The search continues: looking for predictive biomarkers for response to mammalian target of rapamycin inhibition in endometrial cancer.

OBJECTIVE: PI3K/mammalian target of rapamycin (mTOR) pathway aberrations occur in 40% to 80% of endometrial cancer. Prior studies suggest KRAS mutations are associated with resistance to mTOR inhibitors in solid tumors. The objective of this study was to determine if biomarker expression in the PI3K/mTOR pathway or KRAS mutations would predict response to therapy with everolimus, an mTOR inhibitor. METHODS: Specimens from a phase II study of everolimus in recurrent endometrioid endometrial cancer were utilized. The primary end point was clinical benefit rate (CBR: objective response and nonprogression at 20 weeks). Correlative studies evaluating PTEN expression and phospho-S6 ribosomal protein (pS6rp) status by immunohistochemistry and KRAS mutational analysis were performed. RESULTS: Six of 28 evaluable patients achieved prolonged stable disease (SD) at 20 weeks (CBR, 21%). Loss of PTEN expression did not predict CBR (P = 0.62) with a positive predictive value (PPV) of 0.13. Five (83%) of 6 patients with SD maintained PTEN expression. Neither pS6rp expression (P = 0.65) nor KRAS mutation (P = 0.99) predicted CBR; the PPV was 0.14 for each. Eighty percent (4/5) of those with SD were KRAS wild type. Combined analysis of pS6rp expression and KRAS mutation provided 100% PPV (95% confidence interval, 39.6%-100%), suggesting no chance of CBR for these individuals with 100% specificity (95% confidence interval, 46.3%-100%). CONCLUSIONS: S6rp phosphorylation, loss of PTEN expression, and presence of KRAS mutations alone did not correlate with CBR. However, positive pS6rp staining combined with KRAS mutation performed with 100% PPV and specificity to predict nonresponse. Identifying patients who will not benefit from mTOR inhibitors can direct therapy and reduce exposure to agents that add toxicity without clinical benefit.

Rapid clonal shifts in response to kinase inhibitor therapy in chronic myelogenous leukemia are identified by quantitation mutation assays.

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.

Application of COLD-PCR for improved detection of KRAS mutations in clinical samples.

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 80 degrees C (vs 94 degrees C 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 >4.74-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, paraffin-embedded tissue.

Correlation between KIT expression and KIT mutation in melanoma: a study of 173 cases with emphasis on the acral-lentiginous/mucosal type.

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.

Ultra-Rapid Reporting of GENomic Targets (URGENTseq): Clinical Next-Generation Sequencing Results within 48 Hours of Sample Collection.

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.

BRAF and MAP2K1 mutations in Langerhans cell histiocytosis: a study of 50 cases.

Langerhans cell histiocytosis (LCH) is a proliferation of Langerhans cells, often associated with lymphocytes, eosinophils, macrophages, and giant cells. BRAF mutations, usually V600E, have been reported in 40%-70% of cases, and recently, MAP2K1 mutations have been reported in BRAF-negative cases. We assessed 50 cases of LCH for BRAF mutations and assessed a subset of cases for MAP2K1 mutations. The study group included 28 men and 22 women (median age, 36.5 years; range, 1-78 years). BRAF V600E mutation was detected in 8 (16%) cases including 3 (30%) skin, 2 (11%) bone, 1 (50%) colon, 1 (20%) lung, and 1 (33%) extradural, intracranial mass. MAP2K1 mutations were detected in 6 of 13 (46%) BRAF-negative cases including 2 (100%) lymph node, 2 (50%) bone, 1 (25%) skin, and 1 (100%) orbit. Patients with BRAF mutation were younger than patients with wild-type BRAF (median age, 28 versus 38 years; P = .026). The median age of MAP2K1-mutated patients was 34.5 years, similar to patients without MAP2K1 mutation (41 years; P = .368). In agreement with 2 recent studies, we showed a high frequency of MAP2K1 mutations in BRAF-negative LCH cases. Unlike other studies, the overall frequency of BRAF mutation in this cohort is substantially lower than what has been reported in pediatric patients, perhaps because most patients in this study were adults. Moreover, we showed a high concordance between mutational and immunohistochemical analysis for BRAF mutation. There was no statistically significant association between BRAF or MAP2K1 mutation and anatomic site, unifocal versus multifocal presentation, or clinical outcome.

