of 14) of nasal cavity, and 20% (1 of 5) of penile melanomas. No KIT mutations were found in 7 vaginal melanomas. The difference in KIT mutation frequency between vulvar and nonvulvar cases was statistically significant (P = 0.014). The overall frequencies of NRAS and BRAF mutations were 10% and 6%, respectively. Notably, vaginal melanomas showed a NRAS mutation rate of 43%. KIT gene amplification (≥4 copies), as assessed by quantitative real-time PCR, was observed in 19% of cases. KIT expression was associated with KIT mutation status (P < 0.001) and was more common in vulvar than nonvulvar tumors (P = 0.016). Expression of p-ERK and p-AKT was observed in 42% and 59% of tumors, respectively, and occurred irrespective of KIT/NRAS/BRAF mutation status. NRAS mutation was associated with worse overall survival in univariate analysis. Conclusions: Results show that KIT mutations are more common in vulvar melanomas than other types of mucosal melanomas and that both the RAF/MEK/ERK and PI3K/AKT pathways are activated in mucosal melanoma specimens. Clin Cancer Res; 17(12); 3933–42. ©2011 AACR.

UV radiation is a major factor in melanoma genesis, but non-UV linked factors are also operational, since primary malignant melanomas can emerge in body sites that never see the sun. The scarcity of melanomas in sun-shielded body sites reflects only the absolute number of melanomas, not the number of tumours per square unit of the surface in which they emerge. Studies on melanoma density conducted by us and others are here briefly reviewed. The access to reliable numbers along with measurable anatomical areas directed our choice of melanomas at the sun-shielded locations described here. Melanomas at the body surface. Calculations of surface areas bearing melanomas relative to the total body surface included sites on the vulva, subungual tissues, volar and palmar skin, and, for comparison melanomas of the face during the same period of time. The density of vulvar melanomas was identical to that in chronically sun-exposed facial skin. Subungual melanomas were almost nine times denser than expected whereas melanomas of palms and soles showed a lower density than expected. Melanomas beneath the body surface. The densities of melanomas in the vagina, anal canal and uvea, were calculated separately and compared to the average density of cutaneous melanomas (CMMs) during the same period of time. Melanomas of the anal canal displayed a density almost twice the average for CMMs, whereas the vaginal melanomas were similar in density to CMMs. In contrast, the density of the uveal melanomas was calculated as 50 and 41 times (men and women, respectively) the average density of CMMs. Conclusion. The high density of some melanomas in sun-shielded body areas indicates the presence of factors underlying the origins of these tumours that seem to be equivalent in strength to UV radiation and also implies that specific anatomical sites favour the emergence and proliferation of melanomas, independent of UV radiation.

BACKGROUND:Anorectal melanoma is rare and surgery is the recommended primary treatment. There has been some debate whether abdominoperineal resection (APR) or local excision is most appropriate. The aim of this study was to provide a population-based analysis of symptoms, treatment and outcome.METHODS:From the Swedish National Cancer Registry, 251 patients with anorectal melanoma were identified from 1960 to 1999. Medical reports were collected and reviewed retrospectively. R0 resection was defined by clear macroscopic margins and a pathology report showing a margin greater than 10 mm. Survival was compared with the log rank test, and Cox multivariable analysis was performed.RESULTS:APR and local excision was performed in 66 and 86 patients respectively. Median survival after surgery was 14 months, with no statistically significant difference between the two groups. Seventy-two patients in whom an R0 resection was achieved, irrespective of approach, had a significantly better overall 5-year survival rate than patients with involved margins (19 versus 6 per cent; P < 0.001). Multivariable analysis showed resection status and tumour stage to be independent prognostic variables.CONCLUSION:Both APR and LE seem appropriate for anorectal melanoma provided clear margins can be achieved; prognosis is poor regardless of surgical approach.

We have previously demonstrated an association of the human leukocyte antigen (HLA), HLA-A2 allele with ovarian and prostate cancer mortality as well as a segregation of the ancestral HLA haplotype (AHH) 62.1 [(A2) B15 Cw3 DRB1*04] in patients with stage III–IV serous ovarian cancer. The objective of the present study was to determine the role of the HLA phenotype on the prognosis in stage III–IV malignant melanoma patients.

