An attractor state zone precedes neural crest fate in melanoma initiation.

The field cancerization theory suggests that a group of cells containing oncogenic mutations are predisposed to transformation 1, 2 . We previously identified single cells in BRAF V600E ;p53 -/- zebrafish that reactivate an embryonic neural crest state before initiating melanoma 3-5 . Here we show that single cells reactivate the neural crest fate from within large fields of adjacent abnormal melanocytes, which we term the "cancer precursor zone." These cancer precursor zone melanocytes have an aberrant morphology, dysplastic nuclei, and altered gene expression. Using single cell RNA-seq and ATAC-seq, we defined a distinct transcriptional cell attractor state for cancer precursor zones and validated the stage-specific gene expression initiation signatures in human melanoma. We identify the cancer precursor zone driver, ID1, which binds to TCF12 and inhibits downstream targets important for the maintenance of melanocyte morphology and cell cycle control. Examination of patient samples revealed precursor melanocytes expressing ID1, often surrounding invasive melanoma, indicating a role for ID1 in early melanomagenesis. This work reveals a surprising field effect of melanoma initiation in vivo in which tumors arise from within a zone of morphologically distinct, but clinically covert, precursors with altered transcriptional fate. Our studies identify novel targets that could improve early diagnosis and prevention of melanoma.

Triage options for positive high-risk HPV results from HPV-based cervical cancer screening: a review of the potential alternatives to Papanicolaou test cytology.

The American Cancer Society has recommended high-risk human papillomavirus (HPV) testing as the primary screening method for cervical cancer since 2020. Up to this point, the transition from Pap test cytology-based screening or co-testing with cytology and HPV testing has been slow and limited. However, more health systems in the United States are in the process of implementing this change. The transition to HPV-based screening requires a triage strategy for positive results. Genotyping to specifically detect HPV types 16 and 18 in conjunction with reflex cytology for the remaining high-risk HPV genotypes has been the recommended method. Testing options including Dual Stain for p16/Ki-67 and extended HPV genotyping are currently being incorporated into treatment algorithms as alternatives. Methylation testing is another promising method extensively investigated around the world. This review, performed by members of the Clinical Practice Committee of the American Society of Cytopathology, examines the rationale behind the switch away from reliance on Pap test cytology in the cervical cancer screening algorithm and the opportunities and problems associated with the most promising alternative approaches. Published studies that give insight into the performance characteristics of these newer tests are reviewed. At the present time, Pap test cytology remains a viable triage option for positive HPV screening results, but alternative tests have significant appeal and should be considered in tandem with the decision to offer primary HPV screening.

The diagnostic utility of Merkel cell polyoma virus immunohistochemistry in cytology specimens.

OBJECTIVE: Merkel cell carcinoma (MCC) is an aggressive cutaneous neuroendocrine neoplasm that predominantly affects elderly and immunocompromised patients. Merkel cell polyoma virus (MCPyV) is clonally integrated into the majority of MCCs and has been linked to patient outcomes, playing a central role in the pathogenesis of the disease. We aimed to assess the utility of MCPyV immunohistochemistry (IHC) in the diagnosis of MCC in cytology cell block specimens and correlating with clinicopathologic features. METHODS: Fifty-three cytology samples of MCC with sufficient cell block material were stained for MCPyV by IHC and scored semi-quantitatively in extent and intensity. Morphologic mimics of MCC including small cell lung carcinoma (n = 10), non-Hodgkin lymphoma (n = 10), basaloid squamous cell carcinoma (n = 6) and other neuroendocrine carcinomas (n = 8) were stained in parallel. Positive staining was defined as >1% of the tumour cells showing at least moderate staining intensity. RESULTS: The cytologic features of MCC were characterized by high nuclear-cytoplasmic ratios, hyperchromatic nuclei with 'salt and pepper' chromatin, and nuclear moulding. MCPyV was detected in 24 of 53 cases (45%). Staining was strong and diffuse in roughly half of the positive samples. Of the morphologic mimics, one follicular lymphoma showed strong and diffuse staining. In contrast to prior studies, we saw no association between MCPyV status and patient outcomes. CONCLUSION: Merkel cell polyoma virus IHC is highly specific (97%) for the diagnosis of MCC in our cohort, and can serve as a useful diagnostic tool for distinguishing MCC for morphologic mimics.

