Epigenetic reprogramming shapes the cellular landscape of schwannoma.

Mechanisms specifying cancer cell states and response to therapy are incompletely understood. Here we show epigenetic reprogramming shapes the cellular landscape of schwannomas, the most common tumors of the peripheral nervous system. We find schwannomas are comprised of 2 molecular groups that are distinguished by activation of neural crest or nerve injury pathways that specify tumor cell states and the architecture of the tumor immune microenvironment. Moreover, we find radiotherapy is sufficient for interconversion of neural crest schwannomas to immune-enriched schwannomas through epigenetic and metabolic reprogramming. To define mechanisms underlying schwannoma groups, we develop a technique for simultaneous interrogation of chromatin accessibility and gene expression coupled with genetic and therapeutic perturbations in single-nuclei. Our results elucidate a framework for understanding epigenetic drivers of tumor evolution and establish a paradigm of epigenetic and metabolic reprograming of cancer cells that shapes the immune microenvironment in response to radiotherapy.

LZTS2: A novel and independent prognostic biomarker for clear cell renal cell carcinoma.

PURPOSE: Leucine zipper tumor suppressor 2 (LZTS2), a putative tumor suppressor gene, has been demonstrated to be a negative regulator of microtubule severing during cytokinesis and a negative regulator of the Wnt signaling pathway. In a genetically modified mouse model, deletion of Lzts2 altered normal ureteric bud branching morphogenesis and caused cystogenesis in mice. Cyst-lining cells demonstrated atypical features, closely resembling those observed in mouse models of human clear cell renal cell carcinoma (ccRCC), which could represent a preneoplastic lesion. These findings suggest that LZTS2 may play a role in ccRCC tumorigenesis. The aim of this study was to establish an association between LZTS2 differential expression and clinicopathological features of ccRCC and to investigate its prognostic value as well as the underlying mechanisms in ccRCC progression. MATERIALS AND METHODS: Gene expression data by RNA-sequencing for cohorts of 510 ccRCC cases with clinical outcome data were extracted from The Cancer Genome Atlas (TCGA) using cBioPortal. Chi-square test of independence, Kaplan-Meier curves, and Cox regression models were used to investigate the possible relationship between LZTS2 mRNA expression levels and clinicopathological parameters as well as patient survival to establish its prognostic values. To examine its cellular localization, we performed LZTS2 antibody staining and scored the expression levels in a pilot study on a tissue microarray (TMA) containing 31 clear cell RCCs, 32 chromophobe RCCs, 12 papillary RCCs, and 20 adjacent benign renal tissue, as well as placental tissue diagnosed between 2001 and 2007 at the University of California San Francisco Medical Center. Staining was subsequently repeated in 15 ccRCCs on whole section slides to confirm the results. RESULTS: Our analysis of TCGA data demonstrated that LZTS2 expression levels were associated with tumor grade (p = 0.005), T stage (p < 0.001), metastasis status (p < 0.001), and overall clinical stage (p < 0.001). High level of expression was correlated with worse overall survival (p < 0.001), disease-specific survival (p < 0.001), progression-free survival (p < 0.001), and disease-free survival (p < 0.001) compared to low level of expression. Multivariate Cox regression analysis revealed that high LZTS2 expression was an independent poor prognostic factor for overall survival (HR = 2.083, p < 0.001), disease-specific survival (HR = 2.298, p < 0.001), and progression-free survival (HR = 1.896, p < 0.001) in patients with ccRCC. A few known driver mutations in ccRCC pathogenesis, including BAP1, NF2, and RELN, were enriched in LZTS2 high expression tumors. In particular, LZTS2 expression level could be a biomarker for risk stratification of the prognosis of BAP1-mutated ccRCCs. Immunohistochemical staining with anti-LZTS2 antibody was performed to examine its cellular localization in ccRCC and demonstrated centrosomal and acentrosomal distribution in tumors of various Fuhrman nuclear grades. Furthermore, high LZTS2 cytoplasmic expression was associated with centrosomal amplification (p = 0.030) in this small-scale study. CONCLUSIONS: The current study established an independent prognostic value of LZTS2 expression in ccRCC and explored the molecular mechanisms of LZTS2 in predicting the prognosis of ccRCC. Further studies are needed to validate our analysis and to provide a precise understanding of the function of LZTS2 in ccRCC.

