Intratumor Heterogeneity of KIT Gene Mutations in Acral Lentiginous Melanoma.

Melanoma is an aggressive skin malignancy, and the acral lentiginous melanoma (ALM) subtype affects non-sun-exposed sites such as the volar surface of the hands and feet and the subungual region and is most common in Asians, Hispanics, and Afro-descendants. The presence of different clones within the same tumor seems to influence the aggressiveness of tumors. Patients with mutations in the KIT gene have shown a good response to tyrosine kinase inhibitor therapy. We tested the hypothesis of intratumor heterogeneity through analysis of KIT gene mutations in ALM and determined the correlation between KIT mutations and demographic, clinical, and histopathological variables. Twenty-five ALM samples were examined. We selected up to four different regions per tumor for sequencing by the Sanger method for analysis of KIT gene exon 11 and exon 13 mutations. Advanced lesions were predominant, and the main histopathological characteristics of lesions were Breslow index >4.0 mm (17/25, 68%), Clark level IV/V (21/25, 84%), ulceration (16/25, 64%), and >3 mitoses/mm (8/25, 32%). KIT gene mutations were detected in 11/25 cases (44%), and all these 11 cases displayed intratumor heterogeneity, that is, at least 2 tumor regions had different mutational profiles. The predicted effect of most mutations detected was detrimental to protein function. No significant correlations between histopathological variables and either KIT mutations or intratumor heterogeneity were observed. The hypothesis of intratumor heterogeneity of KIT gene mutations in acral lentiginous melanoma was supported.

IMP dehydrogenase rod/ring structures in acral melanomas.

Acral lentiginous melanoma (ALM) is a rare subtype of melanoma with aggressive behavior. IMPDH enzyme, involved in de novo GTP biosynthesis, has been reported to assemble into large filamentary structures called rods/rings (RR) or cytoophidium (cellular snakes). RR assembly induces a hyperactive state in IMPDH, usually to supply a high demand for GTP nucleotides, such as in highly proliferative cells. We investigate whether aggressive melanoma tumor cells present IMPDH-based RR structures. Forty-five ALM paraffin-embedded tissue samples and 59 melanocytic nevi were probed with anti-IMPDH2 antibody. Both the rod- and ring-shaped RR could be observed, with higher frequency in ALM. ROC curve analyzing the proportions of RR-positive cells in ALM versus nevi yielded a 0.88 AUC. Using the cutoff of 5.5% RR-positive cells, there was a sensitivity of 80% and specificity of 85% for ALM diagnosis. In ALM, 36 (80%) showed RR frequency above the cutoff, being classified as RR-positive, compared with only 9 (15%) of the nevi (p < .001). Histopathology showed that 71% of the RR-positive specimens presented Breslow thickness > 4.0mm, compared with only 29% in the RR-low/negative (p = .039). We propose that screening for RR structures in biopsy specimens may be a valuable tool helping differentiate ALM from nevi and accessing tumor malignancy.

Acral Lentiginous Melanomas Harbour Intratumor Heterogeneity in BRAF Exon 15, With Mutations Distinct From V600E/V600K.

The choice of appropriate therapeutic strategies may be influenced by intratumor heterogeneity and makes cancer treatment considerably more challenging. We aimed to evaluate the heterogeneity of BRAF exon 15 mutations in different areas of acral lentiginous melanoma (ALM). The entire exon 15 was sequenced in 4 different areas of paraffin-embedded samples from 26 patients with ALM. A total of 26 of 49 cases of ≥1 mm in depth of ALM identified by clinical, anatomical, and pathological data fulfilled the inclusion and exclusion criteria for this study. Tumors had a mean Breslow depth of 7.2 mm and an average mitotic index of 3 mitosis/mm. Mutations distinct from the common V600E and V600K were detected in 31%, and intratumor heterogeneity was observed in 31% of samples. Interestingly, 63.5% of all mutations had been previously associated with cancer. Most (62.5%) of the missense BRAF exon 15 mutations found in the ALM samples examined here were deemed "detrimental" for protein function according to at least 2 functional prediction programs, and 3 mutations (37.5%) were predicted to be "neutral," with no effect on protein function. BRAF exon 15 mutations were detected frequently in ALM and displayed heterogeneity, a finding to be further investigated.

