Formalin-fixed Paraffin-embedded Tissue Kit Research Articles

Genetic Mutations in Primary Refractory Diffuse Large B-Cell Lymphoma: Next Generation Sequencing Analysis

Introduction Diffuse large B-cell lymphoma (DLBCL) accounts for 30-40% of newly diagnosed adult non-Hodgkin lymphoma. About 60-70% of DLBCL patients could be cured with rituximab, cyclophosphamide, doxorubicin, vincristine and prednisolone (R-CHOP), while the other patients show chemo-refractory or relapse of disease and have poor prognosis. The prognostic difference between DLBCL patients is not fully understood, and many attempts to classify DLBCL have been made. Recently, with the development of the next generation sequencing (NGS) technique, Schmitz et al. identified four prominent genetic subtypes named MCD, BN2, N1, EZB. In this study, we performed targeted deep sequencing in DLBCL patients and analyzed whether there is any genetic difference between the patients who are primary refractory disease and the patients who are not. Methods Totally, 77 patients who was diagnosed with DLBCL and treated with R-CHOP from January 2021 to April 2023 were included in this analysis. Formalin-fixed, paraffin-embedded (FFPE) tumor samples obtained at diagnosis were archived. Genomic DNA was extracted using QIAmp DNA FFPE tissue kit according to the manufacturer's protocol. Genetic profiling included the targeted deep sequencing of 424 genes, which had been selected based on recurrent mutations from previous studies, and sequenced using an Illumina Nextseq. The threshold of mutation positivity as a variant allele frequency ≥2% for SNV/Indel and as a copy number ≥4.0(gain) for CNV. Patients were assigned as germinal center B-cell (GCB) or non-GCB subtype based on the immunohistochemistry (IHC) algorithm. BCL-2 and MYC FISH were conducted in patients with double-expressor DLBCL with non-GCB subtype. Results Median age of the patients were 66 years (24-86 years), and 49 patients (63.6%) were male. Forty-five patients (58.5%) were advanced stage. Twenty-four patients (31.2%) were GCB subtype and 51 patients (66.2%) were non-GCB subtype. Among the GCB type DLBCL patients, 10 patients were double expressor DLBCL, and there was no case of double hit lymphoma. As a result of targeted deep sequencing, 317 mutations were found in at least 1 patient, and the patients had a median number of 16 mutations (4-50). KMT2D was the most commonly detected mutation (22/77, 29.3%), followed by MYD88 (19/77, 25.3%), PCLO (19/77, 25.3%), PIM1 (19/77, 25.3%), TP53 (18/77, 24.0%). According to NGS analysis, 26 patients (33.85%) were assigned to MCD subgroup, 13 patients (16.9%) to BN2, 11 patients (14.3%) to N1, 10 patients (13.0%) to EZB, and 17 patients (17%) were not assigned to any subrgroup. Median follow-up of the patients was 13.8 months (0.4-22.8 months), and 12 patients (15.6%) relapsed and 5 patients (6.5%) died. Median PFS and OS were not reached. Among relapsed patients, 7 patients were primary refractory to R-CHOP. In primary refractory DLBCL patients, TP53 (3/7, 42.9%) and CIITA (3/7, 42.9%) were most commonly detected. CIITA is known to be associated with immune escape and treatment resistance. CIITA, RASSF54, STAT6, SYK significantly more mutated in the primary refractory patients than the other ( CIITA, 42.9% vs. 8.6%, p=0.031; RASSF54, 28.6% vs. 2.9%, p=0.039; STAT6, 28.6% vs. 1.4%, p=0.021; SYK, 28.6% vs. 2.9%, p=0.039) (Table 1). Conclusion CIITA, RASSF54, STAT6, SYK were frequently mutated in primary refractory DLBCL in this study. Because of the small sample size, however, the prognostic significance of these mutations needed to be studied in further researches.

Abstract 6110: Multi-site evaluation of FFPE homologous recombination deficiency reference materials

