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Purpose: MicroRNAs are short noncoding RNAs that regulate gene expression and are over- or underexpressed in most tumors, including colorectal adenocarcinoma. MicroRNAs are potential biomarkers and therapeutic targets and agents, but limited information on microRNAome alterations during progression in the well-known adenoma-adenocarcinoma sequence is available to guide their usage.Experimental Design: We profiled 866 human microRNAs by microarray analysis in 69 matched specimens of microsatellite-stable adenocarcinomas, adjoining precursor adenomas including areas of high- and low-grade dysplasia, and nonneoplastic mucosa.Results: We found 230 microRNAs that were significantly differentially expressed during progression, including 19 not reported previously. Altered microRNAs clustered into two major patterns of early (type I) and late (type II) differential expression. The largest number (n = 108) was altered at the earliest step from mucosa to low-grade dysplasia (subtype IA) prior to major nuclear localization of β-catenin, including 36 microRNAs that had persistent differential expression throughout the entire sequence to adenocarcinoma. Twenty microRNAs were intermittently altered (subtype IB), and six were transiently altered (subtype IC). In contrast, 33 microRNAs were altered late in high-grade dysplasia and adenocarcinoma (subtype IIA), and 63 in adenocarcinoma only (subtype IIB). Predicted targets in 12 molecular pathways were identified for highly altered microRNAs, including the Wnt signaling pathway leading to low-grade dysplasia. β-catenin expression correlated with downregulated microRNAs.Conclusions: Our findings suggest that numerous microRNAs play roles in the sequence of molecular events, especially early events, resulting in colorectal adenocarcinoma. The temporal patterns and complexity of microRNAome alterations during progression will influence the efficacy of microRNAs for clinical purposes. Clin Cancer Res; 17(23); 7283–93. ©2011 AACR.

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CCR Translation for This Article from Complex Patterns of Altered MicroRNA Expression during the Adenoma-Adenocarcinoma Sequence for Microsatellite-Stable Colorectal Cancer

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AimsIn advanced-stage non-small-cell lung cancer (NSCLC), incomplete genotyping for guideline-recommended genomic biomarkers poses a significant challenge to making informed and timely clinical decisions. We report our institution’s experience in assessing the adequacy of small specimens for comprehensive genomic profiling for guideline-recommended lung cancer biomarker testing.MethodsWe performed a retrospective evaluation of all image-guided procedures for NSCLC performed in our institution between October 2016 and July 2018, including core needle biopsy (CNB) and fine-needle aspiration (FNA) in patients who had undergone genomic profiling for lung cancer. Lung cancer biomarker adequacy, defined as successful testing of guideline-recommended biomarkers including, epidermal growth factor receptor (EGFR); serine/threonine protein kinase B-Raf (BRAF); anaplastic lymphoma kinase (ALK); proto-oncogene tyrosine protein kinase ROS (ROS1); Rearranged during Transfection (RET); Tyrosine protein kinase Met (MET); and programmed cell death ligand 1 (PD-L1), was evaluated.ResultsA total of 865 cases were evaluated in this study, 785 of which included testing of all lung cancer biomarkers. Lung tissue was adequate for biomarker testing in 84% of cases; this rate increased to 87% when biomarker testing was combined with concurrently acquired FNA or CNB specimens. Biomarker testing success correlated strongly with DNA concentration (pConclusionThe growing numbers of therapeutic biomarkers in NSCLC requires judicious triage of limited-volume tissue from small specimens. Our study showed that thoracic small tissue specimens can be used successfully to provide prognostic and predictive information for the current guideline-recommended biomarkers for NSCLC in most cases.

Introduction Tumor mutation profiling is standard-of-care in lung carcinoma patients. However, comprehensive molecular profiling of small specimens, including core needle biopsy (CNB) and fine-needle aspiration (FNA) specimens, may often be inadequate due to limited tissue. Centrifuged FNA supernatants, which are typically discarded, have emerged recently as a novel liquid-based biopsy for molecular testing. In this study, we evaluate the use of lung carcinoma FNA supernatants for detecting clinically relevant mutations. Methods Supernatants from lung carcinoma FNA samples (n = 150) were evaluated. Samples were further analyzed using next-generation sequencing (NGS) and ultrasensitive droplet digital PCR (ddPCR). Mutation profiles in a subset of samples were compared with results derived from paired tissue samples from the same patient (n = 67) and available plasma liquid biopsy assay (n = 45). Results All 150 samples yielded adequate DNA and NGS were carried out successfully on 104 (90%) of 116 selected samples. Somatic mutations were detected in 82% of the samples and in 50% of these patients a clinically relevant mutation was identified that would qualify them for targeted therapy or a clinical trial. There was high overall concordance between the mutation profiles of supernatants and the corresponding tissue samples, with 100% concordance with concurrent FNA and 96% with concurrent CNB samples. Comparison of actionable driver mutations detected in supernatant versus plasma samples showed 84% concordance. Conclusions FNA supernatants can provide a valuable specimen source for genotyping lung carcinoma especially in patients with insufficient tumor tissue, thereby reducing multigene mutation profiling failure rates, improving turnaround times, and avoiding repeat biopsies.

