A Multiplexed Amplicon Approach for Detecting Gene Fusions by Next-Generation Sequencing.

Chromosomal rearrangements that result in oncogenic gene fusions are clinically important drivers of many cancer types. Rapid and sensitive methods are therefore needed to detect a broad range of gene fusions in clinical specimens that are often of limited quantity and quality. We describe a next-generation sequencing approach that uses a multiplex PCR-based amplicon panel to interrogate fusion transcripts that involve 19 driver genes and 94 partners implicated in solid tumors. The panel also includes control assays that evaluate the 3'/5' expression ratios of 12 oncogenic kinases, which might be used to infer gene fusion events when the partner is unknown or not included on the panel. There was good concordance between the solid tumor fusion gene panel and other methods, including fluorescence in situ hybridization, real-time PCR, Sanger sequencing, and other next-generation sequencing panels, because 40 specimens known to harbor gene fusions were correctly identified. No specific fusion reads were observed in 59 fusion-negative specimens. The 3'/5' expression ratio was informative for fusions that involved ALK, RET, and NTRK1 but not for BRAF or ROS1 fusions. However, among 37 ALK or RET fusion-negative specimens, four exhibited elevated 3'/5' expression ratios, indicating that fusions predicted solely by 3'/5' read ratios require confirmatory testing.

Molecular profiling and targeted therapy for advanced thoracic malignancies: a biomarker-derived, multiarm, multihistology phase II basket trial.

PURPOSE: We conducted a basket clinical trial to assess the feasibility of such a design strategy and to independently evaluate the effects of multiple targeted agents against specific molecular aberrations in multiple histologic subtypes concurrently. PATIENTS AND METHODS: We enrolled patients with advanced non-small-cell lung cancer (NSCLC), small-cell lung cancer, and thymic malignancies who underwent genomic characterization of oncogenic drivers. Patients were enrolled onto a not-otherwise-specified arm and treated with standard-of-care therapies or one of the following five biomarker-matched treatment groups: erlotinib for EGFR mutations; selumetinib for KRAS, NRAS, HRAS, or BRAF mutations; MK2206 for PIK3CA, AKT, or PTEN mutations; lapatinib for ERBB2 mutations or amplifications; and sunitinib for KIT or PDGFRA mutations or amplification. RESULTS: Six hundred forty-seven patients were enrolled, and 88% had their tumors tested for at least one gene. EGFR mutation frequency was 22.1% in NSCLC, and erlotinib achieved a response rate of 60% (95% CI, 32.3% to 83.7%). KRAS mutation frequency was 24.9% in NSCLC, and selumetinib failed to achieve its primary end point, with a response rate of 11% (95% CI, 0% to 48%). Completion of accrual to all other arms was not feasible. In NSCLC, patients with EGFR mutations had the longest median survival (3.51 years; 95% CI, 2.89 to 5.5 years), followed by those with ALK rearrangements (2.94 years; 95% CI, 1.66 to 4.61 years), those with KRAS mutations (2.3 years; 95% CI, 2.3 to 2.17 years), those with other genetic abnormalities (2.17 years; 95% CI, 1.3 to 2.74 years), and those without an actionable mutation (1.85 years; 95% CI, 1.61 to 2.13 years). CONCLUSION: This basket trial design was not feasible for many of the arms with rare mutations, but it allowed the study of the genetics of less common malignancies.

Novel mutations in neuroendocrine carcinoma of the breast: possible therapeutic targets.

