Nextflow pipeline for Visium and H&E data from patient-derived xenograft samples.

We designed a Nextflow DSL2-based pipeline, Spatial Transcriptomics Quantification (STQ), for simultaneous processing of 10x Genomics Visium spatial transcriptomics data and a matched hematoxylin and eosin (H&E)-stained whole-slide image (WSI), optimized for patient-derived xenograft (PDX) cancer specimens. Our pipeline enables the classification of sequenced transcripts for deconvolving the mouse and human species and mapping the transcripts to reference transcriptomes. We align the H&E WSI with the spatial layout of the Visium slide and generate imaging and quantitative morphology features for each Visium spot. The pipeline design enables multiple analysis workflows, including single or dual reference genome input and stand-alone image analysis. We show the utility of our pipeline on a dataset from Visium profiling of four melanoma PDX samples. The clustering of Visium spots and clustering of H&E imaging features reveal similar patterns arising from the two data modalities.

Spatiotemporal profiling defines persistence and resistance dynamics during targeted treatment of melanoma.

Resistance of BRAF-mutant melanomas to targeted therapy arises from the ability of cells to enter a persister state, evade treatment with relative dormancy, and repopulate the tumor when reactivated. Using spatial transcriptomics in patient derived xenograft models, we capture clonal lineage evolution during treatment, finding the persister state to show increased oxidative phosphorylation, decreased proliferation, and increased invasive capacity, with central-to-peripheral gradients. Phylogenetic tracing identifies intrinsic- and acquired-resistance mechanisms (e.g. dual specific phosphatases, Reticulon-4, CDK2) and suggests specific temporal windows of potential therapeutic efficacy. Using deep learning to analyze histopathological slides, we find morphological features of specific cell states, demonstrating that juxtaposition of transcriptomics and histology data enables identification of phenotypically-distinct populations using imaging data alone. In summary, we define state change and lineage selection during melanoma treatment with spatiotemporal resolution, elucidating how choice and timing of therapeutic agents will impact the ability to eradicate resistant clones. Statement of Significance: Tumor evolution is accelerated by application of anti-cancer therapy, resulting in clonal expansions leading to dormancy and subsequently resistance, but the dynamics of this process are incompletely understood. Tracking clonal progression during treatment, we identify conserved, global transcriptional changes and local clone-clone and spatial patterns underlying the emergence of resistance.

Nextflow Pipeline for Visium and H&E Data from Patient-Derived Xenograft Samples.

Highlights: We have developed an automated data processing pipeline to quantify mouse and human data from patient-derived xenograft samples assayed by Visium spatial transcriptomics with matched hematoxylin and eosin (H&E) stained image. We enable deconvolution of reads with Xenome, quantification of spatial gene expression from host and graft species with Space Ranger, extraction of B-allele frequencies, and splicing quantification with Velocyto. In the H&E image processing sub-workflow, we generate morphometric and deep learning-derived feature quantifications complementary to the Visium spots, enabling multi-modal H&E/expression comparisons. We have wrapped the pipeline into Nextflow DSL2 in a scalable, portable, and easy-to-use framework. Summary: We designed a Nextflow DSL2-based pipeline, Spatial Transcriptomics Quantification (STQ), for simultaneous processing of 10x Genomics Visium spatial transcriptomics data and a matched hematoxylin and eosin (H&E)-stained whole slide image (WSI), optimized for Patient-Derived Xenograft (PDX) cancer specimens. Our pipeline enables the classification of sequenced transcripts for deconvolving the mouse and human species and mapping the transcripts to reference transcriptomes. We align the H&E WSI with the spatial layout of the Visium slide and generate imaging and quantitative morphology features for each Visium spot. The pipeline design enables multiple analysis workflows, including single or dual reference genomes input and stand-alone image analysis. We showed the utility of our pipeline on a dataset from Visium profiling of four melanoma PDX samples. The clustering of Visium spots and clustering of imaging features of H&E data reveal similar patterns arising from the two data modalities.