Validation of targeted next-generation sequencing of cell-free DNA from archival cerebrospinal fluid specimens for the detection of somatic variants in cancer involving the leptomeninges: Cytopathologic and radiographic correlation.

BACKGROUND: Leptomeningeal metastases occur across multiple solid and lymphoid cancers, and patients typically undergo cytopathologic assessment of cerebrospinal fluid (CSF) in this setting. For patients diagnosed with metastatic cancer, the detection of actionable somatic mutations in CSF can provide clinically valuable information for treatment without the need for additional tissue collection. METHODS: The authors validated a targeted next-generation sequencing assay for the detection of somatic variants in cancer (OncoPanel) on cell-free DNA (cfDNA) isolated from archival CSF specimens in a cohort of 25 patients who had undergone molecular testing of a prior tumor specimen. RESULTS: CSF storage time and volume had no impact on cfDNA concentration or mean target coverage of the assay. Previously identified somatic variants in CSF cfDNA were detected in 88%, 50%, and 27% of specimens diagnosed cytologically as positive, suspicious/atypical, and negative for malignancy, respectively. Somatic variants were identified in 81% of CSF specimens from patients who had leptomeningeal enhancement on magnetic resonance imaging compared with 31% from patients without such enhancement. CONCLUSIONS: These data highlight the stability of cfDNA in CSF, which allows for cytopathologic evaluation before triage for next-generation sequencing assays. For a subset of cases in which clinical suspicion is high but cytologic or radiographic studies are inconclusive, the detection of pathogenic somatic variants in CSF cfDNA may aid in the diagnosis of leptomeningeal metastases.

The diagnostic utility of methylthioadenosine phosphorylase immunohistochemistry for pancreatic ductal adenocarcinoma in FNA and small biopsy specimens.

BACKGROUND: Accurate diagnosis of pancreatic lesions by endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) or fine-needle biopsy can be challenging. Although surrogate immunohistochemical markers for genetic alterations associated with pancreatic ductal adenocarcinoma (PDAC) have been identified, they have modest sensitivity. Biallelic loss of CDKN2A occurs in up to 46% of PDACs, and methylthioadenosine phosphorylase (MTAP) immunohistochemistry (IHC) has been identified as a reliable surrogate marker for this alteration. The current study evaluates the utility of MTAP IHC for the diagnosis of PDAC. METHODS: In total, 136 cases of EUS-FNA cell block or core biopsy targeting solid pancreatic masses were identified. MTAP IHC was performed and evaluated for complete loss of expression in neoplastic cells. These results were correlated with available clinical next-generation sequencing that was performed on a subset of cases. RESULTS: Complete loss of MTAP expression was identified in 23 of 80 (29%) PDACs. A subset of cases classified as suspicious (4 of 21) and atypical (4 of 22) showed MTAP loss. All morphologically indeterminate cases with MTAP loss were confirmed as PDAC on resection/additional sampling. No benign samples (n = 13) showed loss of MTAP. In samples that had available clinical next-generation sequencing data (n = 13), copy number loss of CDKN2A was detected in all cases that had loss of MTAP expression (n = 4). CONCLUSIONS: Loss of MTAP was identified in approximately 30% of PDAC small biopsy specimens. As loss of MTAP expression is not expected in nonneoplastic cells, and these findings suggest that MTAP IHC can support a diagnosis of PDAC in small biopsy samples.

Leveraging thoughtful quality metric selection for individual and system improvements: the atypical category and use of dashboards.

Quality management is integral to the practice of cytopathology, especially given the heavily manual workflows and expanding ancillary testing requirements inherent to the cytopathology laboratory. Monitoring quality data like turnaround time, specimen unsatisfactory rates, and diagnostic category utilization rates allows for better understanding of performance with opportunities for targeted improvement if there are variations from that which is expected. However, there are costs to quality monitoring including the time and resources needed, and, in already taxed systems, quality management risks being viewed as just another box to check. While there are mandated quality metrics that must be collected by cytology laboratories, thoughtful selection of key performance indicators can be of tremendous benefit in helping to better understand complex laboratory processes and directing improvement endeavors where needed. The following short communication is a discussion on quality management in the cytopathology laboratory from 3 Cytopathology Quality Management Directors. The discussion focuses on monitoring the atypical reporting category with an emphasis on how trending and visualizing quality metrics can provide laboratories with key data.