BRAFV600E induces reversible mitotic arrest in human melanocytes via microrna-mediated suppression of AURKB.

Benign melanocytic nevi frequently emerge when an acquired BRAFV600E mutation triggers unchecked proliferation and subsequent arrest in melanocytes. Recent observations have challenged the role of oncogene-induced senescence in melanocytic nevus formation, necessitating investigations into alternative mechanisms for the establishment and maintenance of proliferation arrest in nevi. We compared the transcriptomes of melanocytes from healthy human skin, nevi, and melanomas arising from nevi and identified a set of microRNAs as highly expressed nevus-enriched transcripts. Two of these microRNAs-MIR211-5p and MIR328-3p-induced mitotic failure, genome duplication, and proliferation arrest in human melanocytes through convergent targeting of AURKB. We demonstrate that BRAFV600E induces a similar proliferation arrest in primary human melanocytes that is both reversible and conditional. Specifically, BRAFV600E expression stimulates either arrest or proliferation depending on the differentiation state of the melanocyte. We report genome duplication in human melanocytic nevi, reciprocal expression of AURKB and microRNAs in nevi and melanomas, and rescue of arrested human nevus cells with AURKB expression. Taken together, our data describe an alternative molecular mechanism for melanocytic nevus formation that is congruent with both experimental and clinical observations.

Lots of people have small dark patches on their skin known as moles. Most moles form when individual cells known as melanocytes in the skin acquire a specific genetic mutation in a gene called BRAF. This mutation causes the cells to divide rapidly to form the mole. After a while, most moles stop growing and remain harmless for the rest of a person's life. Melanoma is a type of skin cancer that develops from damaged melanocytes. The same mutation in BRAF that is found in moles is also present in half of all cases of melanoma. Unlike in moles, the melanoma-causing mutation makes the melanocytes divide rapidly to form a tumor that keeps on growing indefinitely. It remains unclear why the same genetic mutation in the BRAF gene has such different consequences in moles and melanomas. To address this question, McNeal et al. used genetic approaches to study melanocytes from moles and melanomas. The experiments identified some molecules known as microRNAs that are present at higher levels in moles than in melanomas. Increasing the levels of two of these microRNAs in melanocytes from human skin stopped the cells from growing and dividing by inhibiting a gene called AURKB. This suggested that these microRNAs are responsible for halting the growth of moles. Introducing the mutated form of BRAF into melanocytes also stopped cells from growing and dividing by inhibiting AURKB. However, changing the environment surrounding the cells reversed this effect and allowed the melanocytes to resume dividing. In this way the mutated form of BRAF acts like a switch that allows melanocytes in skin cancers to start growing again under certain conditions. Further experiments found that a drug called barasertib is able to inhibit the growth of melanoma cells with the mutant form of BRAF. Future work will investigate whether it is possible to use this drug and other tools to stop skin cancer tumors from growing, and possibly even prevent skin tumors from forming in the first place.

Human melanocyte development and melanoma dedifferentiation at single-cell resolution.

