Search

Primary cutaneous carcinosarcoma is a rare biphenotypic neoplasm exhibiting both epithelial and sarcomatous elements. Even though its origin and biological aspects remain poorly understood, it has been postulated that this tumor may arise from progenitor cells, which subsequently differentiate into distinct tumor components. We have investigated the histological and immunohistochemical staining patterns of a cutaneous carcinosarcoma case, as well as its ultrastructural aspects. In addition, sarcomatous and epithelial tumor components were separated by laser capture microdissection and subjected to targeted, high-depth, next-generation sequencing of a 46-cancer gene panel to asses the gene mutational pattern amongst both components. There were transitional cells at the epithelial/mesenchymal transition that labeled with putative progenitor cell markers (K19, c-kit, CD34 and Bcl-2). There was shared reactivity to antibodies directed against the progenitor cell marker EpCAM (epithelial cell adhesion molecule) in both components. Ultrastructurally, individual cells were demonstrated to have overlapping features of epithelial and mesenchymal differentiation. The mutational analysis revealed point mutations in exon 5 of TP53, which were identical in both the epithelial and sarcomatous components, and which were concordant with p53 expression at a tissue level. The aforementioned histological, ultrastructural, immunohistochemical and mutational pattern is strongly suggestive of a common clonal origin to the distinct elements of this tumor.

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.

Increasing use of fine needle aspiration for oncological diagnosis, while minimally invasive, poses a challenge for molecular testing by traditional sequencing platforms due to high sample requirements. The advent of affordable benchtop next-generation sequencing platforms such as the semiconductor-based Ion Personal Genome Machine (PGM) Sequencer has facilitated multi-gene mutational profiling using only nanograms of DNA. We describe successful next-generation sequencing-based testing of fine needle aspiration cytological specimens in a clinical laboratory setting. We selected 61 tumor specimens, obtained by fine needle aspiration, with known mutational status for clinically relevant genes; of these, 31 specimens yielded sufficient DNA for next-generation sequencing testing. Ten nanograms of DNA from each sample was tested for mutations in the hotspot regions of 46 cancer-related genes using a 318-chip on Ion PGM Sequencer. All tested samples underwent successful targeted sequencing of 46 genes. We showed 100% concordance of results between next-generation sequencing and conventional test platforms for all previously known point mutations that included BRAF, EGFR, KRAS, MET, NRAS, PIK3CA, RET and TP53, deletions of EGFR and wild-type calls. Furthermore, next-generation sequencing detected variants in 19 of the 31 (61%) patient samples that were not detected by traditional platforms, thus increasing the utility of mutation analysis; these variants involved the APC, ATM, CDKN2A, CTNNB1, FGFR2, FLT3, KDR, KIT, KRAS, MLH1, NRAS, PIK3CA, SMAD4, STK11 and TP53 genes. The results of this study show that next-generation sequencing-based mutational profiling can be performed on fine needle aspiration cytological smears and cell blocks. Next-generation sequencing can be performed with only nanograms of DNA and has better sensitivity than traditional sequencing platforms. Use of next-generation sequencing also enhances the power of fine needle aspiration by providing gene mutation results that can direct personalized cancer therapy.

Transfer of next-generation sequencing technology to a Clinical Laboratory Improvement Amendments–certified laboratory requires vigorous validation. Herein, we validated a next-generation sequencing screen interrogating 740 mutational hotspots in 46 cancer-related genes using the Ion Torrent AmpliSeq cancer panel and Ion Torrent Personal Genome Machine (IT-PGM). Ten nanograms of FFPE DNA was used as template to amplify mutation hotspot regions of 46 genes in 70 solid tumor samples, including 22 archival specimens with known mutations and 48 specimens sequenced in parallel with alternate sequencing platforms. In the archival specimens, the IT-PGM detected expected nucleotide substitutions (n = 29) and four of six insertions/deletions; in parallel, 66 variants were detected. These variants, except a single nucleotide substitution, were confirmed by alternate platforms. Repeated sequencing of progressively diluted DNA from two cancer cell lines with known mutations demonstrated reliable sensitivity at 10% variant frequency for single nucleotide variants with high intrarun and inter-run reproducibility. Manual library preparation yielded relatively superior sequencing performance compared with the automated Ion Torrent OneTouch system. Overall, the IT-PGM platform with the ability to multiplex and simultaneously sequence multiple patient samples using low amounts of FFPE DNA was specific and sensitive for single nucleotide variant mutation analysis and can be incorporated easily into the clinical laboratory for routine testing.