Abstract Homologous recombination deficiency (HRD) arises when defects in DNA repair pathways occur, leading to genomic instability. HRD status is an emerging therapeutic biomarker; NGS assays that measure it can be used to stratify ovarian and breast cancer patients and determine eligibility for clinical trials, and PARP inhibitor- and platinum-based therapies. Seraseq FFPE HRD reference materials were developed to cover a range of genomic instability scores (GIS) to help NGS HRD assay validation and development. Here we discuss a multi-site evaluation across various platforms of our high-positive, low-positive, and negative HRD reference materials. Tumor cell lines were characterized by sequencing and evaluated in collaboration with several IVD partners. Three breast cancer cell lines (along with their SNP-matched normal cell lines) were selected based on their GIS. Tumor cells were blended with their SNP-matched normal cells to achieve ~65% tumor content. Biosynthetic DNA containing mutations in homologous recombination repair (HRR) genes (ATM, BRIP1, RAD51C, RAD51D) were added to the high-positive and negative reference materials targeted at >5% variant allele frequency (VAF) and measured by digital PCR. Formalin fixed paraffin embedded (FFPE) blocks were made and each block was tested for yield per curl using both the Qiagen QIAamp DNA FFPE Tissue Kit and the Maxwell RSC DNA FFPE Kit for extraction and Qubit dsDNA HS kit for concentration analysis. DNA quality was assessed using an Agilent gDNA ScreenTape Assay for the TapeStation. Whole genome shotgun sequencing was performed on extracted DNA using LGC NxSeq AmpFREE Low DNA Library Kit and Roche KAPA UDI adapters and Illumina NextSeq 2000 P1 flow cell. HRD status was evaluated by external collaborators with multiple NGS and microarray assays, including the Illumina TSO500 HRD RUO assay, the SOPHiA DDM HRD Solution and the OncoScan CNV Array A breast cancer cell line with a GIS of ~75 was selected as the HRD high-positive reference material, a second breast cancer cell line with a GIS of ~60 was selected as HRD low-positive, and a third breast cancer cell line with a GIS of ~30 was selected as HRD negative. Representative DNA yields per 10-micron section (determined using the HRD positive FFPE curls extracted by Qiagen QIAamp method) were 165 ± 28 ng/curl. Digital PCR confirmed the presence of the 8 HRR mutations (in 4 genes) at levels >5% VAF. GIS varied for each material across assays, but HRD status was consistent, confirming the wide applicability of the new reference materials We have developed the Seraseq HRD reference materials to meet the needs of laboratories looking to analyze HRD in cancer patient samples. These reference materials facilitate standardization and quality control in HRD testing by clinical labs for current and new PARP inhibitor treatment stratification in expanded patient populations that may include those with WT BRCA1/2 genes Citation Format: Dana Ruminski Lowe, Robert M. Whiting, Matthew G. Butler, Yves Konigshofer, Catherine Huang, Indira Chivukula, Krystyna Nahlik, Dianren Xia, Russell Garlick, Bharathi Anekella. Multi-site evaluation of FFPE homologous recombination deficiency reference materials. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6110.

Next generation sequencing for the formalin-fixed paraffin-embedded samples of ovarian cancer in Vietnam

Ovarian cancer is one of the most popular causes of mortality among women, and the prevalence of ovarian cancer is increased. Early diagnosis of this disease via genetic variant testing is one potential strategy for enhancing treatment and disease outcome. In this study, we used the formalin-fixed, paraffin-embedded (FFPE) samples of ovarian tumor tissues from Vietnamese patients to detect pathogenic variants in BRCA1/BRCA2 via next-generation sequencing. DNA was extracted using QIAamp DNA FFPE Tissue Kit, and then its quality was assessed using BioDrop and Qubit. The BRCAaccuTestTM PLUS kit and Illumina MiSeqDx instrument were used for both library preparation and sequencing. All samples had passed the A260/280 ratio cut-off for DNA purity and the requirement of DNA concentration. Excepted for the 1st time, the percentage of ≥ Q30 was more than 80%, while the density was approximately 1,200 K/mm2, while the phasing and prephasing (%) metrics were satisfied to be less than 0.1%. 5 pathogenic variants in BRCA1/BRCA2, including both single nucleotide polymorphisms and indels were successfully detected using NgeneAnalySysTM software.

Analysis of mRNA Expression in Peripheral, Bone Marrow and Lymph Node Dervied CLL Cells Using the Nanosting Ncounter Platform

BackgroundMicroenvironmental niches within the bone marrow and lymph node have been shown to play a significant role in promoting CLL cell survival and proliferation. Several intracellular signalling pathways have been identified as key mediators of the interaction between CLL cells and accessory cells within these tissues. A better understanding of the underlying biology of the CLL cell and the roles of the tumor microenvironment have provided the rationale for trials of a range of novel therapeutic agents including ibrutinib and idelalisib, which target components of the BCR signaling pathway [Brown et al. Blood 2014, Byrd et al. Blood 2014]. Curative approaches for CLL therapy must target the proliferative, drug-resistant compartments of disease within these microenvironments.The NanoString® nCounter platform enables mRNA profiling of archival samples, including formalin fixed, paraffin embedded tissue (FFPE). A recent study from our laboratory demonstrated the utility of this technology in profiling lymph node and bone marrow sections from patients with diffuse large B-cell lymphoma for determining cell of origin classification [Gifford et al. BJH 2017].We have employed the NanoString® technology to profile and compare the mRNA expression profile of CLL cells derived from the peripheral blood, bone marrow and lymph node. In addition, we profiled PB-derived CLL cells following in vitro co-culture with a human stromal cell line under either normoxic or hypoxic conditions, mimicking these tissue microenvironments.MethodsRNA was extracted from FFPE archival bone marrow trephines (n = 2) and lymph node sections (n = 2), using the QIAGEN RNeasy FFPE Kit. All biopsies analysed were comprised of > 80 % lymphocytes, as determined by microscopic review.RNA from cryopreserved PBMC fractions (n = 4) was isolated either immediately following thawing or following co-culture with HS5 stromal cells for 24 h under normoxic (n = 4) or hypoxic (n = 4) conditions using the QIAGEN RNeasy Mini Kit.RNA from all preparations was quantified using a NanoDrop™ spectrophotometer. A total of 200ng of FFPE-derived RNA and 100ng of PBMC-derived RNA was analysed per sample on the NanoString® platform using a 260 gene panel comprised of the Human Cancer Reference gene panel (NanoString) spiked with an additional 30 genes of interest.Two-fold increases or decreases in mRNA expression were considered significant.ResultsOf the 260 genes profiled, 107 were upregulated in the lymph node samples and 68 in the bone marrow samples compared to expression in peripheral blood-derived CLL cells. Changes were seen in genes encoding surface receptors such as EGFR and NOTCH1, genes coding for intra-cellular signalling proteins such as ATK and WNT and genes associated with metabolism and cell division. 40 genes were downregulated in the lymph node samples and 27 in the bone marrow samples. These changes were seen in genes coding for surface receptors such as BAFF-R and CXCR4, genes coding for intra-cellular signalling proteins such as SYK and PI3K and genes coding for transcription factors such as JUN and FOS.Co-culturing PBMCs with an HS5 stromal layer induced similar changes to those observed in our comparison of the PBMCs and trephine samples; 56.8% and 44.7% of the mRNA expression changes observed in the bone marrow were observed in PBMCs cultured under normoxic and hypoxic conditions respectively. A similar comparison of the lymph node samples identified concordant changes in expression of 46.2% and 36.8% of genes under normoxic and hypoxic conditions respectively. Importantly, changes observed in genes coding for the anti-apoptotic protein MCL1, the surface receptors CXCR4 and ROR1 and the transcription factors ATF, FOS and JUN were consistent across samples from lymph nodes, marrow and the in vitro model.In summary, our data demonstrate that it is possible to of assess mRNA expression levels in FFPE archival tissue from CLL patients using the NanoString® platform. We have utilised this technology to profile PB, bone marrow and lymph node derived CLL cells and have identified a number of genes that are either up or down-regulated in cells derived from these microenvironments or following culture in an in vitro model of the tumour microenvironment. These data increase our understanding of how CLL cells populate and proliferate in the tumour microenvironment and lead to therapeutic strategies that target these cells. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Retrieving high-quality genomic DNA from formalin-fixed paraffin-embedded tissues for multiple molecular analyses