Background The use of RNA-based next-generation sequencing (NGS) assays to detect gene fusions for targeted therapy has rapidly become an essential component of comprehensive molecular profiling. For cytology specimens, the cell block (CB) is most commonly used for fusion testing; however, insufficient cellularity and/or suboptimal RNA quality are often limiting factors. In the current study, the authors evaluated the factors affecting RNA fusion testing in cytology and the added value of smears in cases with a suboptimal or inadequate CB. Methods A 12-month retrospective review was performed to identify cytology cases that were evaluated by a targeted RNA-based NGS assay. Samples were sequenced by targeted amplicon-based NGS for 51 clinically relevant genes on a proprietary platform. Preanalytic factors and NGS quality parameters were correlated with the results of RNA fusion testing. Results The overall success rate of RNA fusion testing was 92%. Of the 146 cases successfully sequenced, 14% had a clinically relevant fusion detected. NGS testing success positively correlated with RNA yield (P = .03) but was independent of the tumor fraction, the tumor size, or the number of slides used for extraction. CB preparations were adequate for testing in 45% cases, but the inclusion of direct smears increased the adequacy rate to 92%. There was no significant difference in testing success rates between smears and CB preparations. Conclusions The success of RNA-based NGS fusion testing depends on the quality and quantity of RNA extracted. The use of direct smears significantly improves the adequacy of cytologic samples for RNA fusion testing for predictive biomarkers.

There is currently no blood-based marker in routine use for endometrial cancer patients. Such a marker could potentially be used for early detection, but it could also help to track tumor recurrence following hysterectomy. This is important, as extra-vaginal recurrence of endometrial endometrioid adenocarcinoma is usually incurable. This proof-of-principle study was designed to determine if tumor-associated mutations could be detected in cell-free DNA from the peripheral blood of early and late stage endometrial endometrioid carcinoma patients. Approximately 90% of endometrioid carcinomas have at least one mutation in the genes CTNNB1, KRAS, PTEN, or PIK3CA. Using a custom panel targeting 30 hotspot amplicons in these four genes, next-generation sequencing was performed on cell-free DNA extracted from plasma obtained from a peripheral blood draw at the time of hysterectomy and the matching tumor DNA from 48 patients with endometrioid endometrial carcinomas. At least one mutation in the tumor was detected in 45/48 (94%) of patients. Fifteen of 45 patients (33%) had a mutation in the plasma that matched a mutation in the tumor. These same mutations were not detected in the matched negative control buffy coat. Presence of a plasma mutation was significantly associated with advanced stage at hysterectomy, deep myometrial invasion, lymphatic/vascular invasion, and primary tumor size. Detecting a plasma-based mutation was independent of the amount of cell-free DNA isolated from the plasma. Overall, 18% of early stage patients had a mutation detected in the plasma. These results demonstrate that mutations in genes relevant to endometrial cancer can be identified in the peripheral blood of patients at the time of surgery. Future studies can help to determine the post-operative time course of mutation clearance from the peripheral blood and if mutation re-emergence is predictive of recurrence.

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.

With the expanding role of targeted therapy in patients with solid tumors, pathologists face the daunting task of having to maximize limited volume tissue obtained by fine needle aspiration for a variety of molecular tests. While most molecular studies on fine needle aspiration samples have been reported using cellular material, recent studies have shown that a substantial amount of DNA can be retrieved from the supernatant fluid of aspirate needle rinses after cell pelleting for cytospin or cell block preparations. In routine clinical workflow, the supernatant is discarded; however this fluid may provide a complementary source of DNA for tumor mutational profiling. In this study, we evaluated the post-centrifuged supernatant from 25 malignant and 10 benign fine needle aspiration needle rinses. The mean and median DNA yields from the supernatants were 445 ng and 176.4 ng (range, 15.1-2958 ng), respectively. Next generation sequencing using the Ion AmpliSeq Cancer Hotspot Panel v2 detected somatic mutations in all 25 malignant samples. No mutations were detected in any of the benign samples tested. When available, mutations detected in the supernatant fluid were compared to the next generation sequencing analysis performed on a prior or concurrent surgical specimen from the same patient and showed 100% concordance. In a subset of cases (n = 19) mutations in EGFR, KRAS, BRAF, PIK3CA, and NRAS were successfully confirmed by droplet digital PCR, providing an orthogonal platform for mutation analysis. In summary, in this study we show that post centrifuged supernatants from fine needle aspiration needle rinses can provide a robust substrate for expanded mutation profiling by next generation sequencing, as well as hotspot mutation testing by droplet digital PCR. The ability to detect somatic mutations from otherwise discarded supernatant fluids offers the ability to triage and effectively utilize limited volume fine needle aspiration samples when multiple molecular tests are requested, without the need to re-biopsy for additional tissue samples.