Deep learning image analysis quantifies tumor heterogeneity and identifies microsatellite instability in colon cancer.

BACKGROUND AND OBJECTIVES: Deep learning utilizing convolutional neural networks (CNNs) applied to hematoxylin & eosin (H&E)-stained slides numerically encodes histomorphological tumor features. Tumor heterogeneity is an emerging biomarker in colon cancer that is, captured by these features, whereas microsatellite instability (MSI) is an established biomarker traditionally assessed by immunohistochemistry or polymerase chain reaction. METHODS: H&E-stained slides from The Cancer Genome Atlas (TCGA) colon cohort are passed through the CNN. Resulting imaging features are used to cluster morphologically similar slide regions. Tile-level pairwise similarities are calculated and used to generate a tumor heterogeneity score (THS). Patient-level THS is then correlated with TCGA-reported biomarkers, including MSI-status. RESULTS: H&E-stained images from 313 patients generated 534 771 tiles. Deep learning automatically identified and annotated cells by type and clustered morphologically similar slide regions. MSI-high tumors demonstrated significantly higher THS than MSS/MSI-low (p < 0.001). THS was higher in MLH1-silent versus non-silent tumors (p < 0.001). The sequencing derived MSIsensor score also correlated with THS (r = 0.51, p < 0.0001). CONCLUSIONS: Deep learning provides spatially resolved visualization of imaging-derived biomarkers and automated quantification of tumor heterogeneity. Our novel THS correlates with MSI-status, indicating that with expanded training sets, translational tools could be developed that predict MSI-status using H&E-stained images alone.

Survival after checkpoint inhibitors for metastatic acral, mucosal and uveal melanoma.

BACKGROUND: Checkpoint inhibitors (CPIs) are thought to be effective against cutaneous melanoma in part because of the large burden of somatic mutations (neoantigens) generated from exposure to ultraviolet radiation. However, rare melanoma subtypes arising from acral skin, mucosal surfaces, and the uveal tract are largely sun-shielded. Genomic studies show these sun-shielded melanomas have a paucity of neoantigens and unique biology; they are thought to be largely resistant to immunotherapy. It has not been definitively shown that CPI improves survival in metastatic sun-shielded melanoma. METHODS: We reviewed a single institutional experience using antibodies against CTLA-4, PD-1 and/or PD-L1 to treat patients with metastatic melanoma. Primary tumor histology was categorized as cutaneous, unknown, acral, mucosal, or uveal. We studied demographic data, treatment characteristics, and overall survival (OS) after CPI. RESULTS: We treated 428 patients with metastatic melanoma from 2007 to 2019. Primary tumors were cutaneous in 283 (66%), unknown in 55 (13%), acral in 22 (5%), mucosal in 38 (9%), and uveal in 30 (7%). Patients with metastatic disease from cutaneous primary tumors had median OS after CPI of 45 months compared with 17 months for acral (p=0.047), 18 months for mucosal (p=0.003), and 12 months for uveal (p<0.001). For all patients with sun-shielded melanoma (n=90), first treatment with anti-PD-1 or anti-PD-L1 was followed by a median OS of 9 months compared with 18 months after anti-CTLA-4 (p=0.010) and 20 months after combination therapy (p=0.003). There were 21 patients who achieved actual 3-year survival; 20 received both anti-CTLA-4 and anti-PD-1, either sequentially or in combination. Over 80% of 3-year survivors with progressive disease were treated with local therapy after CPI. CONCLUSIONS: Long survival in patients with metastatic melanoma from acral, mucosal, and uveal primary tumors was associated with receipt of both anti-CTLA-4 and anti-PD-1 antibodies. Complete responses were rare, and local therapy was frequently employed to control disease progression. While sun-shielded melanomas exhibit worse outcomes after CPI than cutaneous melanomas, with an aggressive multidisciplinary approach, 5-year survival is still possible for 25%-32% of these patients.

