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Assessment of internal tandem duplications in FLT3(FLT3-ITDs) and their allelic ratio (AR) is recommended by clinical guidelines for diagnostic workup of acute myeloid leukemia and traditionally performed through capillary electrophoresis (CE). Although significant progress has been made integrating FLT3-ITD detection within contemporary next-generation sequencing (NGS) panels, AR estimation is not routinely part of clinical NGS practice because of inherent biases and challenges. In this study, data from multiple NGS platforms—anchored multiplex PCR (AMP), amplicon [TruSeq Custom Amplicon (TSCA)], and hybrid-capture—were analyzed through a custom algorithm, including platform-specific measures of AR. Sensitivity and specificity of NGS for FLT3-ITD status relative to CE were 100% (42/42) and 99.4% (1076/1083), respectively, by AMP on an unselected cohort and 98.1% (53/54) and 100% (48/48), respectively, by TSCA on a selected cohort. Primer analysis identified criteria for ITDs to escape detection by TSCA, estimated to occur in approximately 9% of unselected ITDs. Allelic fractions under AMP or TSCA were highly correlated to CE, with linear regression slopes near 1 for ITDs not duplicating primers, and systematically underestimated for ITDs duplicating a primer. Bias was alleviated in AMP through simple adjustments. This article provides an approach for targeted computational FLT3-ITD analysis for NGS data from multiple platforms; AMP was found capable of near perfect sensitivity and specificity with relatively accurate estimates of ARs.

Pleomorphic LCIS (P-LCIS) and florid LCIS (F-LCIS) are morphologic variants distinguished from classic LCIS by marked nuclear pleomorphism and/or an expansile growth pattern with or without necrosis. Given the rarity of these LCIS variants, little data exist regarding their molecular pathogenesis, natural history, and optimal management. The purpose of this study was to genomically profile LCIS variants to gain further insight into their biology. Nineteen cases of pure LCIS variants (17 P-LCIS, 2 F-LCIS) diagnosed on core needle biopsy at our institution from 2006 to 2017 were included, five of which were upgraded to invasive cancer at excision. Macrodissected lesions were analyzed by a hybrid-capture next generation sequencing assay that surveyed exonic sequences of 447 genes for mutations and copy number variations (CNVs) and 191 regions across 60 genes for structural rearrangements. LCIS variants were all confirmed as E-cadherin negative by immunohistochemistry. Receptor profiles among the 17 P-LCIS cases included HR+/HER2− (nine cases), HR+/HER2+ (three cases), HR−/HER2+ (two cases), and HR−/HER2− (three cases). The two F-LCIS cases were HR+/HER2− and HR+/HER2+. All LCIS variants had genetic alterations consistent with a lobular phenotype including 1q gain (16 cases), 16q loss (18 cases), and CDH1mutations (18 cases). Highly recurrent ERBB2alterations were noted including mutations (13 cases) and amplifications (six cases). Other significant alterations included mutations in PIK3CA(six cases), RUNX1(four cases), ERBB3(four cases), and CBFB(three cases), as well as amplification of CCND1(five cases). A TP53mutation was identified in one case of HR−/HER2+ P-LCIS with signet ring cell features that lacked 1q gain and 16q loss. P-LCIS and F-LCIS contain genetic alterations characteristic of lobular neoplasia; however, these LCIS variants are distinguished from classical LCIS reported in the literature by their highly recurrent ERBB2alterations.