Today, DNA sequencing is one of the most important tools for the study of nucleic acid structure and composition. Traditional DNA sequencing methods are based on dideoxy chain termination technology and are limited in both throughput and cost for most future applications. Recently, pyrosequencing has surfaced as a new sequencing methodology for real-time nucleotide sequencing. It is a nonelectrophoretic DNA sequencing method which circumvents the need for labeled nucleotides, labeled primers, and gel electrophoresis, and offers the advantages of high accuracy, flexibility, parallel processing, and easy automation. Thus, it is increasingly being used in DNA sequencing, genotyping, single nucleotide polymorphism analysis, allele quantification, and whole genome sequencing within the areas of clinical genetics, pharmacogenetics, and microbiology. In this chapter, we will briefly review the key aspects of pyrosequencing technology with a focus on its application in hotspot mutation analysis.

To the editor:We thank Dr. Zidar and Dr. Gale for their timely and detailedcomments on our paper [1]. Indeed, epithelial-mesenchymaltransition (EMT) is a multifaceted process driven by an intri-cate network of molecular mechanisms which to date remainincompletelyunderstood,duetotheextraordinarycomplexityoftheirregulation[2].Theseprocessesincludechangesincelladhesionandshape,geneexpression,andextensivecross-talkbetween signaling pathways that create complex biochemicalnetworks [2, 3], of which the final result in cancer is the de-velopment of mesenchymal-like cells with enhanced capacityfor invasion, migration, and dissemination [3].Even though some authors have proposed that phenotypicmodulation in tumor progression is a reflection of genomicinstabilityratherthananEMT-drivenprocess[4],westronglybelievethatthereiscompellingevidencesuggestingakeyroleof EMTin the biology of tumor progression.As we proposedin our paper, primary carcinosarcomas of the skin are rarebiphenotypiccutaneoustumorswhichsimultaneouslyexpressboth epithelial and mesenchymal elements, suggesting that intheirdevelopmentanEMTprocessmightbeinvolved[5].Weexplored this possibility by assessing loss of E-cadherin ex-pression and upregulation of vimentin expression, a patternthat is considered to be the hallmark of EMTchanges in epi-thelial cells [5]. However, we do agree that more stringentcriteria should be applied when assessing EMT-suggestivehistological patterns.As Zidar and Gale state, among the multiple criteria thatshouldbeassessed,thepresenceofspindlecellmorphologyisa sine qua non. Although different epithelial morphotypes ofcutaneous carcinosarcoma exist (squamous cell or basal cellderived), the presence of a spindle cell component is obliga-tory and a feature of all cases we studied. In all cases westudied, β-catenin and E-cadherin were expressed in the cy-toplasmic membrane of benign epithelium, while tumor cellsshowed decreased E-cadherin membranous expression along

Context.—Nucleophosmin 1 (NPM1) is the most commonly mutated gene in acute myeloid leukemia. Detection of NPM1 mutations is useful for stratifying patients for therapy, predicting prognosis, and assessing for minimal residual disease. Several methods have been developed to rapidly detect NPM1 mutations in genomic DNA and/or messenger RNA specimens. Objective.—To directly compare a quantitative real-time polymerase chain reaction (qPCR) assay with a widely used capillary electrophoresis assay for detecting NPM1 mutations. Design.—We adopted and modified a qPCR assay designed to detect the 6 most common NPM1 mutations and performed the assay in parallel with capillary electrophoresis assay in 207 bone marrow aspirate or peripheral blood samples from patients with a range of hematolymphoid neoplasms. Results.—The qPCR assay demonstrated a higher analytical sensitivity than the capillary electrophoresis 1/1000 versus 1/40, respectively. The capillary electrophoresis assay generated 10 equivocal results that needed to be repeated, whereas the qPCR assay generated only 1 equivocal result. After test conditions were optimized, the qPCR and capillary electrophoresis methods produced 100% concordant results, 85 positive and 122 negative. Conclusions.—Given the higher analytical sensitivity and specificity of the qPCR assay, that assay is less likely to generate equivocal results than the capillary electrophoresis assay. Moreover, the qPCR assay is quantitative, faster, cheaper, less prone to contamination, and well suited for monitoring minimal residual disease.