Recurrence and Complications in Pediatric and Adolescent Papillary Thyroid Cancer in a High-Volume Practice.

BACKGROUND: Treatment approaches for pediatric papillary thyroid cancer (PTC) are historically extrapolated from adult experience. However, pediatric PTC demonstrates a greater propensity for lymph node involvement, early metastases, and recurrence, highlighting the need for pediatric-specific treatment paradigms. MATERIALS AND METHODS: A retrospective review included patients with PTC aged ≤21 y, with ≥18 mo of follow-up, treated between 2002 and 2015. Fisher's exact test and Cox proportional hazard were used to estimate the effect of risk factors on disease recurrence. RESULTS: Seventy-two cases of PTC were identified with median age of 17.0 y and median follow-up of 64.1 mo. Disease recurred at a median of 24.6 mo (range 7.8-78.1) in 7 of 51 (13.7%) of patients with disease limited to the thyroid or central neck, 7 of 18 (39%) patients with lateral neck disease at presentation who underwent a compartment-based resection, and three of three patients (100%) with lateral neck disease who sought care after non-compartment-based resection. There were no deaths from disease. Univariate predictors of recurrence included tumor size >2 cm (P = 0.005), lateral neck disease (P = 0.004), lymphovascular invasion (P = 0.017), extracapsular invasion (P < 0.0001), multifocality (P = 0.03), and non-Caucasian race (P = 0.05). Multivariate analysis identified race (P = 0.05) as an independent predictor of recurrence. In patients without lateral neck disease, there was a trend toward lower recurrence in patients undergoing thyroidectomy with central neck dissection compared with thyroidectomy alone (P = 0.07). CONCLUSIONS: Pediatric PTC is associated with excellent survival, although recurrence is common in patients with lateral node involvement. Predictors of recurrence are multifactorial and may be influenced by extent of disease, patient or tumor biology, and aggressiveness of resection. LEVEL OF EVIDENCE: Prognosis study, level IV, retrospective case series.

APC mutational patterns in gastric adenocarcinoma are enriched for missense variants with associated decreased survival.

Adenomatous polyposis coli (APC) mutations are causally associated with familial adenomatous polyposis (FAP) and are recurrent somatic events across numerous tumor types, including gastric adenocarcinoma. Severity of disease in FAP correlates with specific APC mutations, but the impact of given mutations on phenotype in gastric cancer is not well studied. Sequencing data from the Genomic Data Commons (GDC) demonstrate an APC mutational pattern in gastric cancer that differs dramatically from that seen in colon cancer. Exome sequencing data from APC-mutant colon and gastric adenocarcinomas in GDC was filtered for single nucleotide variants (SNVs) using MuTect2 Variant Aggregation and Masking pipeline, Somatic Aggregation Workflow. APC mutations were found in 57/441 gastric (12.9%) and 309/433 colon adenocarcinomas (71.4%). There was a significant difference in the proportion of stopgain, frameshift, and missense mutations between tumor types(P < .00001). Colon tumors were predominated by frameshift and stopgains, comprising 47.7% and 35.7%, respectively. In contrast, 47.1% of gastric mutations were missense. Gastric tumors harboring missense mutations showed decreased overall survival relative to other mutational subtypes(P = .008). In the gastric samples, 25.9% of frameshift and stopgain mutations are in the 3' portion of the gene, compared to 1.4% of colon samples. APC mutations demonstrate different distributions in gastric and colon adenocarcinoma, with a shift toward missense variants in gastric tumors and worse survival in gastric tumors harboring them. As different mutations confer variable degrees of protein dysfunction and resultant clinical manifestation, expanded investigation of specific mutational patterns will prove integral to future-risk stratification strategies.

Next-generation Sequencing in the Management of Gastric and Esophageal Cancers.