Marker Protein Expression Combined With Expression Heterogeneity is a Powerful Indicator of Malignancy in Acral Lentiginous Melanomas.

PATIENTS AND METHODS: Samples of acral lentiginous melanomas (ALMs) were obtained from the Department of Pathology at Escola Paulista de Medicina-Universidade Federal de São Paulo (UNIFESP), São Paulo, Brazil. Demographic, clinical, and follow-up data were obtained from the charts of Hospital São Paulo. From 2 tissue microarrays containing 60 nevi and quadruplicate samples of ≥1.0-mm of 49 ALM, sections were stained to evaluate SCF, KIT, BRAF, CYCLIND1, MYC, and PTEN immunohistochemical protein expression. RESULTS: Nevi and ALM from 2006 to 2010 were reviewed and collected. All specimens were in the vertical growth phase, and histopathological parameters indicated that tumors were at an advanced stage at diagnosis. Average tumor thickness was 6.95 mm, 63% were ulcerated, average mitotic index was 5 mitotic cells per mm, and 43% were at Clark's level V. Compared with nevi, the χ test showed that ALM significantly correlated with SCF protein expression (P = 0.001) and expression heterogeneity (P < 0.000). Similar findings were observed for KIT (P = 0.005, P = 0.003, respectively), MYC (P < 0.000, P < 0.000), and PTEN (P = 0.005, P < 0.000). Malignancy did not correlate with BRAF and CYCLIN D1 expression (P = 0.053 and P = 0.259, respectively), but it did significantly correlate with their heterogeneous expression (P < 0.000, P = 0.024, respectively). Combined protein expression had an odds ratio of greater malignancy when BRAF and MYC were positive and/or heterogeneously expressed (OR of 78 and 95, respectively). DISCUSSION AND CONCLUSION: We show that marker protein expression, when combined with heterogeneous expression as shown by immunohistochemistry, is a powerful indicator of malignancy in ALMs, especially, when protein pairs are combined.

Identificação de Staphylococcus epidermidis em formigas (Hymenoptera: Formicidae) coletadas em uma área de alimentação no município de Guarulhos, São Paulo / Identification of Staphylococcus epidermidis on ants (Hymenoptera: Formicidae) collected in a food court in the city of Guarulhos, São Paulo

Out of the urbanization process, there was an increase in the spread of diseases carried by arthropods, and the most common are the ants. Their presence is related to its size, ease of locomotion and form of social life. Therefore, they can act as mechanical vectors of obligatory endosymbiont bacteria and pathogen ones as food contamination, and contamination of hospital environment. The purposes of this article was to isolate and identify contaminating bacteria of the genera Escherichia sp., Staphylococcus sp. and Salmonella sp. on ants workers circulating around a diner of high movement of people. Ant tracks were collected in the afternoon, sampled at four points to around the patio of the cafe. After the collection, the bacteria were identified, by growing in culture medium for enrichment and Tryptic Soy Broth - specific means. Of the four collected points around the diner, two of them showed growth of Staphylococcus epidermidis. This study identified the presence S. epidermidis on ants workers vectors in a cafeteria located in an area of great movement of persons indicating that the ants can be vectors of contamination in food trade.

Com o processo de urbanização, ocorreu aumento da disseminação de doenças veiculadas por artrópodes, sendo os mais comuns as formigas. A presença delas é mais frequente pelo seu tamanho, por sua facilidade de locomoção e por sua forma de vida social. Assim, podem atuar como vetores mecânicos de bactérias endossimbiontes e patogênicas, ocasionando contaminação em alimentos e no ambiente hospitalar. Os objetivos deste artigo foram isolar e identificar bactérias contaminantes dos gêneros Escherichia sp., Staphylococcus sp. e Salmonella sp. em formigas operárias circulantes no entorno de uma lanchonete de intenso fluxo de pessoas. Foram coletados rastros de formigas no período vespertino, amostradas em quatro pontos do pátio no entorno da lanchonete. Após a coleta, as bactérias foram identificadas por cultivo em meio de cultura Caldo Triptona de Soja para enriquecimento e meios específicos. Dos quatro pontos coletados no entorno da lanchonete, dois apresentaram crescimento de Staphylococcus epidermidis. Este estudo identificou a presença de S. epidermidis em formigas operárias em uma lanchonete localizada em uma área de grande circulação de pessoas, indicando que elas podem ser vetores de contaminação em estabelecimentos de comércio de alimentos.

