QIAamp DNA Mini Kit Research Articles

Determination of O6-Methylguanine in dried blood spot of breast cancer patients after cyclophosphamide administration

Cyclophosphamide is a nitrogen mustard class of drugs that are often used in cancer chemotherapy. However, the use of Cyclophosphamide in high doses over a long period has been shown to increase the risk of developing secondary cancer. This can be indicated by the formation of mutagenic DNA adducts, such as O6-Methylguanine. Therefore, this adduct can be used as a biomarker for secondary cancer in patients receiving Cyclophosphamide. Bio sampling was carried out by using the Dried Blood Spot (DBS) method, followed by DNA extraction by using QIAamp DNA mini kit, and acid hydrolysis to obtain O6-Methylguanine. Analysis of O6-Methylguanine was performed by using the UPLC-MS/MS instrument with the conditions developed by Vianney, Harahap, & Suryadi (2021). Partial validation was carried out before the analysis. The results obtained from the calibration curve, accuracy, and precision validation test met the FDA requirements. The analysis method was then implemented in 16 breast cancer patients who received the Cyclophosphamide regimen. The O6-Methylguanine was successfully detected and quantified in all of the samples in the range of 0.55–6.66 ng/mL. It shows that the O6-Methylguanine accumulation in cancer patients receiving Cyclophosphamide is very likely to occur and the analysis method proposed by Vianney, Harahap, & Suryadi (2021) is potential to be used for Therapeutic Drug Monitoring in this group of patients.

IDH1 and IDH2 : Founding or Progressor Mutations in Myeloid Neoplasms

IDH1/2 genes normally regulate cellular metabolism and epigenetic expression. Mutations in IDH1/2 are believed to play a significant part in leukemogenesis, and inhibitors are in active development. IDH1/2 mutations are rarely the sole mutation in myeloid neoplasms and are frequently found with co-mutations in other genes. However, it is not clear if the IDH1/2 mutations are founder or progressor mutations. Determining whether an IDH1/2 mutation is a founder or progressor may have significant impact on therapy outcome. As founding mutations persist in subclones, targeting founding mutations is likely to result in the eradication of the leukemic clone, whereas targeting progressor mutations can only destroy the subclone and progeny. We used variant allele frequency (VAF) and next generation sequencing (NGS) to determine if IDH1/2 mutations are founding or progressor mutations and to characterize the co-mutational background for 6390 patients who presented with a clinical impression of myeloid neoplasm.Methods: A total of 6390 consecutive bone marrow aspirate samples or peripheral blood samples submitted with clinical impression of AML, MDS, or MPN between January 2014 and mid 2017 were tested for mutations in myeloid genes using NGS. We used the TruSight Myeloid Panel (Illumina, San Diego, CA) for detecting missense mutations and fragment length analysis (FLA) for detecting ITD in FLT3 and large indels in CALR . DNA was extracted from samples using the QIAamp DNA Mini Kit. This NGS testing covers mutations in 54 myeloid-related genes. The average depth of sequencing was 10,000x.Results: A total of 2670 (42%) of the tested samples showed a mutation in one or more of the tested genes. Of the samples with mutations 467 patients (17.5%) patients showed mutations in either IDH1 or IDH2 genes. IDH1 mutations were detected 185 (40%) and IDH2 was detected in 291 (62%) of these patients. Nine patients (2%) had mutations in both IDH1 and IDH2. To distinguish patients with IDH1/2 as a founder mutation vs. subsequent (progressor) mutation, we used a lower VAF of a 10% in IDH1 or IDH2 as compared with the average VAF of all genes as a cut-off. Using this criteria, we found patients 35 patients (19%) as a progressor in the IDH1 mutated group and 48 (16.5%) in the IDH2 mutated group as progressors. Only 12 patients (6.5%) of the IDH1 mutated patients and 11 (4%) of the IDH2 mutated patients had IDH1 or IDH2 as the sole mutated gene.The most commonly co-mutated genes with IDH1/2 were DNMT3A, SRSF2, NPM1, ASXL1, and RUNX1 detected in 34%, 28%, 22%, 22%, and 20%, respectively. Mutations in TP53 and FLT3, which may have significant impact on therapy and outcome, were detected in 7% and 12% of patients, respectively. In patients with an IDH1/2 progressor mutation, there was significantly higher relative mutations in TP53 (P=0.03) and FLT-ITD (P=0.0001). The presence of mutations in TP53 and FLT-3 -ITD signify significantly more aggressive myeloid neoplasm or acute leukemia. In addition to the typical mutations in codon 132 of IDH1 and codons 140 and 132 in IDH2, we detected mutations by NGS in codons 100, 103, 109, and 183 in IDH1 and codon 173 in IDH2 .Conclusion: IDH1/2 mutations are founding mutations in most myeloid neoplasms. However, 19% of patients with IDH1/2 mutations may have acquired their IDH1/2 mutations in a subclone. Majority of these patients have mutations in FLT-3-ITD and TP53 and may represent significantly more aggressive disease. Clinical studies are needed to determine the pattern of response to IDH1/2 inhibitors in patients with subclonal (progressor) mutation in IDH1/2 . The potential of selecting for the leukemic clone that lack IDH1/2 mutation should be considered. Complete molecular profiling rather than testing for mutations in IDH1/2 alone provides important information in the management and therapy of myeloid neoplasms. [Display omitted] DisclosuresMa:NeoGenomics: Employment. De Dios:NeoGenomics: Employment. Funari:NeoGenomics: Employment. Sudarsanam:NeoGenomics: Employment. Jiang:NeoGenomics: Employment. Agersborg:NeoGenomics: Employment. Hummel:NeoGenomics: Employment. Blocker:NeoGenomics: Employment. Albitar:NeoGenomics: Employment.