Next-generation sequencing has enabled genome-wide molecular profiling of gastric and esophageal malignancies at single-nucleotide resolution. The resultant genomic profiles provide information about the specific oncogenic pathways that are the likely driving forces behind tumorigenesis and progression. The abundance of available genomic data has immense potential to redefine management paradigms for these difficult disease processes. The ability to capitalize on the information provided through high-throughput sequencing technologies will define cancer care in the coming decades and could shift the paradigm from current stage-based, organ-specific treatments toward tailored regimens that target the specific culprit pathways driving individual tumors.

Lymph node ratio predicts recurrence in pediatric papillary thyroid cancer.

BACKGROUND: Regional lymph node (LN) metastasis at the time of presentation plays a significant role in predicting recurrence in patients with papillary thyroid cancer (PTC). Multiple studies in the adult population have demonstrated that the lymph node ratio (LNR) in both the central and lateral neck can improve the accuracy of recurrence prediction, but this ratio has not been studied in the pediatric population. In this study, we sought to investigate the LNR in the central and lateral compartments as a prognostic predictor for recurrence in pediatric patients with PTC. METHODS: A retrospective analysis of pediatric patients (≤21 years old) at a single institution between 2002 and 2014 who underwent total thyroidectomy with prophylactic central neck dissection (TTpCND) with at least 3 sampled nodes or total thyroidectomy with unilateral modified radical neck dissection (TTMRND) with at least 10 sampled nodes, and on whom at least 24 months of follow up data were available was performed. The LNR was defined as the ratio of metastatic LNs to total number of investigated LNs. Recurrence after TTpCND and TTMRND was examined separately as a function of LNR, using the value of 0.45 as a cutoff. RESULTS: Forty-eight patients met inclusion criteria. Thirty-two underwent TTpCND, and sixteen underwent TTMRND. Median age at time of operation was 17 years (range 6-20), and median duration of follow-up was 53.5 months (range 24-183). In the TTpCND, LNR ranged from 0 to 1.0. There were two recurrences among the eight patients (25%) undergoing TTpCND in patients with LNRs >0.45 and a single recurrence among the 24 patients (4.2%) undergoing TTpCND with an LNR ≤0.45. In the TTMRND, LNR ranged from 0.1 to 1.0. There were 3 recurrences in 12 patients with LNR ≤0.45 (30.8%%) and 4 recurrences in 4 patients with LNR >0.45 (100%) (p = 0.03). CONCLUSIONS: Although limited by small sample size, LNR may be a useful predictor to stratify the likelihood of recurrence in pediatric patients undergoing TTpCND or TTMRND for pathologic N1a or N1b PTC. TYPE OF STUDY: Prognosis study / retrospective case series. LEVEL OF EVIDENCE: Level IV.

SLC12A7 alters adrenocortical carcinoma cell adhesion properties to promote an aggressive invasive behavior.

BACKGROUND: Altered expression of Solute Carrier Family 12 Member 7 (SLC12A7) is implicated to promote malignant behavior in multiple cancer types through an incompletely understood mechanism. Recent studies have shown recurrent gene amplifications and overexpression of SLC12A7 in adrenocortical carcinoma (ACC). The potential mechanistic effect(s) of SLC12A7 amplifications in portending an aggressive behavior in ACC has not been previously studied and is investigated here using two established ACC cell lines, SW-13 and NCI-H295R. METHODS: SW-13 cells, which express negligible amounts of SLC12A7, were enforced to express SLC12A7 constitutively, while RNAi gene silencing was performed in NCI-H295R cells, which have robust endogenous expression of SLC12A7. In vitro studies tested the outcomes of experimental alterations in SLC12A7 expression on malignant characteristics, including cell viability, growth, colony formation potential, motility, invasive capacity, adhesion and detachment kinetics, and cell membrane organization. Further, potential alterations in transcription regulation downstream to induced SLC12A7 overexpression was explored using targeted transcription factor expression arrays. RESULTS: Enforced SLC12A7 overexpression in SW-13 cells robustly promoted motility and invasive characteristics (p < 0.05) without significantly altering cell viability, growth, or colony formation potential. SLC12A7 overexpression also significantly increased rates of cellular attachment and detachment turnover (p < 0.05), potentially propelled by increased filopodia formation and/or Ezrin interaction. In contrast, RNAi gene silencing of SLC12A7 stymied cell attachment strength as well as migration and invasion capacity in NCI-H295R cells. Transcription factor expression analysis identified multiple signally pathways potentially affected by SLC12A7 overexpression, including osmotic stress, bone morphogenetic protein, and Hippo signaling pathways. CONCLUSIONS: Amplification of SLC12A7 observed in ACCs is shown here, in vitro, to exacerbate the malignant behavior of ACC cells by promoting invasive capacities, possibly mediated by alterations in multiple signaling pathways, including the osmotic stress pathway.