Keratocystic odontogenic tumors (KCOTs) are locally aggressive odontogenic neoplasms with recurrence rates of up to 60%. Approximately 5% of KCOTs are associated with nevoid basal cell carcinoma (Gorlin) syndrome and 90% of these show genomic inactivation of the PTCH1gene encoding Patched 1. Sporadic KCOTs reportedly have PTCH1mutations in 30% of cases, but previous genomic analyses have been limited by low tumor DNA yield. The aim of this study was to identify recurrent genomic aberrations in sporadic KCOTs using a next-generation sequencing panel with complete exonic coverage of sonic hedgehog (SHH) pathway members PTCH1, SMO, SUFU, GLI1, and GLI2. Included were 44 sporadic KCOTs from 23 female and 21 male patients with a median age of 50 years (range, 10 to 82 y) and located in the mandible (N=33) or maxilla (N=11). Sequencing identified PTCH1inactivating mutations in 41/44 (93%) cases, with biallelic inactivation in 35 (80%) cases; 9q copy neutral loss of heterozygosity targeting the PTCH1locus was identified in 15 (34%) cases. No genomic aberrations were identified in other sequenced SHH pathway members. In summary, we demonstrate PTCH1inactivating mutations in 93% of sporadic KCOTs, indicating that SHH pathway alterations are a near-universal event in these benign but locally aggressive neoplasms. The high frequency of complete PTCH1loss of function may provide a rational target for SHH pathway inhibitors to be explored in future studies.

Background:Service-learning (SL) programs have been shown to be effective for college students in developing positive attitudes toward people with disabilities (PWDs). Such positive attitudes are particularly important for Kinesiology (KIN) students as they are trained to become future health and fitness professionals. Purpose:The present study investigated the effect of a 10-week in-person versus virtual SL program on KIN students’ attitudes toward PWD. Methodology/Approach:A pre- postintervention, three-way, analysis of variance was utilized. This 3  ×  2 mixed model design was utilized to assess students’ attitudes toward PWD with three independent variables (in-person program, virtual program, and control) on two dependent variables (pre and post). Findings/Conclusions:There was significant Group × Time interaction. The between-group comparison showed a significant increase in Attitudes Toward Disabled Person scale score in the in-person SL group (p< .05) compared to non-SL group. There were no significant differences in the virtual SL group. Implications:These results indicate that in-person SL is effective for improving students’ attitudes toward PWD, whereas virtual programs may not be as effective as in-person. In-person SL programs for KIN students may positively improve their attitudes toward PWD thus ameliorating interpersonal skills and creating more inclusive health and fitness professionals. KIN programs should consider SL programs with an emphasis on PWDs.

Supplemental Digital Content is available in the text.Female adnexal tumor of probable Wolffian origin (FATWO) is a rare gynecologic neoplasm of low-malignant potential presumed to be derived from mesonephric remnants in the upper female genital tract. Similarly, mesonephric remnants in the lower female genital tract are thought to be the origin for mesonephric carcinoma. Although the molecular alterations in mesonephric carcinoma have been recently reported, the pathogenesis of and molecular alterations in FATWO are not well understood. The aims of this study were to examine the molecular alterations in FATWO and to establish whether these neoplasms are molecularly similar to mesonephric carcinoma. Eight FATWOs underwent massively parallel sequencing to detect single nucleotide variations, copy number variations, and structural variants by surveying exonic DNA sequences of 300 cancer genes and 113 introns across 35 genes. Good quality DNA was isolated from 7 of 8 cases. Novel KMT2Dvariants (1 frameshift, 3 missense) were identified in 4 of 7 cases (57%), but were variants of uncertain biologic significance. STK11mutations (both frameshift) were identified in 2 of 7 cases (29%); one of these was in a patient with a known history of Peutz-Jeghers syndrome. A mutation in the chromatin remodeling gene ARID1Bwas identified in 1 of 7 cases (14%). No cases harbored KRAS, NRAS, TP53, PIK3CA, PTEN, or DICER1mutations. There were relatively low numbers of copy number variations, and no recurrent copy number variations were identified. One case demonstrated moderate copy gain of CCND1. No structural variants were identified. In summary, FATWO is characterized molecularly by the absence of KRAS/NRASmutations (characteristic of mesonephric carcinoma), absence of DICER1mutations (characteristic of Sertoli-Leydig cell tumor) and frequent KMT2Dmutations of unknown biologic significance. FATWOs exhibit a limited number of molecular aberrations that are significantly different from those reported in tumors in the differential diagnosis, and our results question the relationship of mesonephric carcinoma with FATWO. Disease-defining molecular alterations for FATWO have yet to be discovered.