Primary neuroendocrine carcinoma of the breast is a rare variant, accounting for only 2% to 5% of diagnosed breast cancers, and may have relatively aggressive behavior. Mutational profiling of invasive ductal breast cancers has yielded potential targets for directed cancer therapy, yet most studies have not included neuroendocrine carcinomas. In a tissue microarray screen, we found a 2.4% prevalence (9/372) of neuroendocrine breast carcinoma, including several with lobular morphology. We then screened primary or metastatic neuroendocrine breast carcinomas (excluding papillary and mucinous) for mutations in common cancer genes using polymerase chain reaction-mass spectroscopy (643 hotspot mutations across 53 genes), or semiconductor-based next-generation sequencing analysis (37 genes). Mutations were identified in 5 of 15 tumors, including 3 with PIK3CA exon 9 E542K mutations, 2 of which also harbored point mutations in FGFR family members (FGFR1 P126S, FGFR4 V550M). Single mutations were found in each of KDR (A1065T) and HRAS (G12A). PIK3CA mutations are common in other types of breast carcinoma. However, FGFR and RAS family mutations are exceedingly rare in the breast cancer literature. Likewise, activating mutations in the receptor tyrosine kinase KDR (VEGFR2) have been reported in angiosarcomas and non-small cell lung cancers; the KDR A1065T mutation is reported to be sensitive to VEGFR kinase inhibitors, and fibroblast growth factor receptor inhibitors are in trials. Our findings demonstrate the utility of broad-based genotyping in the study of rare tumors such as neuroendocrine breast cancer.

Oncogenic mutations in melanomas and benign melanocytic nevi of the female genital tract.

BACKGROUND: The genetic heterogeneity of melanomas and melanocytic nevi of the female genital tract is poorly understood. OBJECTIVE: We aim to characterize the frequency of mutations of the following genes: BRAF, NRAS, KIT, GNA11, and GNAQ in female genital tract melanomas. We also characterize the frequency of BRAF mutations in female genital tract melanomas compared with melanocytic nevi. METHODS: Mutational screening was performed on the following female genital tract melanocytic neoplasms: 25 melanomas, 7 benign melanocytic nevi, and 4 atypical melanocytic nevi. RESULTS: Of the 25 female genital tract melanoma specimens queried, KIT mutations were detected in 4 (16.0%), NRAS mutations in 4 (16.0%), and BRAF mutations in 2 (8.0%) samples. Two of the tumors with KIT mutations harbored double mutations in the same exon. No GNAQ or GNA11 mutations were identified among 11 melanomas screened. BRAF V600E mutations were detected in 7 of 7 benign melanocytic genital nevi (100%) and 3 of 4 atypical genital nevi (75%). LIMITATIONS: Our study is limited by the small sample size of this rare subset of melanomas. CONCLUSION: KIT, NRAS, and BRAF mutations are found in a subset of female genital tract melanomas. Screening for oncogenic mutations is important for developing and applying clinical therapies for melanomas of the female genital tract.

Mutation profiling in gallbladder cancer in Indian population.

AIM: Gallbladder cancer is an aggressive malignancy usually diagnosed at late stage. The molecular genetics of this cancer is heterogeneous and not well established. Mutation profiling of gallbladder cancer was performed through massarray technology with an aim to identify molecular markers involved in the tumor pathogenesis that can be helpful as markers for early diagnosis and targets for therapy. MATERIALS AND METHODS: Forty nine cases of gallbladder cancer were screened through Sequenom Massarray technology for 390 mutations across 30 genes in formalin fixed paraffin embedded archived tissues and the results of mutation profiling was correlated with tumor characteristics. Mutations were observed in 9 of 49 cases across four genes--TP53 (four cases), CTNNB1 (two cases), PIK3CA (two cases), and KRAS (one case). Six of these cases were well differentiated but of eight of them belonged to stage II to IV disease. Six cases had associated gallstones. CONCLUSION: The mutation frequency found in gallbladder cancer is comparable to the data available in literature. Identification of PIK3CA and KRAS mutations would help in formulating more efficacious targeted approach for management. Studies with large number of cases would help in exploring more targets and better classification of these cancers at genetic level.

Adoption of a clinical pharmacogenomics implementation program during outpatient care--initial results of the University of Chicago "1,200 Patients Project".