A suitable clinical-grade platform is required for detection of somatic mutations with high sensitivity in cell-free DNA (cfDNA) of patients with solid tumors. In this study, we evaluated in parallel ultra-deep NGS with MassARRAY and allele-specific droplet digital PCR (ddPCR) for cfDNA genotyping and correlated cfDNA yield and mutation status with overall survival (OS) of patients. We assessed plasma samples from 46 patients with various advanced metastatic solid tumors and known mutations by deep sequencing using an Ampliseq cancer hotspot panel V2 on Ion Proton. A subset of these samples with DNA availability was tested by ddPCR and UltraSEEK MassARRAY for mutation detection in 5 genes (IDH1, PIK3CA, KRAS, BRAF, and NRAS). Sixty one of 104 expected tissue mutations and 6 additional mutations not present in the tissue were detected in cfDNA. ddPCR and MassARRAY showed 83% and 77% concordance with NGS for mutation detection with 100% and 79% sensitivity, respectively. The median OS of patients with lower cfDNA yield (74 vs 50 months; P < 0.03) and cfDNA negative for mutations (74.2 vs 53 months; p < 0.04) was significantly longer than in patients with higher cfDNA yield and positive for mutations. A limit-of-detection of 0.1% was demonstrated for ddPCR and MassARRAY platforms using a serially diluted positive cfDNA sample. The MassARRAY and ddPCR systems enable fast and cost-effective genotyping for a targeted set of mutations and can be used for single gene testing to guide response to chemotherapy or for orthogonal validation of NGS results.

Detection of mutations in plasma circulating cell-free DNA (cfDNA) by next-generation sequencing (NGS) has opened up new possibilities for monitoring treatment response and disease progression in patients with solid tumors. However, implementation of cfDNA genotyping in diagnostic laboratories requires systematic assessment of preanalytical parameters and analytical performance of NGS platforms. We assessed the effects of peripheral blood collection tube and plasma separation time on cfDNA yield and integrity and performance of the Ion PGM, Proton, and MiSeq NGS platforms. cfDNA from 31 patients with diverse advanced cancers and known tumor mutation status was deep sequenced using targeted hotspot panels. Forty-five of 52 expected mutations and two additional mutations (KRAS p.Q61H and EZH2 p.Y646F) were detected in plasma through a custom bioinformatics pipeline. We observed comparable cfDNA concentration/integrity between collection tubes within 16 hours of plasma separation and equal analytical performance among NGS platforms, with 1% detection sensitivity for cfDNA genotyping.

RNA-seq was used to identify the partner gene and confirm the presence of a BCR-PDGFRB fusion. Identification of this fusion product resulted in successful treatment and long-term remission of this myeloid neoplasm. Based on our results, we suggest that despite current WHO recommendations, screening for PDGFRB rearrangement in cases of leukocytosis with eosinophilia and no other etiologic explanation is necessary, even if the karyotype is normal.

Background Molecular testing is recommended as an adjunct to improve the preoperative diagnosis of fine-needle aspiration (FNA) of thyroid nodules. Centrifuged supernatants from FNA samples, which are typically discarded, have recently emerged as a novel liquid-based biopsy for molecular testing. This study evaluates the use of thyroid FNA supernatants for detecting clinically relevant mutations. Methods Supernatants from thyroid FNA samples (n = 156) were evaluated. A 50-gene next-generation sequencing (NGS) assay was used, and mutation analysis results from a subset of samples were further compared with those of paired FNA smears and/or cell blocks. Results All 156 samples yielded adequate DNA (median, 135 ng; range, 11-3180 ng), and 129 of these samples (83%) were successfully sequenced by NGS. The most frequently detected somatic mutations included BRAF and RAS mutations, which were followed by RET, TP53, PTEN, CDKN2A, and PIK3CA mutations. Eleven of 31 cases with an indeterminate cytologic diagnosis and 9 of 12 cases that were suspicious for malignancy had somatic mutations, including the BRAF V600E mutation, which is highly definitive for papillary thyroid carcinoma (PTC). Seven of the 9 indeterminate and suspicious cases with the BRAF V600E mutation had surgical follow-up, and they were all confirmed to be PTC. A comparison of the mutation profiles derived from supernatants with those of paired smears and/or cell blocks in a small subset of cases (n = 8) showed 100% concordance. Conclusions This study provides evidence that FNA supernatants can be used as a surrogate for thyroid molecular testing to improve diagnostic accuracy in indeterminate nodules, provide prognostic/predictive information, and improve overall patient management.

Objectives: Clinical laboratories are rapidly implementing next-generation sequencing (NGS) tests for mutation analysis, but there are few guidelines regarding sample quality for successful results. Methods: We aimed to establish tissue quality parameters for successful NGS in solid tumors and to improve NGS performance. Results: Analysis of 614 clinical cases tested in 2013 using a 50-gene hotspot mutation panel identified the major cause for unsuccessful NGS analysis was DNA less than 10 ng (91%, 67/74) associated with extremely small and low cellularity samples. High success rates were associated with resection procedures (333/342, 97%) and biopsied tumor larger than 10 mm2 (77/77, 100%). NGS can be successfully performed on bone specimens processed with formic acid–based decalcification procedures (8/11, 73%). Tumor type and paraffin block age did not affect success. We demonstrated that NGS can be carried out on samples with less than 10 ng DNA. Analysis of 408 cases tested in 2014 using an optimized workflow showed improved NGS success rates from 88% to 95% (387/408) with pronounced improvement among tiny (