Modeling Spitz melanoma in zebrafish using sequential mutagenesis.

Spitz neoplasms are a diverse group of molecularly and histologically defined melanocytic tumors with varying biologic potentials. The precise classification of Spitz neoplasms can be challenging. Recent studies have revealed recurrent fusions involving multiple kinases in a large proportion of Spitz tumors. In this study, we generated a transgenic zebrafish model of Spitz melanoma using a previously identified ZCCHC8-ROS1 fusion gene. Animals developed grossly apparent melanocytic proliferations as early as 3 weeks of age and overt melanoma as early as 5 weeks. By 7 weeks, ZCCHC8-ROS1 induced a histologic spectrum of neoplasms ranging from hyperpigmented patches to melanoma. Given the swift onset of these tumors during development, we extended this approach into adult fish using a recently described electroporation technique. Tissue-specific expression of ZCCHC8-ROS1 in adults led to melanocyte expansion without overt progression to melanoma. Subsequent electroporation with tissue-specific CRISPR, targeting only tp53 was sufficient to induce transformation to melanoma. Our model exhibits the use of sequential mutagenesis in the adult zebrafish, and demonstrates that ZCCHC8-ROS1 induces a spectrum of melanocytic lesions that closely mimics human Spitz neoplasms.

Quantifying the hidden impact of COVID-19 pandemic: The cytology perspective.

BACKGROUND: The burden of the COVID-19 pandemic is often enumerated in lives lost, but the strain on health care resources and mobility limitations contributed to the burden of non-COVID related disease. In this study, we evaluated the impact of the pandemic through a time series review of cytology samples. METHODS: Pathology reports for all cytology specimens received from January 2019 through April 2021 at our institution were reviewed. Time series analysis was performed using moving averages, time trend analysis, cross-correlation, and tests of homogeneity. RESULTS: During the first peak of the pandemic (March-June 2020), breakpoint analysis showed a downward shift in the number of gynecologic (-89.4%) and non-gynecologic (-70.4%) cytology specimens within a week of declaration of an emergency. Cross-correlation analysis showed a relationship between sample numbers and COVID-19 cases during the initial phase of the pandemic (April-June 2020). During the second surge (October 2020-April 2021), despite the higher incidence of COVID-19, there was a smaller impact on cytology samples (-20.1% and - 24.8% for gynecologic and non-gynecologic samples, respectively). During the first 3 months of the pandemic, 154 fewer malignant cases were identified compared with the prior year. Although specimen numbers slowly returned to baseline following the first wave of the pandemic, the earlier decline in malignant diagnoses was not offset during the study period. CONCLUSIONS: The deleterious effects of COVID-19 extend beyond direct mortality attributed to the disease. The significant decrease in diagnostic cytology specimens during this period has profound implications including delayed care and missed disease.

Genomic catastrophe, the peritoneal cavity and ovarian cancer prevention.

The current theory of carcinogenesis for the deadliest of 'ovarian' cancers-high-grade serous carcinoma (HGSC)-holds that the malignancy develops first in the fallopian tube and spreads to the ovaries, peritoneum, and/or regional lymph nodes. This is based primarily on the observation of early forms of serous neoplasia (serous tubal intraepithelial lesions [STILs], and serous tubal intraepithelial carcinomas [STICS]) in the fimbria of women undergoing risk reduction surgery. However, these lesions are uncommon in the general population, confer a low risk (5%) of HGSC following their removal in at-risk women with germ-line BRCA1/2 mutations, and require 4 or more years to recur as intraperitoneal HGSC. These features suggest that isolated STILs and STICs behave as precursors, with uncertain cancer risk rather than carcinomas. Their evolution to HGSC within, or after, escape from the tube could proceed stepwise with multiple biologic events; however, it is unclear whether tubal or ovarian HGSCs encountered in the setting of advanced disease evolved in the same fashion. The latter scenario could also be explained by a 'catastrophic' model in which STICs suddenly develop with invasive and metastatic potential, overwhelming or obscuring the site of origin. Moreover, a similar model might explain the sudden emergence of HGSC in the peritoneal cavity following escape of precursor cells years before. Long-term follow-up data from opportunistic or prophylactic salpingectomy should shed light on where malignant transformation occurs, as well as the timeline from precursor to metastatic HGSC. © 2022 The Pathological Society of Great Britain and Ireland.

