Evidence for three tumor suppressor loci on chromosome 9p involved in melanoma development

Cytogenetic and loss of heterozygosity (LOH) studies have long indicated the presence of a tumor suppressor gene (TSG) on 90 involved in the development of melanoma, Although LOH at 90 has been reported in approximately 60% of melanoma tumors, only 5-10% of these tumors have been shown to carry CDKN2A mutations, raising the possibility that another TSG involved in melanoma maps to chromosome 90. To investigate this possibility, a panel of 37 melanomas derived from 35 individuals was analyzed for CDKN2A mutations hy single-strand conformation polymorphism analysis and sequencing. The melanoma samples were then typed for 15 markers that map to 9p13-24 to investigate LOH trends in this region. In those tumors demonstrating retention of heterozygosity at markers flanking CDKN2A and LOH on one or both sides of the gene, multiplex microsatellite PCR was performed to rule out homozygous deletion of the region encompassing CDKN2A. CDKN2A mutations were found in tumors from 5 patients [5 (14%) of 35], 4 of which demonstrated LOH across the entire region examined. The remaining tumor with no observed LOH carried two point mutations, one on each allele, Although LOH was identified at one or more markers in 22 (59%) of 37 melanoma tumors corresponding to 20 (57%) of 35 individuals, only 11 tumors from 9 individuals [9 (26%) of 35] demonstrated LOH at D9S942 and D9S1748, the markers closest to CDKN2A. Of the remaining 11 tumors with LOH, 9 demonstrated LOH at two or more contiguous markers either centromeric and/or telomeric to CDKN2A while retaining heterozygosity at several markers adjacent to CDKN2A. Multiplex PCR revealed one tumor carried a homozygous deletion extending from D9S1748 to the IFN-alpha locus. In the remaining eight tumors, multiplex PCR demonstrated that the observed heterozygosity was not attributable to homozygous deletion and stromal contamination at D9S1748, D9S942, or D9S974, as measured by comparative amplification strengths, which indicates that retention of heterozygosity with flanking LOH does not always indicate a homozygous deletion, This report supports the conclusions of previous studies that at least two TSGs involved in melanoma development in addition to CDKN2A may reside on chromosome 9p.

No evidence of a role for activating CDK2 mutations in melanoma

Inactivation of p16(INK4a) and/or activation of cyclin-dependent kinase-4 (CDK4) are strongly associated with both susceptibility and progression in melanoma. Activating CDK4 mutations prevent the binding and inhibition of CDK4 by p16(INK4a). A second, more indirect role for CDK4 is in late G(1), where It may sequester the inhibitors p27(KIP1) or p21(CIP1) away from CDK2, and in doing so upregulate the CDK2 activity necessary for cells to proceed completely through G(1) into S phase. As the pivotal residues around the most predominant R24C activating CDK4 mutation are invariant between CDK2 and CDK4, we speculated that the pivotal arginine (position 22 in CDK2), or a nearby residue, may be mutated in some melanomas, resulting in the diminution of its binding and inhibition by p27(KIP1) or p21(CIP1). However, except for a silent polymorphism, we detected no variants within this region of the CDK2 gene in 60 melanoma cell lines. Thus, if CDK2 activity is dysregulated in melanoma it is likely to occur by a means other than mutations causing loss of direct inhibition. We also examined the expression of the CDK2 gene in melanoma cell lines, to assess its possible co-regulation with the gene for the melanocyte-lineage antigen pmel17, which maps less than 1 kb away in head to head orientation with CDK2 and may be transcribed off the same bidirectional promoter. However, expression of the genes is not co-regulated. (C) 2001 Lippincott Williams & Wilkins.

