Search

We report a case of membranous conjunctivitis and erythema multiforme major (EMM) after a coronavirus disease 2019 (COVID-19) diagnosis. A previously well 18-year-old man presented with increasingly erythematous eyes and oral and genital ulceration 2 weeks after confirmation of COVID-19 infection. Clinical examination showed sloughy membranous conjunctivitis with normal visual acuity. He was reviewed by dermatology and diagnosed with EMM secondary to severe acute respiratory syndrome coronavirus 2 infection. The symptoms resolved with oral and topical steroids, lubricants and chloramphenicol eye drops. Erythema multiforme has been reported in association with COVID-19, although the major form is rare. Ophthalmologists should consider current or previous COVID-19 infection in patients presenting with conjunctivitis or pseudomembrane formation. Prompt initiation of steroids aids resolution.

Objective Malignant melanoma of the female genital tract is a rare disease with poor prognosis, with controversies remaining in its staging and management. In this study, we investigate clinical, pathological, and outcome data for patients referred to a tertiary cancer center with female genital tract melanoma over a decade. Methods Patients were retrospectively identified using a search of pathology reports to identify all cases of female genital tract melanoma from 2007 to 2019. Electronic patient records were used to record clinical information. Histopathology specimens were reviewed by a gynecological and dermatological pathology specialist. Results We identified 30 cases of genital tract melanoma, of which 19 were vulvar, 10 were vaginal, and 1 cervical. Overall survival at 1, 3, and 5 years was found to be 80%, 60%, and 57%. Patients who died were not significantly older at presentation than patients who survived (62 y vs 69 y, p = .215). No association was found between mortality and microscopic ulceration, lymphovascular invasion, pigmentation, resection margins, or radical versus local surgery.Nonvulvar lesions were significantly associated with mortality compared with vulvar lesions (p = .0018), despite similar age and Breslow thickness. Five patients were diagnosed at in situ stage, all of these were vulvar. Even after excluding these melanomas in situ, nonvulvar melanomas still had a significantly worse mortality rate (p = .048). A higher proportion of nonvulvar lesions than vulvar lesions displayed loss of pigmentation (p = .026). Conclusions Nonvulvar genital tract melanomas carry a significantly worse prognosis. Survival was not related to resection margins, supporting the use of more conservative surgical margins.

ADAPTeR is a prospective, phase II study of nivolumab (anti-PD-1) in 15 treatment-naive patients (115 multiregion tumor samples) with metastatic clear cell renal cell carcinoma (ccRCC) aiming to understand the mechanism underpinning therapeutic response. Genomic analyses show no correlation between tumor molecular features and response, whereas ccRCC-specific human endogenous retrovirus expression indirectly correlates with clinical response. T cell receptor (TCR) analysis reveals a significantly higher number of expanded TCR clones pre-treatment in responders suggesting pre-existing immunity. Maintenance of highly similar clusters of TCRs post-treatment predict response, suggesting ongoing antigen engagement and survival of families of T cells likely recognizing the same antigens. In responders, nivolumab-bound CD8+ T cells are expanded and express GZMK/B. Our data suggest nivolumab drives both maintenance and replacement of previously expanded T cell clones, but only maintenance correlates with response. We hypothesize that maintenance and boosting of a pre-existing response is a key element of anti-PD-1 mode of action.