A phase II trial of all-trans retinoic acid (ATRA) in advanced adenoid cystic carcinoma.

BACKGROUND: Effective therapies are lacking for recurrent, metastatic adenoid cystic carcinoma (R/M ACC) and preclinical models suggest retinoic acid agonists inhibit ACC growth. This phase II trial evaluated all-trans retinoic acid (ATRA) as a novel therapy for ACC. METHODS: Patients with R/M ACC (any site) with clinical and/or radiographic progression ≤12 months prior to study entry were eligible. Cohort 1 (CH1) received ATRA 45 mg/m2 split oral daily dosing on days 1-14 of a 28-day cycle; Cohort 2 (CH2) received the same dosing continuously. Primary endpoint was best overall response rate (CR + PR) (RECIST v1.1). Secondary endpoints: safety and progression-free survival (PFS). Exploratory analyses: ATRA impact on MYB expression and genomic predictors of response. RESULTS: Eighteen patients enrolled. There were no responses, but 61% (11/18) had stable disease (SD) and 28% (5/18) progression as best response; 11% (2/18) unevaluable. Median duration of stability: 3.7 months (95%CI, 1.9-3.9). One patient (CH1) remains on drug with SD approaching 1 year. Half of those who received prior VEGFR therapy achieved SD (4/8). At median follow up of 7.9 months, median PFS was 3.2 months (95%CI, 1.8-3.9). N = 1 required dose adjustment; N = 1 came off drug for toxicity. There were no grade 3-4 adverse events. NOTCH1 and PI3K pathway alterations were most frequent. Low MYB protein expression was associated with longer duration of stability on ATRA (P < 0.01). CONCLUSION(S): While the trial did not meet its prespecified response endpoint, ATRA alone or in combination may be a low toxicity treatment for disease growth stabilization in R/M ACC.

SATB2 induction of a neural crest mesenchyme-like program drives melanoma invasion and drug resistance.

Recent genomic and scRNA-seq analyses of melanoma demonstrated a lack of recurrent genetic drivers of metastasis, while identifying common transcriptional states correlating with invasion or drug resistance. To test whether transcriptional adaptation can drive melanoma progression, we made use of a zebrafish mitfa:BRAFV600E;tp53-/- model, in which malignant progression is characterized by minimal genetic evolution. We undertook an overexpression-screen of 80 epigenetic/transcriptional regulators and found neural crest-mesenchyme developmental regulator SATB2 to accelerate aggressive melanoma development. Its overexpression induces invadopodia formation and invasion in zebrafish tumors and human melanoma cell lines. SATB2 binds and activates neural crest-regulators, including pdgfab and snai2. The transcriptional program induced by SATB2 overlaps with known MITFlowAXLhigh and AQP1+NGFR1high drug-resistant states and functionally drives enhanced tumor propagation and resistance to Vemurafenib in vivo. In summary, we show that melanoma transcriptional rewiring by SATB2 to a neural crest mesenchyme-like program can drive invasion and drug resistance in autochthonous tumors.

Recurrent co-alteration of HDGF and SETDB1 on chromosome 1q drives cutaneous melanoma progression and poor prognosis.

