Label-free DNA imaging is highly desirable in biology and medicine to perform live imaging without affecting cell function and to obtain instant histological tissue examination during surgical ...procedures. Here we show a label-free DNA imaging method with stimulated Raman scattering (SRS) microscopy for visualization of the cell nuclei in live animals and intact fresh human tissues with subcellular resolution. Relying on the distinct Raman spectral features of the carbon-hydrogen bonds in DNA, the distribution of DNA is retrieved from the strong background of proteins and lipids by linear decomposition of SRS images at three optimally selected Raman shifts. Based on changes on DNA condensation in the nucleus, we were able to capture chromosome dynamics during cell division both in vitro and in vivo.We tracked mouse skin cell proliferation, induced by drug treatment, through in vivo counting of the mitotic rate. Furthermore, we demonstrated a label-free histology method for human skin cancer diagnosis that provides comparable results to other conventional tissue staining methods such as H&E. Our approach exhibits higher sensitivity than SRS imaging of DNA in the fingerprint spectral region. Compared with spontaneous Raman imaging of DNA, our approach is three orders of magnitude faster, allowing both chromatin dynamic studies and label-free optical histology in real time.
The yield of skin biopsies in the evaluation of intravascular lymphoma (IVL) is largely unknown in Western patients. Most data supporting this test come from Asian populations, in which both ...prevalence and disease presentation seem to differ.
To determine the yield and diagnostic properties of skin biopsy in the evaluation of IVL.
We reviewed skin biopsy pathology reports of 50 patients being evaluated for IVL to calculate the diagnostic yield of this test. An additional 6 patients, who underwent skin biopsies after the diagnosis of IVL was made by other means, were included to calculate the sensitivity and specificity of our index test.
Skin biopsy samples were positive for 5 of the 50 patients being investigated for IVL. Sensitivity was 50% and specificity was 100%.
Only pathology reports containing IVL as an indication for the biopsy were retrieved. This might have excluded patients in whom the disease was considered but was not deemed likely enough to be listed as the indication for the test, inflating our estimative of skin biopsy yield.
A relatively high diagnostic yield was found in the evaluation of IVL among patients with a diverse presentation in a Western hospital.
People with pale skin, red hair, freckles and an inability to tan--the 'red hair/fair skin' phenotype--are at highest risk of developing melanoma, compared to all other pigmentation types. ...Genetically, this phenotype is frequently the product of inactivating polymorphisms in the melanocortin 1 receptor (MC1R) gene. MC1R encodes a cyclic AMP-stimulating G-protein-coupled receptor that controls pigment production. Minimal receptor activity, as in red hair/fair skin polymorphisms, produces the red/yellow pheomelanin pigment, whereas increasing MC1R activity stimulates the production of black/brown eumelanin. Pheomelanin has weak shielding capacity against ultraviolet radiation relative to eumelanin, and has been shown to amplify ultraviolet-A-induced reactive oxygen species. Several observations, however, complicate the assumption that melanoma risk is completely ultraviolet-radiation-dependent. For example, unlike non-melanoma skin cancers, melanoma is not restricted to sun-exposed skin and ultraviolet radiation signature mutations are infrequently oncogenic drivers. Although linkage of melanoma risk to ultraviolet radiation exposure is beyond doubt, ultraviolet-radiation-independent events are likely to have a significant role. Here we introduce a conditional, melanocyte-targeted allele of the most common melanoma oncoprotein, BRAF(V600E), into mice carrying an inactivating mutation in the Mc1r gene (these mice have a phenotype analogous to red hair/fair skin humans). We observed a high incidence of invasive melanomas without providing additional gene aberrations or ultraviolet radiation exposure. To investigate the mechanism of ultraviolet-radiation-independent carcinogenesis, we introduced an albino allele, which ablates all pigment production on the Mc1r(e/e) background. Selective absence of pheomelanin synthesis was protective against melanoma development. In addition, normal Mc1r(e/e) mouse skin was found to have significantly greater oxidative DNA and lipid damage than albino-Mc1r(e/e) mouse skin. These data suggest that the pheomelanin pigment pathway produces ultraviolet-radiation-independent carcinogenic contributions to melanomagenesis by a mechanism of oxidative damage. Although protection from ultraviolet radiation remains important, additional strategies may be required for optimal melanoma prevention.