The rare benign giant cell tumour of bone (GCTB) is defined by an almost unique mutation in the H3.3 family of histone genes H3-3A or H3-3B; however, the same mutation is occasionally found in primary malignant bone tumours which share many features with the benign variant. Moreover, lung metastases can occur despite the absence of malignant histological features in either the primary or metastatic lesions. Herein we investigated the genetic events of 17 GCTBs including benign and malignant variants and the methylation profiles of 122 bone tumour samples including GCTBs. Benign GCTBs possessed few somatic alterations and no other known drivers besides the H3.3 mutation, whereas all malignant tumours harboured at least one additional driver mutation and exhibited genomic features resembling osteosarcomas, including high mutational burden, additional driver event(s), and a high degree of aneuploidy. The H3.3 mutation was found to predate the development of aneuploidy. In contrast to osteosarcomas, malignant H3.3-mutated tumours were enriched for a variety of alterations involving TERT, other than amplification, suggesting telomere dysfunction in the transformation of benign to malignant GCTB. DNA sequencing of the benign metastasising GCTB revealed no additional driver alterations; polyclonal seeding in the lung was identified, implying that the metastatic lesions represent an embolic event. Unsupervised clustering of DNA methylation profiles revealed that malignant H3.3-mutated tumours are distinct from their benign counterpart, and other bone tumours. Differential methylation analysis identified CCND1, encoding cyclin D1, as a plausible cancer driver gene in these tumours because hypermethylation of the CCND1 promoter was specific for GCTBs. We report here the genomic and methylation patterns underlying the rare clinical phenomena of benign metastasising and malignant transformation of GCTB and show how the combination of genomic and epigenomic findings could potentially distinguish benign from malignant GCTBs, thereby predicting aggressive behaviour in challenging diagnostic cases. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland. ispartof: JOURNAL OF PATHOLOGY vol:252 issue:4 pages:433-440 ispartof: location:England status: published

International audience; Although thousands of solid tumors have been sequenced to date, a fundamental under-sampling bias isinherent in current methodologies. This is caused by a tissue sample input of fixed dimensions (e.g., 6 mmbiopsy), which becomes grossly under-powered as tumor volume scales. Here, we demonstrate representative sequencing (Rep-Seq) as a new method to achieve unbiased tumor tissue sampling. Rep-Seq uses fixed residual tumor material, which is homogenized and subjected to next-generation sequencing. Analysis of intratumor tumor mutation burden (TMB) variability shows a high level of misclassification using current single-biopsy methods, with 20% of lung and 52% of bladder tumors having at least one biopsy with high TMB butlow clonal TMB overall. Misclassification rates by contrast are reduced to 2% (lung) and 4% (bladder) when a more representative sampling methodology is used. Rep-Seq offers an improved sampling protocol for tumor profiling, with significant potential for improved clinical utility and more accurate deconvolution of clonal structure.

A 3-tier histopathologic scoring system, the chemotherapy response score (CRS), was previously devised for reporting the histologic response to neoadjuvant chemotherapy in interval debulking surgery specimens of stage IIIc/IV tuboovarian high-grade serous carcinoma. This has been shown to predict the outcome and offer additional information to other methods of assessing the treatment response. In the present study, the reproducibility of this scoring system was assessed by determining the interobserver agreement among reporting pathologists. A total of 5 groups each comprising 3 pathologists with different levels of expertise were selected. The participants underwent an online tutorial on how to apply the CRS system. 40 cases (38 cases in 2 appraiser groups) were scored individually by each of the 15 pathologists. The interobserver reproducibility was calculated using Fleiss' κ, Kendall's coefficient of concordance, and the absolute agreement between (a) individual pathologists within 1 group, (b) with the majority score agreement between all groups, and (c) with all individual scores. The CRS system was found to be highly reproducible among all the pathologists' groups (κ=0.761). The agreement in identifying the group of patients with the best response to chemotherapy was exceptionally high (κ=0.926). We conclude that CRS has a high interobserver reproducibility, especially in identifying the subgroup of patients with the best chemotherapy response, justifying its inclusion in clinical trials and reporting practice.

Recent studies indicate that 40% of chronic myeloid leukemia patients who achieve sustained undetectable BCR-ABL1 transcripts on tyrosine kinase inhibitor therapy remain disease-free after drug discontinuation. In contrast, 60% experience return of detectable disease and have to restart treatment, thus highlighting the need for an improved method of identifying patients with the lowest likelihood of relapse. Here we describe the validation of a personalized DNA-based digital PCR (dPCR) approach for quantifying very low levels of residual disease, which involves the rapid identification of t(9;22) fusion junctions using targeted next-generation sequencing coupled with the use of a dPCR platform. t(9;22) genomic breakpoints were successfully mapped in samples from 32 of 32 patients with early stage disease. Disease quantification by DNA-based dPCR was performed using the Fluidigm BioMark platform on 46 follow-up samples from 6 of the 32 patients, including 36 samples that were in deep molecular remission. dPCR detected persistent disease in 81% of molecular-remission samples, outperforming both RT-dPCR (25%) and DNA-based quantitative PCR (19%). We conclude that dPCR for BCR-ABL1 DNA is the most sensitive available method of residual-disease detection in chronic myeloid leukemia and may prove useful in the management of tyrosine kinase inhibitor withdrawal.

