Whole-Exome Sequencing and cfDNA Analysis Uncover Genetic Determinants of Melanoma Therapy Response in a Real-World Setting.

Although several studies have explored the molecular landscape of metastatic melanoma, the genetic determinants of therapy resistance are still largely unknown. Here, we aimed to determine the contribution of whole-exome sequencing and circulating free DNA (cfDNA) analysis in predicting response to therapy in a consecutive real-world cohort of 36 patients, undergoing fresh tissue biopsy and followed during treatment. Although the underpowered sample size limited statistical analysis, samples from non-responders had higher copy number variations and mutations in melanoma driver genes compared to responders in the BRAF V600+ subset. In the BRAF V600- subset, Tumor Mutational Burden (TMB) was twice that in responders vs. non-responders. Genomic layout revealed commonly known and novel potential intrinsic/acquired resistance driver gene variants. Among these, RAC1, FBXW7, GNAQ mutations, and BRAF/PTEN amplification/deletion were present in 42% and 67% of patients, respectively. Both Loss of Heterozygosity (LOH) load and tumor ploidy were inversely associated with TMB. In immunotherapy-treated patients, samples from responders showed higher TMB and lower LOH and were more frequently diploid compared to non-responders. Secondary germline testing and cfDNA analysis proved their efficacy in finding germline predisposing variants carriers (8.3%) and following dynamic changes during treatment as a surrogate of tissue biopsy, respectively.

Ataxia-Telangiectasia Mutated Loss of Heterozygosity in Melanoma.

ATM germline pathogenic variants were recently found enriched in high-risk melanoma patients. However, ATM loss of heterozygosity (LOH) has never been investigated in melanoma and, therefore, a causal association with melanoma development has not been established yet. The purpose of this study was to functionally characterize 13 germline ATM variants found in high-risk melanoma patients-and classified by in silico tools as pathogenic, uncertain significance, or benign-using multiple assays evaluating ATM/pATM expression and/or LOH in melanoma tissues and cell lines. We assessed ATM status by Immunohistochemistry (IHC), Western Blot, Whole-Exome Sequencing/Copy Number Variation analysis, and RNA sequencing, supported by Sanger sequencing and microsatellite analyses. For most variants, IHC results matched those obtained with in silico classification and LOH analysis. Two pathogenic variants (p.Ser1135_Lys1192del and p.Ser1993ArgfsTer23) showed LOH and complete loss of ATM activation in melanoma. Two variants of unknown significance (p.Asn358Ile and p.Asn796His) showed reduced expression and LOH, suggestive of a deleterious effect. This study, showing a classic two-hit scenario in a well-known tumor suppressor gene, supports the inclusion of melanoma in the ATM-related cancer spectrum.

Two-year-old children's processing of two-word sequences occurring 19 or more times per million and their influence on subsequent word learning.

We know that 8-month-old infants track the statistical properties of a series of syllables and that 2- and 3-year-old children process familiar phrases more efficiently than unfamiliar phrases, but less is known about the intermediary level of two-word sequences. In Study 1, 2-year-olds (N = 45, mean age = 651 days) heard two-word sequences consisting of a prime word followed by a noun, with two pictures appearing on the screen (depicting the noun and a distractor). Eye tracking showed that children looked more quickly at the noun picture for two-word sequences occurring an average of 19 times per million and 206 times per million in child-directed speech than for novel sequences. In Study 2, corpus analyses showed that 2-year-olds' noun learning increased in line with the frequency of the two-word sequence that preceded it in caregiver speech utterances. This effect holds even after controlling nouns for frequency in caregiver speech, phonemic length, neighborhood density, phonotactic probability, and concreteness and after removing nouns produced in isolation by caregivers and nouns produced by children before being produced by caregivers. These studies show that young children's language processing is facilitated by known two-word sequences, allowing children to focus on more novel aspects of the utterance. Such efficiencies are far-reaching because nearly two thirds of child-directed utterances contain two-word sequences with frequencies of 19 or more per million.

Atypical Spitz Tumor Arising on a Congenital Linear Plaque-Type Blue Nevus: A Case Report With a Review of the Literature on Plaque-Type Blue Nevus.