Metastatic lesions in the joint associated with acute inflammatory arthritis after dendritic cell immunotherapy for metastatic melanoma

A 47 year old man undergoing immunotherapy for metastatic melanoma with autologous dendritic cells pulsed with autologous tumour peptide and hepatitis a surface antigen developed acute left ankle arthritis. Gout and acute infection were excluded, and an autoimmune aetiology or occult metastasis were considered. The arthritis initially subsided with indomethacin, but the symptoms recurred 2 months later, and magnetic resonance imaging demonstrated metastatic melanoma of the left talus. Immunohistochemical staining of a cerebral metastatic deposit biopsied 1 week after the onset of arthritis demonstrated T-cell and macrophage infiltration of the tumour. In addition, the patient developed melanoma-specific delayed type hypersensitivity and cytotoxic T-cell responses after vaccination. Thus, the monoarthritis represented an 'appropriate' inflammatory response directed against metastatic melanoma. (C) 2001 Lippincott Williams & Wilkins.

Melanoma in adolescents: A case-control study of risk factors in Queensland, Australia

The incidence of melanoma increases markedly in the second decade of life but almost nothing is known of the causes of melanoma in this age group. We report on the first population-based case-control study of risk factors for melanoma in adolescents (15-19 years). Data were collected through personal interviews with cases, controls and parents. A single examiner conducted full-body nevus counts and blood samples were collected from cases for analysis of the CDKN2A melanoma predisposition gene. A total of 201 (80%) of the 250 adolescents with melanoma diagnosed between 1987 and 1994 and registered with the Queensland Cancer Registry and 205 (79%) of 258 age-, gender- and location-matched controls who were contacted agreed to participate. The strongest risk factor associated with melanoma in adolescents in a multivariate model was the presence of more than 100 nevi 2 mm or more in diameter (odds ratio [OR] = 46.5, 95% confidence interval [Cl] = 11.4-190.8). Other risk factors were red hair (OR = 5.4, 95%Cl = 1.0-28.4); blue eyes (OR = 4.5, 95%Cl = 1.5- 13.6); inability to tan after prolonged sun exposure (OR = 4.7, 95%Cl = 0.9-24.6); heavy facial freckling (OR = 3.2, 95% Cl = 0.9-12.3); and family history of melanoma (OR = 4.0, 95%Cl = 0.8-18.9). Only 2 of 147 cases tested had germline variants or mutations in CDKN2A. There was no association with sunscreen use overall, however, never/rare use of sunscreen at home under the age of 5 years was associated with increased risk (OR = 2.2, 95%Cl = 0.7-7.1). There was no difference between cases and controls in cumulative sun exposure in this high-exposure environment. Factors indicating genetic susceptibility to melanoma, in particular, the propensity to develop nevi and freckles, red hair, blue eyes, inability to tan and a family history of the disease are the primary determinants of melanoma among adolescents in this high solar radiation environment. Lack of association with reported sun exposure is consistent with the high genetic susceptibility in this group. (C) 2002 Wiley-Liss, Inc.

Loss of p16 expression is associated with histological features of melanoma invasion

While mutations of CDKN2A are associated with melanoma predisposition, the precise role of its gene product p16 in the development of sporadic melanoma is less clearly understood. We sought to determine the prevalence of p16 expression using immunohistochemical analysis in a population-based sample of melanoma tumours, and also to identify histological, phenotypic and environmental factors associated with the presence or absence of p16 expression. We conducted face-to-face interviews with 108 patients newly diagnosed with melanoma to ascertain their history of sun exposure, and recorded various phenotypic parameters. Paraffin sections of tumours from these patients were stained with an anti-p16 monoclonal antibody following antigen retrieval. Overall, 52 (48%) tumours expressed p16; nodular melanomas had significantly lower levels of p16 immunoreactivity than superficial spreading melanomas (P = 0.015). While no association was found between p16 expression and host phenotype, loss of p16 staining was associated with thicker lesions (p = 0.084) and a high mitotic index (P = 0.013). Taken together, these findings are consistent with loss of p16 being a late event in the progression of sporadic primary melanomas, being associated with tumours of a more aggressive nature. (C) 2002 Lippincott Williams Wilkins.