Formalin-fixed paraffin-embedded (FFPE) tissues represent the biggest source of archival materials for molecular biology research and pathology investigations. Nevertheless, fixation by formalin may cause denaturation and modification of macromolecules constraining DNA quality and its downstream applications. In this study, we developed a fast, simple, and cost-effective phenol/chloroform-based protocol for the extraction of high-quality DNA from 101 FFPE colorectal cancer tissue blocks that can be used in multiple molecular studies. DNA samples extracted using this phenol/chloroform protocol and the QIAamp DNA FFPE Tissue kit were evaluated for the quantity, quality, and amplificability. Spectrophotometer analyses revealed significantly higher quality and quantity of DNA samples obtained with the phenol/chloroform protocol as compared to those of the QIAamp DNA FFPE Tissue kit. In addition, the amplificability of these samples as assessed by conventional and multiplex polymerase chain reaction (PCR), followed by sequencing and fragment analyses presented an absolute success rate. Additionally, it is able to amplify a DNA fragment of 725 base-pairs at an adequate amount for downstream applications. This fast, simple, and cost-effective method may facilitate the molecular analyses of a large number of FFPE specimens that best suits the needs of the overall study design in terms of the quality and quantity of the extracted DNA.

Investigation of JC Polyomavirus (JCV) Genome in Colorectal Cancer Patients from Iran

Background: JC polyomavirus (JCV) is an epitheliotropic and neurotropic virus that identified in relationship with some devastating complications such as progressive multifocal leukoencephalopathy (PML) and linked to colorectal cancer. The aim of current study was to identify the prevalence of JCV in colorectal cancer for the first time in Iran. Methods: This retrospective case-control study was conducted by the hospitals affiliated to Iran University of Medical Sciences, Tehran, Iran from 2011 to 2016. Formalin-fixed paraffin-embedded (FFPE) blocks were used for DNA extraction by QIAamp® DNA FFPE Tissue Kit. The SYBER Green Real-time PCR assay performed by specific primers for JCV T-Large Ag. Melting curve analysis used for evaluation of amplification specificity. Positive control cloned in pTZ57R/T plasmid by Generay Biotechnology system. Results: Of 157 specimens 66 were colorectal cancer by the mean age (y) ± std. deviation 59.35±14.48 and 91 healthy control by the mean age (y) ± std. deviation 57.21±14.66. All 157 specimens tested for JCV T-Large Ag gene by Real-time PCR method and we found that there was not any positive result although the melting analysis showed specificity of positive control amplification. Conclusion: Low prevalence of JCV infection in Iranian CRC population confirmed by the current study results; there was not any JCV positive result in CRC and healthy control groups. Further studies by broader and different populations are recommended.

Mutational Landscape of Relapsed or Refractory Extranodal Natural Killer (NK)/T-Cell Lymphoma in Chinese Patients