Summary BRAF mutation is seen in a variety of human neoplasms including cutaneous malignant melanoma, papillary thyroid carcinoma, colorectal carcinoma, non–small cell lung carcinoma, pleomorphic ...xanthoastrocytoma, and others. Currently, there are 2 commercially available monoclonal antibodies for the detection of BRAF V600E mutation; however, a full and practical comparison of their performance in various tumor types on an automated staining platform has not been done. We investigated their sensitivity and specificity in detecting the BRAF V600E mutation in a series of 152 tumors including 31 malignant melanomas, 25 lung carcinomas, 32 gastrointestinal carcinomas, 23 thyroid carcinomas, 35 gliomas, and 6 other malignancies. In this series, the concordance rate between immunohistochemistry (IHC) and mutational analyses was 97% (148/152) for VE1 and 88% (131/149) for anti–B-Raf. The sensitivity and specificity were 98% (60/61) and 97% (88/91) for monoclonal VE1 and 95% (58/61) and 83% (73/88) for anti–B-Raf, respectively. There were 4 cases with discordant IHC and mutational results for monoclonal VE1 in contrast to 18 cases for anti–B-Raf. Our studies showed that IHC with monoclonal VE1 has a better performance compared with anti–B-Raf in an automated staining platform and confirmed that clone VE1 provides excellent sensitivity and specificity for detecting the BRAF V600E mutation in a variety of tumor types in a clinical setting.
Two accepted possible pathways for Merkel cell carcinoma (MCC) pathogenesis include the clonal integration of the Merkel cell polyomavirus (MCPyV) into the neoplastic cells and by UV irradiation. We ...hypothesize that, in UV etiology, the expression of genes associated with epithelial-mesenchymal transition (EMT) would be higher in MCPyV-negative MCCs. We compared RNA expression in 16 MCPyV-negative with that in 14 MCPyV-positive MCCs in 30 patients using NanoString panel of 760 gene targets as an exploratory method. Subsequently, we confirmed the findings with a publicly available RNA sequencing data set. The NanoString method showed that 29 of 760 genes exhibited significant deregulation. Ten genes (CD44, COL6A3, COL11A1, CXCL8, INHBA, MMP1, NID2, SPP1, THBS1, and THY1) were part of the EMT pathway. The expression of CDH1/E-cadherin, a key EMT gene, and TWIST1, regulator gene of EMT, was higher in MCPyV-negative tumors. To further investigate the expression of EMT genes in MCPyV-negative MCCs, we analyzed publicly available RNA sequencing data of 111 primary MCCs. Differential expression and gene set enrichment analysis of 35 MCPyV-negative versus 76 MCPyV-positive MCCs demonstrated significantly higher expression of EMT-related genes and associated pathways such as Notch signaling, TGF-β signaling, and Hedgehog signaling, and UV response pathway in MCPyV-negative MCCs. The significance of the EMT pathway in MCPyV-negative MCCs was confirmed independently by a coexpression module analysis. One of the modules (M3) was specifically activated in MCPyV-negative MCCs and showed significant enrichment for genes involved in EMT. A network analysis of module M3 revealed that CDH1/E-cadherin was among the most connected genes (hubs). E-cadherin and LEF1 immunostains demonstrated significantly more frequent expression in MCPvV-negative versus MCPyV-positive tumors (P < .0001). In summary, our study showed that the expression of EMT-associated genes is higher in MCPyV-negative MCC. Because EMT-related proteins can be targeted, the identification of EMT pathways in MCPyV-negative MCCs is of potential therapeutic relevance.
SOX10 Sy, Albert L; Hoang, Mai P
Journal of clinical pathology,
10/2023, Volume:
76, Issue:
10
Journal Article
Peer reviewed
The SOX gene encodes for transcription factors that are involved in embryogenesis and cell differentiation. Specifically, SOX10 aids with neural crest shuttling and development. In diagnostic ...histopathology, Sox10 immunostain is a helpful ancillary test due to its high sensitivity for melanocytic and peripheral nerve sheath neoplasms, and its role in distinguishing triple-negative breast carcinomas from gynaecological carcinoma, cutaneous adnexal neoplasms and salivary glands neoplasms from histological mimics.