FLT3 Mutation in Acute Myeloid Leukemia: Primary or Secondary Mutation?

The receptor tyrosine kinase FLT3 gene is commonly mutated in acute myeloid leukemia (AML) and its mutation is associated with significantly more aggressive disease and poor outcome. However, in the co-presence of NPM1 mutation, the disease is less aggressive and is associated with better outcome. FLT3 inhibitor midostaurin (Rydapt; Novartis Pharmaceuticals, Inc.) was recently approved for patients with FLT3 mutations. While the presence ofa FLT3 mutation is commonly associated with AML,it can develop in less acute myeloid neoplasms, such as MDS and CMML, and implies transformation into more acute phase. In this context, the FLT3 mutation is typically detected in a subclone of the original chronic myeloid clone. The prevalence of subclonal FLT3 mutations is unknown, as is the clinical relevance of subclonal FLT3, especially when treated with FLT3 inhibitors. We used variant allele frequency (VAF) and next generation sequencing (NGS) to determine if FLT3 mutations are founding or secondary mutations and to characterize the co-mutational background in large number of consecutive patients who presented with a clinical impression of myeloid neoplasm.Methods: A total of 6390 consecutive bone marrow aspirate samples or peripheral blood samples were submitted with clinical impression of AML, MDS, or MPN between January 2014 and mid 2017 were tested for mutations in myeloid genes using NGS. We used the TruSight Myeloid panel (Illumina, San Diego, CA) for detecting missense mutations and fragment length analysis (FLA) for detecting internal tandem repeat (ITD) in FLT3 . DNA was extracted from samples using the QIAamp DNA Mini Kit. This NGS testing covers mutations in 54 myeloid-related genes. The average depth of sequencing was 10,000x.Results: A total of 2670 (42%) of the samples showed a mutation in one or more of the tested genes. Of the samples with mutations, FLT3 ITD and kinase domain (KD) mutations were detected in 313 (12%) patients. Of these patients, 208 (66.5%) had ITD and 117 (37.4%) had KD mutations. Twelve patients (3.8%) had both ITD and KD mutations. NPM1 mutation was detected in 138 (44%) of the patients. Most of FLT3 -KD mutations were in codon 835, but mutations in codons 836, 839, 842 and 845 were also found. Additionally, missense mutations in codons 576, 592, and 593 at the site of ITD were detected. To distinguish a FLT3 mutation as a founding mutation rather than a secondary mutation, we used a lower VAF of 10% as a cut-off. Using this approach, we found that 27% (115/313) of patients had secondary FLT3 mutations. Of these subclonal FLT3 mutants, NPM1 co-mutation was detected in 61 (53%) patients, but was not statistically significant (P=0.06). Patients with FLT3 mutation as a subclone had a significantly higher number of co-mutated genes (P-value <0.0001). This group of patients had higher (P≤0.01) mutations in TET2, DNMT3A, NRAS, IDH2, EZH2, and BCOR, consistent with an MDS/CMML background. On the other hand, patients with both FLT3 and NPM1 mutations had a significantly lower number of co-mutant genes (P<0.0001) and particularly lower (P≤0.01) mutation rate in U2AF1, SF3B1, SRSF2, RUNX1, IDH1, BCOR, and AXL1 .Conclusion: FLT3 mutations are frequently (27%) a secondary mutation in a subclone of the neoplastic founding mutations. This appears to be more common when FLT3 mutation is developing in a background of MDS/CMML. The co-presence of an NPM1 mutation is more commonly associated with fewer co-mutations in MDS-related genes. This data suggests that in addition to detecting ITD by FLA, using NGS for profiling mutations in FLT3 -KD and other myeloid genes should be considered whenever FLT3 mutations are evaluated. Further, the clinical decision for combination therapy should consider the entire mutation profile and not only mutations in FLT3 . [Display omitted] DisclosuresMa:NeoGenomics: Employment. De Dios:NeoGenomics: Employment. Funari:NeoGenomics: Employment. Sudarsanam:NeoGenomics: Employment. Jiang:NeoGenomics: Employment. Agersborg:NeoGenomics: Employment. Hummel:NeoGenomics: Employment. Blocker:NeoGenomics: Employment. Albitar:NeoGenomics: Employment.