Introduction: Extranodal natural killer/T-cell lymphoma (ENKTL), which is a very aggressive disease with poor prognosis, is prevalent in East Asian populations and accounts for 5-10% of all lymphomas in the Chinese population. The pathogenesis and molecular characteristics of different subgroups of ENKTL have largely remained elusive. Methods: Samples from 12 patients, aged 13 to 79, who were diagnosed with ENKTL in southwest China and received chemotherapy as first-line therapy, were collected for whole-exome sequencing (WES). Samples included 5 relapsed/refractory (rel/ref) patients and 7 treatment effective patients. The inclusion criteria for rel/ref group were that the patient progressed after 2 cycles of standard or conventional first-line treatment, or failed to achieve complete remission after 4 cycles (complete response (CR), or CR after treatment, but relapsed within 1 year after treatment). The inclusion criteria for the treatment effective group were that patient achieved CR after treatment, confirmed by long-term survival and no recurrence found after follow-up. In the rel/ref group, 3 cases occurred in the nasal cavity and 2 cases occurred outside the nasal cavity. In the treatment effective group, 3 cases occurred in the nasal cavity and 4 cases occurred outside the nasal cavity. The median overall survival (OS) in the rel/ref group and treatment effective group was 36 weeks and 80 weeks, respectively. Genomic DNA of tumors was extracted from the formalin-fixed, paraffin-embedded (FFPE) samples by using QIAamp DNA FFPE Tissue Kit. Along with virus infection status, somatic genomic alterations, including single nucleotide variations (SNV), short and long insertions and deletions (Indel), copy number variations (CNV), and gene rearrangements, were analyzed. Results: Common gene mutations (frequencies ≥25%) that occurred in both the rel/ref and treatment effective group included TP53, CD274, CSMD2, CUL9, EPPK1, PCDHA6, PCSK5, PKHD1, SDK1, STAT3 and TNXB. Relatively specific genes to the rel/ref group were ABCA13, COL22A1, NOTCH1, NDN, OBSL1, PPFIA2, SDK2, ZFP36L2, and ZNF860. In addition, we found that the most frequently mutated genes were different in patients with ENKTL occurring in the nasal cavity compared to those outside the nasal cavity. The high frequency mutated genes relatively specific to ENKTL in the nasal cavity were TP53 (50% vs. 17%), PKHD1 (50% vs. 0%), SDK1 (50% vs. 0%) and TNXB (50% vs. 0%), while in ENKTL outside the nasal cavity, EPPK1 (0% vs. 50%) was the most mutated gene. Meanwhile, we detected the wildly concerned biomarker CD274 (33% vs. 17%) and CHD8 (33% vs. 0%) in ENKTL occurring in the nasal cavity, and STAT3 (17% vs. 33%) in ENKTL outside the nasal cavity. Furthermore, we revealed that gene rearrangements were more common in the treatment effective group than in the rel/ref group (43% vs. 20%, respectively). A CD274 rearrangement and two TARP rearrangement were detected in 3/7 treatment effective patients, however, in the rel/ref ENKTL group, only a CD274 rearrangement was found. In this cohort, one ENKTL patient (1/12) exhibited a high tumor mutational burden (TMB) (15.4 Muts/Mb). Conclusion: Our findings revealed the molecular characteristics of different ENKTL subgroups, especially in the rel/ref subgroup, which might provide useful information for targeted/immunotherapy of ENKTL patients. Disclosures No relevant conflicts of interest to declare.

EP1.01-76 Oncomine Tumor Mutation Load Assay in NSCLC Patients from Argentina: Study Design and Feasibility

High throughput next generation sequencing has improved the understanding of the genomic landscape of cancer. Somatic mutations throughout the exome derive in novel peptide sequences that can be recognized by the host immune system and drive immune responses in patients. The tumor mutational load (TML) is the number of somatic mutations per megabase of DNA and is an emerging biomarker to predict response to immune checkpoint inhibitors. The objective of this study was to evaluate the feasibility of assessing TML in tumors from patients with advanced NSCLC, treated with Nivolumab in second with Ion Torrent™ Oncomine™ TML Assay and compare it with TML from WES. The Oncomine TML is a targeted NGS assay that provides quantification of somatic mutations, from limited formalin-fixed, paraffin-embedded (FFPE) samples. DNA was purified with QIAamp DNA FFPE Tissue Kit (QIAGEN). We used a new Ion AmpliSeq targeted panel, derived from the Comprehensive Cancer Panel which covers approximately 1.7 Mb of genomic DNA and 409 genes, OTML in S5, Ion Proton (Life Technologies); and WES were performed in NovaSeq 6000 Sequencing System (Illumina). We included 40 patients with advanced NSCLC. 27 (68%) of patients had both, tumor and normal tissue to perform WES. FFPE samples were obtained from lungs (N=21), lymph nodes (N=11), pleura (N= 3), brain (N=1), skin (N= 1), bone marrow (N = 1), soft tissue (N=1). We used 20 ng of DNA to develop the manual library and templating. We covered a large genomic footprint to accurately measure somatic mutations, replacing the need for whole-exome sequencing (WES). Pipeline for analysis of the NGS output will be presented. TML estimation with low DNA input requirements from FFPE samples is feasible. Up to 8 samples can be sequenced in a Ion 540™ Chip. Mutation load assessment can be done within 2–3 days. This assay was highly reproducible in FFPE samples. A detailed report provides normalized mutation count per MB as well as mutation signatures.

122P - Prevalence of EGFR mutations and its correlation with Egyptian patients’ human kinetics (PEEK Study)