BACKGROUND The use of cytology specimens for next-generation sequencing (NGS) is particularly challenging because of the unconventional substrate of smears and the often limited sample volume. An analysis of factors affecting NGS testing in cytologic samples may help to increase the frequency of successful testing. METHODS This study reviewed variables associated with all in-house cytology cases (n = 207) that were analyzed by NGS with the Ion Torrent platform during a 10-month interval. A statistical analysis was performed to measure the effects of the DNA input threshold, specimen preparation, slide type, tumor fraction, DNA yield, and cytopathologist bias. RESULTS One hundred sixty-four of 207 cases (79%) were successfully sequenced by NGS; 43 (21%) failed because of either a low DNA yield or a template/library preparation failure. The median estimated tumor fraction and DNA concentration for the successfully sequenced cases were 70% and 2.5 ng/μL, respectively, whereas they were 60% and 0.2 ng/μL, respectively, for NGS failures. Cell block sections were tested in 91 cases, and smears were used in 116 cases. NGS success positively correlated with the DNA yield but not the tumor fraction. Cell block preparations showed a higher success rate than smears. Frosted-tip slides yielded significantly more DNA than fully frosted slides. Lowering the input DNA concentration below the manufacturer's recommended threshold of 10 ng (>0.85 ng/μL) resulted in a marked increase in the NGS success rate from 58.6% to 89.8%. CONCLUSIONS The failure of NGS with cytology samples is usually a result of suboptimal DNA due to multiple pre-analytical factors. Knowledge of these factors will allow better selection of cytology material for mutational analysis. Cancer (Cancer Cytopathol) 2015;123:659–668. © 2015 American Cancer Society.

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.

Anaplastic large cell lymphomas (ALCL) are tumors of T/null-cell lineage characterized by uniform CD30 expression. The 2008 World Health Organization classification subdivided ALCLs into 2 groups: anaplastic lymphoma kinase (ALK)-positive (established entity) and ALK-negative (proposed new entity) ALCL. The genetic basis for the pathogenesis of newly categorized ALK- ALCL is poorly understood. In this study, we used microRNA microarray analysis to identify differentially expressed microRNAs in ALK+ and ALK- ALCL. ALK- ALCL showed significantly higher expression of miR-155 (0.888 ± 0.228) compared with ALK+ ALCL (0.0565 ± 0.009) on microarray and by quantitative real-time polymerase chain reaction in ALK- ALCL compared with ALK+ ALCL (P < .05) with a strong correlation between the 2 platforms (R = 0.9, P < .0003). A novel in situ hybridization method allows direct visualization of expression patterns and relative quantitation of miR-155 (mean score, 2.3 versus 1.3; P = .01) for the first time in tissue sections of ALCL. Among computationally predicted targets of miR-155, we identified ZNF652 (r = -0.57, P = .05), BACH1 (r = 0.88, P = .02), RBAK (r = 0.81, P = .05), TRIM32 (r = 0.92, P = .01), E2F2 (r = 0.81, P = .05), and TP53INP1 (r = -0.31, P = .03) as genes whose expression by quantitative real-time polymerase chain reaction correlated significantly with the level of miR-155 in ALCL tumor tissue.

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.

Objectives: Identification of tumor-specific somatic mutations has had a significant impact on both disease diagnosis and therapy selection. The ability of next-generation sequencing (NGS) to provide a quantitative assessment of mutant allele burden, in numerous target genes in a single assay, provides a significant advantage over conventional qualitative genotyping platforms. Methods: We assessed the quantitative capability of NGS and a primer extension–based matrix-assisted laser desorption ionization–time-of-flight (PE-MALDI) assay and directly correlated NGS mutant allele burden determination to morphologic assessment of tumor percentage in H&E-stained slides. Results: Our results show a 100% concordance between NGS and PE-MALDI in mutant allele detection and a significant correlation between NGS and PE-MALDI for determining mutant allele burden when mutant allele burden is 10% or more. Conclusions: NGS-based mutation screening provides a quantitative assessment comparable to that of PE-MALDI. In addition, NGS also allows for a high degree of multiplexing and uses nanogram quantities of DNA, thereby preserving precious material for future analysis. Furthermore, this study provides evidence that H&E-based morphologic assessment of tumor burden does not correlate to actual tumor mutant allele burden frequency.