The plaque-type blue nevus (PTBN) is a rare variant of blue nevus, of which only a few reports are described. A nodular growth within a preexistent PTBN should always alert to the possibility of malignant transformation. The authors report the first case of an atypical Spitz tumor arising on a congenital linear PTBN in a 60-year-old woman. The diagnosis of "atypical Spitz tumor" is here used to describe a microscopic "gray zone" in which it is not possible to differentiate with adequate certainty between a Spitz nevus and a spitzoid melanoma. This report adds to and summarizes the small body of literature describing PTBN and discusses diagnostic and clinical implications.

Perforated Meckel's diverticulitis on the mesenteric side: MDCT findings.

Meckel's diverticulum (MD) is the most common congenital anomaly of the gastro-intestinal tract (approximately 2% of population), and arises from improper closure and absorption of the omphalomesenteric duct. Very few cases of Meckel's diverticulitis on the mesenteric side have been reported in the surgical literature, and no reported cases have been documented on preoperative imaging. We report a 65-year-old woman presenting symptoms and signs of acute abdomen with an initial suspicion of acute appendicitis. MDCT imaging revealed a mesenteric abscess in the right lower quadrant at the level of the distal ileum as a complication of Meckel's diverticulitis on the mesenteric side. The patient recovered after a diverticulectomy without the need for a small bowel resection. This case demonstrates that MDCT is a fast imaging technique that may be helpful in the emergency setting for the preoperative diagnosis of an unusual complicated MD on the mesenteric side.

P53 staining index and zonal staining patterns in actinic keratoses.

Actinic keratoses (AKs) are common dysplastic lesions resulting from chronic excessive ultraviolet exposure. Neither the clinical grade of thickness nor the histological grade of dysplasia seems valid predictors of aggressive potential of AKs. Instead, the mutational status in AKs appears to predict well the clinical course. TP53 gene mutations result in a non-functional protein resistant to degradation, thus immunohistochemical staining for p53 can suggest mutation status. Increased p53 was associated with progression from AK to squamous cell carcinoma. To investigate how the intensity of p53 staining (p53 staining index) varies according to body site, histological subtype and grade dysplasia of AKs. Secondly, we sought to investigate the distribution in the epidermal layers of non-functional p53 (zonal staining patterns). p53 staining index was greater than 50% in 90.7% of AKs. p53 staining index was significantly higher in older age (p < 0.0093) and in facial AKs compared to other body areas (p = 0.03). A significant correlation between p53 staining index and grade of dysplasia was observed (p = 0.006) and between p53 staining index and zonal p53 staining pattern (p = 0.003). No significant differences in p53 staining index among the various histological AK types were observed. No correlation between clinical and histological grade. All AKs, independently from their clinical appearance, should be treated but special attention is required for AKs on severely photodamaged skin on the face and in older patients.

Does short-term memory develop?

Such is the consistency by which performance on measures of short-term memory (STM) increase with age that developmental increases in STM capacity are largely accepted as fact. However, our analysis of a robust but almost ignored finding - that span for digit sequences (the traditional measure of STM) increases at a far greater rate than span for other verbal material - fundamentally undermines the assumption that increased performance in STM tasks is underpinned by developmental increases in capacity. We show that this digit superiority with age effect is explained by the relatively greater linguistic exposure to random sequences of digits versus other stimuli such as words. A simple associative learning process that learns incrementally from exposure to language accounts for the effect, without any need to invoke an STM mechanism, much less one that increases in capacity with age. By extension, using corpus data directed at 2-3 year old children, 4-6 year old children, and adults, we show that age-related performance increases with other types of verbal material are equally driven by the same basic associative learning process operating on the expanding exposure to language experienced by the child. Our results question the idea that tests such as digit span are measuring a dedicated system for the temporary maintenance and manipulation of verbal material, and as such have implications for our understanding of those aspects of typical and atypical development that are usually accounted for with respect to the operation of such a system.

Solitary nodule of the penis as unique manifestation of Rosai-Dorfman disease.