Clinical validation of a multipurpose assay for detection and genotyping of CALR mutations in myeloproliferative neoplasms.

OBJECTIVES: To develop a polymerase chain reaction (PCR)-based approach to detect CALR mutations in myeloproliferative neoplasms (MPNs) in a clinical laboratory. METHODS: DNA was extracted from bone marrow aspirate samples of 67 JAK2 wild-type MPNs (22 with matched peripheral blood), 54 cases of unclassifiable myelodysplastic syndrome/MPN, and 16 cases of atypical chronic myeloid leukemia. We used genomic DNA to detect somatic mutations in exon 9 of CALR and PCR with fluorescently labeled and M13-tagged primers and subjected the products to capillary electrophoresis (CE) followed by Sanger sequencing. Detailed assay performance characteristics were established. RESULTS: We identified CALR mutations in 19 (28.4%) of 67 JAK2-negative MPNs, including 14 type I (52-base pair [bp] deletion), four type II (5-bp insertions), and one type III (18-bp deletion). All mutations were confirmed by Sanger sequencing. Sensitivity studies showed 2.5% and 5% mutation detection levels by CE and Sanger sequencing, respectively, with high reproducibility. CONCLUSIONS: This assay allows for rapid, convenient screening for CALR mutations in MPNs, thereby reducing the number of cases that require assessment by Sanger sequencing, reducing labor and improving turnaround time.

Cutaneous carcinosarcoma: further insights into its mutational landscape through massive parallel genome sequencing.

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.

High-level microsatellite instability in appendiceal carcinomas.

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% to 3% of CRCs overall. In contrast to CRC, MSI-high in appendiceal adenocarcinomas is rare. Only 4 MSI-high appendiceal carcinomas and 1 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 MD 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 1 was MSI-low. The 3 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 tumors, 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 with the right colon and suggest that MLH1 promoter methylation is not a mechanism for MSI in this location.

Frequency and spectrum of BRAF mutations in a retrospective, single-institution study of 1112 cases of melanoma.

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 = 774), acral (n = 111), mucosal (n = 26), uveal (n = 23), leptomeningeal (n = 1), and metastatic melanomas of unknown primary site (n = 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.

Distinct patterns of cytogenetic and clinical progression in chronic myeloproliferative neoplasms with or without JAK2 or MPL mutations.

Chronic myeloproliferative neoplasms (MPN), including essential thrombocythemia (ET) and primary myelofibrosis (PMF), result from interactions between initiating growth factor mutations and secondary genomic changes. Codon 617 mutation of the JAK2 kinase is found in 40-50% of ET/PMF, whereas the mutation of codon 515 in the JAK2-linked thrombopoietin receptor MPL is found in approximately 20% of JAK2-unmutated cases of ET and PMF. Using quantitative mutation assays, we compared patterns of clinical and cytogenetic progression in MPL-mutated MPN (n=21) to those with JAK2 V617F mutation (n=383) or neither mutation (n=109). Among patients with MPL mutations, ET was seen in 9 and PMF in 12. Median mutation levels in pretreatment ET samples were significantly higher for MPL-mutated cases (60%) than for JAK2-mutated cases (24%; P=0.01), as was presentation with anemia. Differential genomic changes included +9 in JAK2-mutated cases and chromosome 1 alterations in MPL-mutated ones, implicating dosage effects related to gene copy number. Decreases in the levels of MPL mutation were seen in sequential marrow samples from some patients under treatment with biologic therapies, but not in those treated with kinase inhibitors, consistent with selective response of the MPL-mutated clone similar to the responses seen in JAK2-mutated MPN.