BackgroundLung cancer is a leading cause of cancer-related deaths. Knowledge is evolving regarding cancer driver genes. In Egypt, very limited data exist about cancer driver genes for non-small cell lung cancer. EGFR mutation testing was initiated in Egypt through a collaboration between Colors lab and AstraZeneca. Data collection was planned to detect the prevalence of EGFR and determine consequent medical decisions. In this real-world evidence context, we tried to sketch out the Egyptian EGFR gene mutations and correlatie them to patient demographics regarding gender, age, smoking history and pathological sub-types. MethodsA total of 2017 formalin-fixed, paraffin-embedded (FFPE) tumors from patients diagnosed with non-small cell lung cancer were tested for EGFR mutation during the period between 2016 & 2018, following all aspects of GLP procedures including human rights, legal regulatory requirements, and AstraZeneca’s policy on bioethics. Patients were consented to collect their demographic and clinico-pathological data anonymously. DNA extraction was carried out using QIAamp DNA FFPE Tissue Kit. Extracted samples were tested by use of Therascreen EGFR RGQ PCR Kit on Rotorgene®, Qiagen. ResultsA total of 353 (17.5%) out of 2017 tested samples were positive for EGFR mutations. Mutations subtypes were detected in EGFR mutations presenting 151 (42.78%) for ex19 del, 106 (30.0%) for L858R point mutation, 39 (11.05%) for T790M mutations, 12 (3.4%) for insertions in exon 20, 8 (2.27%) for L861Q mutation, 8 (2.27%) for S768I mutation and 29 (8.22%) for other mutations. The older the patient age showed higher significance in prevalence rate (P<0.001) than in younger age. EGFR prevalence was numerically (NS) higher among females than males. Both current and formerly smoking patients showed highly significant (P<0.001) mutation frequencies compared with non-smokers. Adenocarcinoma showed a frequency of 55.8% with higher significance than other pathological cell types. ConclusionsThis is the first and largest study on EGFR mutation prevalence among Egyptian patients showing a slightly higher percentage than in European patients. Age, smoking status and pathological reports define significant impact on EGFR mutation pattern in Egyptian patients. Legal entity responsible for the studyAstraZeneca Egypt. FundingAstraZeneca Egypt. DisclosureAll authors have declared no conflicts of interest.

DNA extraction from FFPE tissue samples - a comparison of three procedures.

Aim of the studyOne of the critical steps in molecular oncology diagnostics is obtaining high quality genomic DNA. Therefore, it is important to evaluate and compare the techniques used to extract DNA from tissue samples. Since formalin-fixed, paraffin-embedded (FFPE) tissues are routinely used for both retrospective and prospective studies, we compared three commercially available methods of nucleic acid extraction in terms of quantity and quality of isolated DNA.Material and methodsSlides prepared from 42 FFPE blocks were macro-dissected. Resulting material was divided and processed simultaneously using three extraction kits: QIAamp DNA FFPE Tissue Kit (QIAGEN), Cobas DNA Sample Preparation Kit (Roche Molecular Systems) and Maxwell 16 FFPE Plus LEV DNA Purification Kit (Promega). Subsequently, quantity and quality of obtained DNA samples were analysed spectrophotometrically (NanoDrop 2000, Thermo Scientific). Results of quantitative analysis were confirmed by a fluorometric procedure (Qubit 3.0 Fluorometer, Life Technologies).ResultsThe results demonstrated that the yields of total DNA extracted using either Maxwell or Cobas methods were significantly higher compared to the QIAamp method (p < 0.001). The Maxwell Extraction Kit delivered DNA samples of the highest quality (p < 0.01). However, the highest total yield of extracted DNA was achieved with the Cobas technique, which may be due to a higher volume of eluate compared to the Maxwell method.ConclusionsTo our knowledge, this is the first paper which directly compares three extraction methods: Cobas, Maxwell and QIAamp. The data herein provide information required for the selection of a protocol that best suits the needs of the overall study design in terms of the quantity and quality of the extracted DNA.

Molecular survey on Merkel cell polyomavirus in patients with colorectal cancer

Background: Merkel cell polyomavirus (MCV) has been associated with Merkel cell carcinoma (MCC) in humans, and its role in other human cancers is under investigation. The aim of this study was to investigate MCV genome infection in patients with colorectal cancer (CRC).Methods: This retrospective, case-control study used archived formalin-fixed, paraffin-embedded (FFPE) tissue samples from colorectal cancer patients (cases) and matched healthy subjects (controls) diagnosed by an expert pathologist from hospitals affiliated with Iran University of Medical Sciences, Tehran, Iran from 2011 to 2016. After DNA extraction with a QIAamp® DNA FFPE Tissue Kit, real-time polymerase chain reaction (PCR) was used for diagnosis. A positive control was produced by cloning with the Generay Biotechnology system. SPSS v.22 was used for analysis of demographic variables.Results: There were 157 participants included in the study: 66 were cases and 91 were controls. Their mean ages (±SD) were 59.35±14.48 and 57.21±14.66, respectively. The proportion of males was 57.6% in the case group and 57.1% in the control group. None of the samples were positive for MCV expression by real-time PCR assay. Association was detected between males with CRC and tumor location in the rectum and between males with CRC and the mucinous tumor type.Conclusion: None of the tissues from the CRC or non-cancerous control groups were positive for MCV genome infection, although a low viral load, the sample type, or the method of use should not be neglected. Further studies are recommended to obtain more comprehensive results.