Loss of PTEN (phosphatase and tensin homolog) expression and microsatellite instability are two of the more common molecular alterations in endometrial carcinoma. From the published literature, it is controversial as to whether there is a relationship between these different molecular mechanisms. Therefore, a cohort of 187 pure endometrioid and non-endometrioid endometrial carcinomas, carefully characterized as to clinical and pathological features, was examined for PTEN sequence abnormalities and the immunohistochemical expression of PTEN and the DNA mismatch repair proteins MLH1, MSH2, MSH6, and PMS2. MLH1 methylation analysis was performed when tumors had loss of MLH1 protein. Mismatch repair protein loss was more frequent in endometrioid carcinomas compared with non-endometrioid carcinomas, a difference primarily attributable to the presence of MLH1 methylation in a greater proportion of endometrioid tumors. Among the non-endometrioid group, mixed endometrioid/non-endometrioid carcinomas were the histotype that most commonly had loss of a mismatch repair protein. In endometrioid tumors, the frequency of PTEN loss measured by immunohistochemistry and mutation did not differ significantly between the mismatch repair protein intact or mismatch repair protein loss groups, suggesting that PTEN loss is independent of mismatch protein repair status in this group. However, in non-endometrioid carcinomas, both intact positive PTEN immunohistochemical expression and PTEN wild type were highly associated with retained positive expression of mismatch repair proteins in the tumor. Relevant to screening endometrial cancers for Lynch Syndrome, an initial PTEN immunohistochemistry determination may be able to replace the use of four mismatch repair immunohistochemical markers in 63% of patients with non-endometrioid endometrial carcinoma. Therefore, PTEN immunohistochemistry, in combination with tumor histotype, is a useful adjunct in the clinical evaluation of endometrial carcinomas for Lynch Syndrome.

Minimally invasive procedures, such as fine needle aspiration and core needle biopsy, are commonly used for the diagnosis in solid organ malignancies. In the era of targeted therapy, it is crucial for molecular testing to be performed on these limited volume specimens. Although several recent studies have demonstrated the utility of small biopsy specimens for molecular testing, there remains debate as to whether core needle biopsy specimens are more reliable than fine needle aspiration for molecular studies. In this study, we reviewed concurrently acquired fine needle aspiration and core needle biopsy samples (n=24), and compared overall cellularity, tumor fraction, and the results of next-generation sequencing. All somatic mutations detected in core needle biopsy samples were also detected in fine needle aspiration samples. The estimated tumor fraction was significantly higher in fine needle aspiration smears than core needle biopsy samples (P=0.003), whereas the overall DNA yield from smears was significantly lower than that obtained from the core needle biopsy specimens (P=0.01). The normalized average amplicon coverage for the genes analyzed was significantly higher in cytology smears than paired core needle biopsy samples, with lower numbers of failed amplicons and higher overall mutation allelic frequencies seen in the former. We further evaluated 100 malignant fine needle aspiration and core needle biopsy samples, acquired concurrently, for overall cellularity and tumor fraction. Overall cellularity and tumor fraction of fine needle aspiration samples was significantly higher than concurrently acquired core needle biopsy samples (P

DNA methyltransferase 3A (DNMT3A) is mutated in a subset of de novo acute myeloid leukemia patients and is associated with poor overall and event-free survival. Because routine Sanger sequencing of the 23 DNMT3A exons is impractical in clinical laboratories, we developed a high-throughput method using high-resolution melting (HRM) analysis, which identifies sequence variants by detecting subtle changes in the melting patterns of mutant DNA in comparison with WT sequences. DNA from 104 acute myeloid leukemia patients was tested for mutations in 12 exons encoding 3 major functional domains of DNMT3A: the PWWP (proline-tryptophan-tryptophan-proline) domain (exons 8 to 10), the ADD (ATM-DNMT3-DNMT3L) zinc finger, and the methyltransferase domains encoded by exons 15 to 23. HRM analysis identified 20 of 104 patient samples as variants, which we confirmed by Sanger sequencing. Codon 882 of exon 23 was mutated at the highest frequency with an occurrence rate of 11.5%. All HRM WT calls were confirmed to be devoid of mutations by Sanger sequencing. We also identified seven novel and previously unreported DNMT3A mutations. Structural modeling showed seven of the eight missense mutations detected in our study increased the free energy, destabilized protein, and altered solvent accessibility, suggesting their loss-of-function nature. These data demonstrate HRM analysis to be a higher throughput, sensitive, and efficient alternative to Sanger sequencing for detecting DNMT3A mutations in the clinical diagnostic laboratory.

PTEN (phosphatase and tensin homolog) is a tumor suppressor that negatively regulates the PI3K-AKT signaling pathway, which is implicated in the pathogenesis of endometrial carcinoma. Sanger sequencing has been considered to be the gold standard for detection of PTEN sequence abnormalities. However, this approach fails to address the epigenetic mechanisms that contribute to functional PTEN loss. Using a study cohort of 154 endometrioid and non-endometrioid endometrial carcinomas, we performed full-length PTEN sequencing and PTEN immunohistochemistry on each tumor. PTEN sequence abnormalities were detected in a significantly lower proportion of cases (43%) than PTEN protein loss (64%, P=0.0004). Endometrioid tumors had a significantly higher proportion of PTEN sequence abnormalities and PTEN protein loss than non-endometrioid tumors. Within the latter group, PTEN sequence abnormalities and PTEN protein loss were most frequent in undifferentiated carcinomas, followed by mixed carcinomas; they were least frequent in carcinosarcomas. Overall, at least one PTEN sequence abnormality was detected in each exon, and the greatest number of sequence abnormalities was detected in exon 8. Pure-endometrioid tumors had a significantly higher frequency of sequence abnormalities in exon 7 than did the non-endometrioid tumors (P=0.0199). Importantly, no mutational hotspots were identified. While PTEN protein loss by immunohistochemistry was identified in 89% of cases with a PTEN sequence abnormality, PTEN protein loss was detected by immunohistochemistry in 44% of cases classified as PTEN wild type by sequencing. For the first time, we demonstrate that PTEN immunohistochemistry is able to identify the majority of cases with functional PTEN loss. However, PTEN immunohistochemistry also detects additional cases with PTEN protein loss that would otherwise be undetected by gene sequencing. Therefore, for clinical purposes, immunohistochemistry appears to be a preferable technique for identifying endometrial tumors with loss of PTEN function.