Epithelioid malignant peripheral nerve sheath tumors (EMPNST) are characterized by diffuse S-100 and SOX10 positivity, frequent immunohistochemical loss of SMARCB1 expression (70%), and rare association with neurofibromatosis type 1. Some cases arise in a preexisting epithelioid schwannoma (ESCW), which also show SMARCB1 loss in 40% of cases. To date, little is known about the genomic landscape of this distinctive variant of malignant peripheral nerve sheath tumor. The aim of this study was to use targeted next-generation sequencing to identify recurrent genomic aberrations in EMPNST and a subset of ESCW, including the basis of SMARCB1 loss. Sixteen EMPNSTs (13 SMARCB1-lost, 3 SMARCB1-retained) and 5 ESCWs with SMARCB1 loss were selected for the cohort. Sequencing identified SMARCB1gene inactivation in 12/16 (75%) EMPNST and all 5 (100%) ESCW through homozygous deletion (N=8), nonsense (N=7), frameshift (N=2), or splice site (N=2) mutations; 2 EMPNSTs harbored 2 concurrent mutations each. SMARCB1 immunohistochemistry status and SMARCB1alterations were concordant in 20/21 of the sequenced tumors. Additional genetic alterations in a subset of EMPNST included inactivation of CDKN2Aand gain of chromosome 2q. Among SMARCB1-wild-type EMPNSTs there were single cases each with NF1and NF2mutations. No cases had SUZ12or EEDmutations. In summary, we identified recurrent SMARCB1alterations in EMPNST (and all 5 SMARCB1-negative ESCWs tested), supporting loss of SMARCB1tumor suppressor function as a key oncogenic event. SMARCB1-retained EMPNSTs lack SMARCB1mutations and harbor different driver events.

Supplemental digital content is available in the text.

Human papillomavirus-negative keratinizing vulvar cancers typically harbor TP53mutations as do their precursors, differentiated vulvar intraepithelial neoplasia. However, atypical verruciform proliferations are also associated with these malignancies and their pathogenesis is poorly understood. This study compared 11 atypical verruciform lesions, including atypical verruciform hyperplasia, vulvar acanthosis with altered differentiation, and verruciform lichen simplex chronicus, with 14 human papillomavirus-negative keratinizing squamous cell carcinomas. Extracted tissue DNA was subjected to targeted massively parallel sequencing of the exonic regions of 300 genes. Eight (73%) and six (55%) of eleven atypical verruciform lesions contained mutations in PIK3CAand ARID2, respectively. No TP53mutations were identified. Eleven (79%) and five (36%) of fourteen keratinizing squamous cell carcinomas tested contained TP53and CDKN2Amutations, respectively. Keratinizing squamous cell carcinomas displayed the majority of copy number variations with some variations (7p gain and 8p loss) shared by some cases in both groups. One patient developed atypical verruciform lesions with PIK3CAmutations followed by a keratinizing carcinoma with mutations in both PIK3CAand TP53. This study, for the first time segregates atypical verruciform lesions by virtue of a unique genotype (PIK3CAmutant/TP53wild type) illustrating an example of progression to a TP53-mutated keratinizing carcinoma. The findings indicate that although PIK3CAmutations are found in <10% of vulvar squamous cell carcinomas, they may be specific for a particular pathway involving atypical verruciform lesions, which could function as either a direct precursor or a risk factor for vulvar squamous cell carcinoma. Given the presence of a molecular signature, we propose the term ‘differentiated exophytic vulvar intraepithelial lesion’ for this group. Whether they function as direct precursors to a less common form of squamous cell carcinoma will require further study, but carcinomas associated with these lesions might warrant testing for PIK3CAmutations to address this question.

Mucinous differentiation is observed in a subset of lung adenocarcinomas with unique clinical and pathological features, but the biology of these neoplasms is poorly understood.