Purpose. To analyze 251 patients (101 males and 150 females) diagnosed with ano-rectal malignant melanoma (ARMM) reported to the Swedish National Cancer Registry during 1960-1999. Methods. Incidence, gender and age profiles, primary anatomical sites and density of the melanomas along with geographic distribution, and prognosis were investigated. Results. The age-standardized incidence of ARMM was significantly higher for females (1.0 per 10(6) females) than for males (0.7 per 10 6 males) throughout the 40-year-period. The incidence increased with age peaking at 75-84 years in both genders. 54% of the tumours were primary in the anal canal, 24% engaged the whole ano-rectal unit and 10% were located at the anal verge (11% unknown primary site). Although ARMM were rare in absolute numbers, their density (number of tumours/square unit) was higher than that of cutaneous malignant melanomas (CMM) on average. No linkage between the geographic distribution of ARMM and population density was found. The prognosis was very poor albeit with a significant gender difference with a five-year survival rate of 10.6% for males and 15.7% for females. The survival rates for both genders improved during the 40-year-period but significantly more for females than males. Conclusion. The reason(s) for the difference in incidence and prognosis according to gender is unknown. The majority of ARMM emerged primary in the anal canal and a primary location exclusively in the colonic mucosa of the rectum is questionable. The higher density of ARMM as compared to the average density of CMM tallies with the result of our previous studies on vulvar melanoma and might be instrumental in exploring non-UV light associated factors in melanoma genesis. The concentration of patients with anal squamous cell carcinoma to population-dense urban areas, as previously reported, was not found in cases of ARMM.

ABSTRACT The single most important causative factor for malignant melanomas of the skin is UV radiation. However, this is not true for melanomas on body surfaces sheltered from the sun; thus, it is important to seek new causative factors of melanoma genesis. Human papillomaviruses and gammaherpesviruses are associated with human skin cancer; for example, human papillomavirus types 5 and 8 are associated with epidermodysplasia verruciformis, and human herpesvirus 8 is associated with Kaposi's sarcoma. Recently, a newly described human polyomavirus, Merkel cell polyomavirus (MCPyV), has been associated with Merkel cell carcinoma, an unusual form of neurotropic skin cancer. Moreover, melanocytes are of neuroepithelial origin. This background impelled us to investigate if human polyomavirus DNA could play a role in the development of extracutaneous melanomas. Sixty-four extracutaneous melanomas were initially collected and dissected. Of these, 38 could be successfully used for further testing for the presence of the five human polyomaviruses known so far—BK virus (BKV), JC virus (JCV), KI polyomavirus (KIPyV), WU polyomavirus (WUPyV), and MCPyV—and of simian virus 40 (SV40). No polyomavirus DNA could be detected in any of the samples tested by use of a nested PCR detecting BKV, JCV, and SV40; a newly designed PCR detecting KIPyV and WUPyV; or a newly designed PCR for MCPyV. We conclude that since no human polyomavirus DNA was detected in primary malignant melanomas on non-sun-exposed body surfaces, these polyomaviruses presumably are not major factors for the development of extracutaneous melanomas.

PURPOSE. To investigate the occurrence of other primary malignancies before and after diagnosis of uveal melanoma in a Swedish population.METHODS. In the Swedish Cancer Registry 2995 patients with uveal melanoma were notified during the period 1960 to 1998. In the same registry, a search for additional malignancies among these patients was performed. A matched case-control study with 2,916 patients and 14,577 population control subjects was set up for malignancies before diagnosis of uveal melanoma. Malignancies after diagnosis of uveal melanoma were evaluated in 2,995 patients through standardized incidence ratios (SIRs), based on the expected rates in the Swedish population.RESULTS. Before the diagnosis of uveal melanoma, the odds ratio (OR) for the risk of cancer was 1.25 (95% CI: 0.98 - 1.59). No significantly increased risk was found for any specific malignancy. The OR for cutaneous melanoma was 1.74 (95% CI: 0.78 - 3.89). The risk of subsequent cancers was increased, SIR 1.13 (95% CI: 1.02 - 1.26). After reevaluation of archival specimens, the SIR of a cutaneous melanoma's developing after a uveal melanoma was found to be 1.75 (95% CI: 0.87 - 3.12).CONCLUSIONS. An increased risk of second primary cancers was observed among Swedish patients with uveal melanoma. Metastases from uveal melanoma were found to be misclassified as cutaneous melanoma or as primary liver cancer.