Purpose: MicroRNAs are short noncoding RNAs that regulate gene expression and are over- or underexpressed in most tumors, including colorectal adenocarcinoma. MicroRNAs are potential biomarkers and therapeutic targets and agents, but limited information on microRNAome alterations during progression in the well-known adenoma-adenocarcinoma sequence is available to guide their usage. Experimental Design: We profiled 866 human microRNAs by microarray analysis in 69 matched specimens of microsatellite-stable adenocarcinomas, adjoining precursor adenomas including areas of high- and low-grade dysplasia, and nonneoplastic mucosa. Results: We found 230 microRNAs that were significantly differentially expressed during progression, including 19 not reported previously. Altered microRNAs clustered into two major patterns of early (type I) and late (type II) differential expression. The largest number (n = 108) was altered at the earliest step from mucosa to low-grade dysplasia (subtype IA) prior to major nuclear localization of β-catenin, including 36 microRNAs that had persistent differential expression throughout the entire sequence to adenocarcinoma. Twenty microRNAs were intermittently altered (subtype IB), and six were transiently altered (subtype IC). In contrast, 33 microRNAs were altered late in high-grade dysplasia and adenocarcinoma (subtype IIA), and 63 in adenocarcinoma only (subtype IIB). Predicted targets in 12 molecular pathways were identified for highly altered microRNAs, including the Wnt signaling pathway leading to low-grade dysplasia. β-catenin expression correlated with downregulated microRNAs. Conclusions: Our findings suggest that numerous microRNAs play roles in the sequence of molecular events, especially early events, resulting in colorectal adenocarcinoma. The temporal patterns and complexity of microRNAome alterations during progression will influence the efficacy of microRNAs for clinical purposes. Clin Cancer Res; 17(23); 7283–93. ©2011 AACR.

Isocitrate dehydrogenase 1 (IDH1) and IDH2 mutations and polymorphism are reported in 5% to 15% of acute myeloid leukemia (AML) cases, with G105 and R132 of IDH1 and R140 and R172 of IDH2 known to be clinically significant. Current Sanger sequencing assays to detect IDH mutations are labor intensive and not cost effective for clinical testing of low-frequency mutations. Therefore, we developed clinical assays using high-resolution melting (HRM) analysis to screen for all four variants listed above, followed by Sanger sequencing confirmation. The sensitivities of the assays were 7.3% and 7.9% for the detection of IDH2 and IDH1 variants, respectively, against the background of wild-type transcripts. Comparison of HRM to Sanger sequencing on 146 AML bone marrow samples for validation showed near-perfect concordance for all positive and negative results for IDH1 (98%) and IDH2 (94%). Postvalidation clinical implementation of upfront HRM screening (N = 106), using a more conservative algorithm to avoid false-negative results, reduced the number of Sanger sequencing tests by 73% (IDH1) and 78% (IDH2). Of the variant calls made by HRM in postvalidation clinical samples, Sanger confirmed the presence of a variant in 62% (IDH1) and 44% (IDH2) of the samples. In conclusion, our HRM assays are rapid, convenient, and versatile assays for screening and confirmation of alterations in IDH1 and IDH2.

BACKGROUND: Isolated isochromosome (17q) is a rare cytogenetic abnormality in Philadelphia chromosome-negative myeloid neoplasms, usually myelodysplastic and/or myeloproliferative neoplasms (MDS/MPN). De novo acute myeloid leukemia (AML) with isochromosome 17q has rarely been reported. The frequency of genetic mutations is unknown. METHODS: The authors assessed clinicopathologic, immunophenotypic, and molecular genetic features of 22 myeloid neoplasms with isolated isochromosome 17q. RESULTS: Fourteen patients presented as MDS/MPN; 8 as de novo AML. Most presented with leukocytosis, anemia, thrombocytopenia, and splenomegaly. Morphologically, all showed myelodysplastic and myeloproliferative features, including pseudo-Pelger-Huet-like neutrophils, micromegakaryocytic hyperplasia, hypercellularity, fibrosis, and osteosclerosis. Blasts were increased (median, 40% in de novo AML; 9% in MDS/MPN). Immunohistochemical assessment of proliferation and apoptosis rates in AML were similar to a matched group without isochromosome 17q. In most patients, isochromosome 17q occurred at time of blast transformation or disease progression. DNA sequencing revealed no mutation in the uninvolved TP53 allele. Mutational analyses showed rare mutations in NRAS (3 of 10), FLT3 (2 of 16), and JAK2 (1 of 18), and no mutations in NPM1 (0 of 15), KIT (0 of 4), and CEBPA (0 of 4). The median overall survival was 14.5 months for de novo AML, and 11.0 months for MDS/MPN. With a median follow-up of 8.5 months (range, 1.5-107 months), 15 died of disease, 6 had persistent disease, and 1 was in remission after bone marrow transplantation. CONCLUSIONS: The authors conclude that myeloid neoplasms with isolated isochromosome 17q represent a distinct clinicopathologic entity with myelodysplastic and myeloproliferative features, high risk of leukemic transformation, and wild-type TP53. Cancer 2011;. © 2011 American Cancer Society.