Identificação de Staphylococcus epidermidis em formigas (Hymenoptera: Formicidae) coletadas em uma área de alimentação no município de Guarulhos, São Paulo / Identification of Staphylococcus epidermidis on ants (Hymenoptera: Formicidae) collected in a food court in the city of Guarulhos, São Paulo

Com o processo de urbanização, ocorreu aumento da disseminação de doenças veiculadas por artrópodes, sendo os mais comuns as formigas. A presença delas é mais frequente pelo seu tamanho, por sua facilidade de locomoção e por sua forma de vida social. Assim, podem atuar como vetores mecânicos de bactérias endossimbiontes e patogênicas, ocasionando contaminação em alimentos e no ambiente hospitalar. Os objetivos deste artigo foram isolar e identificar bactérias contaminantes dos gêneros Escherichia sp., Staphylococcus sp. e Salmonella sp. em formigas operárias circulantes no entorno de uma lanchonete de intenso fluxo de pessoas. Foram coletados rastros de formigas no período vespertino, amostradas em quatro pontos do pátio no entorno da lanchonete. Após a coleta, as bactérias foram identificadas por cultivo em meio de cultura Caldo Triptona de Soja para enriquecimento e meios específicos. Dos quatro pontos coletados no entorno da lanchonete, dois apresentaram crescimento de Staphylococcus epidermidis. Este estudo identificou a presença de S. epidermidis em formigas operárias em uma lanchonete localizada em uma área de grande circulação de pessoas, indicando que elas podem ser vetores de contaminação em estabelecimentos de comércio de alimentos.(AU)

Out of the urbanization process, there was an increase in the spread of diseases carried by arthropods, and the most common are the ants. Their presence is related to its size, ease of locomotion and form of social life. Therefore, they can act as mechanical vectors of obligatory endosymbiont bacteria and pathogen ones as food contamination, and contamination of hospital environment. The purposes of this article was to isolate and identify contaminating bacteria of the genera Escherichia sp., Staphylococcus sp. and Salmonella sp. on ants workers circulating around a diner of high movement of people. Ant tracks were collected in the afternoon, sampled at four points to around the patio of the cafe. After the collection, the bacteria were identified, by growing in culture medium for enrichment and Tryptic Soy Broth - specific means. Of the four collected points around the diner, two of them showed growth of Staphylococcus epidermidis. This study identified the presence S. epidermidis on ants workers vectors in a cafeteria located in an area of great movement of persons indicating that the ants can be vectors of contamination in food trade.(AU)

DNA extraction and molecular analysis of non-tumoral liver, spleen, and brain from autopsy samples: the effect of formalin fixation and paraffin embedding.

The use of molecular biology in combination with morphological analysis is increasing because of the treatments by target therapies. However, to improve the methods for obtaining DNA for molecular analyses from formalin-fixed, paraffin-embedded (FFPE) tissue is a challenge. The aim of this study was to evaluate the DNA extracted from FFPE tissue blocks (non-tumoral liver, spleen, and brain), obtained from autopsy, 8-24 h post mortem, using three methods of DNA extraction. PCR of the ß-actin (136 pb) and human amelogenin (AMEL 212-218 bp/106-112 bp) genes, as well as short tandem repeat (STR) (100-400 bp fragments), reported in forensic scientific analysis, was performed to evaluate the effectiveness of the methods of DNA extraction. We used 28 archived (1 and 5 years) and 12 recent autopsy cases. The commercial kit showed reproducible and consistent results in the PCR amplification of the ß-actin and AMEL genes and in analysis by STR used in forensic analysis. This is the first report using non-tumoral samples from FFPE autopsy tissues, comparing the three most common methods of DNA extraction and using the STR previously described in forensics. Our study has clarified the challenges for pathologists in applying the molecular biology approach in combination with methods suited for morphology, which must be improved. The data provided here should be used in other molecular studies in FFPE samples.