Clonal Hematopoiesis in Normal Individuals Is Not Random and Likely Reflects Early MDS

Introduction: Clonal hematopoiesis, as determined by the presence of TET2, ASXL1, and DNMT3 mutation, is believed to be increasingly common in normal individuals as they age. This phenomenon is currently referred to as clonal hematopoiesis of indeterminate potential (CHIP). It has been reported that a few of these patients will develop myeloid neoplasms that begin as MDS and may evolve into AML. However, the clinical relevance of this particular subset of gene mutations (TET2, ASXL1, and DNMT3A) in healthy individuals is unclear as there are more than 50 genes that have been reported to be involved in myeloid neoplasms.FLT3 and NPM1 mutations have been reported in de novo AML patients and may occur as a secondary mutations on an MDS background. In order to clarify the significance of TET2, ASXL1, and DNMT3 in CHIP and their relationship to MDS and AML, we assessed the prevalence of TET2, ASXL1, and DNMT3 genes in patients with NPM1 and FLT3 mutations. In addition, since IDH1/2 gene are rarely mutated with TET2, we also studied the frequency of mutations of various myeloid genes in this group of patients.Methods: A total of 6390 consecutive bone marrow aspirate samples or peripheral blood samples submitted with clinical impression of AML, MDS, or MPN between January 2014 and mid 2017 were tested for mutations in myeloid genes using NGS. We used the TruSight Myeloid Panel (Illumina, San Diego, CA) for detecting missense mutations and fragment length analysis (FLA) for detecting ITD in FLT3 and large indels in CALR . DNA was extracted from samples using the QIAamp DNA Mini Kit. This NGS testing covers mutations in 54 myeloid-related genes. The average depth of sequencing was 10,000x.Results: In our database of consecutively tested patients, there were 311 patients with FLT3 mutations, 318 patients with NPM1 mutation, and 467 patients with IDH1/2 mutation. In addition, there were 308 patients diagnosed as MDS. All these patients tested for mutations in all 54 myeloid-related genes. The median age of patients in FLT3, MDS, IDH1/2, and NPM1 groups was 65, 75, 69, and 66, respectively. The top 4 most frequently mutated in genes in the FLT3 -positive patients, which present AML, were NPM1, DNMT3, WT1, and RUNX1 . In patients with NPM1 mutations, also representing AML, the top 4 most mutated genes were FLT3, IDH2, NRAS, and PTPN11 . In the IDH1/2 mutated patients, the top 4 mutated genes were DNMT3A, SRSF2, ASXL1, and NPM1 . As expected in the MDS group, the top 4 mutated genes were TET2, DNMT3A, ASXL1, and SRSF2 .Conclusions: The demonstration that mutations in TET2, DNMT3A, and ASXL1 are most common in patients presenting with MDS, as well as in CHIP, but not in patients presenting with acute myeloid neoplasms, suggests that CHIP likely represents early MDS. It is possible that mutations in these genes in few hematopoietic cells may not be adequate for manifestation of clinical MDS, but the presence of these mutations in large number of cells (high VAF) or the accumulation of mutations in additional genes is necessary for clinical disease. Therefore the clinical relevance of CHIP should always be considered in conjunction with other mutations and with VAF. [Display omitted] DisclosuresMa:NeoGenomics: Employment. De Dios:NeoGenomics: Employment. Funari:NeoGenomics: Employment. Sudarsanam:NeoGenomics: Employment. Jiang:NeoGenomics: Employment. Agersborg:NeoGenomics: Employment. Hummel:NeoGenomics: Employment. Blocker:NeoGenomics: Employment. Albitar:NeoGenomics: Employment.