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 (IDH1(R)¹³²) and IDH2 (IDH2(R)¹⁷²) were assessed by Sanger sequencing in 199 AML cases. Point mutations in IDH1(R)¹³² were detected in 12 (6.0%) of 199 cases and in IDH2(R)¹⁷² 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%. IDH1(R)¹³² and IDH2(R)¹⁷² mutations were mutually exclusive. Concurrent mutations in NPM1, FLT3, CEBPA, and NRAS were detected only in AML with the IDH1(R)¹³² 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 IDH1(R)¹³² and IDH2(R)¹⁷² mutations occur most often in cytogenetically normal AML cases with an overall frequency of approximately 11.8%.

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.

1633 Poster Board I-659 Background The presence of abnormal cytogenetic findings at diagnosis is an independent indicator of the outcome in patients with acute myeloid leukemias (AML). Recent availability of high-resolution analysis by aCGH technology has facilitated rapid detection of cytogenetic abnormalities previously undetected by the conventional G-banded karyotyping. Aim: To determine whether specific cytogenetic abnormalities, as detected by the aCGH analysis, are associated with differences in clinical outcomes in a group of patients with AML treated uniformly with the standard chemotherapy regimen of Idarubicin and Cytarabine. Methods A total of 111 patients with newly diagnosed AML (Median age: 55 years; range, 22 to 73 years) were enrolled in the study. All patients were treated with Idarubicin 12 mg/m 2 IV daily x 3 days and Cytarabine 1.5 g/m 2 by continuous IV infusion daily x 4 days (3 days in patients older than 60). The diagnostic bone marrow samples from 48 of these patients were analyzed by aCGH using a 44K CGH array with a spatial resolution of 50-75 kb (Agilent Inc). The aberrations detected by aCGH using CGH Analytics (Agilent Inc, Santa Clara, CA) and the Nexus Copy Number Software (Biodiscovery Inc, El Segundo, CA) were compared to conventional G-banded karyotyping results. Correlation of aCGH-detected aberrations with clinical outcome was performed by Kaplan-Meier analysis and log-rank test. Results Complete remission (CR) was achieved in 34/48 (71%) patients, out of which 16 (47%) patients relapsed in less than 1 year (Median: 18.5 weeks, range: 1-44 weeks) and the remaining 18 (53%) patients sustained CR beyond one year (Median: 89 weeks, range: 58-132 weeks). The comparison of aCGH findings between the patients achieving CR and the resistant patients showed significant association of loss of a 155 kilobase region on 5q33.3 with achievement of CR (p TP53 gene in 3/12 (25%) patients, although these three patients showed genetic instability and poor clinical outcome. Overall, the aCGH findings were in concordance with the conventional karyotyping results. The aCGH analysis was able to detect aberrations in samples containing blast count as low as 5%. In addition, aCGH identified previously undetected aberrations, as small as 5 kb, of currently unknown significance. Conclusion The aCGH analysis indicates that the loss of 5q33.3 is associated with achievement of complete remission and the loss of 17p11.2-q11.1 is associated with poor overall survival in AML patients treated with Idarubicin and Cytarabine. Array CGH analysis provides a useful and rapid diagnostic tool for identifying these high-risk patients. The biologic roles of EBF1 and RNF145 in therapy response warrant further investigation. Disclosures No relevant conflicts of interest to declare.