Effects of tissue handling and processing steps on PCR for detection of Mycobacterium tuberculosis in formalin-fixed paraffin-embedded samples.

Development and standardization of reliable methods for detection of Mycobacterium tuberculosis in clinical samples is an important goal in laboratories throughout the world. In this work, lung and spleen fragments from a patient who died with the diagnosis of miliary tuberculosis were used to evaluate the influence of the type of fixative as well as the fixation and paraffin inclusion protocols on PCR performance in paraffin embedded specimens. Tissue fragments were fixed for four h to 48 h, using either 10% non-buffered or 10% buffered formalin, and embedded in pure paraffin or paraffin mixed with bee wax. Specimens were submitted to PCR for amplification of the human beta-actin gene and separately for amplification of the insertion sequence IS6110, specific from the M. tuberculosis complex. Amplification of the beta-actin gene was positive in all samples. No amplicons were generated by PCR-IS6110 when lung tissue fragments were fixed using 10% non-buffered formalin and were embedded in paraffin containing bee wax. In conclusion, combined inhibitory factors interfere in the detection of M. tuberculosis in stored material. It is important to control these inhibitory factors in order to implement molecular diagnosis in pathology laboratories.

Effects of tissue handling and processing steps on PCR for detection of Mycobacterium tuberculosis in formalin-fixed paraffin-embedded samples / Efeitos das etapas de tratamento e processamento do tecido na PCR para detecção de Mycobacterium tuberculosis em amostras fixadas em formalina e incluídas em parafina

Development and standardization of reliable methods for detection of Mycobacterium tuberculosis in clinical samples is an important goal in laboratories throughout the world. In this work, lung and spleen fragments from a patient who died with the diagnosis of miliary tuberculosis were used to evaluate the influence of the type of fixative as well as the fixation and paraffin inclusion protocols on PCR performance in paraffin embedded specimens. Tissue fragments were fixed for four h to 48 h, using either 10 percent non-buffered or 10 percent buffered formalin, and embedded in pure paraffin or paraffin mixed with bee wax. Specimens were submitted to PCR for amplification of the human beta-actin gene and separately for amplification of the insertion sequence IS6110, specific from the M. tuberculosis complex. Amplification of the beta-actin gene was positive in all samples. No amplicons were generated by PCR-IS6110 when lung tissue fragments were fixed using 10 percent non-buffered formalin and were embedded in paraffin containing bee wax. In conclusion, combined inhibitory factors interfere in the detection of M. tuberculosis in stored material. It is important to control these inhibitory factors in order to implement molecular diagnosis in pathology laboratories.

O desenvolvimento e a padronização de métodos confiáveis para a detecção de Mycobacterium tuberculosis em amostras clínicas é um objetivo importante nos laboratórios de todo o mundo. Neste trabalho, fragmentos de pulmão e baço de paciente que morreu com o diagnóstico de tuberculose miliar foram usados para avaliar a influência do tipo de fixador e dos protocolos de fixação e inclusão em parafina na performance da PCR. Fragmentos de tecido foram fixados por quatro h a 48 h, usando formalina não tamponada a 10 por cento ou formalina tamponada a 10 por cento e incluídos em parafina pura ou misturada a cera de abelha. As amostras foram submetidas a PCR para amplificação do gene da beta-actina humana e, separadamente, para amplificação da sequência de inserção IS6110, específica do complexo M. tuberculosis. O resultado da amplificação do gene da beta-actina foi positivo em todas as amostras. Não foram gerados amplicons na PCR-IS6110 em amostras de tecido pulmonar fixadas usando formalina não tamponada a 10 por cento e incluídas em parafina com cera de abelha. Em conclusão, fatores inibitórios combinados interferiram na detecção de M. tuberculosis em material de arquivo. É importante controlar estes fatores inibitórios para poder implementar o diagnóstico molecular em laboratórios de patologia.