MRNA Profiling of CLL Cells Derived from the Blood, Bone Marrow and Lymph Node

BackgroundChemo-immunotherapy remains the backbone of therapy for patients with chronic lymphocytic leukaemia (CLL), with evidence pointing towards long term remission and even cure in patients with mutated IGHV status receiving this therapy frontline (1). However, relapse is common especially in those harbouring abnormalities in TP53 or ATM, unmutated IGHV status or a complex karyotype (2).It has become increasingly apparent that the bone marrow (BM) and lymph node (LN) play important roles in promoting the survival and proliferation of CLL cells. Signalling pathways triggered by interactions within these niches, such as the B cell receptor (BCR) pathway, and intracellular proteins such as Bcl-2 are vitally important in the biology of CLL. Novel therapeutic agents, such as ibrutinib, which target components of the BCR pathway, and the Bcl-2 inhibitor venetoclax, have demonstrated the potential of targeted therapies in CLL (3, 4). Novel therapeutic approaches must target the proliferative, drug-resistant compartments of disease within these microenvironments.The NanoString® nCounter platform enables mRNA profiling of archival samples, including formalin-fixed, paraffin-embedded tissue (FFPE). We have previously demonstrated the utility of this technology by comparing the mRNA expression profile of CLL cells derived from the peripheral blood (PB), archival BM and LN tissue as well as PB-derived CLL cells following in vitro co-culture with a human stromal cell line under either normoxic or hypoxic conditions. Here we present an update on our previous work with increased sample numbers in each of the tissues or culture conditions.MethodsRNA was extracted from FFPE BM trephines (n = 5) and LN sections (n = 5), using the QIAGEN RNeasy FFPE Kit. All biopsies analysed were comprised of > 80 % lymphocytes, as determined by microscopic review.RNA from PBMC fractions (n = 5) was isolated either immediately or following co-culture with HS5 stromal cells for 24 h under normoxic (n = 5) or hypoxic (n = 5) conditions using the QIAGEN RNeasy Mini Kit.RNA from all preparations was quantified using a NanoDrop™ spectrophotometer. A total of 200ng of FFPE-derived RNA and 100ng of PBMC-derived RNA was analysed per sample on the NanoString® platform using a 260 gene panel.Three-fold changes in mRNA expression were considered significant.ResultsOf the 260 genes profiled, 89 were upregulated in the BM samples and 52 in the LN samples compared to expression in PB-derived CLL cells. Changes were seen in genes encoding for proteins involved in chemotaxis (CXCL9), the regulation of apoptosis (BCL2L1), surface receptors (FLT3) and genes associated with intracellular signalling, metabolism and cell division. 35 genes were downregulated in the LN samples and 31 in the BM samples. These changes were seen in genes coding for surface receptors (ROR1 and CXCR4), genes coding for intracellular signalling proteins (RAF1) and genes coding for transcription factors (JUN and FOS).Co-culture of PBMCs with HS5 cells induced similar changes to those observed in our comparison of the PBMCs and BM samples; genes coding for 61.5% and 50.0% of the mRNA expression changes observed in the LN were observed in PBMCs cultured under normoxic and hypoxic conditions respectively. A similar comparison of the BM samples identified concordant changes in expression of 46.5% and 39.2% of genes under normoxic and hypoxic conditions respectively. Importantly, changes observed in genes coding for the anti-apoptotic protein MCL1, the surface receptors CXCR4 and ROR1 and the transcription factors ATF, FOS and JUN were consistent across samples from LN, BM and the in vitro model.In summary, we have utilised the NanoString® nCounter platform to profile PB, BM and LN-derived CLL cells and have identified panels of genes that are either up or down-regulated in cells derived from these microenvironments. Furthermore, the high concordance between RNA changes in the in vitro model and the primary tissue suggest the HS5 co-culture system mimics aspects of the tumour microenvironment. These data provide a better understanding of how CLL cells populate and proliferate in the tumour microenvironment and may lead to novel therapeutic strategies. DisclosuresNo relevant conflicts of interest to declare.

Qproteome FFPE Tissue Kit is not suitable for protein analysis using Agilent 2100 Bioanalyzer

Introduction: The chip-based protein separation using Agilent 2100 Bioanalyzer is a promising tool for proteomic analysis. However, it has not been defined if the above-mentioned device is suitable for analysis of formalin-fixed paraffin-embedded (FFPE) proteins extracts.&lt;br&gt;The aim of the study: Therefore we performed the analysis aimed at testing if extracts from FFPE tissues are suitable for proteins detection using Agilent 2100 Bioanalyzer.&lt;br&gt;Material and methods: FFPE tissues and cell cultures were used for experiments. Proteins were extracted from them using Qproteome FFPE Tissue Kit and RIPA buffer, respectively. For protein analysis Bioanalyzer Instrument, Western Blot and immunohistochemistry (IHC) were applied.&lt;br&gt;Results: In FFPE extracts, using Bioanalyzer we failed to detect β-actin. However, Western Blot analysis proved β-actin presence in them. One of possible explanations of this phenomenon might be lack of antibody-antigen interaction during immunoprecipitation preceding Bioanalyzer analysis. We suspected that, Extraction Buffer (from Qproteome FFPE Tissue Kit) or β-mercaptoethanol (both present in FFPE protein extracts) might be responsible for the above-mentioned blockade. Therefore, we applied IHC with detection of CD34 because, CD34 staining is robust against small methodological variations and this marker is always present in tumor tissue sections. Anti-CD34 antibody was diluted in Tris saline buffer with Extraction Buffer, β-mercaptoethanol or without. We did not observe CD34 immunopositivity only in the presence of Extraction Buffer.&lt;br&gt;Conclusion: Qproteome FFPE Tissue Kit is not suitable for protein analysis using Agilent 2100 Bioanalyzer, because the Extraction Buffer from the kit prevents an interaction between antibody and antigen during immunoprecipitation procedure.