In humans, epidermal melanocytes are responsible for skin pigmentation, defence against ultraviolet radiation and the deadliest common skin cancer, melanoma. Although there is substantial overlap in melanocyte development pathways between different model organisms, species-dependent differences are frequent and the conservation of these processes in human skin remains unresolved. Here, we used a single-cell enrichment and RNA-sequencing pipeline to study human epidermal melanocytes directly from the skin, capturing transcriptomes across different anatomical sites, developmental age, sexes and multiple skin tones. We uncovered subpopulations of melanocytes that exhibit anatomical site-specific enrichment that occurs during gestation and persists through adulthood. The transcriptional signature of the volar-enriched subpopulation is retained in acral melanomas. Furthermore, we identified human melanocyte differentiation transcriptional programs that are distinct from gene signatures generated from model systems. Finally, we used these programs to define patterns of dedifferentiation that are predictive of melanoma prognosis and response to immune checkpoint inhibitor therapy.

Primary Cilia Are Preserved in Cellular Blue and Atypical Blue Nevi and Lost in Blue Nevus-like Melanoma.

Distinguishing cellular blue nevi (CBNs) and atypical CBNs from blue nevus-like melanoma (BNLM) can be diagnostically challenging. Immunohistochemistry may inform the diagnosis in a subset of cases but is not always diagnostic. Further, ancillary molecular testing is expensive and often requires significant tissue to complete. Primary cilia are cell-surface organelles with roles in signal transduction pathways and have been shown to be preserved in conventional melanocytic nevi but lost in melanoma. Immunofluorescence staining of primary cilia can be performed using a single standard-thickness formalin-fixed paraffin-embedded tissue section and has a turnaround time similar to immunohistochemistry. The percentage of tumoral melanocytes retaining a primary cilium is quantified and reported as the ciliation index. In the current study, we explored the utility of the ciliation index in a series of 31 blue nevus-like lesions, including CBNs (12), atypical CBNs (15), and BNLM (4). The average ciliation index for the CBNs was 59±18%, with a median of 60 (range: 28 to 87). The average ciliation index for atypical CBNs was 59±23, with a median of 59 (range: 20 to 93). The average ciliation index for BNLM was 4±3, with a median of 3 (range: 1 to 8). There was no significant difference in ciliation index between the CBN and atypical CBN categories. There was a significant difference between CBN and BNLM and between atypical CBNs and BNLM (P<0.001 for each). Here, we show that ciliation index is a quantitative diagnostic tool useful in the setting of blue nevus-like neoplasms, with benefits including cost and time efficiency.

BAP1-inactivated melanocytic tumors show prominent centrosome amplification and associated loss of primary cilia.

BACKGROUND: BRCA1-associated protein (BAP1) is a tumor suppressor whose loss is associated with various malignancies. The primary cilium is an organelle involved in signal transduction and cell cycle progression. Primary cilia have been shown to be absent in melanoma but retained to some extent in melanocytic nevi, and the severity of dysplasia influences the degree of cilia loss. Additionally, studies have revealed roles for BAP1 in centrosome and mitotic spindle formation. Because the primary cilium is nucleated on the mother centriole, we examined the connection between the presence of primary cilia and the formation of centrosomes in BAP1-inactivated melanocytic tumors (BIMTs). METHODS: We evaluated the cilia and centrosomes in 11 BIMTs and five conventional melanocytic nevi using immunofluorescence staining of acetylated alpha-tubulin and gamma-tubulin. RESULTS: We found that, compared to nevi, BIMTs show loss of primary cilia and amplification of centrosomes. Occasional nevi also showed increased centrioles; however, these foci of amplification were more likely to be ciliated than those in BIMTs. CONCLUSIONS: Although centrosome amplification does not absolutely correlate with loss of primary cilia in melanocytic neoplasms, absence of BAP1 exacerbates the phenotype. Moreover, aberrant centrosome and cilia formation are likely critical in the pathogenesis of other BAP1-inactivated tumors.

Meningioma cells express primary cilia but do not transduce ciliary Hedgehog signals.