Mesonephric carcinoma is a rare form of gynecologic cancer derived from mesonephric remnants usually located in the lateral wall of the uterine cervix. An analogous tumor occurs in the adnexa, female adnexal tumor of probable Wolffian origin. The pathogenesis and molecular events in mesonephric carcinoma are not known. The aim of this study was to examine the molecular alterations in mesonephric carcinoma to identify driver mutations and therapeutically targetable mutations. This study consisted of 19 tumors from 17 patients: 18 mesonephric carcinomas (15 primary tumors and three metastatic tumors) and 1 female adnexal tumor of probable Wolffian origin. In two patients, both primary and metastatic tumors were available. Genomic DNA was isolated and targeted next-generation sequencing was performed to detect mutations, copy number variations, and structural variants by surveying full exonic regions of 300 cancer genes and 113 selected intronic regions across 35 genes. Fluorescence in situhybridization (FISH) for 1p and 1q was performed in two cases. Eighty-one percent (13/16) of mesonephric carcinomas had either a KRAS(n=12) or NRAS(n=1) mutation. Mutations in chromatin remodeling genes (ARID1A, ARID1B, or SMARCA4) were present in 62% of mesonephric carcinomas. All mesonephric carcinomas lacked mutations in PIK3CAand PTEN. The most common copy number alteration was 1q gain, found in 12 (75%) mesonephric carcinomas; this was confirmed by FISH in two cases. Mesonephric carcinoma is characterized by molecular alterations that differ from those of more common variants of cervical and endometrial adenocarcinoma, which harbor KRAS/NRASmutations in 7% and 25% of cases, respectively. KRAS/NRASmutations are common in mesonephric carcinoma and are often accompanied by gain of 1q and mutations in chromatin remodeling genes. Targeting inhibitors of the RAS/MAPK pathway may be useful in the treatment of mesonephric carcinoma.

Mesonephric carcinoma is a rare form of gynecologic cancer derived from mesonephric remnants usually located in the lateral wall of the uterine cervix. An analogous tumor occurs in the adnexa, female adnexal tumor of probable Wolffian origin. The pathogenesis and molecular events in mesonephric carcinoma are not known. The aim of this study was to examine the molecular alterations in mesonephric carcinoma to identify driver mutations and therapeutically targetable mutations. This study consisted of 19 tumors from 17 patients: 18 mesonephric carcinomas (15 primary tumors and three metastatic tumors) and 1 female adnexal tumor of probable Wolffian origin. In two patients, both primary and metastatic tumors were available. Genomic DNA was isolated and targeted next-generation sequencing was performed to detect mutations, copy number variations, and structural variants by surveying full exonic regions of 300 cancer genes and 113 selected intronic regions across 35 genes. Fluorescence in situ hybridization (FISH) for 1p and 1q was performed in two cases. Eighty-one percent (13/16) of mesonephric carcinomas had either a KRAS (n=12) or NRAS (n=1) mutation. Mutations in chromatin remodeling genes (ARID1A, ARID1B, or SMARCA4) were present in 62% of mesonephric carcinomas. All mesonephric carcinomas lacked mutations in PIK3CA and PTEN. The most common copy number alteration was 1q gain, found in 12 (75%) mesonephric carcinomas; this was confirmed by FISH in two cases. Mesonephric carcinoma is characterized by molecular alterations that differ from those of more common variants of cervical and endometrial adenocarcinoma, which harbor KRAS/NRAS mutations in 7% and 25% of cases, respectively. KRAS/NRAS mutations are common in mesonephric carcinoma and are often accompanied by gain of 1q and mutations in chromatin remodeling genes. Targeting inhibitors of the RAS/MAPK pathway may be useful in the treatment of mesonephric carcinoma.