Mutations in the isocitrate dehydrogenase 1 (IDH1) and IDH2 genes are reported in acute myeloid leukemia (AML). We studied the frequency and the clinicopathologic features of IDH1 and IDH2 mutations in AML. Mutations in IDH1 (IDH1R132) and IDH2 (IDH2R172) were assessed by Sanger sequencing in 199 AML cases. Point mutations in IDH1R132 were detected in 12 (6.0%) of 199 cases and in IDH2R172 in 4 (2.0%) of 196 cases. Of the 16 mutated cases, 15 (94%) were cytogenetically normal, for an overall frequency in this group of 11.8%. IDH1R132 and IDH2R172 mutations were mutually exclusive. Concurrent mutations in NPM1, FLT3, CEBPA, and NRAS were detected only in AML with the IDH1R132 mutation. The clinical and laboratory variables of patients with AML with IDH mutations showed no significant differences compared with patients with wild-type IDH. We conclude that IDH1R132 and IDH2R172 mutations occur most often in cytogenetically normal AML cases with an overall frequency of approximately 11.8%.

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.

Suppression of annexin A1 (ANXA1), a mediator of apoptosis and inhibitor of cell proliferation, is well documented in various cancers but the underlying mechanism remains unknown. We investigated whether decreased ANXA1 expression was mediated by microRNAs (miRNAs), which are small, non-coding RNAs that negatively regulate gene expression. Using Sanger miRBase, we identified miR-584, miR-196a and miR-196b as potential miRNAs targeting ANXA1. Only miRNA-196a showed significant inverse correlation with ANXA1 mRNA levels in 12 cancer cell lines of esophageal, breast and endometrial origin (Pearson's correlation -0.66, P=0.019), identifying this as the candidate miRNA targeting ANXA1. Inverse correlation was also observed in 10 esophageal adenocarcinomas (Pearson's correlation -0.64, P=0.047). Analysis of paired normal/tumor tissues from additional 10 patients revealed an increase in miR-196a in the cancers (P=0.003), accompanied by a decrease in ANXA1 mRNA (P=0.004). Increasing miR-196a levels in cells by miR-196a mimics resulted in decreased ANXA1 mRNA and protein. In addition, miR-196a mimics inhibited luciferase expression in luciferase plasmid reporter that included predicted miR-196a recognition sequence from ANXA1 3'-untranslated region confirming that miR-196a directly targets ANXA1. miR-196a promoted cell proliferation, anchorage-independent growth and suppressed apoptosis, suggesting its oncogenic potential. This study demonstrated a novel mechanism of post-transcriptional regulation of ANXA1 expression and identified miR-196a as a marker of esophageal cancer.

Objectives Waldenstrom macroglobulinemia (WM) is a B-cell lymphoma characterized by the accumulation of lymphocytes and plasmacytic cells in the bone marrow and by excess production of immunoglobulin M in serum. WM has been closely linked with the MYD88 L265P mutation. Whole genome sequencing has identified somatic mutations in the CXCR4 gene in ∼29% of WM cases with MYD88 L265P . CXCR4 mutations may interfere with treatment response to ibrutinib. The goal of this study was to design and validate a clinical assay to detect CXCR4 mutations. Methods Thirty-three low-grade B-cell lymphomas with plasmacytic differentiation (23 MYD88 L265P and 10 MYD88 WT ) involving various samples types (fresh and formalin-fixed tissues) formed the study group. We designed and validated Sanger sequencing and pyrosequencing assays to detect mutations in CXCR4 in a Clinical Laboratory Improvement Amendments-approved clinical laboratory. Results We identified 8 cases with CXCR4 mutations, including 5 single nucleotide substitutions (3 resulting in p.S338* and 1 in p.R334*), and 3 insertion/deletions. Seven of 8 CXCR4 mutated cases were also MYD88 L265P mutant. Among the single nucleotide substitutions, we identified a novel missense variant (p.L326P) and a previously reported variant (G335S) of uncertain clinical significance. Conclusions We successfully validated a set of clinical assays to detect mutations in CXCR4 mutations in a clinical laboratory.

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.