Genetics of melanoma predisposition

Predisposition to melanoma is genetically heterogeneous. Two high penetrance susceptibility genes, CDKN2A and CDK4, have so far been identified and mapping is ongoing to localize and identify others. With the advent of a catalogue of millions of potential DNA polymorphisms, attention is now also being focused on identification of genes that confer a more modest contribution to melanoma risk, such as those encoding proteins involved in pigmentation, DNA repair, cell growth and differentiation or detoxification of metabolites. One such pigmentation gene, MC1R, has not only been found to be a low penetrance melanoma gene but has also been shown to act as a genetic modifier of melanoma risk in individuals carrying CDKN2A mutations. Most recently, an environmental agent, ultraviolet radiation, has also been established as a modifier of melanoma risk in CDKN2A mutation carriers. Hence, melanoma is turning out to be an excellent paradigm for studying gene-gene and gene-environment interactions.

PET/CT com 18-fluor-fluorodeoxiglucose no seguimento do melanoma maligno cutâneo

Introdução – O melanoma maligno cutâneo (MMC) é considerado uma das mais letais neoplasias e no seu seguimento recorre-se, para além dos exames clínicos e da análise de marcadores tumorais, a diversos métodos imagiológicos, como é o exame Tomografia por Emissão de Positrões/Tomografia Computorizada (PET/CT, do acrónimo inglês Positron Emission Tomography/Computed Tomography) com 18fluor-fluorodeoxiglucose (18F-FDG). O presente estudo tem como objetivo avaliar a utilidade da PET/CT relativamente à análise da extensão e à suspeita de recidiva do MMC, comparando os achados imagiológicos com os descritos em estudos CT. Metodologia – Estudo retrospetivo de 62 estudos PET/CT realizados em 50 pacientes diagnosticados com MMC. Excluiu-se um estudo cujo resultado era duvidoso (nódulo pulmonar). As informações relativas aos resultados dos estudos anatomopatológicos e dos exames imagiológicos foram obtidas através da história clínica e dos relatórios médicos dos estudos CT e PET/CT. Foi criada uma base de dados com os dados recolhidos através do software Excel e foi efetuada uma análise estatística descritiva. Resultados – Dos estudos PET/CT analisados, 31 foram considerados verdadeiros positivos (VP), 28 verdadeiros negativos (VN), um falso positivo (FP) e um falso negativo (FN). A sensibilidade, especificidade, o valor preditivo positivo (VPP), o valor preditivo negativo (VPN) e a exatidão da PET/CT para o estadiamento e avaliação de suspeita de recidiva no MMC são, respetivamente, 96,9%, 96,6%, 96,9%, 96,6% e 96,7%. Dos resultados da CT considerados na análise estatística, 14 corresponderam a VP, 12 a VN, três a FP e cinco a FN. A sensibilidade, especificidade, o VPP e o VPN e a exatidão da CT para o estadiamento e avaliação de suspeita de recidiva no MMC são, respetivamente, 73,7%, 80,0%, 82,4%, 70,6% e 76,5%. Comparativamente aos resultados CT, a PET/CT permitiu uma mudança na atitude terapêutica em 23% dos estudos. Conclusão – A PET/CT é um exame útil na avaliação do MMC, caracterizando-se por uma maior acuidade diagnóstica no estadiamento e na avaliação de suspeita de recidiva do MMC comparativamente à CT isoladamente.

Histoplasmosis presenting with multiple pulmonary nodules. A case mimicking radiological features of pulmonary metastasis

We present a case of histoplasmosis with multiple pulmonary nodules in a patient with a history of melanoma. This case closely simulated malignancy, including the presence of feeding vessel sign, which occurs in pulmonary metastasis. We emphasize the need to be aware of this infection in areas where histoplasmosis is endemic.

Attempts using cryotherapy to achieve more rapid healing in patients with cutaneous leishmaniasis due to L. braziliensis braziliensis

The use of cryotherapy as an adjunct to systemic antimonial therapy (Clucantime) was studied in 17 patients with a total of23 skin lesions of leishmaniasis in an area where L. braziliensis braziliensis is the species in circulation. Cryotherapy did not speed healing and has been discarded as an auxiliary therapeutic measure in our practice. However this technique may be suitable for species o/Leishmania causing more limited superficial lesions in man without the danger of metastasis.