Bortezomib, the first of a new class of agents called proteasome inhibitors, has shown promising activity in several tumor types, especially in hematological malignancies. To further characterize its activity in Non Hodgkin Lymphoma (NHL), we determined the efficacy of bortezomib, in vitro in 19 cell lines representing five subtypes of lymphoma and in ex vivo culture of patients samples. Using an MTS cell cytotoxicity assay, bortezomib was found to induce apoptosis at lower doses (IC50 ranging from 13.2 to 19.4 nM) in Mantle Cell Lymphoma (MCL) and Diffuse Large B-Cell Lymphoma (DLBCL) cells than in Burkitt lymphoma, Primary Effusion Lymphoma (PEL) and Anaplastic Large Cell Lymphoma (ALCL). In ex vivo culture of patients samples treated with 5 or 10 nM of bortezomib, the percentage of cells undergoing apoptosis as determined by annexin V-FITC staining was greater in MCL (+28.2% ± 12 with 10nM; +12.5% ± 13.8 with 5 nM) than in DLCL samples (+13.4% ± 6.2 with 10nM; +4.9% ± 1.5 with 5nM) when compared to spontaneous apoptosis. Bortezomib also showed a much lower IC50 than conventional cytotoxic agents (doxorubicin, vincristine and 4-hydroperoxycyclophosphamide) in 4 MCL cell lines. The potential synergy between cytotoxic agents and bortezomib was studied using annexin FITC staining as well as PARP cleavage analysis. Co-treatment of MCL cell lines with bortezomib and any of these chemotherapy agents showed a synergistic effect. However, the synergy was greater if the cells were sequentially treated with doxorubicin or vincristine followed by bortezomib. On the opposite the synergistic effect was greatly reduced or abolished when the cells were pretreated with bortezomib before exposure to doxorubicin or vincristine. We then used gene expression profiling to characterize the molecular consequences of bortezomib treatment in both MCL and DLBCL lymphoma cell lines. Using a 2 fold cut-off and a p value of

We tested the feasibility of performing gene expression profiling using amplified RNA from Fine Needle Aspiration (FNA) obtained from lymph nodes. Twenty-four samples from patients with a diagnosis of Follicular Lymphoma (FL) or Large B-Cell Lymphoma (LBCL) were obtained after informed consent. The diagnosis were performed by two pathologists and classified into two groups (10 LBCL and 14 FL) using conventional morphology and immunophenotyping. Total RNA was extracted using RNaqueous (Ambion, Austin, TX). Total RNA (100ng /per sample) was subjected to 2 cycles of standard double-stranded cDNA synthesis using Superscript Choice System (Invitrogen, Grand Island, NY) and in vitro transcription for target amplification as per GeneChip’s Eukaryotic Small Sample Target Labeling protocol (Affymetrix, Inc, Santa Clara, CA). The biotinylated cRNA from each sample was hybridized to Affymetrix HG-U133A chips. Gene expression profiling results were first analyzed by Principal Component Analysis (PCA) using a list of 146 probe sets representing 62 genes characteristic of Acivated B-Cell (ABC) or Germinal Center (GC) signature. This analysis identified 5 LBCL cases with ABC cell signature (Fig 1). Using a list of 207 probe sets representing 113 genes involved in FL transformation (K Elenitoba-Johnson, PNAS 2003), PCA analysis identified two overlapping clusters corresponding to FL and GC-DLBCL. To further improve this classification, we generated a list of 82 genes differentially expressed between FL and GC-LBCL. Using this list of genes, PCA analysis demonstrated a clear separation between FL and GC-LBCL (Fig 2). These results demonstrate that comprehensive transcription profiles can be performed in patients with NHL using RNA obtained from FNA and that one can distinguish among FL and LBCL lymphoma genetic subtypes. Figure Figure

Establishment and Characterization by Gene Expression Profiling of a New Diffuse Large B-Cell Lymphoma Cell Line, EJ-1, Carrying t(14;18) and t(8;14) Translocations