Jill C. Rubinstein

Incidence of the V600K mutation among melanoma patients with BRAF mutations, and potential therapeutic response to the specific BRAF inhibitor PLX4032

Activating mutations in BRAF kinase are common in melanomas. Clinical trials with PLX4032, the mutant-BRAF inhibitor, show promising preliminary results in patients selected for the presence of V600E mutation. However, activating V600K mutation is the other most common mutation, yet patients with this variant are currently excluded from the PLX4032 trials. Here we present evidence that a patient bearing the BRAF V600K mutation responded remarkably to PLX4032, suggesting that clinical trials should include all patients with activating BRAF V600E/K mutations.

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.

Loss of epigenetic silencing in tumors preferentially affects primate-specific retroelements.

Close to 50% of the human genome harbors repetitive sequences originally derived from mobile DNA elements, and in normal cells, this sequence compartment is tightly regulated by epigenetic silencing mechanisms involving chromatin-mediated repression. In cancer cells, repetitive DNA elements suffer abnormal demethylation, with potential loss of silencing. We used a genome-wide microarray approach to measure DNA methylation changes in cancers of the head and neck and to compare these changes to alterations found in adjacent non-tumor tissues. We observed specific alterations at thousands of small clusters of CpG dinucleotides associated with DNA repeats. Among the 257,599 repetitive elements probed, 5% to 8% showed disease-related DNA methylation alterations. In dysplasia, a large number of local events of loss of methylation appear in apparently stochastic fashion. Loss of DNA methylation is most pronounced for certain members of the SVA, HERV, LINE-1P, AluY, and MaLR families. The methylation levels of retrotransposons are discretely stratified, with younger elements being highly methylated in healthy tissues, while in tumors, these young elements suffer the most dramatic loss of methylation. Wilcoxon test statistics reveals that a subset of primate LINE-1 elements is demethylated preferentially in tumors, as compared to non-tumoral adjacent tissue. Sequence analysis of these strongly demethylated elements reveals genomic loci harboring full length, as opposed to truncated elements, while possible enrichment for functional LINE-1 ORFs is weaker. Our analysis suggests that, in non-tumor adjacent tissues, there is generalized and highly variable disruption of epigenetic control across the repetitive DNA compartment, while in tumor cells, a specific subset of LINE-1 retrotransposons that arose during primate evolution suffers the most dramatic DNA methylation alterations.

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.

Primary Low-Grade Fibromyxoid Sarcoma of the Kidney in a Child with the Alternative EWSR1-CREB3L1 Gene Fusion

We present the case of a 6-year-old boy with a deceptively bland spindle cell renal neoplasm found to harbor the EWSR1-CREB3L1 gene fusion. This fusion has recently been described as a variant translocation in low-grade fibromyxoid sarcoma (LGFMS), a tumor more typically characterized by a recurrent t(7;16) chromosomal translocation, resulting in the fusion of FUS and CREB3L2 genes. LGFMS is an indolent tumor with late metastatic potential and a propensity for long-term disease recurrence. The tumor is rare in children, with only 33 published cases. In the pediatric population, it has not previously been reported arising in the kidney.

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.

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.

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.

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.