Pharmacogenomic testing is viewed as an integral part of precision medicine. To achieve this, we originated The 1,200 Patients Project which offers broad, preemptive pharmacogenomic testing to patients at our institution. We analyzed enrollment, genotype, and encounter-level data from the first year of implementation to assess utility of providing pharmacogenomic results. Results were delivered via a genomic prescribing system (GPS) in the form of traffic lights: green (favorable), yellow (caution), and red (high risk). Additional supporting information was provided as a virtual pharmacogenomic consult, including citation to relevant publications. Currently, 812 patients have participated, representing 90% of those approached; 608 have been successfully genotyped across a custom array. A total of 268 clinic encounters have occurred at which results were accessible via the GPS. At 86% of visits, physicians accessed the GPS, receiving 367 result signals for medications patients were taking: 57% green lights, 41% yellow lights, and 1.4% red lights. Physician click frequencies to obtain clinical details about alerts varied according to color severity (100% of red were clicked, 72% yellow, 20% green). For 85% of visits, clinical pharmacogenomic information was available for at least one drug the patient was taking, suggesting relevance of the delivered information. We successfully implemented an individualized health care model of preemptive pharmacogenomic testing, delivering results along with pharmacogenomic decision support. Patient interest was robust, physician adoption of information was high, and results were routinely utilized. Ongoing examination of a larger number of clinic encounters and inclusion of more physicians and patients is warranted.

Frequent phosphatidylinositol-3-kinase mutations in proliferative breast lesions.

The phosphatidylinositol-3-kinase pathway is one of the most commonly altered molecular pathways in invasive breast carcinoma, with phosphatidylinositol-3-kinase catalytic subunit (PIK3CA) mutations in 25% of invasive carcinomas. Ductal carcinoma in situ (DCIS), benign papillomas, and small numbers of columnar cell lesions harbor an analogous spectrum of PIK3CA and AKT1 mutations, yet there is little data on usual ductal hyperplasia and atypical ductal and lobular neoplasias. We screened 192 formalin-fixed paraffin-embedded breast lesions from 75 patients for point mutations using a multiplexed panel encompassing 643 point mutations across 53 genes, including 58 PIK3CA substitutions. PIK3CA point mutations were identified in 31/62 (50%) proliferative lesions (usual ductal hyperplasia and columnar cell change), 10/14 (71%) atypical hyperplasias (atypical ductal hyperplasia and flat epithelial atypia), 7/16 (44%) lobular neoplasias (atypical lobular hyperplasia and lobular carcinoma in situ), 10/21 (48%) DCIS, and 13/37 (35%) invasive carcinomas. In genotyping multiple lesions of different stage from the same patient/specimen, we found considerable heterogeneity; most notably, in 12 specimens the proliferative lesion was PIK3CA mutant but the concurrent carcinoma was wild type. In 11 additional specimens, proliferative epithelium and cancer contained different point mutations. The frequently discordant genotypes of usual ductal hyperplasia/columnar cell change and concurrent carcinoma support a role for PIK3CA-activating point mutations in breast epithelial proliferation, perhaps more so than transformation. Further, these data suggest that proliferative breast lesions are heterogeneous and may represent non-obligate precursors of invasive carcinoma.

Genetic profiling to determine risk of relapse-free survival in high-risk localized prostate cancer.

PURPOSE: The characterization of actionable mutations in human tumors is a prerequisite for the development of individualized, targeted therapy. We examined the prevalence of potentially therapeutically actionable mutations in patients with high-risk clinically localized prostate cancer. EXPERIMENTAL DESIGN: Forty-eight samples of formalin-fixed paraffin-embedded prostatectomy tissue from a neoadjuvant chemotherapy trial were analyzed. DNA extracted from microdissected tumor was analyzed for 643 common solid tumor mutations in 53 genes using mass spectroscopy-based sequencing. In addition, PTEN loss and erythroblast transformation-specific-related gene (ERC) translocations were examined using immunohistochemistry (IHC) in associated tissue microarrays. Association with relapse during 5 years of follow-up was examined in exploratory analyses of the potential clinical relevance of the genetic alterations. RESULTS: Of the 40 tumors evaluable for mutations, 10% had point mutations in potentially actionable cancer genes. Of the 47 tumors evaluable for IHC, 36% had PTEN loss and 40% had ERG rearrangement. Individual mutations were not frequent enough to determine associations with relapse. Using Kaplan-Meier analysis with a log-rank test, the 16 patients who had PTEN loss had a significantly shorter median relapse-free survival, 19 versus 106 months (P = 0.01). CONCLUSIONS: This study confirms that point mutations in the most common cancer regulatory genes in prostate cancer are rare. However, the PIK3CA/AKT pathway was mutated in 10% of our samples. Although point mutations alone did not have a statistically significant association with relapse, PTEN loss was associated with an increased relapse in high-risk prostate cancer treated with chemotherapy followed by surgery.