The association of immune markers and clinicopathologic features and patient outcome has not been extensively studied in Merkel cell carcinoma (MCC). We correlated tumoral PD-L1 and IDO1 expression, ...and intratumoral CD8+ and FoxP3+ lymphocytes count with clinicopathologic variables, Merkel cell polyomavirus (MCPyV) status, and patient outcomes in a series of 132 MCC. By univariate analyses, tumoral PD-L1 expression >1% and combined tumoral PD-L1 >1% and high intratumoral FoxP3+ lymphocyte count correlated with improved overall survival (OS) (p = 0.016, 0.0072), MCC-specific survival (MSS) (p = 0.019, 0.017), and progression-free survival (PFS) (p = 0.043, 0.004, respectively). High intratumoral CD8+ and FoxP3+ lymphocyte count correlated with longer MSS (p = 0.036) and improved PFS (p = 0.047), respectively. Ulceration correlated with worse OS and worse MSS. Age, male gender, and higher stage (3 and 4) significantly correlated with worse survival. MCPyV positivity correlated with immune response. By multivariate analyses, only ulceration and age remained as independent predictors of worse OS; gender and stage remained for shorter PFS. Tumoral PD-L1 expression and increased density of intratumoral CD8+ lymphocytes and FoxP+ lymphocytes may represent favorable prognosticators in a subset of MCCs. Tumoral PD-L1 expression correlated with intratumoral CD8+ and FoxP3+ lymphocytes, which is supportive of an adaptive immune response.
Brain metastases are associated with a dismal prognosis. Whether brain metastases harbor distinct genetic alterations beyond those observed in primary tumors is unknown. We performed whole-exome ...sequencing of 86 matched brain metastases, primary tumors, and normal tissue. In all clonally related cancer samples, we observed branched evolution, where all metastatic and primary sites shared a common ancestor yet continued to evolve independently. In 53% of cases, we found potentially clinically informative alterations in the brain metastases not detected in the matched primary-tumor sample. In contrast, spatially and temporally separated brain metastasis sites were genetically homogenous. Distal extracranial and regional lymph node metastases were highly divergent from brain metastases. We detected alterations associated with sensitivity to PI3K/AKT/mTOR, CDK, and HER2/EGFR inhibitors in the brain metastases. Genomic analysis of brain metastases provides an opportunity to identify potentially clinically informative alterations not detected in clinically sampled primary tumors, regional lymph nodes, or extracranial metastases.
Decisions for individualized therapies in patients with brain metastasis are often made from primary-tumor biopsies. We demonstrate that clinically actionable alterations present in brain metastases are frequently not detected in primary biopsies, suggesting that sequencing of primary biopsies alone may miss a substantial number of opportunities for targeted therapy.
Since molecular assays are not accessible to all uveal melanoma patients, we aim to identify cost-effective prognostic tool in risk stratification using machine learning models based on routine ...histologic and clinical variables.
We identified important prognostic parameters in a discovery cohort of 164 enucleated primary uveal melanomas from 164 patients without prior therapies. We then validated the prognostic prediction of top important parameters identified in the discovery cohort using 80 uveal melanomas from the Tumor Cancer Genome Atlas database with available gene expression prognostic signature (GEPS). The performance of three different survival analysis models (Cox proportional hazards (CPH), random survival forest (RSF), and survival gradient boosting (SGB)) was compared against GEPS using receiver operating curves (ROC).
In all three selection methods, BAP1 status, nucleoli size, age, mitotic rate per 1 mm
, and ciliary body infiltration were identified as significant overall survival (OS) predictors; and BAP1 status, nucleoli size, largest basal tumor diameter, tumor-infiltrating lymphocyte density, and tumor-associated macrophage density were identified as significant progression-free survival (PFS) predictors. ROC plots for the median survival time point showed that significant parameters in SGB studied model can predict OS better than GEPS. For PFS, SGB model performed similarly to GEPS. The time-dependent area under the curve (AUC) showed SGB model performing better than GEPS in predicting OS and metastatic risk.
Our study shows that routine histologic and clinical variables are adequate for patient risk stratification in comparison with not readily accessible GEPS.
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