Temozolomide is a rapidly absorbed chemotherapeutic agent, achieving significant central nervous system penetration. Previous clinical trials suggested that temozolomide in sequence with low-dose recombinant human interleukin-2 might be an efficacious and relatively non-toxic chemo-immunotherapeutic treatment, which may synergistically eliminate tumours. The primary objective was to determine the safety and tolerance of temozolomide administered orally 200 mg/m(2) days 1-5, in sequential combination with subcutaneous injections of 4.5 x 10(6) IU recombinant human interleukin-2 on days 8-11, 15-18 and 22-25 in patients with measurable, progressive metastatic malignant melanoma without radiological signs of central nervous system metastases. The secondary objectives were to determine tumour response and time to progression. Twenty-seven patients were included, of which four were non-evaluable for response. Twenty-three patients tolerated the regimen with side effects below grade 3 according to the World Health Organization (WHO) scale. Three patients suspended the treatment because of WHO grade 3 side effects already during the first 3 days of the first course of temozolomide. Seven patients showed no tumour progression during the first four treatment cycles. Two patients had complete responses, three partial responses and two stable disease at the end of the four cycles defined by the protocol and they continued the treatment until signs of relapse or a maximum of 21 courses. Five of these patients are still alive. Thrombocytopenia was significantly more pronounced in patients with objective response and stable disease than in non-responders to therapy. The median time to progression for all patients was 3.1 months and for responding and stable disease patients was 15 months. Five of 23 treated patients (22%) developed brain metastases during follow-up. Temozolomide in combination with recombinant human interieukin-2 is a well-tolerated regimen for outpatient treatment and the bio-chemotherapy combination induced durable clinical responses. Thrombocytopenia might be a positive predictive factor for response to therapy.

BACKGROUND:UV-radiation is the most important causative factor for malignant melanomas of the skin. However, this is not the case for melanomas on sun-sheltered body surfaces. The aim of this study was to investigate if human herpes virus DNA could be found in malignant melanomas in sun-sheltered body areas and if these viruses play a role in the development of extracutaneous melanomas.MATERIALS AND METHODS:Forty-one extracutaneous melanomas were dissected and used for further analysis. Quantitative PCR methods were used for detection of the eight human herpes viruses in melanoma samples.RESULTS:Human herpes virus DNA was absent in 37/41 melanomas, however, cytomegalovirus DNA was detected in two samples, and one sample each exhibited presence of Epstein-Barr virus and Human Herpes virus-6 DNA respectively.CONCLUSION:Human herpes virus DNA is rarely detected in primary malignant melanomas in non-sun exposed body surfaces and is not a major factor for the development of extracutaneous melanomas.

Human papillomavirus (HPV) has been associated with some types of human cancer. The aim of this study was to investigate if HPV could be associated with human primary malignant melanoma in non sun-exposed body areas like mucous membranes. Through the Swedish National Cancer Registry, in compliance with the rules of the Human Ethical Committee, histopathological specimens were collected from different pathological laboratories throughout Sweden. The histopathological diagnosis was reviewed, and from 45 primary melanomas, tumour tissue was micro-dissected and analysed further. A protocol for detection of HPV DNA using general HPV primers GP5 + /GP6 + or CPI/ IIG, which together identify 36 different HPV subtypes, was developed. This protocol could detect presence of HPV DNA in less than 10 ng of DNA of a control cell that contained 1{2 copies of HPV type 16/cell. Before HPV testing the melanoma samples were examined for amplifiable DNA by a beta-microglobulin PCR and 39 were positive. Thirty-five of these could be evaluated for HPV DNA and no samples were positive according to all five defined criteria for HPV positivity although two were positive according to 4/5 criteria. In conclusion, HPV is rarely detected in primary malignant melanomas of non-sun exposed body areas.

Malignant melanomas appear in such sun-shielded areas as the vulva, challenging conventional knowledge that they are associated with UV radiation. Based on 1 442 patients with vulvar melanomas the tumors’ epidemiology, clinical manifestations, histopathology, molecular genetics, treatment strategies, and prognosis were surveyed. Despite their sun-shielded location and rare incidence, vulvar melanomas were, on average, more dense than melanomas on the body surface and nearly the density of melanomas in chronically sun-exposed skin of the head and neck. Vulvar melanomas differed markedly from cutaneous melanomas, as evidenced by histopathological lesions and molecular genetics. Most melanomas were located on the glabrous skin as opposed to the hairy skin within the vulva and differed significantly in biological properties. The prognosis for the patients was poor, and in the 11 largest studies of surgical strategies, none offered a significant survival advantage. Tumor thickness and ulceration were usually significant predictors of (poor) prognosis in multivariate analyses along with macroscopic amelanosis, angioinvasion or DNA non-diploidy in some reports. Clear-cut biological differences between vulvar and cutaneous melanomas and between melanomas within different vulvar sites provide new paths for extensive research on melanomagenesis and for potential therapies. Additionally, studies of vulvar and other extracutaneous melanomas should characterize subgroups of cutaneous melanomas and identify their cause(s), which are apparently not linked to UV radiation.