Potential Familial Predisposition to Hematologic Neoplasm Detected Incidentally in Adult Patients Presenting with Myeloid Neoplasms

Introduction: Germline mutations in CEBPA, ETV6, RUNX1, and GATA2 have been reported to be associated with predisposition to hematologic malignancies. Next Generation Sequencing (NGS), used to identify somatic mutations to aid in targeted therapies, may incidentally uncover inherited germline mutations. Recognizing familial predisposition is important as it may impact management and surveillance. We assessed the prevalence of potentially clinically significant germline mutations detected incidentally by NGS in samples from patients investigated for myeloid disorders.Methods: NGS was performed to identify somatic mutations in 54 genes, on 6100 consecutive bone marrow aspirates or peripheral blood specimens between January 2014 and mid-2017. The average depth of sequencing was 10,000x. DNA was extracted using the QIAamp DNA Mini Kit and subjected to the TruSight Myeloid panel (Illumina, San Diego, CA). Most (92%) of the common dbSNP variants (>10% allele frequency in dbSNP) were found between 45-55% variant allele frequency (VAF) or 98-100% VAF for hetero- and homozygous states. This suggests that common SNPs are mostly found between those frequencies; hence, alleles with these frequencies were defined as candidate germline variants. However, to further enrich for germline variants, we only analyzed those cases with at least one other somatic variant with VAF of 5-40%; the rational being that it is unlikely that a sample would have two true independent somatic mutation events in different subclones. Following this enrichment process, 2951 specimens were eligible for further analysis for potential germline mutations. The ClinVar and dbSNP databases were used to assess allele frequencies and clinical significance.Results: In 2951 of 6100 specimens, we reported at least one somatic mutation with a variant allele frequency in the range of 5-40%. A total of 5322 variants were identified in this subset of specimens. Among the candidate germline mutations in 4 genes implicated in myeloid neoplasm predisposition, we identified 93 unique putative loss of function variants that met our germline criteria. Nine of these mutations were identified in more than 3 cases and more than half of these were represented in ClinVar as germline loss of function variants. One of them, RUNX1 : p.Arg201Gln, identified in 5 patients, is described as a germline pathogenic variant. In contrast, the other 84 variants identified in the 4 genes were rare (observed in 1 or 2 cases) and few (7/84) had been characterized in dbSNP. Most loss of function germline variants are unique and rare with few hotspots, which is consistent with our data.Analysis of germline mutations in genes, which have not yet been implicated in predisposition to myeloid neoplasm, revealed 4858 candidate mutations in 23 genes: 15.4% (750/4858) were unique, 13.6% (102/750) were more common (> 3 cases), while most (86.4%; 648/750) were found in only 1 or 2 cases. Some of the more common variants were identified in ClinVar as pathogenic germline variants, including IDH2 (p.Arg140Gln; MIM: 613657), identified in 84 cases, and a TP53 loss of function mutation (p.Pro72Arg) identified in 424 cases.Conclusion: Germline mutations, GATA2, RUNX1, CEBPA, and ETV6, known to predispose to myeloid malignancies, may be identified incidentally during testing for somatic mutations in myeloid malignancies.In addition, if the NGS panel includes genes other than those known to predispose towards myeloid malignancies, pathogenic variants of wide ranging clinical implication may be uncovered, as observed in the cases with mutation in IDH2 or TP53 . Although the specific mutation observed in IDH2 has been identified most often as a somatic mutation, it has been reported to be inherited in one instance. Inheriting this allele can confer D-2-hydroxyglutaric aciduria-2 (D2HGA2). The TP53 mutation observed is known to be involved in other hereditary cancers (e.g. Li-Fraumeni syndrome) and could affect the recommended therapy of antineoplastic agents.Rare incidentally identified potential germline mutations remain a challenge as their clinical significance may be unknown. These mutations require additional investigation to determine if they could contribute to clinical management. DisclosuresFunari:NeoGenomics: Employment. Thangavelu:NeoGenomics: Employment. Ma:NeoGenomics: Employment. De Dios:NeoGenomics: Employment. Agersborg:NeoGenomics: Employment. Blocker:NeoGenomics: Employment. Albitar:NeoGenomics: Employment.