Frequent PIK3CA mutations in radial scars.

Radial scars are breast lesions of uncertain pathogenesis that are associated with a 2-fold increased risk of breast cancer compared with that in controls. Activating point mutations in PIK3CA are found in 25% to 30% of invasive breast cancers; however, they have not previously been investigated in radial scars. We sought to evaluate radial scars for known activating point mutations commonly seen in invasive breast cancer. Sixteen surgical cases containing 22 radial scars were identified from pathology archives. Lesional tissue was macrodissected from unstained paraffin sections; genomic DNA was then extracted and screened for a panel of known hotspot mutations using polymerase chain reaction and mass spectroscopy analysis. Of the 22 radial scars, 14 (63.6%) had PIK3CA mutations (10 with H1047R mutations, 2 G1049R mutations, 1 E542K, 1 E545K). The remaining 8 lesions were wild type for all of the screened genes. Of the radial scars without epithelial atypia, 9/16 (56.3%) had PIK3CA mutations; furthermore, 5/6 (83.3%) radial scars with atypia had mutations detected. In this study, the frequency of PIK3CA mutations was notably higher than the 25% to 30% mutation frequency of invasive breast cancer. This finding raises interesting questions as to the role of PIK3CA mutations in breast cancer development. Additional larger studies are indicated to confirm and extend these observations in understanding the pathogenesis of radial scars and their relationship to breast cancer.

MDM2 Amplification and PI3KCA Mutation in a Case of Sclerosing Rhabdomyosarcoma.

A rare sclerosing variant of rhabdomyosarcoma characterized by prominent hyalinization and pseudovascular pattern has recently been described as a subtype biologically distinct from embryonal, alveolar, and pleomorphic forms. We present cytogenetic and molecular findings as well as experimental studies of an unusual case of sclerosing rhabdomyosarcoma. The primary lesion arose within the plantar subcutaneous tissue of the left foot of an otherwise healthy 23-year-old male who eventually developed pulmonary nodules despite systemic chemotherapy. Two genetic abnormalities identified in surgical and/or autopsy samples of the tumor were introduced into 10T1/2 murine fibroblasts to determine whether these genetic changes cooperatively facilitated transformation and growth. Cytogenetic analysis revealed a complex abnormal hyperdiploid clone, and MDM2 gene amplification was confirmed by fluorescence in situ hybridization. Cancer gene mutation screening using a combination of multiplexed PCR and mass spectroscopy revealed a PIK3CA exon 20 H1047R mutation in the primary tumor, lung metastasis, and liver metastasis. However, this mutation was not cooperative with MDM2 overexpression in experimental assays for transformation or growth. Nevertheless, MDM2 and PIK3CA are genes worthy of further investigation in patients with sclerosing rhabdomyosarcoma and might be considered in the enrollment of these patients into clinical trials of targeted therapeutics.

Novel method for PIK3CA mutation analysis: locked nucleic acid--PCR sequencing.

Somatic mutations in PIK3CA are commonly seen in invasive breast cancer and several other carcinomas, occurring in three hotspots: codons 542 and 545 of exon 9 and in codon 1047 of exon 20. We designed a locked nucleic acid (LNA)-PCR sequencing assay to detect low levels of mutant PIK3CA DNA with attention to avoiding amplification of a pseudogene on chromosome 22 that has >95% homology to exon 9 of PIK3CA. We tested 60 FFPE breast DNA samples with known PIK3CA mutation status (48 cases had one or more PIK3CA mutations, and 12 were wild type) as identified by PCR-mass spectrometry. PIK3CA exons 9 and 20 were amplified in the presence or absence of LNA-oligonucleotides designed to bind to the wild-type sequences for codons 542, 545, and 1047, and partially suppress their amplification. LNA-PCR sequencing confirmed all 51 PIK3CA mutations; however, the mutation detection rate by standard Sanger sequencing was only 69% (35 of 51). Of the 12 PIK3CA wild-type cases, LNA-PCR sequencing detected three additional H1047R mutations in "normal" breast tissue and one E545K in usual ductal hyperplasia. Histopathological review of these three normal breast specimens showed columnar cell change in two (both with known H1047R mutations) and apocrine metaplasia in one. The novel LNA-PCR shows higher sensitivity than standard Sanger sequencing and did not amplify the known pseudogene.