The use of cytology specimens for next-generation sequencing (NGS) is particularly challenging because of the unconventional substrate of smears and the often limited sample volume. An analysis of factors affecting NGS testing in cytologic samples may help to increase the frequency of successful testing.This study reviewed variables associated with all in-house cytology cases (n = 207) that were analyzed by NGS with the Ion Torrent platform during a 10-month interval. A statistical analysis was performed to measure the effects of the DNA input threshold, specimen preparation, slide type, tumor fraction, DNA yield, and cytopathologist bias.One hundred sixty-four of 207 cases (79%) were successfully sequenced by NGS; 43 (21%) failed because of either a low DNA yield or a template/library preparation failure. The median estimated tumor fraction and DNA concentration for the successfully sequenced cases were 70% and 2.5 ng/μL, respectively, whereas they were 60% and 0.2 ng/μL, respectively, for NGS failures. Cell block sections were tested in 91 cases, and smears were used in 116 cases. NGS success positively correlated with the DNA yield but not the tumor fraction. Cell block preparations showed a higher success rate than smears. Frosted-tip slides yielded significantly more DNA than fully frosted slides. Lowering the input DNA concentration below the manufacturer's recommended threshold of 10 ng (0.85 ng/μL) resulted in a marked increase in the NGS success rate from 58.6% to 89.8%.The failure of NGS with cytology samples is usually a result of suboptimal DNA due to multiple pre-analytical factors. Knowledge of these factors will allow better selection of cytology material for mutational analysis.

A new mantle cell lymphoma cell line, M-1, was established from peripheral blood mononuclear cells of a patient with a diagnosis of blastoid variant of mantle cell lymphoma in leukemic phase. This cell line showed cell surface antigens identical to the original tumor and demonstrated the profile of a mature B-cell phenotype typical of mantle cell lymphoma: positive for CD5, CD19, CD20, sIgM and FMC7, and negative for CD3, CD10 and CD23. Cytogenetically, the M-1 cell line showed chromosomal alterations similar to the initial clinical specimen, among which a translocation t(11;14) (q13;q32) resulting in the overexpression of cyclin D1 as well as additional abnormalities involving chromosomes 3, 9 and 10. This cell line was used as a model to investigate the activity of the three drugs doxorubicin, cyclophosphamide and vincristine, commonly used in the treatment of mantle cell lymphoma patients. The effect of the drugs was evaluated by a 24 h cytotoxicity test and a 7-days anti-proliferation test using a microculture tetrazolium-based assay (MTT). Both assays indicated a higher sensitivity of the cell line to vincristine when compared to doxorubicin and cyclophosphamide. The characterization of a new mantle cell lymphoma cell line is a unique tool for studying the biology of this subtype of lymphoma for which only a few cell lines have been established.

Background: In a clinical diagnostic laboratory, we evaluated the applicability of the Ion Proton sequencer for screening 409 cancer-related genes in solid tumours. Methods: DNA was extracted from formalin-fixed, paraffin-embedded (FFPE) tissue biopsy specimens of 55 solid tumours (20 with matched normal tissue) and four cell lines and screened for mutations in 409 genes using the Ion Proton system. The mutation profiles of these samples were known based on prior testing using the Ion Torrent Personal Genome Machine (46-gene hotspot panel), Sanger sequencing, or fluorescence in situ hybridisation (FISH). Concordance with retrospective findings and additional mutations were evaluated. Assay sensitivity and reproducibility were established. Gene copy number variations (CNVs) detected were confirmed by molecular inversion probe (MIP) array. Results: The average Ion Proton (409-gene panel) sequencing output per run was 8 gigabases with 128 million sequencing reads. Of the 15,992 amplicons in the 409-gene panel, 90% achieved a minimum average sequencing depth of 100X. In 59 samples, the Ion Proton detected 100 of 105 expected single-nucleotide variants (SNVs) and all expected deletions (n=8), insertions (n=5), and CNVs (n=7). Five SNVs were not detected due to failed amplification of targeted regions. In 20 tumours with paired normal tissue, Ion Proton detected 37 additional somatic mutations, several in genes of high prognostic or therapeutic significance, such as MET, ALK, TP53, APC, and PTEN. MIP array analysis confirmed all CNVs detected by Ion Proton. Conclusions: The Ion Proton (409-gene panel) system was found to be well suited for use in a clinical molecular diagnostic laboratory. It can simultaneously screen 409 genes for a variety of sequence variants in multiple samples using a low input of FFPE DNA with high reproducibility and sensitivity.

Quantitative profiling of codon 816 KIT mutations can aid in the classification of systemic mast cell disease

Array-based comparative genomic hybridization (aCGH) chromosomal analysis facilitates rapid detection of cytogenetic abnormalities previously undetectable by conventional cytogenetics. In this study, we analyzed 48 uniformly treated patients with acute myeloid leukemia (AML) by 44K aCGH and correlated the findings with clinical outcome. aCGH identified previously undetected aberrations, as small as 5 kb, of currently unknown significance. The 36.7 Mb minimally deleted region on chromosome 5 lies between 5q14.3 and 5q33.3 and contains 634 genes and 15 microRNAs, whereas loss of chromosome 17 spans 3194 kb and involves 342 genes and 12 microRNAs. Loss of a 155 kb region on 5q33.3 (p < 0.05) was associated with achievement of complete remission (CR). In contrast, loss of 17p11.2-q11.1 was associated with a lower CR rate and poorer overall survival (Kaplan-Meier analysis, p < 0.0096). aCGH detected loss of 17p in 12/48 patients as compared to 9/48 by conventional karyotyping. In conclusion, aCGH analysis adds to the prognostic stratification of patients with AML.