Human tumor genomics and zebrafish modeling identify SPRED1 loss as a driver of mucosal melanoma.

Melanomas originating from mucosal surfaces have low mutation burden, genomic instability, and poor prognosis. To identify potential driver genes, we sequenced hundreds of cancer-related genes in 43 human mucosal melanomas, cataloging point mutations, amplifications, and deletions. The SPRED1 gene, which encodes a negative regulator of mitogen-activated protein kinase (MAPK) signaling, was inactivated in 37% of the tumors. Four distinct genotypes were associated with SPRED1 loss. Using a rapid, tissue-specific CRISPR technique to model these genotypes in zebrafish, we found that SPRED1 functions as a tumor suppressor, particularly in the context of KIT mutations. SPRED1 knockdown caused MAPK activation, increased cell proliferation, and conferred resistance to drugs inhibiting KIT tyrosine kinase activity. These findings provide a rationale for MAPK inhibition in SPRED1-deficient melanomas and introduce a zebrafish modeling approach that can be used more generally to dissect genetic interactions in cancer.

Drug induced pseudolymphoma.

Atypical lymphocytic infiltrates of the skin comprise a broad spectrum of entities ranging from benign infiltrates to those that are malignant. Many of these infiltrates are in fact reactive lymphomatoid ones related to drug therapy falling under the general category of drug associated pseudolymphoma. Within this nosologic umbrella are nodular and diffuse infiltrates resembling low grade T and B cell lymphoma consistent with lymphocytoma cutis, drug associated reversible T cell dyscrasias which draw a strong morphologic and phenotypic parallel with mycosis fungoides and the various pre-lymphomatous T cell dyscrasias, and angiocentric CD30 positive infiltrates mirroring lymphomatoid papulosis. The implicated drug classes are quite varied and include antidepressants, antihistamines, calcium channel blockers, statins, anticonvulsants, and various biologic drugs. The drugs from these various drug classes exert certain effects on lymphoid function including evoking overzealous responses to other antigenic stimuli. As the adverse effect on lymphocyte function may be cumulative over years and or reflect the interplay of other drugs, a temporal association may not exist between the onset of the rash/lesion and the initiation of the drug. In certain lymphomatoid reactions however such as DRESS syndrome the drug may function as both an antigen as well as an immune dysregulating agent. It is critical that the pathologist works carefully with the clinician in the evaluation of all atypical cutaneous lymphoid infiltrates where the distinction between pseudolymphoma versus lymphoma cannot be reliably made based on pathologic analysis alone.

Radiation-associated angiosarcoma in the setting of breast cancer mimicking radiation dermatitis: A diagnostic pitfall.

BACKGROUND: We have encountered cases of post-radiation angiosarcoma (PRAS) histologically mimicking radiation dermatitis. METHODS: Cases of PRAS from institutional/consultation archives from 2006 to 2016 were reviewed. For inclusion, tumors had to have inapparent/subtle tumor at low magnification and scattered individual tumor cells resembling radiation fibroblasts. Prior ancillary studies were reviewed, with additional immunostains performed as needed. RESULTS: 10 cases met criteria. All occurred in women treated for breast cancer (mean age 71 years). All had similar findings: in particular, scattered single atypical cells with pleomorphic nuclei associated with microscopic hemorrhage. They also had narrow, slightly wavy "worm-like" vascular channels lined by atypical endothelial cells that lacked architectural complexity. Four cases showed focal areas of more conventional angiosarcoma. One case was an excision of a large mass that showed the "radiation dermatitis-like" pattern radiating out from the central mass. All were positive for vascular markers (CD31, CD34, and/or ERG) and MYC. MYC amplification was demonstrated by FISH in both cases tested. In 3 of 3 cases with available re-excision specimens, more obvious angiosarcoma was seen. CONCLUSIONS: PRAS can be very subtle and histologically mimic radiation dermatitis. Careful attention to histologic features and ancillary tests allow accurate diagnosis in subtle PRAS.

T-Cell-Mediated Inflammatory Myopathies in HIV-Positive Individuals: A Histologic Study of 19 Cases.

T cell-mediated inflammatory myopathies (polymyositis [PM] and inclusion body myositis [IBM]) sometimes arise in conjunction with HIV infection; however, it is not understood whether PM and IBM arising in the context of HIV (HIV-PM and HV-IBM) differ from PM and IBM arising sporadically in HIV-negative individuals (sPM and sIBM). Here, we report the largest series of T cell-mediated inflammatory myopathies from HIV-infected patients (19 biopsies from 15 subjects); 5 cases were pathologically classified as PM (HIV-PM) and 14 as IBM (HIV-IBM). As with sporadic cases, quantitative immunohistochemistry for LC3, p62, and TDP-43 showed significantly greater percentage of stained fibers (% FS) in HIV-IBM compared to HIV-PM samples; however, there was no significant difference in % FS for any of the three markers between HIV-associated and sporadic cases. Despite histologic similarities between HIV-IBM and sIBM but in concordance with prior case reports, patients with HIV-IBM were significantly younger at diagnosis than patients with sIBM; in contrast, the mean age of HIV-PM and sPM patients was not significantly different. In summary, HIV-PM and HIV-IBM are morphologically similar to sPM and sIBM; thus, it remains unclear why patients with HIV-IBM, in contrast to patients with sIBM, sometimes show clinical improvement in response to immunosuppressive therapy.