We report the case of a 53-year-old man with a solitary nodule located on the inner surface of the prepuce. The lesion was removed and the final diagnosis was Rosai-Dorfman disease (RDD/sinus histiocytosis with massive lymphadenopathy). This report, being the first case of RDD presenting as a single nodule of the penis, broadens the spectrum of the cutaneous expression of RDD.

Primary cutaneous melanoma: correlation between clinical and histologic aspects in a retrospective Italian study.

BACKGROUND: In Italy, the incidence of new cases of melanoma is roughly 10,000 cases per year, with an average rate of mortality of 5-6 per 100,000 population per year respectively. The objective of this retrospective study was to evaluate the epidemiological incidence of primitive melanoma, including the incidence of multiple melanomas occurring in the same patient. Furthermore, we studied all histological different types of melanoma focusing on the presence of an association nevus-melanoma. METHODS: A clinical epidemiologic retrospective study from January 2010 to March 2015 was recorded. For each lesion, mitotic rate, Breslow's index, ulceration, presence of regression, vascular and perineural invasion, lymphocytic infiltrate, microsatellitosis and presence of pre-existencing nevus were also studied. RESULTS: Five hundred eighty primitive cutaneous melanomas (CMs) were removed from 525 patients with an incidence of 18-20 new melanomas/100,000 habitants/year. Eighty percent of these were at stage T0-1. Among other melanomas, SSM was the predominant subtype (85% of cases). Only 18 cases had lymph node metastases and 13 (2%) lymph node and/or distant metastases (stage IV) at time of diagnosis. Mitotic figures were present in 25% of cases (143 cases out of 580) without significant gender differences. CONCLUSIONS: The incidence of new melanomas founded is close to the CM's incidence in US population in 2016 with a high percentage of superficial melanomas highlighting the importance of prevention campaigns. The presence of melanoma on a preexisting nevus in only 16% of cases allow to conclude that this association is overestimated in literature. On the contrary the high incidence of a second melanoma in the 7% of cases in a relatively short period of survey leads to the conclusion that this data is underestimated.

Correction: Combining molecular and immunohistochemical analyses of key drivers in primary melanomas: interplay between germline and somatic variations.

[This corrects the article DOI: 10.18632/oncotarget.23204.].

Combining molecular and immunohistochemical analyses of key drivers in primary melanomas: interplay between germline and somatic variations.

Due to the high mutational somatic burden of Cutaneous Malignant Melanoma (CMM) a thorough profiling of the driver mutations and their interplay is necessary to explain the timing of tumorigenesis or for the identification of actionable genetic events. The aim of this study was to establish the mutation rate of some of the key drivers in melanoma tumorigenesis combining molecular analyses and/or immunohistochemistry in 93 primary CMMs from an Italian cohort also characterized for germline status, and to investigate an interplay between germline and somatic variants. BRAF mutations were present in 68% of cases, while CDKN2A germline mutations were found in 16 % and p16 loss in tissue was found in 63%. TERT promoter somatic mutations were detected in 38% of cases while the TERT -245T>C polymorphism was found in 51% of cases. NRAS mutations were found in 39% of BRAF negative or undetermined cases. NF1 was expressed in all cases analysed. MC1R variations were both considered as a dichotomous variable or scored. While a positive, although not significant association between CDKN2A germline mutations, but not MC1R variants, and BRAF somatic mutation was found, we did not observe other associations between germline and somatic events. A yet undescribed inverse correlation between TERT -245T>C polymorphism and the presence of BRAF mutation was found. It is possible to hypothesize that -245T>C polymorphism could be included in those genotypes which may influence the occurrence of BRAF mutations. Further studies are needed to investigate the role of -245T>C polymorphism as a germline predictor of BRAF somatic mutation status.

Heterogeneity and frequency of BRAF mutations in primary melanoma: Comparison between molecular methods and immunohistochemistry.