PIK3CA-AKT pathway mutations in micropapillary breast carcinoma.

Micropapillary carcinoma of the breast is associated with increased rates of lymph node metastasis and lymphovascular invasion. While activating point mutations in PIK3CA (encoding phosphatidylinositol-3-kinase catalytic subunit) or AKT1 are found in 25% to 30% of invasive ductal carcinomas, the mutational profile of invasive micropapillary carcinomas has not been characterized in detail. Micropapillary carcinomas, concurrent metastatic and precursor breast lesions from 19 patients were identified. Lesional tissue was punched from paraffin-tissue blocks, and genomic DNA was extracted and screened for a large panel of known hotspot mutations using multiplex polymerase chain reaction and mass-spectroscopy analysis (643 mutations in 53 genes). Hotspot point mutations were identified in 35% (7/20) of micropapillary breast carcinomas, including PIK3CA exons 7, 9 and 20 hotspots, as well as the AKT1 plekstrin homology domain mutation (E17K); mutations in TP53 and KRAS were each found in a single patient. In 6 patients, micropapillary and non-micropapillary components of the same tumor were separately tested, yielding concordant results in five; one had a wild type micropapillary component, but a PIK3CA mutation in the invasive ductal component. Concurrent lymph node metastases were mostly wild type (2/8 mutant). Accompanying ductal carcinoma in situ had point mutations in 45% (5/11), mostly concordant with invasive carcinoma; however, mutational status of other breast proliferative lesions was generally discordant with accompanying carcinoma. The rate of PIK3CA mutations in this series of micropapillary carcinomas is similar to invasive ductal carcinomas; however, there may be an enrichment of AKT1 mutations (10%). The non-micropapillary components and precursor lesions occasionally had different mutations.

Biphasic papillary and lobular breast carcinoma with PIK3CA and IDH1 mutations.

Morphologic "special types" of breast carcinomas have been recognized for many years, and their molecular and genetic properties have not been specifically studied until recently. Lobular carcinoma lacks functional E-cadherin expression but shares molecular similarities with low-grade invasive ductal carcinomas. Papillary carcinoma is relatively rare, and molecular features are just being elucidated. We report a case of concurrent invasive lobular and papillary carcinoma, the latter with extensive nodal involvement. Multiplex screening for activating point mutations identified different point mutations in the distinct morphologic components: lobular PIK3CA H1047R, papillary; PIK3CA Q546P, and IDH1 R132H. These molecular data favor coincidental "collision tumors" over clonal evolution. The IDH1 R132H point mutation is common in gliomas and acute myelogenous leukemia, but this has not been previously reported in breast carcinoma. The characterization of activating point mutations in morphologic special types of breast carcinoma may suggest avenues amenable to targeted therapy.

Mucinous breast carcinomas lack PIK3CA and AKT1 mutations.

Activating point mutations in the phosphatidylinositol-3-kinase catalytic subunit (PIK3CA) are among the most common molecular defects in invasive breast cancer. Point mutations in the downstream kinase AKT1 are seen in a minority of carcinomas. These mutations are found preferentially in estrogen receptor-positive and Her2-positive breast carcinomas; however, special morphologic types of breast cancer have not been well studied. Twenty-nine cases of pure invasive mucinous carcinoma and 9 cases of ductal carcinoma with mucinous differentiation were screened for a panel of point mutations (>321 mutations in 30 genes) using a multiplex polymerase chain reaction panel with mass spectroscopy readout. In addition, associated ductal carcinoma in situ, hyperplasia, or columnar cell lesions were separately tested where available (25 lesions). In 3 invasive cases and 15 ductal carcinoma in situ/proliferative lesions, PIK3CA hotspot mutations were, instead, tested by direct sequencing. No point mutations were identified in invasive mucinous breast carcinoma. This contrasts with the 35% frequency of PIK3CA mutations in a comparative group of invasive ductal carcinomas of no special type. Interestingly, PIK3CA hotspot point mutations were identified in associated ductal carcinoma in situ (3/14) and hyperplasia (atypical ductal hyperplasia [2/3], usual ductal hyperplasia [2/3], columnar cell change [1/5]), suggesting that PIK3CA mutations may play a role in breast epithelial proliferation. This series represents the largest study, to date, of PIK3CA genotyping in mucinous carcinoma and supports the unique pathogenetics of invasive mucinous breast carcinoma.