Comparative utility of LC3, p62 and TDP-43 immunohistochemistry in differentiation of inclusion body myositis from polymyositis and related inflammatory myopathies.

BACKGROUND: Inclusion body myositis (IBM) is a slowly progressive inflammatory myopathy of the elderly that does not show significant clinical improvement in response to steroid therapy. Distinguishing IBM from polymyositis (PM) is clinically important since PM is steroid-responsive; however, the two conditions can show substantial histologic overlap. RESULTS: We performed quantitative immunohistochemistry for (1) autophagic markers LC3 and p62 and (2) protein aggregation marker TDP-43 in 53 subjects with pathologically diagnosed PM, IBM, and two intermediate T cell-mediated inflammatory myopathies (polymyositis with COX-negative fibers and possible IBM). The percentage of stained fibers was significantly higher in IBM than PM for all three immunostains, but the markers varied in sensitivity and specificity. In particular, both LC3 and p62 were sensitive markers of IBM, but the tradeoff between sensitivity and specificity was smaller (and diagnostic utility thus greater) for LC3 than for p62. In contrast, TDP-43 immunopositivity was highly specific for IBM, but the sensitivity of this test was low, with definitive staining present in just 67% of IBM cases. CONCLUSIONS: To differentiate IBM from PM, we thus recommend using a panel of LC3 and TDP-43 antibodies: the finding of <14% LC3-positive fibers helps exclude IBM, while >7% of TDP-43-positive fibers strongly supports a diagnosis of IBM. These data provide support for the hypothesis that disruption of autophagy and protein aggregation contribute to IBM pathogenesis.

LC3 and p62 as diagnostic markers of drug-induced autophagic vacuolar cardiomyopathy: a study of 3 cases.

Autophagic vacuolar cardiomyopathy is an underrecognized, but potentially fatal, complication of treatment with chloroquine (CQ) and its derivative hydroxychloroquine (HCQ), which are used as therapy for malaria and common connective tissue disorders. Currently, the diagnosis of autophagic vacuolar cardiomyopathy is established through an endomyocardial biopsy and requires electron microscopy, which is not widely available and has a significant potential for sampling error. Recently, we have reported that immunohistochemistry for autophagic markers LC3 and p62 can replace electron microscopy in the diagnosis of HCQ-induced and colchicine-induced autophagic vacuolar skeletal myopathies. In the current study, we use 3 cases of CQ-induced or HCQ-induced cardiomyopathy and 1 HCQ-treated control case to show that the same two markers can be used to diagnose autophagic vacuolar cardiomyopathies by light microscopy. CQ-induced or HCQ-induced autophagic vacuolar cardiomyopathy is not universally fatal, but successful treatment requires early detection. By lowering the barriers to diagnosis, the application of these immunohistochemical markers will decrease the number of misdiagnosed patients, thus increasing the likelihood of favorable clinical outcomes.

Clinical utility of LC3 and p62 immunohistochemistry in diagnosis of drug-induced autophagic vacuolar myopathies: a case-control study.

BACKGROUND: Some patients treated with chloroquine, hydroxychloroquine, or colchicine develop autophagic vacuolar myopathy, the diagnosis of which currently requires electron microscopy. The goal of the current study was to develop an immunohistochemical diagnostic marker for this pathologic entity. METHODOLOGY: Microtubule-associated protein light chain 3 (LC3) has emerged as a robust marker of autophagosomes. LC3 binds p62/SQSTM1, an adapter protein that is selectively degraded via autophagy. In this study, we evaluated the utility of immunohistochemical stains for LC3 and p62 as diagnostic markers of drug-induced autophagic vacuolar myopathy. The staining was performed on archival muscle biopsy material, with subject assignment to normal control, drug-treated control, and autophagic myopathy groups based on history of drug use and morphologic criteria. PRINCIPAL FINDINGS: In all drug-treated subjects, but not in normal controls, LC3 and p62 showed punctate staining characteristic of autophagosome buildup. In the autophagic myopathy subjects, puncta were coarser and tended to coalesce into linear structures aligned with the longitudinal axis of the fiber, often in the vicinity of vacuoles. The percentage of LC3- and p62-positive fibers was significantly higher in the autophagic myopathy group compared to either the normal control (p<0.001) or the drug-treated control group (p<0.05). With the diagnostic threshold set between 8% and 15% positive fibers (depending on the desired level of sensitivity and specificity), immunohistochemical staining for either LC3 or p62 could be used to identify subjects with autophagic vacuolar myopathy within the drug-treated subject group (p ≤ 0.001). SIGNIFICANCE: Immunohistochemistry for LC3 and p62 can facilitate tissue-based diagnosis of drug-induced autophagic vacuolar myopathies. By limiting the need for electron microscopy (a time consuming and costly technique with high specificity, but low sensitivity), clinical use of these markers will improve the speed and accuracy of diagnosis, resulting in significantly improved clinical care.