The US Food and Drug Administration (FDA) approved vemurafenib to treat patients with metastatic melanoma harboring the BRAF c.1799T>A (p.V600E) mutation. However, a subset of melanomas harbor non-p.V600E BRAF mutations, and these data are of potential importance regarding the efficacy of current targeted therapies. To better understand the BRAF mutation profile in melanomas, we retrospectively analyzed data from 1112 primary and metastatic melanomas at our institution. The cohort included nonacral cutaneous (n Z 774), acral (n Z 111), mucosal (n Z 26), uveal (n Z 23), leptomeningeal (n Z 1), and metastatic melanomas of unknown primary site (n Z 177). BRAF mutation hotspot regions in exons 11 and 15 were analyzed by pyrosequencing or with the primer extension MassARRAY system. A total of 499 (44.9%) specimens exhibited BRAF mutations, involving exon 15 [497 (99.6%)] or exon 11 [2 (0.4%)]. p.V600E was detected in 376 (75.4%) cases; the remaining 123 (24.6%) cases exhibited non-p.V600E mutations, of which p.V600K was most frequent [86 (17.2%)]. BRAF mutations were more frequent in nonacral cutaneous (51.4%) than acral melanomas [18 (16.2%)] (P < 0.001); however, there was no significant difference among cutaneous histological subtypes. All mucosal, uveal, and leptomeningeal melanomas were BRAF wild type (WT). The high frequency of non-p.V600E BRAF mutations in melanoma has important implications because the FDA-approved companion diagnostic test for p.V600E detects some but not all non-p.V600E mutations. However, the therapeutic efficacy of vemurafenib is not well established in these lesions. (J Mol Diagn 2013, 15: 220e226; http://dx

The role of immunohistochemistry in the assessment of KIT status in melanomas, especially acral lentiginous/mucosal, is not well established. Although the reported prevalence of KIT mutations in acral lentiginous/ mucosal melanomas is relatively low, detection of mutations in KIT can have profound therapeutic implications. We evaluated the efficacy of immunohistochemistry to predict mutations in KIT. One hundred seventy-three tumors, comprising primary and metastatic melanomas (141 acral lentiginous/mucosal, 5 nodular, 4 lentigo maligna, 3 superficial spreading, 2 uveal, 1 melanoma of soft parts, 8 metastases from unclassified primaries, and 9 metastases from unknown primaries) were studied. Immunohistochemical expression of KIT using an anti-CD117 antibody and KIT mutational analysis by gene sequencing of exons 11, 13, and 17 were performed. Eighty-one percent of acral lentiginous/mucosal melanomas, primary and metastatic, showed KIT expression by at least 5% of the tumor cells. The overall frequency of activating KIT gene mutations in acral lentiginous/ mucosal melanomas was 15% (14 out of 91 cases), being the L576P mutation in exon 11 the most frequently detected (4 of 14 cases). Cases showing less than 10% positive tumor cells were negative for KIT mutations. Eighty-two percent (12 of 14) of cases positive for KIT mutation showed KIT expression in more than 50% of the cells. An association between immunohistochemical expression of KIT and mutation status was found (P= 0.007). Immunohistochemical expression of KIT in less than 10% of the cells of the invasive component of acral lentiginous/mucosal melanomas appears to be a strong negative predictor of KIT mutation and therefore can potentially be used to triage cases for additional KIT genotyping.

KRAS mutations have been detected in approximately 30% of all human tumors, and have been shown to predict response to some targeted therapies. The most common KRAS mutation-detection strategy consists of conventional PCR and direct sequencing. This approach has a 10-20% detection sensitivity depending on whether pyrosequencing or Sanger sequencing is used. To improve detection sensitivity, we compared our conventional method with the recently described co-amplification-at-lower denaturation-temperature PCR (COLD-PCR) method, which selectively amplifies minority alleles. In COLD-PCR, the critical denaturation temperature is lowered to 801C (vs 941C in conventional PCR). The sensitivity of COLD-PCR was determined by assessing serial dilutions. Fifty clinical samples were used, including 20 fresh bone-marrow aspirate specimens and the formalin-fixed paraffin-embedded (FFPE) tissue of 30 solid tumors. Implementation of COLD-PCR was straightforward and required no additional cost for reagents or instruments. The method was specific and reproducible. COLD-PCR successfully detected mutations in all samples that were positive by conventional PCR, and enhanced the mutant-to-wild-type ratio by 44.74-fold, increasing the mutation detection sensitivity to 1.5%. The enhancement of mutation detection by COLD-PCR inversely correlated with the tumor-cell percentage in a sample. In conclusion, we validated the utility and superior sensitivity of COLD-PCR for detecting KRAS mutations in a variety of hematopoietic and solid tumors using either fresh or fixed, paraffinembedded tissue.