Hyperparathyroidism-Jaw Tumor Syndrome Associated With Large-Scale 1q31 Deletion.

Hyperparathyroidism-jaw tumor syndrome (HPT-JT) is a rare autosomal dominant cause of familial hyperparathyroidism associated with benign, ossifying fibromas of the maxillofacial bones and increased risk of parathyroid carcinoma. The putative tumor suppressor gene CDC73 has been implicated in the syndrome, with a multitude of inactivating mutations identified; however, HPT-JT due to large-scale deletion of the chromosomal region containing the gene is exceedingly rare, and the clinical significance of this variant remains unclear. We report the case of a 32-year-old woman with a history of mandibular ossifying fibroma who presented with primary hyperparathyroidism and was found to harbor a large-scale, germline deletion on chromosome 1q31, including the CDC73 locus. HPT-JT is associated with loss of function of the putative tumor suppressor gene CDC73. Over 100 mutations and small insertions/deletions have been identified within the gene, the majority of which result in premature truncation of the parafibromin protein product. We report a case of HPT-JT associated with a large chromosomal deletion (4.1 Mb) encompassing the CDC73 gene locus. In the future, molecular testing in this autosomal dominant disorder should use techniques that allow for the detection of large-scale deletions in addition to the more commonly observed mutations and smaller-scale copy number alterations. Further investigation is needed to determine whether HPT-JT associated with a large-scale deletion carries increased risk of malignancy relative to the more common truncating mutations and what the implications are for genetic counseling.

Overexpression of cytochrome P450 2A6 in adrenocortical carcinoma.

BACKGROUND: Cytochrome P450-mediated metabolism of chemotherapeutic agents contributes to chemotherapy resistance in multiple malignancies. Adrenocortical carcinoma is known to have a poor response to adjuvant therapies; however, the mechanism remains unknown. Recent comprehensive genetic analyses of adrenocortical carcinomas demonstrated recurrent copy number gains in multiple cytochrome P450 genes prompting investigation into whether cytochrome P450 overexpression potentiates adrenocortical carcinoma chemoresistance. METHODS: We determined the expression patterns of 6 cytochrome P450 genes (CYP2A6, CYP2A7, CYP2A13, CYP2B6, CYP2S1, and CYP4F2) predicted to be amplified in adrenocortical carcinoma (n = 29) relative to normal adrenal cortex (n = 10). Gene copy numbers were determined with the TaqMan copy number assay. Gene silencing was performed via small interfering RNA (siRNA) in the adrenocortical carcinoma cell line NCI-H295R and treated with mitotane and cisplatin. RESULTS: Of the 6 cytochrome P450 genes tested, CYP2A6 was overexpressed with a 55-fold mean increase compared to normal adrenal samples (P < .05). Immunohistochemical analysis confirmed protein overexpression. Copy gains of CYP2A6 were found in 26% (7/27) of adrenocortical carcinoma specimens. Silencing of CYP2A6 in NCI-H295R cells resulted in decreased cell viability and increased chemosensitivity (P < .05). CONCLUSION: Frequent upregulation in adrenocortical carcinomas and the reversal of chemoresistance in adrenocortical carcinoma cells via enforced silencing suggest a role for CYP2A6 in adrenocortical malignancy.