Finding the best technique to identify BRAF mutations with a high sensitivity and specificity is mandatory for accurate patient selection for target therapy. BRAF mutation frequency ranges from 40 to 60% depending on melanoma clinical characteristics and detection technique used.Intertumoral heterogeneity could lead to misinterpretation of BRAF mutational status; this is especially important if testing is performed on primary specimens, when metastatic lesions are unavailable.Aim of this study was to identify the best combination of methods for detecting BRAF mutations (among peptide nucleic acid - PNA-clamping real-time PCR, immunohistochemistry and capillary sequencing) and investigate BRAF mutation heterogeneity in a series of 100 primary melanomas and a subset of 25 matched metastatic samples.Overall, we obtained a BRAF mutation frequency of 62%, based on the combination of at least two techniques. Concordance between mutation status in primary and metastatic tumor was good but not complete (67%), when agreement of at least two techniques were considered. Next generation sequencing was used to quantify the threshold of detected mutant alleles in discordant samples. Combining different methods excludes that the observed heterogeneity is technique-based. We propose an algorithm for BRAF mutation testing based on agreement between immunohistochemistry and PNA; a third molecular method could be added in case of discordance of the results. Testing the primary tumor when the metastatic sample is unavailable is a good option if at least two methods of detection are used, however the presence of intertumoral heterogeneity or the occurrence of additional primaries should be carefully considered.

Lichen scrofulosorum caused by Mycobacterium leprae: first report.

BACKGROUND: Tuberculides are skin lesions caused by the hematogeneous dissemination of Mycobacterium tuberculosis. Bacilli are rapidly destroyed in the skin and are thus neither visible histologically nor identifiable by culture. Diagnosis depends on previous knowledge of systemic and/or cutaneous tuberculosis. Lichen scrofulosorum (LS), the most uncommon variant of tuberculid, is usually associated with M. tuberculosis infection of lymph nodes or bone but was also reported in association with other mycobacterioses. OBJECTIVES: We report a case of LS in a patient with M. leprae infection. METHODS: In 2008, a 51-year-old woman from the Philippines was diagnosed with tuberculoid leprosy and treated. In 2010 the leprosy was considered to have been cured, and treatment was stopped. In 2011 the patient presented with lesions on the trunk and legs. Biopsy specimens were obtained for histopathologic examination and DNA detection for polymerase chain reaction (PCR). RESULTS: Histopathology in the biopsy from the trunk revealed the dermis to be diffusely occupied by granulomas with perineural and periadnexal disposition. Granulomas were composed of epithelioid cells and lymphocytes. Fite-Faraco staining revealed a few solid acid-fast bacilli within nerve fascicles. Reinfection or the re-reactivation of multibacillary borderline tuberculoid leprosy was diagnosed. Histopathology in the biopsy taken from the leg showed superficial, well-formed granulomas in the vicinity of hair follicles and sweat ducts. No acid-fast bacilli were seen. Analysis by PCR revealed M. leprae DNA in specimens from both the leg and trunk. The clinical features of the papular eruption and the histopathologic findings and concomitant mycobacterial infection with M. leprae led to a diagnosis of LS. Treatment was commenced with dapsone 100 mg/day, clofazimine 50 mg/day and 300 mg/month, and rifampicin 600 mg/day. The lichenoid eruption on the legs disappeared at one month of therapy, whereas the other skin lesions resolved in one year leaving residual hypochromic macules. CONCLUSIONS: Infection with M. leprae may cause LS. The use of PCR in skin biopsies from granulomatous dermatitis of unknown origin can help to identify the responsible agents.

Descemet membrane air-bubble separation in donor corneas.

We describe a technique to obtain Descemet-endothelium disks from donors. To detach Descemet membrane, an air bubble was introduced in the deep stroma of human donor corneas mounted on an artificial chamber. In Group A (n = 5), the bubble was left inflated. In Group B (n = 4), the bubble was deflated immediately after the membrane was detached. In Group C (n = 7), the Descemet-endothelium disk was trephined and separated from the stroma after the bubble was deflated. All tissues were stored at 4°C. Descemet detachment was achieved in 89% of the tissues. After 48 hours, the mean endothelial loss was 83% ± 10% (SD), 15% ± 11%, and 3% ± 3% in the 3 groups, respectively. With this technique, Descemet-endothelium disks were obtained without significant alterations in the endothelial layer.