Recent studies indicate that 40% of CML patients on imatinib who achieve complete molecular remission (CMR; equivalent to MR4 or better in current terminology) remain disease-free after drug discontinuation, raising the possibility of an “operational cure”. However, the safe introduction of TKI withdrawal into clinical practice would require a method of evaluating risk of relapse, which is likely to be related to the presence of residual disease undetectable by RT-qPCR. A PCR method based on the use of genomic DNA has been shown to be more sensitive for the detection of residual disease than one that relies on cDNA and may, therefore, help to predict outcome post-withdrawal. However, the former method is arduous requiring a customised patient-specific assay. We describe here a method based on targeted next-generation sequencing (NGS) which allows the rapid identification of BCR-ABL1 breakpoints and the generation of DNA-based qPCR assays. The location of the BCR-ABL1 fusion junction was mapped in disease samples from 32 CML patients using Illumina's MiSeq platform. A custom TruSeq DNA target enrichment system (Illumina) was used to enrich for fragments containing sequences from the BCR and ABL1 genes using probes covering both genes including an additional 50kb of sequence in both 5’ and 3’ directions. All 32 patients’ t(9;22) translocation junctions were successfully mapped using a custom designed bioinformatics algorithm. To date we have designed and validated DNA qPCR assays for 26 of these. On testing consecutive clinical samples from all 26 patients after achievement of MR4 or better (previously defined as CMR; mean 6 samples per patient), the DNA-qPCR assays detected residual disease in 8 out of 26 patients (31%), demonstrating that a DNA-based method is capable of identifying the presence of residual leukaemia in a proportion of patients who would be defined as “disease-free” using conventional RNA-based methodology. One area in which inaccuracy may be introduced into the DNA qPCR technique is in the use of a standard curve generated using serial dilutions of patient’s diagnostic material. We therefore sought to further enhance the sensitivity of a DNA-based approach by optimising this technique for use on a digital quantitative PCR (dqPCR) platform, which provides absolute molecular quantification without the need for standard curve. Using DNA dqPCR on a Fludigm BioMark HD instrument, we measured the level of BCR-ABL1 in 48 samples from 6 CML patients after achievement of a sustained response of MR4 or better. At the time point at which patients were first determined to have achieved at least MR4, all samples tested positive by DNA dqPCR. However, in subsequent samples collected two years following achievement of at least an MR4 (the point of entry into ongoing withdrawal trials, at which point all six patients had achieved MR4.5 or better), we found that patients fell into two distinct categories: 1) those whose disease continued to be detectable by DNA dqPCR (n=3), and 2) those whose disease had fallen to a level that was no longer detectable by DNA dqPCR (n=3). In contrast the conventional BCR-ABL1 DNA qPCR method detected disease in only one of these six patients at this time point, suggesting superior sensitivity of a digital PCR-based screening platform. From this pilot study we conclude that NGS-facilitated DNA dqPCR may be used to stratify patients with RT-qPCR-undetectable disease according to residual disease burden and may therefore prove valuable in the identification of patients for whom TKI therapy could be safely reduced or stopped. Disclosures: Milojkovic: BMS: Honoraria; Pfizer: Honoraria; Ariad: Honoraria; Novartis: Honoraria. Apperley:Novartis: Honoraria, Research Funding; Bristol Myers Squibb: Honoraria; Pfizer, Ariad: Honoraria (not direct from company), Honoraria (not direct from company) Other.

A leaf protein concentrate prepared from the young leaves of the common nettle (Urtica dioica) failed to support normal growth in young rats; supplementation with methionine produced a protein efficiency ratio greater than that of casein. A nettle leaf wholemeal, providing 66% of the dietary protein, supported normal growth in young guinea-pigs, mice and rats. Chicks receiving the whole nettle meal as their sole dietary protein grew at only half the rate of chicks given a standard commercial diet. A nettle meal-induced hypercholesterolaemia in mature guinea-pigs and kidney hypertrophy in mice and guinea-pigs should perhaps be interpreted as a caution against the indiscriminate introduction of nettles into the human diet.