Regression in thin melanoma is associated with nodal recurrence after a negative sentinel node biopsy.

Prognostic markers for nodal metastasis in thin melanoma patients are debated. We present a single institution study looking at factors predictive of nodal disease in thin melanoma patients. Retrospective review from 1997 to 2012 identified 252 patients with thin melanoma (≤1 mm) who underwent a sentinel lymph node biopsy (SLNB). Node-positive patients included positive SLNB patients and negative SLNB patients who developed a nodal recurrence (false-negative SLNB). Clinicopathologic characteristics were correlated with nodal status and outcome. Median follow-up was 45.5 months. Twelve of 252 patients (4.8%) were node-positive including six positive SLNB (2.4%) and six false-negative SLNB (2.4%) patients. No clinicopathologic factors were significantly correlated with nodal disease. For the six false-negative SLNB patients, median time to nodal recurrence was 37.5 months. Regression was seen in only 16% of cases, but the rate increased to 60% for false-negative SLNB cases. Both age (odds ratio [OR]: 1.09, 95% CI: 1.01-1.17; P = 0.02) and regression (OR: 8.33, 95% CI: 1.34-52.63; P = 0.02) were significantly associated with nodal recurrence after a negative SLNB on univariable analysis. Nodal disease in thin melanoma patients was seen in 4.8% of cases. Although regression was not correlated with nodal metastasis, it was correlated with a false-negative SLNB. Patients with thin melanoma and regression may need more intensive surveillance after a negative SLNB. Further study is needed to determine if the same immune mechanisms that result in regression in primary tumors also lead to regression in lymph nodes, which may decrease detection of melanoma nodal metastases.

Shifting patterns of genomic variation in the somatic evolution of papillary thyroid carcinoma.

BACKGROUND: Cancer is increasingly understood to arise in the context of dynamically evolving genomes with continuously generated variants subject to selective pressures. Diverse mutations have been identified in papillary thyroid carcinoma (PTC), but unifying theories underlying genomic change are lacking. Applying a framework of somatic evolution, we sought to broaden understanding of the PTC genome through identification of global trends that help explain risk of tumorigenesis. METHODS: Exome sequencing was performed on 53 PTC and matched adjacent non-tumor thyroid tissues (ANT). Single nucleotide substitution (SNS) signatures from each sample pair were divided into three subsets based on their presence in tumor, non-tumor thyroid, or both. Nine matched blood samples were sequenced and SNS signatures intersected with these three subsets. The intersected genomic signatures were used to define branch-points in the evolution of the tumor genome, distinguishing variants present in the tissues' common ancestor cells from those unique to each tissue type and therefore acquired after genomic divergence of the tumor, non-tumor, and blood samples. RESULTS: Single nucleotide substitutions shared by the tumor and the non-tumor thyroid were dominated by C-to-T transitions, whereas those unique to either tissue type were enriched for C-to-A transversions encoding non-synonymous, predicted-deleterious variants. On average, SNSs of matched blood samples were 81 % identical to those shared by tumor and non-tumor thyroid, but only 12.5 % identical to those unique to either tissue. Older age and BRAF mutation were associated with increased SNS burden. CONCLUSIONS: The current study demonstrates novel patterns of genomic change in PTC, supporting a theory of somatic evolution in which the zygote's germline genome undergoes continuous remodeling to produce progressively differentiated, tissue-specific signatures. Late somatic events in thyroid tissue demonstrate shifted mutational spectra compared to earlier polymorphisms. These late events are enriched for predicted-deleterious variants, suggesting a mechanism of genomic instability in PTC tumorigenesis.

DNA copy amplification and overexpression of SLC12A7 in adrenocortical carcinoma.