Leishmania mexicana in Proechimys iheringi denigratus Moojen (Rodentia, Echimyidae) in a region endemic for American cutaneous leishmaniasis

Three isolates of Leishmania were recovered from five of 27 specimens of the rodent Proechimys iheringi denigratus Moojen captured near Três Braços in the Atlantic Forest region of Bahia, Brazil. Two of these isolates were recovered from hamsters inoculated with a pooled triturate of liver, spleen and skin tissue from apparently healthy P. i. denigratus. The third isolate was recovered from a triturate of only skin tissue from another. Metastasis was observed in the inoculated hamsters, the parasites grew abundantly in artificial media and a typical suprapylarial pattern of infection in Lutzomyia longipalpis was produced indicating that the parasites belong to the Leishmania mexicana complex. All isolates reacted with Leishmania mexicana mexicana and Leishmania mexicana amazonensis monoclonal antibodies. The isoenzyme analysis differentiated these isolates from standard isolates of L. m. mexicana, L. m. amazonensis, L. m. aristedesi, L. m. pifanoi, L. m. garnhami and L. m. ssp.(Goiás-W. Barbosa). These isolates seem to be a subspecies of L. mexicana very closely related to L. m. amazonensis from which they differ by decreased electrophoretic mobility of GPI, PEP and ALAT. This is the first record of the isolation of a parasite of thegenus Leishmania in a rodent captured in the State of Bahia.

Detection of micrometastases in sentinel lymph nodes from melanoma patients: direct comparison of multimarker molecular and immunopathological methods.

The technique of sentinel lymph node (SLN) dissection is a reliable predictor of metastatic disease in the lymphatic basin draining the primary melanoma. Reverse transcription-polymerase chain reaction (RT-PCR) is emerging as a highly sensitive technique to detect micrometastases in SLNs, but its specificity has been questioned. A prospective SLN study in melanoma patients was undertaken to compare in detail immunopathological versus molecular detection methods. Sentinel lymphadenectomy was performed on 57 patients, with a total of 71 SLNs analysed. SLNs were cut in slices, which were alternatively subjected to parallel multimarker analysis by microscopy (haematoxylin and eosin and immunohistochemistry for HMB-45, S100, tyrosinase and Melan-A/MART-1) and RT-PCR (for tyrosinase and Melan-A/MART-1). Metastases were detected by both methods in 23% of the SLNs (28% of the patients). The combined use of Melan-A/MART-1 and tyrosinase amplification increased the sensitivity of PCR detection of microscopically proven micrometastases. Of the 55 immunopathologically negative SLNs, 25 were found to be positive on RT-PCR. Notably, eight of these SLNs contained naevi, all of which were positive for tyrosinase and/or Melan-A/MART-1, as detected at both mRNA and protein level. The remaining 41% of the SLNs were negative on both immunohistochemistry and RT-PCR. Analysis of a series of adjacent non-SLNs by RT-PCR confirmed the concept of orderly progression of metastasis. Clinical follow-up showed disease recurrence in 12% of the RT-PCR-positive immunopathology-negative SLNs, indicating that even an extensive immunohistochemical analysis may underestimate the presence of micrometastases. However, molecular analyses, albeit more sensitive, need to be further improved in order to attain acceptable specificity before they can be applied diagnostically.