個人識別を目的としたパラフィン包埋組織からのDNA抽出法の比較

Formalin-fixed paraffin-embedded (FFPE) tissues are one of the most effective tools for human identification in forensic science. To evaluate the effectiveness for human identification, we compared the following six methods for the extraction of DNA from FFPE tissues; EZ1 DNA Investigator Kit, ReliaPrep FFPE gDNA Miniprep System, DNA Isolator PS-Rapid Reagent, QIAamp DNA FFPE Tissue Kit, NucleoSpin DNA FFPE XS and phenol-chloroform method. DNA extracted using each method from two FFPE tissue blocks were quantified by three different DNA quantification methods (Quant-it dsDNA HS Assay Kit and quantitative PCR based on 207 bp and 98 bp regions in the D17Z1 locus), and short tandem repeat (STR) typing using AmpFlSTR Identifiler Plus PCR Amplification Kit was conducted. All extracts from the FFPE tissues were highly degraded, and large differences were observed among total DNA yields determined by these three quantification methods. DNA obtained using QIAamp DNA FFPE Tissue Kit had the highest total DNA yield among all the quantification methods. In STR typing with 1 ng DNA template estimated by quantitative PCR based on 98 bp region in the D17Z1 locus, the greatest number of detected alleles was observed using QIAamp DNA FFPE Tissue Kit. In contrast, EZ1 DNA Investigator Kit and DNA Isolator PS-Rapid Reagent were inferior to the other methods in terms of the DNA yield and the number of detected alleles via STR typing. In conclusion, QIAamp DNA FFPE Tissue Kit was the most effective method for human identification from FFPE tissues among the other DNA extraction methods, considering the results of this study, its cost-effectiveness, and universal use for forensic science.

Human Papillomavirus Investigation in Head and Neck Squamous Cell Carcinoma: Initial Report from the Low Risk HPV Types Associations

Background:Head and neck squamous cell carcinomas (HNSCC) are a major health issue in many parts of the world. Recently, attention has focused on the human papilloma virus (HPV) as a potential causative agent for HNSCC. This study aimed to survey HPV occurrence in HNSCCs as part of a comprehensive molecular epidemiology approach.Methods:In this retrospective study, patients were recruited from hospitals affiliated to the Iran University of Medical Sciences, Tehran, Iran. Formalin-fixed paraffin-embedded (FFPE) blocks were subjected to DNA isolation by QIAamp® DNA FFPE Tissue Kit and nested PCR, HPV-16 specific conventional PCR, and extra INNO-LiPA HPV genotyping assays were subsequently performed. PCR products were purified with a High Pure PCR Product Purification Kit and sequenced with an ABI 3730 XL sequencer. CLC Main Workbench 5 and MEGA5 bioinformatics software was used to analyze the raw data and to create the phylogenetic tree. SPSS v.20 was applied for statistical analysis.Results:A total of 156 FFPE blocks were collected from 2011 to 2017. Total mean age (y) of participants was 60.5 ± 12.6; 77.6 % (121/156) being men and 22.4% (35/156) e women. Overall, 5/156 (3.2%) patients (3 females and 2 males) were found to be HPV positive using the three methods. HPV genotyping revealed HPV types 16, 2, 27, and 43 in these malignancies. Tumor location and lymph node involvement indicated significant differences between the sexes.Conclusion:Although high risk HPV genotypes have been associated with HNSCCs, our findings indicate a potential of low risk HPV types to also contribute to such malignancies.

Optimization of Urea Based Protein Extraction from Formalin-Fixed Paraffin-Embedded Tissue for Shotgun Proteomics.

Urea based protein extraction of formalin-fixed paraffin-embedded (FFPE) tissue provides the most efficient workflow for proteomics due to its compatibility with liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). This study optimizes the use of urea for proteomic analysis of clinical FFPE tissue. A series of protein extraction conditions manipulating temperature and buffer composition were compared to reduce carbamylation introduced by urea and increase protein detection. Each extraction was performed on a randomized pair of serial sections of homogenous FFPE tissue and analyzed with LC-ESI-MS/MS. Results were compared in terms of yield, missed cleavages, and peptide carbamylation. Lowering extraction temperature to 60°C decreased carbamylation at the cost of decreased protein detection and yield. Protein extraction for at least 20 minutes at 95°C followed by 60°C for 2 hours maximized total protein yield while maintaining protein detection and reducing carbamylation by 7.9%. When accounting for carbamylation during analysis, this modified extraction temperature provides equivalent peptide and protein detection relative to the commercially available Qproteome® FFPE Tissue Kit. No changes to buffer composition containing 7 M urea, 2 M thiourea, and 1 M ammonium bicarbonate resulted in improvements to control conditions. Optimized urea in-solution digestion provides an efficient workflow with maximized yields for proteomic analysis of clinically relevant FFPE tissue.

Clinical utility of reverse phase protein array for molecular classification of breast cancer.