BACKGROUND: Overexpression of Solute carrier family 12 member 7 (SLC12A7) promotes tumor aggressiveness in various cancers. Previous studies have identified the 5p15.33 region, containing the SLC12A7 locus, as being amplified frequently in adrenocortical carcinoma (ACC). Copy number amplifications (CNAs) may alter gene expression levels and occur frequently in ACC; however, SLC12A7 gene amplifications or expression levels have not been studied in ACC. METHODS: Fifty-five cases of clinically well-characterized ACCs were recruited for this study. Whole-exome sequencing was used to predict CNAs in 19 samples. CNA analysis was performed on an expanded cohort of 26 samples with the use of TaqMan Copy Number Assays. SLC12A7 mRNA expression was analyzed in 32 samples with real-time quantitative polymerase chain reaction and protein expression was assessed by immunohistochemistry. SLC12A7 CNAs and expression patterns were evaluated for correlation with patient and tumor characteristics. RESULTS: Whole-exome sequencing and TaqMan Copy Number Assays demonstrated SLC12A7 amplifications in 68.4% and 65.4% of ACCs tested, respectively. Furthermore, SLC12A7 copy gains were associated with increased gene expression (P < .05) and non-functional tumors (P < .05). SLC12A7 gene expression levels were increased in ACCs compared with normal adrenal tissue (P < .05). CONCLUSION: SLC12A7 gene amplification and overexpression occurs frequently in ACCs and may represent a novel molecular event associated with ACC.

Chromosome 19 amplification correlates with advanced disease in adrenocortical carcinoma.

BACKGROUND: Familial syndromes with specific genetic drivers account for a subset of adrenocortical carcinomas (ACCs), but the genomic underpinnings of sporadic cases remain poorly understood. Recent advances in copy number variation (CNV) prediction from exome sequencing are facilitating exploration of genomic rearrangements common to these carcinomas. METHODS: ACC and matched, nontumor samples underwent exome sequencing. CNVs were predicted using coverage-depth comparison. Clinicopathologic characteristics of amplification- and deletion-dominant samples were compared and pathway enrichment analysis performed for regions with significant variation. RESULTS: CNVs are distributed broadly across the ACC genome. Individual signatures demonstrate amplification or deletion dominance. Areas of recurrent amplification include chromosomes 5, 12, 19, and 20, whereas chromosomes 1, 10, 18, and 22 are deletion prone. Large-scale amplification of chromosome 19 occurred in 12 of 19 cases (63%), including 6 of 8 amplification-dominant samples (75%) and was associated with stage III/IV disease (P = .002). Genes within this amplified region are overrepresented among the adrenal hyperplasia and steroid biosynthesis pathways (P = 4.2(-5) and 2.5(-5), respectively). CONCLUSION: CNV detection via exome sequencing allows high-resolution cataloging of structural variations in ACC. Large-scale, recurrent amplifications encompassing known adrenal-specific gene pathways correlate with tumor stage. Further functional analysis of individual genes within these regions could provide mechanistic insight into specific drivers underlying pathogenesis and progression of ACC.

Whole-exome sequencing defines the mutational landscape of pheochromocytoma and identifies KMT2D as a recurrently mutated gene.