Multiplex high-throughput gene mutation analysis in acute myeloid leukemia.

Classification of acute myeloid leukemia increasingly depends on genetic analysis. However, the number of known mutations in acute myeloid leukemia is expanding rapidly. Therefore, we tested a high-throughput screening method for acute myeloid leukemia mutation analysis using a multiplex mass spectrometry-based approach. To our knowledge, this is the first reported application of this approach to genotype leukemias in a clinical setting. One hundred seven acute myeloid leukemia cases were screened for mutations using a panel that covers 344 point mutations across 31 genes known to be associated with leukemia. The analysis was performed by multiplex polymerase chain reaction for mutations in genes of interest followed by primer extension reactions. Products were analyzed on a Sequenom MassARRAY system (San Diego, CA). The multiplex panel yielded mutations in 58% of acute myeloid leukemia cases with normal cytogenetics and 21% of cases with abnormal cytogenetics. Cytogenetics and routine polymerase chain reaction-based screening of NPM1, CEBPA, FLT3-ITD, and KIT was also performed on a subset of cases. When combined with the results of these standard polymerase chain reaction-based tests, the mutation frequency reached 78% in cases with normal cytogenetics. Of these, 42% harbored multiple mutations primarily involving NPM1 with NRAS, KRAS, CEBPA, PTPN11, IDH1, or FLT3. In contrast, cases with abnormal cytogenetics rarely harbored more than 1 mutation (1.5%), suggesting different underlying biology. This study demonstrates the feasibility and utility of broad-based mutation profiling of acute myeloid leukemia in a clinical setting. This approach will be helpful in defining prognostic subgroups of acute myeloid leukemia and contribute to the selection of patients for enrollment into trials with novel inhibitors.

Phosphatidylinositol-3-kinase pathway mutations are common in breast columnar cell lesions.

The phosphatidylinositol-3-kinase pathway is one of the most commonly mutated pathways in invasive breast carcinoma, with PIK3CA mutations in ∼25% of invasive carcinomas, and AKT1 mutations in up to 5%. Ductal carcinoma in situ, and benign papillomas harbor similar mutations. However, activating point mutations in breast columnar cell lesions have been infrequently studied. Twenty-three breast resection specimens containing columnar cell lesions were identified; 14 with associated invasive carcinoma or carcinoma in situ. DNA extracts were prepared from formalin-fixed paraffin-embedded tissue and screened for a panel of point mutations (321 mutations in 30 genes) using a multiplex PCR panel with mass-spectroscopy readout. PIK3CA mutations were identified in 13/24 columnar cell lesions (54%) and 3/8 invasive carcinomas (37%). The mutation status of columnar cell lesions and associated carcinoma was frequently discordant. Of the 14 cases, only 5 demonstrated the same genotype in matched samples of columnar cell lesions and carcinoma (4 wild type, 1 PIK3CA H1047R). Interestingly, five patients had mutations in columnar cell lesions with wild-type carcinoma; two patients had different point mutations in columnar cell lesions and carcinoma. Only three cases had wild-type columnar cell lesion and mutated carcinoma. The 50% PIK3CA mutation prevalence in columnar cell lesions is greater than reported in most studies of invasive breast cancer. Further, columnar cell lesions and carcinoma were frequently discordant for PIK3CA/AKT1 mutation status. These findings raise interesting questions about the role of PIK3CA/AKT pathway in breast carcinogenesis, and the biologic/precursor potential of columnar cell lesions.

BRAF and KRAS mutations in sporadic glomus tumors.