Molecular testing of tumors from formalin-fixed paraffin-embedded (FFPE) tissue blocks is central to clinical practice; however, it requires histology support and increases test turnaround time. Prospective fresh frozen tissue collection requires special handling, additional storage space, and may not be feasible for small specimens. Filter paper–based collection of tumor DNA reduces the need for histology support, requires little storage space, and preserves high-quality nucleic acid. We investigated the performance of tumor smears on filter paper in solid tumor genotyping, as compared with paired FFPE samples. Whatman FTA Micro Card (FTA preps) smears were prepared from 21 fresh tumor samples. A corresponding cytology smear was used to assess tumor cellularity and necrosis. DNA was isolated from FTA preps and FFPE core samples using automated methods and quantified using SYBR green dsDNA detection. Samples were genotyped for 471 mutations on a mass spectrophotometry–based platform (Sequenom). DNA concentrations from FTA preps and FFPE correlated for untreated carcinomas but not for mesenchymal tumors (Spearman =0.39 and =−0.1, respectively). Average DNA concentrations were lower from FTA preps as compared with FFPE, but DNA quality was higher with less fragmentation. Seventy-six percent of FTA preps and 86 of FFPE samples generated adequate DNA for genotyping. FTA preps tended to perform poorly for collection of DNA from pretreated carcinomas and mesenchymal neoplasms. Of the 16 paired DNA samples that were genotyped, 15 (94) gave entirely concordant results. Filter paper–based sample preservation is a feasible alternative to FFPE for use in automated, high-throughput genotyping of carcinomas.

IMPORTANCE: Port-wine stains (PWSs) are common congenital cutaneous capillary malformations. A somatic GNAQ mutation was recently identified in patients with sporadic PWSs and Sturge-Weber syndrome. However, subsequent studies to confirm or extend this observation are lacking. OBSERVATIONS: We report a long-standing, unilateral facial PWS of a man in his early 70s confirmed by histopathological analysis. Staged surgical excision of the vascular malformation was performed, and genomic DNA was extracted from the vascular malformation specimen and normal skin. Targeted next-generation sequencing of the coding sequence of 275 known cancer genes including GNAQ was performed in both specimens. A single-nucleotide variant (c.548G>A, p.Arg183Gln) in GNAQ was identified in the PWS-affected tissue but not in the normal skin sample. In addition, this sequencing approach uncovered several additional novel somatic mutations in the genes SMARCA4, EPHA3, MYB, PDGFR-β, and PIK3CA. CONCLUSIONS AND RELEVANCE: Our findings confirm the presence of somatic mutations in GNAQ in the affected skin of a patient with congenital PWS, as well as alterations in several other novel genes of possible importance in the pathogenesis of PWS that may also offer substantial therapeutic targets.

Ongoing cancer genome characterization studies continue to elucidate the spectrum of genomic abnormalities that drive many cancers, and in the clinical arena assessment of the driver genetic alterations in patients is playing an increasingly important diagnostic and/or prognostic role for many cancer types. However, the landscape of genomic abnormalities is still unknown for less common cancers, and the influence of specific genotypes on clinical behavior is often still unclear. To address some of these deficiencies, we developed Profile, a prospective cohort study to obtain genomic information on all patients at a large tertiary care medical center for cancer-related care. We enrolled patients with any cancer diagnosis, and, for each patient (unselected for cancer site or type) we applied mass spectrometric genotyping (OncoMap) of 471 common recurrent mutations in 41 cancer-related genes. We report the results of the first 5000 patients, of which 26% exhibited potentially actionable somatic mutations. These observations indicate the utility of genotyping in advancing the field of precision oncology.