A progressive increase in copy number variation (CNV) characterizes the natural history of cutaneous melanoma progression toward later disease stages, but our understanding of genetic drivers underlying chromosomal arm-level CNVs remains limited. To identify candidate progression drivers, we mined the TCGA SKCM dataset and identified HDGF as a recurrently amplified gene whose high mRNA expression correlates with poor patient survival. Using melanocyte-specific overexpression in the zebrafish BRAFV600E -driven MiniCoopR melanoma model, we show that HDGF accelerates melanoma development in vivo. Transcriptional analysis of HDGF compared to control EGFP tumors showed the activation of endothelial/angiogenic pathways. We validated this observation using an endothelial kdrl:mCherry reporter line which showed HDGF to increases tumor vasculature. HDGF is frequently co-altered with the established melanoma driver SETDB1. Both genes are located on chromosome 1q, and their co-amplification is observed in up to 13% of metastatic melanoma. TCGA patients with both genes amplified and/or overexpressed have a worse melanoma specific survival. We tested co-expression of HDGF and SETDB1 in the MiniCoopR model, which resulted in faster and more aggressive melanoma development than either gene individually. Our work identifies the co-amplification of HDGF and SETDB1 as a functional driver of melanoma progression and poor patient prognosis.

Radiation-associated sarcomas other than malignant peripheral nerve sheath tumours demonstrate loss of histone H3K27 trimethylation†.

BACKGROUND AND AIMS: Complete loss of histone H3 lysine 27 trimethylation (H3K27me3) has recently emerged as a biomarker for malignant peripheral nerve sheath tumours (MPNST). Loss of H3K27me3 staining has also been reported in post-radiation MPNST; however, it has not been evaluated in a large series of radiation-associated sarcomas of different histological subtypes. The aim of this study was to assess H3K27me3 labelling by immunohistochemistry in radiation-associated sarcomas and to determine the prevalence of H3K27me3 loss in these tumours. METHODS AND RESULTS: Radiation-associated sarcomas (n = 119) from two tertiary care referral centres were evaluated for loss of H3K27me3, defined as complete loss of staining within tumour cells in the presence of a positive internal control. Twenty-three cases (19%) showed H3K27me3 loss, including nine of 10 (90%) MPNST, seven of 77 (9%) undifferentiated spindle cell/pleomorphic sarcomas, five of 25 (20%) angiosarcomas, one of five (20%) leiomyosarcomas and one of two (50%) osteosarcomas. CONCLUSIONS: Complete H3K27me3 loss was present in 19% of radiation-associated sarcomas in our series. Our findings demonstrate that loss of H3K27me3 is not specific for radiation-associated MPNST and may also occur in other histological subtypes of RAS, including radiation-associated undifferentiated spindle cell/pleomorphic sarcoma, angiosarcoma, leiomyosarcoma and osteosarcoma.

MYC expression has limited utility in the distinction of undifferentiated radiation-associated sarcomas from sporadic sarcomas and sarcomatoid carcinoma.

AIMS: MYC is a proto-oncogene that is frequently dysregulated in various malignancies, through translocation or amplification. Radiation-associated angiosarcoma frequently shows MYC amplification, and immunohistochemical expression has been shown to be a reliable surrogate marker for amplification, but less is known about MYC expression in other sarcoma types, despite reports of MYC amplification in some undifferentiated/unclassified radiation-associated sarcomas (RASs). Distinguishing putative RAS from non-radiation-associated sarcoma or sarcomatoid carcinoma can be difficult. The aim of this study was to determine the prevalence and potential diagnostic utility of MYC in this context, by evaluating MYC expression in a cohort of RASs, non-radiation-associated sarcomas, and sarcomatoid carcinomas. METHODS AND RESULTS: Three hundred and eighty-five neoplasms were evaluated, including 81 RASs (18 angiosarcomas; 57 undifferentiated sarcomas; three leiomyosarcomas; and three malignant peripheral nerve sheath tumours), 267 non-radiation-associated sarcomas, and 37 sarcomatoid carcinomas. Immunohistochemistry was performed with a monoclonal anti-MYC antibody. Staining in tumour cells was scored on the basis of extent (focal, 1-4%; multifocal, 5-49%; and diffuse, ≥50%) and intensity (strong, moderate, and weak). One hundred percent of radiation-associated angiosarcomas expressed MYC diffusely. Expression was infrequent among other types of RAS (9.5%), and the frequency was similar to that in non-radiation-associated sarcomas (9.7%). MYC expression was more common in sarcomatoid carcinomas, occurring in 43%. The extent and intensity of staining were variable in all groups. CONCLUSION: MYC expression is infrequent among RASs other than angiosarcoma, and has a similar prevalence in sporadic sarcomas. Given the frequency of expression in sarcomatoid carcinomas, MYC expression outside the context of radiation-associated angiosarcoma is of limited diagnostic utility, and should be interpreted with caution after exclusion of sarcomatoid carcinoma where relevant.