Mutations in the p53 tumour suppressor gene (TP53) have been linked to several types of cancer. We therefore investigated whether such mutations occur in malignant melanomas and, if so, whether they are linked to ultraviolet (sun) light exposure. For the first time, TP53 mutations in mucosal membranes and adjacent tissues shielded from sunlight were compared with those in cutaneous melanomas from sun-exposed skin. Archival tissues were obtained from 35 patients with a primary melanoma taken from unexposed mucosal areas and from 34 patients with a primary melanoma located in chronically sun-exposed head and neck skin. TP53 was characterized by means of polymerase chain reaction amplification and single-strand conformation polymorphism assay followed by nucleotide sequencing. The results showed that 17.6% of the primary cutaneous and 28.6% of the primary mucosal melanomas had point mutations in TP53. Among the cutaneous melanomas, one showed three mutations in exon 7, and one had two mutations in exon 5; the mutation was in the same allele in both cases. One mucosal melanoma had two mutations in exon 7, both in the same allele, and another had two mutations, one in exon 7 and one in intron 6, both in the same allele. C←T mutations at dipyrimidine sites, considered fingerprints for ultraviolet light-induced mutations, were about equally distributed among patients with melanomas from chronically sun-exposed areas (six out of nine; 67%) and those with melanomas from unexposed mucosal areas and adjacent skin (eight out of 14; 57%). Our data, demonstrating the presence of such mutations even in melanomas from mucosal membranes, clearly suggest that factors other than, or additional to, ultraviolet radiation are operational in the induction of TP53 mutations in melanomas.

PURPOSE. To investigate the incidence of uveal melanoma in Sweden during the period from 1960 to 1998, with respect to age distribution, gender, and changes in incidence over time.METHODS. The Swedish Cancer Registry was searched for patients with uveal melanoma and cross-checked against hospital files over patients where an eye-sparing treatment had been applied, to ensure inclusion in the Registry even when no histologic specimen was available. The crude and age-standardized incidence was estimated separately for each gender. The Swedish population of 1970 to 1974 was used as a standard, and the annual change in incidence was calculated by using a regression model with logarithmic incidence numbers.RESULTS. In total, 2997 patients met the criteria, of whom 1542 were males and 1455 females, During the 39-year period, the age-standardized incidence of uveal melanoma declined significantly in the male population, from 117 cases/million to 8.4 cases/million (P = 0.002). The trend toward reduced incidence in females. from 10.3 to 87 cases/million did not reach statistical significance (P = 0.108). The annual relative change in incidence was 1% (95% CI, 0.8%-1.2%) in males and 0.7% (95% CI, 0%-1.3%) in females. The age-specific incidence revealed a significantly higher incidence among men older than 45 years (23.5 cases/million) compared with the incidence in women of the same age group (19.2 cases/million; P < 0.001).CONCLUSIONS. A Swedish national survey performed to establish the incidence of uveal melanoma during the period from 1960 to 1998 revealed a decreasing incidence in the male and a stable incidence in the female population.

BACKGROUND. In an epidemiologic study of 219 Swedish females with vulvar melanoma, the authors previously established the incidence of this disease as 0.19 per 100,000 women, with a 3% annual decrease from 1960 to 1984 and a 5-year relative survival rate of 47%. After reviewing the medical histories of all of the 219 patients, the authors documented their precise clinical and histopathologic features, which, along with treatment, are assessed herein as predictors of survival.METHODS. Of 219 consecutive cases of vulvar melanoma collected from the Swedish National Cancer Registry, 21 were excluded because of inadequate data. Clinical and histopathologic materials from the remaining 198 cases were then reexamined. With a clinical three-stage system, lesion types and treatment modalities were assessed as survival factors in univariate and multivariate analyses.RESULTS. In univariate analysis, significant predictors of survival for patients at Stage I were tumor thickness, ulceration, number of mitoses, macroscopic amelanosis, preexisting nevi, extent of tumor invasion (lateral labia majora), and patient age. The mode of treatment was not significant. In multivariate analysis, staging (Stage I vs. II and III) and tumor thickness were independent predictors of survival. For Stage I only, tumor thickness, ulceration, and clinical amelanosis independently predicted survival time.CONCLUSIONS. To the authors' knowledge, this is the largest series of patients with vulvar melanoma ever reviewed, and an ethnically homogeneous and nationwide female population is represented. In this series, clinical stage, macroscopic amelanosis, and tumor characteristics such as tumor thickness and ulceration, rather than treatment mode, were the best factors for predicting the outcome of these patients. Cancer 1999;86:1285-93. (C) 1999 American Cancer Society.