Meningiomas are the most common primary intracranial tumors, but treatment options for meningioma patients are limited due to incomplete understanding of tumor biology. A small percentage of meningiomas harbor somatic variants in the Hedgehog pathway, a conserved gene expression program that is essential for development and adult stem cell homeostasis. Hedgehog signals are transduced through primary cilia, and misactivation of the Hedgehog pathway is known to underlie cancer. Nevertheless, the mechanisms of Hedgehog signaling in meningioma are unknown. Here, we investigate mechanisms of ciliary Hedgehog signaling in meningioma using tissue microarrays containing 154 human meningioma samples, NanoString transcriptional profiling, primary meningioma cells, pharmacology, and CRISPR interference. Our results reveal that meningiomas of all grades can express primary cilia, but that cilia are less prevalent among anaplastic tumors. Moreover, we find that expression of Smoothened alleles that are oncogenic in other contexts fail to activate the Hedgehog transcriptional program or promote proliferation in primary meningioma cells. These data reveal that meningiomas can express the subcellular structure necessary for canonical Hedgehog signaling, but suggest that they do not transduce ciliary Hedgehog signals.

Clinical Implications of Primary Cilia in Skin Cancer.

The primary cilium is a cell surface organelle that is an important component of cellular biology. While it was once believed to be a vestigial structure without biologic function, it is now known to have essential roles in critical cellular signaling pathways such as Hedgehog (HH) and Wnt. The HH and Wnt pathways are involved in pathogenesis of basal cell carcinoma and melanoma, respectively, and this knowledge is now beginning to inform therapeutic and diagnostic options for patients. The purpose of this review is to familiarize clinicians with primary cilia biology and how this complex cellular organelle has started to translate into clinical care.

Ciliation Index Is a Useful Diagnostic Tool in Challenging Spitzoid Melanocytic Neoplasms.

The loss of primary cilia on melanocytes is a useful biomarker for the distinction of melanoma from conventional melanocytic nevi. It is unknown whether ciliation status is beneficial for diagnosing spitzoid tumors-a subclass of melanomas that present inherently ambiguous histology and are challenging to classify. We evaluated the Ciliation Index (CI) in 68 cases of spitzoid tumors ranging from Spitz nevi and atypical Spitz tumors to spitzoid melanoma. We found a significant decrease in CI within the spitzoid melanoma group when compared with either the Spitz nevi or atypical Spitz tumors groups. In addition, we used a machine-learning-based algorithm to determine the value of CI when considered in combination with other histopathologic and molecular features commonly used for diagnosis. We found that a low CI was consistently ranked as a top predictive feature in the diagnosis of malignancy. Predictive models trained on only the top four predictive features (CI, asymmetry, hyperchromatism, and cytologic atypia) outperformed standard histologic assessment in an independent validation cohort of 56 additional cases. The results provide an alternative approach to evaluate diagnostically challenging melanocytic lesions, and further support the use of CI as an ancillary diagnostic test.

Use of the Ciliation Index to Distinguish Invasive Melanoma From Associated Conventional Melanocytic Nevi.

Our understanding of melanoma precursors and progression to melanoma has developed as a result of advances in the field of molecular diagnostics. We now better understand the potential for genetic heterogeneity within a single lesion. Combined tumors can pose a diagnostic challenge when deciding the line between benign and malignant, which in turn has direct implications for patient management. Primary cilia (PC) are ubiquitous sensory organelles that have essential functions in cellular proliferation, differentiation, and development. The ciliation index (percentage of ciliated melanocytes) has been shown to reliably differentiate melanoma, which fail to ciliate, from melanocytic nevi, which retain PC. We therefore analyzed the potential for using the ciliation index to differentiate benign and malignant components in combined melanocytic lesions. We collected patient samples (n = 10) of unequivocal combined lesions with both melanoma and associated nevus components. Melanocytes were highlighted with SOX10 and costained with gamma-Tubulin and acetylated alpha-Tubulin to highlight the basal body and cilium, respectively. The number of melanocytes retaining cilia under high-power microscopy was examined. The melanoma component had average of 4% ciliation (SD: 7%), whereas the associated nevus component was significantly higher with 59% ciliation (SD: 17%). These data show that PC may be a reliable means of distinguishing benign from malignant components within a single tumor. The ciliation index may be a helpful tool in distinguishing challenging cases of combined lesions of melanoma in situ with a dermal nevus component from invasive melanoma, thus promoting improved staging and clinical management.