Reverse Phase Protein Array (RPPA) represents a sensitive and high-throughput technique allowing simultaneous quantitation of protein expression levels in biological samples. This study aimed to confirm the ability of RPPA to classify archival formalin-fixed paraffin-embedded (FFPE) breast cancer tissues into molecular classes used in the Nottingham prognostic index plus (NPI+) determined by immunohistochemistry (IHC). Proteins were extracted from FFPE breast cancer tissues using three extraction protocols: the Q-proteome FFPE Tissue Kit (Qiagen, Hilden, Germany) and two in-house methods using Laemmli buffer with either incubation for 20min or 2h at 105°C. Two preparation methods, full-face sections and macrodissection, were used to assess the yield and quality of protein extracts. Ten biomarkers used for the NPI+ (ER, PgR, HER2, Cytokeratins 5/6 and 7/8, EGFR, HER3, HER4, p53 and Mucin 1) were quantified using RPPA and compared to results determined by IHC. The Q-proteome FFPE Tissue Kit produced significantly higher protein concentration and signal intensities. The intra- and inter-array reproducibility assessment indicated that RPPA using FFPE lysates was a highly reproducible and robust technique. Expression of the biomarkers individually and in combination using RPPA was highly consistent with IHC results. Macrodissection of the invasive tumour component gave more reliable results with the majority of biomarkers determined by IHC, (80% concordance) compared with full-face sections (60% concordance). Our results provide evidence for the technical feasibility of RPPA for high-throughput protein expression profiling of FFPE breast cancer tissues. The sensitivity of the technique is related to the quality of extracted protein and purity of tumour tissue. RPPA could provide a quantitative technique alternative to IHC for the biomarkers used in the NPI+.

Two methods for proteomic analysis of formalin-fixed, paraffin embedded tissue result in differential protein identification, data quality, and cost.

Formalin-fixed paraffin-embedded (FFPE) tissue is a rich source of clinically relevant material that can yield important translational biomarker discovery using proteomic analysis. Protocols for analyzing FFPE tissue by LC-MS/MS exist, but standardization of procedures and critical analysis of data quality is limited. This study compared and characterized data obtained from FFPE tissue using two methods: a urea in-solution digestion method (UISD) versus a commercially available Qproteome FFPE Tissue Kit method (Qkit). Each method was performed independently three times on serial sections of homogenous FFPE tissue to minimize pre-analytical variations and analyzed with three technical replicates by LC-MS/MS. Data were evaluated for reproducibility and physiochemical distribution, which highlighted differences in the ability of each method to identify proteins of different molecular weights and isoelectric points. Each method replicate resulted in a significant number of new protein identifications, and both methods identified significantly more proteins using three technical replicates as compared to only two. UISD was cheaper, required less time, and introduced significant protein modifications as compared to the Qkit method, which provided more precise and higher protein yields. These data highlight significant variability among method replicates and type of method used, despite minimizing pre-analytical variability. Utilization of only one method or too few replicates (both method and technical) may limit the subset of proteomic information obtained.

Evaluation of five DNA extraction methods for detection of H. pylori in formalin-fixed paraffin-embedded (FFPE) liver tissue from patients with hepatocellular carcinoma

Since Helicobacter spp. DNA was identified in liver tissue resected from patients with hepatocelullar carcinoma (HCC), researchers have suggested a role of this bacterium in hepatic carcinogenesis. Archives of formalin-fixed, paraffin-embedded (FFPE) tissues represent an extraordinary source for clinical studies providing many advantages. However, DNA extraction from FFPE tissues is laborious, time-consuming and still remains a challenge. The aim of this study was to evaluate five protocols for DNA extraction from FFPE liver obtained from patients with HCC in order to detect Helicobacter pylori DNA. These methods were: (1) QIAamp FFPE Tissue Kit, (2) QIAamp DNA Mini Kit, (3) Wizard SV Genomic DNA Purification System, (4) RealiaPrep FFPE gDNA Miniprep System and (5) phenol–chloroform. H. pylori detection was performed using 16S rRNA gene amplification by PCR. The highest total amount of DNA was obtained using the phenol–chloroform method. Analyses of 16S rRNA gene amplification did not show statistically significant differences among the methods (p=0.466), although the highest percentage of positive cases (70%) was found in samples extracted with phenol-chloroform. We suggest that of the five methods evaluated, phenol/chloroform is the most suitable for detection of H. pylori in FFPE liver from patients with HCC.

Analysis of mutations in formalin-fixed paraffin-embedded (FFPE) tumor samples from two phase II clinical trials using a Sequenom-based approach.

1548 Background: Analysis of tumour samples for biomarker discovery, personalised health care and disease stratification is becoming increasingly important. Methods to detect somatic mutations are often limited by the requirement for a significant amount of input DNA, low sensitivity or low sample throughput. The aim of this work was to obtain a comprehensive mutation profile of tumour samples taken from patients enrolled on two Phase II clinical trials. A Sequenom based approach was used to determine mutation profiles for the samples so that these could be correlated with response at some future date. Methods: After successfully piloting the Sequenom MassArray 4 platform on formalin fixed paraffin embedded (FFPE) tumour samples and tumour cell line admixes, we ran an exploratory analysis of 177 melanoma and 230 non small cell lung cancer (NSCLC) FFPE samples collected from two phase II clinical trials. DNA was extracted from the FFPE samples using the QIAmp DNA FFPE Tissue Kit (Qiagen). We designed a panel of 86 assays in 14 genes covering approximately 160+ mutations. Mutations were chosen based on mutation frequency and biological significance in these tumour types. Results: Using this approach we were able to detect concomitant mutation profiles in a significant percentage of samples using only approximately 20uL of FFPE DNA (500 copies/µL average). Mutation status concordance was over 97% in BRAF and KRAS when compared to data generated via other approaches (sequencing, ARMS). Conclusions: Using Sequenom to screen tumour material, collected during clinical trials, is a feasible, rapid and comprehensive approach. Such an approach can be used to identify mutation profiles which correlate with patient response, potentially identify further patient stratification and help generate new hypotheses for further exploration.