A Comparison of Outcomes and Prognostic Features for Radiation-Associated Angiosarcoma of the Breast and Other Radiation-Associated Sarcomas.

PURPOSE: Radiation-associated sarcomas (RAS) are considered to have a poor prognosis. Although the incidence is anticipated to rise, contemporary data regarding predictors of outcomes are few. We performed a retrospective analysis to identify RAS prognostic factors and subset analyses for radiation-associated angiosarcoma arising after treatment for breast cancer (RAAB) and other RAS subtypes (other-RAS). METHODS AND MATERIALS: Patients with localized RAS evaluated at an institutional multidisciplinary sarcoma clinic were identified. Clinical and histologic review was performed, and outcomes were assessed to identify prognostic features. A subset of cases underwent molecular analysis by next-generation sequencing. RESULTS: Among 176 patients, histologic subtypes of RAS included angiosarcoma (41%), undifferentiated/unclassified sarcoma (40%), leiomyosarcoma (8%), malignant peripheral nerve sheath tumor (6%), and osteosarcoma (2%). Sixty-seven patients (38%) had RAAB, and 109 (62%) had other-RAS. RAAB had significantly shorter latency from time of initial radiation compared with other-RAS (8 vs. 15 years; P < .001). Treatment approaches included surgery (91%), chemotherapy (44%), and radiation therapy (27%). Median follow-up was 3.2 years; 3-year overall survival (OS) was 74%. On multivariate analysis, positive margins (P < .0001), deep tumor location (intrathoracic/intra-abdominal, P = .002), and high grade (P < .0001) were associated with worse OS. In particular, 3-year OS with negative versus positive margins was 90% versus 66%. Patients with RAAB versus other-RAS showed a trend for higher 3-year OS (84% vs 68%; P = .09), significantly higher 3-year metastasis-free survival (82% vs 67%; P = .001), but similar 3-year local recurrence-free survival (54% vs 61%; P = .28). Next-generation sequencing identified overall low tumor mutational burden, recurrent MYC amplification in RAAB, and few clinically actionable mutations. CONCLUSIONS: Margin negative excision, superficial tumor location, and low tumor grade are determinants of improved OS for RAS, suggesting that complete surgical excision, when possible, is an optimal component of treatment. RAAB is a clinicopathologically distinct type of RAS with shorter latency from initial RT, different recurrence patterns, and when aggressively managed has potentially better outcomes compared with other-RAS.

Radiation-Associated Sarcomas: An Update on Clinical, Histologic, and Molecular Features.

Approximately half of all cancer patients receive radiation therapy as part of their oncologic treatment. Radiation-associated sarcomas occur in fewer than 1% of patients who receive radiation therapy but account for up to 5% of all sarcomas. As the use of radiation has increased in the past few decades and overall oncologic outcomes are improving, the incidence of radiation-associated sarcomas is also expected to increase. Historically, radiation-associated sarcomas have been associated with poor outcomes but recent data suggest the prognosis is improving. Distinguishing the sarcoma from the primary malignancy is a major diagnostic criterion.

Neural crest state activation in NRAS driven melanoma, but not in NRAS-driven melanocyte expansion.

NRAS mutations are frequently found in many deadly malignancies and are the second most common oncogene driving malignant melanoma. Here, we generate a rapid transient transgenic zebrafish model of NRASQ61R-mutant melanoma. These fish develop extensive melanocytic proliferation in approximately 4 weeks. The majority of these lesions do not engraft upon transplantation and lack overt histologic features of malignancy. Our previous work demonstrated that activation of a neural crest cell transcriptional program is a key initiating event in zebrafish BRAF/p53-driven melanomas using the fluorescent reporter crestin:EGFP. By 8-12 weeks of age, some lesions progress to malignant melanoma and have cytologic atypia, destructive tissue invasion, and express neural crest progenitor markers, including crestin:EGFP. Our studies demonstrate that NRASQ61R induces extensive melanocyte expansion, which arise during zebrafish development and lack a transformed phenotype. These early lesions are highly predisposed to reactivate a neural crest progenitor fate and form malignant melanomas.