Prognostic value of sentinel node biopsy in 327 prospective melanoma patients from a single institution

AIM: To confirm the accuracy of sentinel node biopsy (SNB) procedure and its morbidity, and to investigate predictive factors for SN status and prognostic factors for disease-free survival (DFS) and disease-specific survival (DSS). MATERIALS AND METHODS: Between October 1997 and December 2004, 327 consecutive patients in one centre with clinically node-negative primary skin melanoma underwent an SNB by the triple technique, i.e. lymphoscintigraphy, blue-dye and gamma-probe. Multivariate logistic regression analyses as well as the Kaplan-Meier were performed. RESULTS: Twenty-three percent of the patients had at least one metastatic SN, which was significantly associated with Breslow thickness (p<0.001). The success rate of SNB was 99.1% and its morbidity was 7.6%. With a median follow-up of 33 months, the 5-year DFS/DSS were 43%/49% for patients with positive SN and 83.5%/87.4% for patients with negative SN, respectively. The false-negative rate of SNB was 8.6% and sensitivity 91.4%. On multivariate analysis, DFS was significantly worsened by Breslow thickness (RR=5.6, p<0.001), positive SN (RR=5.0, p<0.001) and male sex (RR=2.9, p=0.001). The presence of a metastatic SN (RR=8.4, p<0.001), male sex (RR=6.1, p<0.001), Breslow thickness (RR=3.2, p=0.013) and ulceration (RR=2.6, p=0.015) were significantly associated with a poorer DSS. CONCLUSION: SNB is a reliable procedure with high sensitivity (91.4%) and low morbidity. Breslow thickness was the only statistically significant parameter predictive of SN status. DFS was worsened in decreasing order by Breslow thickness, metastatic SN and male gender. Similarly DSS was significantly worsened by a metastatic SN, male gender, Breslow thickness and ulceration. These data reinforce the SN status as a powerful staging procedure

Resistance to oxidative stress is associated with metastasis in mucocutaneous leishmaniasis.

Mucocutaneous leishmaniasis (MCL) in South and Central America is characterized by the dissemination (metastasis) of Leishmania Viannia subgenus parasites from a cutaneous lesion to nasopharyngeal tissues. Little is known about the pathogenesis of MCL, especially with regard to the virulence of the parasites and the process of metastatic dissemination. We previously examined the functional relationship between cytoplasmic peroxiredoxin and metastatic phenotype using highly, infrequently, and nonmetastatic clones isolated from an L. (V.) guyanensis strain previously shown to be highly metastatic in golden hamsters. Distinct forms of cytoplasmic peroxiredoxin were identified and found to be associated with the metastatic phenotype. We report here that peroxidase activity in the presence of hydrogen peroxide and infectivity differs between metastatic and nonmetastatic L. (V.) guyanensis clones. After hydrogen peroxide treatment or heat shock, peroxiredoxin was detected preferentially as dimers in metastatic L. (V.) guyanensis clones and in L. (V.) panamensis strains from patients with MCL, compared with nonmetastatic parasites. These data provide evidence that resistance to the first microbicidal response of the host cell by Leishmania promastigotes is linked to peroxiredoxin conformation and may be relevant to intracellular survival and persistence, which are prerequisites for the development of metastatic disease.

Sentinel lymph node involvement and a high Breslow index are independent factors of risk for early relapse of melanoma.

AIM: The clinical relevance of sentinel lymph node (SLN) analysis was evaluated prospectively and compared with other known risk factors of relapse in early stage melanoma. METHODS: Surgery was guided by lymphoscintigraphy, blue dye and gamma probe detection. SLN were analysed by haematoxylin eosin (HE) histochemistry and multimarker immunohistochemistry (IHC). Disease free survival (DFS) was evaluated with Kaplan-Meier plots according to different parameters and Cox analyses of variance. RESULTS: From 210 patients a total of 381 SLN were excised. Lymphoscintigraphy identified all excised SLN with only 2 false positive lymphatic lakes. Fifty patients (24%) had tumour positive SLN. With a mean follow-up of 31.3 months, 29 tumour recurrences were observed, 19 (38%) in 50 SLN positive and 10 (6%) in 160 SLN negative patients. Strong predictive factors for early relapse (p < 0.0005) were SLN positivity and a high Breslow index. CONCLUSION: SLN tumour positivity is an independent factor of high risk for early relapse with a higher power of discrimination than the Breslow index.