MicroRNA Ratios Distinguish Melanomas from Nevi.

The use of microRNAs as biomarkers has been proposed for many diseases, including the diagnosis of melanoma. Although hundreds of microRNAs have been identified as differentially expressed in melanomas as compared to benign melanocytic lesions, a limited consensus has been achieved across studies, constraining the effective use of these potentially useful markers. In this study, we applied a machine learning-based pipeline to a dataset consisting of genetic features, clinical features, and next-generation microRNA sequencing from micro-dissected formalin-fixed paraffin embedded melanomas and their adjacent benign precursor nevi. We identified patient age and tumor cellularity as variables that frequently confound the measured expression of potentially diagnostic microRNAs. By employing the ratios of microRNAs that were either enriched or depleted in melanoma compared to the nevi as a normalization strategy, we developed a model that classified all the available published cohorts with an area under the receiver operating characteristic curve of 0.98. External validation on an independent cohort classified lesions with 81% sensitivity and 88% specificity and was uninfluenced by the tumor content of the sample or patient age.

Suppression of Exosomal PD-L1 Induces Systemic Anti-tumor Immunity and Memory.

PD-L1 on the surface of tumor cells binds its receptor PD-1 on effector T cells, thereby suppressing their activity. Antibody blockade of PD-L1 can activate an anti-tumor immune response leading to durable remissions in a subset of cancer patients. Here, we describe an alternative mechanism of PD-L1 activity involving its secretion in tumor-derived exosomes. Removal of exosomal PD-L1 inhibits tumor growth, even in models resistant to anti-PD-L1 antibodies. Exosomal PD-L1 from the tumor suppresses T cell activation in the draining lymph node. Systemically introduced exosomal PD-L1 rescues growth of tumors unable to secrete their own. Exposure to exosomal PD-L1-deficient tumor cells suppresses growth of wild-type tumor cells injected at a distant site, simultaneously or months later. Anti-PD-L1 antibodies work additively, not redundantly, with exosomal PD-L1 blockade to suppress tumor growth. Together, these findings show that exosomal PD-L1 represents an unexplored therapeutic target, which could overcome resistance to current antibody approaches.

Depletion of primary cilium in acral melanoma.

BACKGROUND: A eukaryotic cell's primary cilium (PC) is critical for cell signaling, migration and homeostasis. Primary cilium dysfunction has been demonstrated in several malignancies, but whether primary cilia loss occurs in acral melanoma has remained unknown. To address this, we examined the ciliation index (% melanocytes containing a PC) of patient-derived, biopsy-proven acral melanoma and compared these to benign acral nevi. METHODS: We generated a pilot initiative study that included six acral melanomas and seven acral nevi derived from the foot. Using fluorescent immunohistochemistry, we calculated ciliation indexes of Sox10+ melanocytes. RESULTS: Average ciliation index for acral nevi was 74.0% (SE of the mean [SEM] 3.3%) vs 9.3% for acral melanoma (SEM 5.7%), finding a statistically significant difference between the groups (P-value <.001, two tailed t test). CONCLUSION: The data show a significant loss of primary cilia in malignant acral melanoma vs benign acral nevi, suggesting that cilia may play an important role during acral melanoma formation. Our data, which should be validated by a larger study with longer follow-up period, suggest that examining ciliation index may be a useful diagnostic test when distinguishing benign acral nevi from melanoma.

Bi-allelic Loss of CDKN2A Initiates Melanoma Invasion via BRN2 Activation.

