cral melanoma is a rare melanocytic tumor that arises on the non-hair-bearing skin of the palms, soles, and nail beds. 1 Unlike cutaneous melanoma, acral melanoma is not linked to UV radiation exposure; it exhibits a much lower point mutation burden than cutaneous melanoma but a higher frequency of structural variants and copy number alterations. 2,3 The incidence of acral melanoma is uniform across all populations, making it the most common melanoma subtype in individuals of African, Asian, and Hispanic descent. 1 Acral melanomas differ in their mutational profiles from cutaneous melanomas, with a lower incidence of BRAF mutations (18%) and NF1 mutations (11%) and the presence of mutations in TYRP1 (8%) and NOTCH2 (4%) and amplification/ mutation in the receptor tyrosine kinase c-KIT (approximately 2%-3%). 3 Acral melanomas with BRAF mutations harbor fewer genomic amplifications and are more common in patients with European ancestry, possibly constituting a unique subset. 4 Melanocytic nevi are benign proliferations of melanocytes; melanomas sometimes arise in a small proportion of melanocytic nevi. [5][6][7] Genomic analyses have shown that 100% of nevi on sun-exposed skin harbor mutually exclusive mutations in melanoma driver oncogenes, such as BRAF and NRAS. [8][9][10] Although the development of nevi has been linked to sun exposure, 10,11 they can also arise on skin with low levels of UV radiation exposure, such as the palms and soles. At this time, little is known about the mutational profiles of acral nevi, which arise on skin with little UV radiation exposure. Methods Patient SamplesThis study was approved by the University of South Florida institutional review board (protocol No. PRO00036516). Deidentified archival samples were collected under a waiver of informed consent under the Common Rule (45 CFR 46). After institutional review board approval, the pathology databases at 2 institutions were queried for a diagnosis containing the words melanocytic nevus at any acral location (palm, sole, finger, toe, foot, hand). Slides were reviewed and diagnoses verified by the study pathologist (J.L.M.); 49 of 50 cases with greater than 10% nevus cellularity and 1 case with 5% nevus cellularity were submitted for analysis. DNA SequencingTargeted DNA sequencing was performed on 151 cancerassociated genes (eTable 1 in the Supplement) (Agilent Sure-Select XT ClearSeq Comprehensive Cancer Panel). For each sample, 75-base paired-end sequence reads were generated IMPORTANCE Acral skin may develop nevi, but their mutational status and association with acral melanoma is unclear.OBJECTIVE To perform targeted next-generation sequencing on a cohort of acral nevi to determine their mutational spectrum.DESIGN, SETTING, AND PARTICIPANTS Acral nevi specimens (n = 50) that had been obtained for diagnostic purposes were identified from the pathology archives of a tertiary care academic cancer center and a university dermatology clinic. Next-generation sequencing was performed on DNA extracted from the specimens, and mutations calle...
Cutaneous metastasis may be the initial sign of internal malignancy but more often represents a late manifestation of widely disseminated disease. Breast carcinoma is the most common malignancy to metastasize to the skin. Although several studies have detailed the histopathologic patterns of cutaneous metastasis from internal malignancies, very little has been published regarding metastases of breast carcinoma to the skin. Furthermore, the histopathologic and clinical features observed in the cases of breast carcinoma with local skin involvement as opposed to cases exhibiting distant cutaneous metastases have not been adequately investigated. We have reviewed 232 cases of breast carcinoma with cutaneous metastases from 2 large institutions. All cases of carcinoma of the breast with involvement of the skin of the anterior chest wall were compared with those with distant cutaneous metastases. Two hundred thirty-two cases in 199 patients were included, of which 126 had skin involvement exclusively involving the ipsilateral anterior chest, and 106 had biopsy-proven distant cutaneous metastases. Twelve patients had both local and distal spread. Distant cutaneous metastases showed a predilection for the contralateral anterior chest wall area, followed by the head and neck, back, and abdomen. Histologically, most of the tumors presented in this series showed features of infiltrating ductal carcinoma. In both ipsilateral and distant metastases, the tumors demonstrated little change in histologic features from the primary lesion; however, the distant metastases showed a tendency to display more poorly differentiated features. The mean patient survival when cutaneous involvement was localized to the skin of the anterior chest wall was 23 months as compared with 20.6 months when distant sites were affected. A comparison of the clinicopathologic features of the patients presented in this series suggests that alternate biological mechanisms may apply for local and distant skin metastases from breast carcinoma.
Although Illumina shot-gun reads cover most genomes almost completely, sequences with extreme base compositions are often underrepresented or missing. Bias can potentially be introduced at any step during the library construction in the lab, on the Illumina instrument, in data processing or at the sequence analysis stage. Here we set out to evaluate sources of bias and ameliorate the effects.To dissect the library construction process, we developed a panel of qPCR assays for loci ranging from 6% to 90% GC that work well in a pool of three microbial DNA samples of different base composition: Plasmodium falciparum (19% GC), Escherichia coli (51% GC) and Rhodobacter sphaeroides (69% GC). We also developed qPCR assays for loci in the human genome that represent four categories of underrepresented sequence motifs as well as GC-rich promoters known to be underrepresented or missing in 'whole' genome sequencing data sets.We tracked the relative abundance of these loci throughout the standard Illumina library protocol and saw no significant introduction of bias in the initial steps including shearing, end repair, adaptor ligation and size selection. However, GC-rich and extremely GC-poor sequences were depleted during the subsequent PCR-enrichment step. Using qPCR as a readout, we tested different PCR enzymes, the addition of betaine and/or DMSO, and thermocycling profile variations. The choice of PCR instrument itself and the ramp rate had a significant effect on the GC profile of the PCR product, especially when using the recommended amplification conditions (Phusion HF and 10s denaturation per cycle).Our optimized conditions produce PCR-amplified libraries that display little systematic bias between 15% and 80% GC that resulted during sample preparation. We saw significantly improved representation of challenging human sequence motifs both in the PCR-amplified library (qPCR assay) and in the final Illumina reads. Our conditions are also more reliable and robust because they minimize the effect of PCR instrument and ramp rate. These conditions are currently being implemented in the Sequencing Platform at the Broad Institute. Finally, we still observe some bias in the sequencing readout, which is introduced by steps subsequent to sample preparation, including cluster generation and sequencing. These sources of bias are the object of ongoing investigations.
Background: We present a case of RCM evaluation of ALM surgical margins demonstrating intracorneal melanocytic bodies overlying subsequently confirmed melanoma in situ by histopathology. Case Presentation: A 73-year-old male with a history of acral lentiginous melanoma (ALM) of the right great toe presented to our clinic for evaluation of positive surgical margins. The positive margin was localized for examination and subsequent biopsy with reflectance confocal microscopy (RCM) which allowed targeted reresection of the area of concern. Three punch biopsies were obtained in the area of concern, which confirmed residual melanoma in situ. Immunostains confirmed the cellular remnants in the stratum corneum were melanocytic. To correlate the intra stratum corneum findings seen with confocal to the histopathology, a 3D rendering of a stack of images was used to demonstrate the location. Discussion: Typically, acral surfaces are challenging to examine with RCM due to the limited ability of light to penetrate thickened stratum corneum; however, we observed unique cellular features with confocal. Scattered hyper-reflective pleomorphic cells consistent with melanocytes were observed in the stratum corneum, although the visualized underlying epidermis appeared normal. Confocal microscopy may aid in diagnosis and management of ALM, especially in the context of positive surgical margins.
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