Metastasis to multiple organs is the primary cause of mortality in breast cancer patients. The poor prognosis for patients with metastatic breast cancer and toxic side effects of currently available treatments necessitate the development of effective tumor‐selective therapies. Neural stem cells (NSCs) possess inherent tumor tropic properties that enable them to overcome many obstacles of drug delivery that limit effective chemotherapy strategies for breast cancer. We report that increased NSC tropism to breast tumor cell lines is strongly correlated with the invasiveness of cancer cells. Interleukin 6 (IL‐6) was identified as a major cytokine mediating NSC tropism to invasive breast cancer cells. We show for the first time in a preclinical mouse model of metastatic human breast cancer that NSCs preferentially target tumor metastases in multiple organs, including liver, lung, lymph nodes, and femur, versus the primary intramammary fat pad tumor. For proof‐of‐concept of stem cell‐mediated breast cancer therapy, NSCs were genetically modified to secrete rabbit carboxylesterase (rCE), an enzyme that activates the CPT‐11 prodrug to SN‐38, a potent topoisomerase I inhibitor, to effect tumor‐localized chemotherapy. In vitro data demonstrate that exposure of breast cancer cells to conditioned media from rCE‐secreting NSCs (NSC.rCE) increased their sensitivity to CPT‐11 by 200‐fold. In vivo, treatment of tumor‐bearing mice with NSC.rCE cells in combination with CPT‐11 resulted in reduction of metastatic tumor burden in lung and lymph nodes. These data suggest that NSC‐mediated enzyme/prodrug therapy may be more effective and less toxic than currently available chemotherapy strategies for breast cancer metastases. STEMCELLS2012; 30:314–325.

Proton and C-13 NMR spectroscopic studies of possible interactions of the copper electroplating accelerant disodium bis(3-sulfopropyl)disulfide (SPS) and its reduced form sodium 3-mercaptopropylsulfonate (MPS) with both cuprous chloride and cupric chloride were carried out in with reagent concentrations between millimolar and under anaerobic conditions. It was confirmed that MPS reacted with CuCl to yield Cu(I)-thiolate products. No evidence for formation of a solution Cu(I)-SPS complex from CuCl and SPS was found. Instead, a redox reaction yielded a Cu(I)-thiolate along with Cu(II) with an estimated equilibrium constant of . The previously reported reaction of aqueous cupric chloride with MPS was investigated further under anaerobic conditions. In addition to SPS, Cu(I)-thiolate products similar to those from the CuCl/MPS reaction were identified. The reaction was found to be complete only upon addition of over 2 equiv of MPS. No evidence for any interaction between and SPS was observed.

Objective The goal of this study was to confirm the reported association between a noncoding SNP (rs642961) in IRF6 and nonsyndromic cleft lip and palate.Design, Setting, and Participants Two SNPs in IRF6 (rs2235371 and rs64296) were genotyped in Hispanic and non-Hispanic white multiplex (122) and simplex (308) nonsyndromic cleft lip and palate families. Linkage and family-based association analyses were performed on the individual SNPs as well as the 2-SNP haplotype.Results Only modest evidence was found for an association with rs642961 and the 2-SNP haplotype. In contrast, strong evidence was found for the association with rs2235371; this was most evident in the non-Hispanic white simplex families.Conclusions Although it is confirmed that variation in IRF6 is associated with nonsyndromic cleft lip and palate, the results do not support the reported association with SNP rs64296. Importantly, the association varies between ethnic groups. This finding underscores the need for evaluating additional variations in IRF6 across multiple populations to better determine its role in nonsyndromic cleft lip and palate.

BACKGROUNDNonsyndromic cleft lip with or without cleft palate is a common birth defect. Although a number of susceptibility loci have been reported, replication has often been lacking. This is likely due, in part, to the heterogeneity of datasets and methodologies. Two independent genomewide association studies of individuals of largely western European extraction have identified a possible susceptibility locus on 8q24.21.METHODSTo determine the overall effect of this locus, we genotyped six of the previously associated single nucleotide polymorphisms in our Hispanic and nonHispanic white familybased datasets and evaluated them for linkage and association. In addition, we genotyped a large African American family with nonsyndromic cleft lip with or without cleft palate that we had previously mapped to the 8q21.324.12 region to test for linkage.RESULTSThere was no evidence for linkage to this region in any of the three ethnic groups. Nevertheless, strong evidence for association was noted in the nonHispanic white group, whereas none was detected in the Hispanic dataset.CONCLUSIONThese results confirm the previously reported association and provide evidence suggesting that there is ethnically based heterogeneity for this locus. Birth Defects Research Part A, 2010. © 2010 WileyLiss, Inc.