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High-level microsatellite instability (MSI-high) is found in approximately 15% of all colorectal adenocarcinomas (CRCs) and in at least 20% of right-sided cancers. It is most commonly due to somatic hypermethylation of the MLH1 gene promoter region, with familial cases (Lynch syndrome) representing only 2–3% of CRCs overall. In contrast to CRC, MSI-high in appendiceal adenocarcinomas is rare. Only four MSI-high appendiceal carcinomas and one MSI-high appendiceal serrated adenoma have been previously reported, and the prevalence of MSI in the appendix is unknown. We identified 108 appendiceal carcinomas from M. D. Anderson Cancer Center in which MSI status had been assessed by immunohistochemistry for the DNA mismatch repair proteins MLH1, MSH2, MSH6, and PMS2 (n=83), polymerase chain reaction (n=7), or both (n=18). Three cases (2.8%) were MSI-high and one was MSI-low. The three MSI-high cases included: 1) a poorly differentiated nonmucinous adenocarcinoma with loss of MLH1/PMS2 expression, lack of MLH1 promoter methylation, and lack of BRAF gene mutation, but no detected germline mutation in MLH1 from a 39-year-old man; 2) an undifferentiated carcinoma with loss of MSH2/MSH6, but no detected germline mutation in MSH2 or TACSTD1, from a 59-year-old woman; and 3) a moderately differentiated mucinous adenocarcinoma arising in a villous adenoma with loss of MSH2/MSH6 expression, in a 38-year-old man with a strong family history of CRC who declined germline testing. When the overall group of appendiceal carcinomas was classified according to histologic features and precursor lesions, the frequencies of MSI-high were: 3 of 108 (2.8%) invasive carcinomas, 3 of 96 (3.1%) invasive carcinomas that did not arise from a background of goblet cell carcinoid, and 0 of 12 (0%) signet ring and mucinous carcinomas arising in goblet cell carcinoid tumors. These findings, in conjunction with the previously reported MSI-high appendiceal carcinomas, highlight the low prevalence of MSI in the appendix as compared to the right colon and suggest that MLH1 promoter methylation is not a mechanism for microsatellite instability in this location.

Cutaneous carcinosarcoma (CCS) is an extraordinarily rare neoplasm with a biphasic morphological pattern exhibiting both epithelial and sarcomatoid components. Although its histogenesis and biological aspects remain poorly understood, previous studies have postulated that this tumor may arise from single cancer stem cells which subsequently differentiate into distinct tumor lineages. In this study, we explored a wide array of mutational hot spot regions, through high-depth next-generation sequencing of 47 cancer-associated genes in order to assess the mutational landscape of these tumors and investigate whether the epithelial and mesenchymal components shared the same genetic signatures. Results from this study confirm that despite their striking phenotypic differences, both elements of this infrequent tumor indeed share a common clonal origin. Additionally, CCS appears to embrace a heterogeneous spectrum with specific underlying molecular signatures correlating with the defining epithelial morphotype, with those carcinosarcomas exhibiting a squamous cell carcinoma epithelial component exhibiting diverse point mutations and deletions in the TP53 gene, and those with a basal cell carcinoma morphotype revealing a more complex mutational landscape involving several genes. Also, the fact that our findings involve several targetable gene pathways suggests that the underlying molecular events driving the pathogenesis of CCS may represent future potential targets for personalized therapies.

Treatment of CML with the tyrosine kinase inhibitor (TKI) imatinib mesylate results in the emergence of point mutations within the kinase domain (KD) of the BCR-ABL1 fusion transcript. The introduction of next-generation TKIs that can overcome the effects of some BCR-ABL1 KD mutations requires quantitative mutation profiling methods to assess responses. We report the design and validation of such quantitative assays, using pyrosequencing and mutation-specific RT-PCR techniques, to allow sequential monitoring and illustrate their use in tracking specific KD mutations (e.g. G250E, T315I, and M351T) following changes in therapy. Pyrosequencing and mutation-specific RT-PCR allows sequential monitoring of specific mutations and identification of rapid clonal shifts in response to kinase inhibitor therapy in CML. Rapid reselection of TKI-resistant clones occurs following therapy switch in CML.

Next-generation sequencing (NGS)-based mutation panels profile multiple genes simultaneously, allowing the reporting of numerous genes while saving labor and resources. However, one drawback of using NGS is that the turnaround time is often longer than conventional single gene tests. This delay can be problematic if molecular results are required to guide therapy in patients with clinically aggressive diseases, such as acute myeloid leukemia. To overcome this limitation, we developed a novel custom platform designated as Ultra-rapid Reporting of GENomic Targets (URGENTseq), an integrated solution that includes workflow optimization and an innovative custom bioinformatics pipeline to provide targeted NGS results on fresh peripheral blood and bone marrow samples within an actionable time period. URGENTseq was validated for clinical use by determining mutant allelic frequency and minimum coverage in silico to achieve 100% concordance for all positive and negative calls between the URGENTseq and conventional sequencing approach. URGENTseq enables the reporting of selected genes useful for immediate diagnosis (CALR, CSF3R, JAK2, KRAS, MPL, NPM1, NRAS, SF3B1) and treatment decisions (IDH1, IDH2) in hematologic malignancies within 48 hours of specimen collection. In addition, we summarize the molecular findings of the first 272 clinical test results performed using the URGENTseq platform.