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.

Immunohistochemical Detection and Molecular Characterization of IDH-mutant Sinonasal Undifferentiated Carcinomas.

Recent studies have identified recurrent isocitrate dehydrogenase 2 (IDH2) mutations in a subset of sinonasal undifferentiated carcinomas (SNUCs); however, the true frequency of IDH mutations in SNUC is unknown. We evaluated the utility of mutation-specific IDH1/2 immunohistochemistry (IHC) in a large multi-institutional cohort of SNUC and morphologic mimics. IHC using a multispecific antibody for IDH1/2 (R132/R172) mutant protein was performed on 193 sinonasal tumors including: 53 SNUCs, 8 poorly differentiated carcinomas (PDCARs) and 132 histologic mimics. Mutant IDH1/2 IHC was positive in 26/53 SNUCs (49%; 20 strongly positive and 6 weak) and 3/8 PDCARs (37.5%; 2 strong; 1 weak) but was absent in all other tumor types (0/132). Targeted next-generation sequencing (NGS) on a subset of SNUC/PDCAR (6 strong and 3 weak positive for IDH1/2 IHC; 7 negative) showed frequent IDH2 R172X mutations (10/16) and a single IDH1 R132C mutation. All 6 cases with strong positive mutant IDH1/2 staining and NGS had IDH2 R172S/G mutations. The 3 IHC-weak cases all had IDH2 R172T mutations. Among the 7 tested cases that were negative for mutant IDH1/2 IHC, NGS detected 1 case each with IDH2 R172T and IDH1 R132C mutation. IDH-mutant carcinomas also had frequent mutations in TP53 (55%) and activating mutations in KIT (45%) or the PI3K pathway (36%). Mutation-specific IDH1/2 IHC identifies IDH mutations in SNUC, however, it lacks sensitivity for the full range of IDH mutations. These findings suggest that IDH-mutant sinonasal carcinoma may represent a distinct pathobiological entity with therapeutic implications that can be identified by a combined approach of multispecific IDH1/2 IHC and sequencing.

SOX10/keratin dual-color immunohistochemistry: An effective first-line test for the workup of epithelioid malignant neoplasms in FNA and small biopsy specimens.

BACKGROUND: The characterization of poorly differentiated neoplasms in fine-needle aspiration (FNA) and small biopsy specimens usually requires immunohistochemistry (IHC) with a panel of markers. Because of an increasing need to preserve limited diagnostic material for tumor genotyping and a mounting demand for cost containment, the authors investigated the usefulness of dual-color IHC with antibodies directed against broad-spectrum keratins and SOX10, a neuroectodermal transcription factor consistently expressed in melanoma, in the workup of epithelioid malignant neoplasms. METHODS: A total of 107 cases of FNA cell blocks (49 cases) and small biopsies (58 cases) were selected, including 34 melanomas, 31 epithelioid/pleomorphic sarcomas, and 42 carcinomas. IHC was performed on all specimens using a peroxidase-based brown chromogen for SOX10 and an alkaline phosphatase-based red chromogen for keratins AE1/AE3. The presence or absence of staining in lesional cells was scored. RESULTS: The majority of tumors demonstrated 1 of 3 distinct patterns: 1) malignant melanomas with nuclear SOX10 (sensitivity of 94% and specificity of 95%); 2) epithelioid/pleomorphic sarcomas negative for both SOX10 and AE1/AE3 (sensitivity of 84% and specificity of 88%); and 3) carcinomas with cytoplasmic AE1/AE3 (sensitivity of 76% and specificity of 98%). In addition, a fourth pattern with cytoplasmic AE1/AE3 and nuclear SOX10 was observed in a subset of carcinomas, most notably triple-negative breast cancers. CONCLUSIONS: SOX10/keratin dual-color IHC appears to be an effective, sensitive, and specific test to distinguish between melanoma, sarcoma, and carcinoma. This approach can identify melanoma, prioritize additional studies, and limit the number of markers needed to workup an epithelioid malignant neoplasm, thereby potentially reducing costs and preserving valuable tissue for ancillary studies with which to guide therapy. Cancer Cytopathol 2018;126:179-89. © 2018 American Cancer Society.