As subsets of pheochromocytomas (PCCs) lack a defined molecular etiology, we sought to characterize the mutational landscape of PCCs to identify novel gene candidates involved in disease development. A discovery cohort of 15 PCCs wild type for mutations in PCC susceptibility genes underwent whole-exome sequencing, and an additional 83 PCCs served as a verification cohort for targeted sequencing of candidate mutations. A low rate of nonsilent single nucleotide variants (SNVs) was detected (6.1/sample). Somatic HRAS and EPAS1 mutations were observed in one case each, whereas the remaining 13 cases did not exhibit variants in established PCC genes. SNVs aggregated in apoptosis-related pathways, and mutations in COSMIC genes not previously reported in PCCs included ZAN, MITF, WDTC1, and CAMTA1. Two somatic mutations and one constitutional variant in the well-established cancer gene lysine (K)-specific methyltransferase 2D (KMT2D, MLL2) were discovered in one sample each, prompting KMT2D screening using focused exome-sequencing in the verification cohort. An additional 11 PCCs displayed KMT2D variants, of which two were recurrent. In total, missense KMT2D variants were found in 14 (11 somatic, two constitutional, one undetermined) of 99 PCCs (14%). Five cases displayed somatic mutations in the functional FYR/SET domains of KMT2D, constituting 36% of all KMT2D-mutated PCCs. KMT2D expression was upregulated in PCCs compared to normal adrenals, and KMT2D overexpression positively affected cell migration in a PCC cell line. We conclude that KMT2D represents a recurrently mutated gene with potential implication for PCC development.

Pathologic leadpoint is uncommon in ileo-colic intussusception regardless of age.

PURPOSE: Historically, the rate of pathologic leadpoints in older children with intussusception is quoted as 20%-25%. Our anecdotal experience suggested a lower rate. We therefore compiled a case series to examine the actual incidence of pathologic leadpoint, and treatment success, by age. METHODS: A retrospective review was performed of all patients admitted with intussusception between 1998 and 2012 and tested for differences in anatomic location, presence of pathologic leadpoint, and need for operative intervention, on the basis of age. RESULTS: In total, 154 cases of intussusception were diagnosed in 141 patients (136 ileo-colic), 38 of which were in children older than 3 (29 ileo-colic). Considering all anatomic locations, older children were more likely to have a pathologic leadpoint (p-value 0.01); however subgroup analysis of ileo-colic intussusception demonstrated no difference (p-value 0.38). Additionally, there was no difference in the success of pneumatic or barium enema reduction on the basis of age (p-value 0.56). CONCLUSION: Despite historical reports of increased pathologic leadpoints in ileo-colic intussusception in older children, in this series the majority were idiopathic. Non-operative management was successful approximately 75% of the time, irrespective of age. In older age groups, there was an increased frequency of pathologic leadpoints in small bowel-small bowel intussusception.

Characterization of the mutational landscape of anaplastic thyroid cancer via whole-exome sequencing.

Anaplastic thyroid carcinoma (ATC) is a frequently lethal malignancy that is often unresponsive to available therapeutic strategies. The tumorigenesis of ATC and its relationship to the widely prevalent well-differentiated thyroid carcinomas are unclear. We have analyzed 22 cases of ATC as well as 4 established ATC cell lines using whole-exome sequencing. A total of 2674 somatic mutations (121/sample) were detected. Ontology analysis revealed that the majority of variants aggregated in the MAPK, ErbB and RAS signaling pathways. Mutations in genes related to malignancy not previously associated with thyroid tumorigenesis were observed, including mTOR, NF1, NF2, MLH1, MLH3, MSH5, MSH6, ERBB2, EIF1AX and USH2A; some of which were recurrent and were investigated in 24 additional ATC cases and 8 ATC cell lines. Somatic mutations in established thyroid cancer genes were detected in 14 of 22 (64%) tumors and included recurrent mutations in BRAF, TP53 and RAS-family genes (6 cases each), as well as PIK3CA (2 cases) and single cases of CDKN1B, CDKN2C, CTNNB1 and RET mutations. BRAF V600E and RAS mutations were mutually exclusive; all ATC cell lines exhibited a combination of mutations in either BRAF and TP53 or NRAS and TP53. A hypermutator phenotype in two cases with >8 times higher mutational burden than the remaining mean was identified; both cases harbored unique somatic mutations in MLH mismatch-repair genes. This first comprehensive exome-wide analysis of the mutational landscape of ATC identifies novel genes potentially associated with ATC tumorigenesis, some of which may be targets for future therapeutic intervention.