Glomus tumors are rare soft tissue neoplasms resembling the normal glomus body, which is a specialized form of arteriovenous anastomosis that regulates heat. The molecular genetics of sporadic glomus tumors has not been studied. We genotyped tumors from 28 patients (16 female patients and 12 male patients) ranging from 13 to 77 years and correlated the results with the tumor site (15 finger/1 hand/4 arm/7 leg/1 eyelid), Ki-67 index, and clinical follow-up. Tumor DNA from paraffin-embedded tissue was screened by multiplex polymerase chain reaction and mass spectroscopy, using a panel covering 370 mutations across 30 genes, including AKT1, BRAF, CTNNB1, EGFR, ERBB2, FGFR1/2/3, HRAS, KIT, KRAS, MEK1/2, NRAS, PDGFRA, and PIK3CA. A BRAF V600E mutation was identified in 3 cases, all of which occurred in proximal locations (upper shin, thigh, and upper arm). Two of the patients with BRAF-mutated tumors were quite young (21 and 13 years) and one of the BRAF-mutated tumors recurred in 3 years. A KRAS G12A mutation was found in tumor removed from the finger. Ki-67 index did not correlate with genotype. To our knowledge, this is the first report of oncogenic mutations in sporadic glomus tumors.

Multiplex mutation screening by mass spectrometry evaluation of 820 cases from a personalized cancer medicine registry.

There is an immediate and critical need for a rapid, broad-based genotyping method that can evaluate multiple mutations simultaneously in clinical cancer specimens and identify patients most likely to benefit from targeted agents now in use or in late-stage clinical development. We have implemented a prospective genotyping approach to characterize the frequency and spectrum of mutations amenable to drug targeting present in urothelial, colorectal, endometrioid, and thyroid carcinomas and in melanoma. Cancer patients were enrolled in a Personalized Cancer Medicine Registry that houses both clinical information and genotyping data, and mutation screening was performed using a multiplexed assay panel with mass spectrometry-based analysis to detect 390 mutations across 30 cancer genes. Formalin fixed, paraffin-embedded specimens were evaluated from 820 Registry patients. The genes most frequently mutated across multiple cancer types were BRAF, PIK3CA, KRAS, and NRAS. Less common mutations were also observed in AKT1, CTNNB1, FGFR2, FGFR3, GNAQ, HRAS, and MAP2K1. Notably, 48 of 77 PIK3CA-mutant cases (62%) harbored at least one additional mutation in another gene, most often KRAS. Among melanomas, only 54 of 73 BRAF mutations (74%) were the V600E substitution. These findings demonstrate the diversity and complexity of mutations in druggable targets among the different cancer types and underscore the need for a broad-spectrum, prospective genotyping approach to personalized cancer medicine.

Immunohistochemical and molecular markers in breast phyllodes tumors.

Phyllodes tumors of the breast are diagnostically and managerially enigmatic, as their malignant potential is difficult to predict based on the standard morphologic criteria. Thus, there is a need for additional markers of biologic potential. Although a number of ancillary tests have been reported, consensus in the literature is lacking. We studied 38 cellular fibroadenomas and phyllodes tumors of various grade (World Health Organization benign, borderline, and malignant) with a panel of immunohistochemical stains (p53, CD117, phospho-Histone3, mdm2, cdk4) and screened 26 of the tumors for mutations across 30 cancer-related genes using PCR and mass-spectrometry based methods. p53 and phospho-Histone3 (mitotic marker) showed increased staining in higher grade phyllodes tumors. CD117, mdm2, and cdk4 showed no difference in expression across different grades of phyllodes tumors. Mutational analysis revealed an S8R substitution in FBX4 (an E3 ubiquitin ligase) in 3 cases: 1 benign and 2 borderline. The S8R substitution seems to be more common in phyllodes tumors (11.5%) as compared with other cancers. FBX4 S8R cases had high cyclin D1 expression, but this finding was not specific. Our data support earlier studies showing that p53 has potential use in pathologic assessment of phyllodes tumors, and we newly characterized phospho-Histone3 for this application. Further studies are needed to characterize the molecular pathogenesis of the phyllodes tumors, as we were unable to identify activating mutations despite screening for a large panel of activating hotspot mutations. The significance of the FBX4 substitution deserves further investigation.