Loss of the CDKN2A tumor suppressor is associated with melanoma metastasis, but the mechanisms connecting the phenomena are unknown. Using CRISPR-Cas9 to engineer a cellular model of melanoma initiation from primary human melanocytes, we discovered that a lineage-restricted transcription factor, BRN2, is downstream of CDKN2A and directly regulated by E2F1. In a cohort of melanocytic tumors that capture distinct progression stages, we observed that CDKN2A loss coincides with both the onset of invasive behavior and increased BRN2 expression. Loss of the CDKN2A protein product p16INK4A permitted metastatic dissemination of human melanoma lines in mice, a phenotype rescued by inhibition of BRN2. These results demonstrate a mechanism by which CDKN2A suppresses the initiation of melanoma invasion through inhibition of BRN2.

Combined activation of MAP kinase pathway and ß-catenin signaling cause deep penetrating nevi.

Deep penetrating nevus (DPN) is characterized by enlarged, pigmented melanocytes that extend through the dermis. DPN can be difficult to distinguish from melanoma but rarely displays aggressive biological behavior. Here, we identify a combination of mutations of the ß-catenin and mitogen-activated protein kinase pathways as characteristic of DPN. Mutations of the ß-catenin pathway change the phenotype of a common nevus with BRAF mutation into that of DPN, with increased pigmentation, cell volume and nuclear cyclin D1 levels. Our results suggest that constitutive ß-catenin pathway activation promotes tumorigenesis by overriding dependencies on the microenvironment that constrain proliferation of common nevi. In melanoma that arose from DPN we find additional oncogenic alterations. We identify DPN as an intermediate stage in the step-wise progression from nevus to melanoma. In summary, we delineate specific genetic alterations and their sequential order, information that can assist in the diagnostic classification and grading of these distinctive neoplasms.Deep penetrating nevi (DPN) are unusual melanocytic neoplasms with unknown genetic drivers. Here the authors show that majority of DPN harbor activating mutations in the ß-catenin and the MAP-kinase pathways; this characteristic can help in the classification and grading of these distinctive neoplasms.

Molecular Melanoma Diagnosis Update: Gene Fusion, Genomic Hybridization, and Massively Parallel Short-Read Sequencing.

Molecular evaluation of melanocytic tumors can be diagnostically useful to confirm malignancy or benignancy. Molecular tools are ancillary and supplemental to histopathologic evaluation and do not replace conventional microscopy. Immunohistochemistry, fluorescence in situ hybridization, array comparative genomic hybridization, and massively parallel short-read sequencing, often referred to as next-generation sequencing, each provide varied (and often incomplete) additional information, and careful planning is necessary if tissue is limited.

Loss of primary cilia correlates with cytologic severity in dysplastic melanocytic nevi.

BACKGROUND: Primary cilia are ubiquitously present in cell surface organelles with essential functions in cellular proliferation, differentiation and development. We have previously shown that cilia in melanoma in situ, invasive melanoma and metastatic melanoma are nearly completely lost, whereas benign nevi are ciliated. Dysplastic nevi (DN) have a wide range of histopathologic features from mild (low-grade) to severe (high-grade) cytologic atypia and represent a key clinical and histopathologic marker for melanomagenesis. We sought to identify whether cilia are retained in these melanocytic lesions and whether increasing degree of dysplasia correlates with loss of ciliation. METHODS: We measured the percentage of ciliated melanocytes (ciliation index) in DN with mild (n = 9) and severe dysplasia (n = 10). We identified the primary cilium and basal bodies by immunofluorescence staining of sections with acetylated alpha-tubulin and gamma-tubulin, respectively. RESULTS: Our results showed a significant decrease in the ciliation index from mild dysplastic (55%) to severe DN (14%) (p = 0.005). CONCLUSIONS: These data support the hypothesis that primary cilium loss may play a role in the underlying biology of severe DN. The ciliation index is a novel quantitative tool that may increase the reproducibility in grading severity of dysplasia for diagnostic and clinical management of melanocytic neoplasms.