Liquid Biopsy As a Diagnostic Test for Myelodysplastic Syndrome and Other Acute and Chronic Myeloid Neoplasms

Cytopenia is common in cause for considering the diagnosis of myelodysplastic syndrome (MDS), especially in the elderly population. To diagnose the presence or absence of MDS, bone marrow biopsy is performed on most patients with cytopenia. Performing bone marrow biopsy is a painful procedure and can be associated with bleeding and other complications. Moreover, bone marrow morphology is unreliable for the diagnosis of MDS. Cytogenetics and molecular studies can provide conclusive evidence for the diagnosis of MDS. Liquid biopsy and testing for molecular abnormalities in cfDNA in peripheral blood plasma is significantly less invasive than traditional bone marrow testing. We evaluated the diagnostic value of liquid biopsy in determining the presence of MDS in 640 patients presented in community setting practice with cytopenia.Methodology: A total of 640 peripheral blood samples from patients with cytopenia were submitted for cfDNA testing. We used the TruSight Myeloid panel (Illumina, San Diego, CA) for detecting missense mutations and fragment length analysis (FLA) for detecting ITD in FLT3 and large indels in CALR . DNA was extracted from samples using the QIAamp DNA Mini Kit. This next generation sequencing (NGS) testing covers mutations in 54 myeloid-related genes. The average depth of sequencing was 10,000x.Results: Of the 640 tested patients, 310 (48.4%) showed mutations in one or more genes: 38% of these had mutations in one gene, 25.5% had mutations in two genes, and 36.5% had mutations in more than two genes. However, 14% of cases with mutation in one gene had two or more mutant subclones. The median age of all patients was 69 (range: 15-99) and 55% were males. Patients with mutations were significantly older (75 vs. 66 years old, P<0.0001) and more likely to be males (60% vs. 46% males, P=0.004). The most commonly mutated genes were TET2, DNMT3A, ASXL1, SRSF2, SF3B1, and RUNX1 detected in 31%, 28%, 25%, 17%, 15%, and 10% of the patients with mutations, respectively. Interestingly, 32 patients (10% of patients with any mutation) had mutations in TP53, 3% had mutations in FLT3, and 4% had mutations in NPM1 genes, indicating aggressive or acute disease. The average variant allele frequency (VAF) was <10% in 22% of cases with any mutation, between 10-20% in 19% of cases, between 20-30% in 16% of cases, and >40% in 43% of the cases. There was a significantly higher number of mutated genes in patients with average VAF >20% (P<0.0001). The combination of VAF, type of mutated gene, and number of mutated genes were considered in determining the significance of abnormal neoplastic clone and recommendation for management. The presence of mutations in NPM1, FLT3, TP53 as well as SF3B1 were considered diagnostic irrespective of the VAF. For patients with VAF at <20% in genes such as TET2, DNMT3A, and ASXL1 were not considered diagnostic for clinically significant myeloid neoplasm.Conclusions: Liquid biopsy for evaluating patients with pancytopenia can be used to evaluate the presence of abnormal hematopoietic neoplasm clones and the severity of the disease. Liquid biopsy can reduce the need for bone marrow biopsy in more than 50% of patients presenting with cytopenia. VAF, mutated gene types, and the combination of mutated genes should be considered for making management decisions based on liquid biopsy. [Display omitted] DisclosuresMa:NeoGenomics: Employment. De Dios:NeoGenomics: Employment. Funari:NeoGenomics: Employment. Jiang:NeoGenomics: Employment. Agersborg:NeoGenomics: Employment. Hummel:NeoGenomics: Employment. Blocker:NeoGenomics: Employment. Albitar:NeoGenomics: Employment.

Evaluation of DNA extraction methods for quantifying Helicobacter pylori in water with PCR inhibitors

Abstract DNA extraction methods were evaluated to reduce PCR inhibitors and quantify Helicobacter pylori directly from water samples using real-time PCR. Three nucleic acid extraction methods were evaluated for different types of water samples. While the QIAamp DNA mini kit for tissue was suitable for DNA extraction from treated water, the QIAamp DNA stool mini kit was still efficient in analyzing samples from river water after heavy rain and with high concentration of PCR inhibitors. The FastDNA SPIN Kit for Soil could extract DNA effectively from microbes in river and stream waters without heavy rain. Immunomagnetic separation (IMS) was used prior to DNA extraction and was a useful tool for reducing PCR inhibitors in influent and stream samples. H. pylori in various waters could be quantified directly by real-time PCR while minimizing the effect of PCR inhibitors by an appropriate method through the evaluation of DNA extraction methods considering the characteristics of the matrix water. The findings of the present study suggest that the types or characteristics of water sample by source and precipitation are an important factor in detecting H. pylori and they can be applied when detecting and monitoring of other pathogens in water.

Distribution of BRCA1/2 germline and somatic alterations across cancer type.

10590 Background: PARP inhibitors (e.g. Olaparib or niraparib) have been approved by FDA as a targeted therapy for many tumors harboring germline or somatic BRCA1/2 (g or sBRCA1/2), including ovarian cancer, prostate cancer, breast cancer and pancreases cancer. It is imperative to study the distribution of BRCA1/2 across cancer type. In this study, we aim to assess the landscape of BRCA1/2 alterations in solid tumors and evaluate the feasibility of circulating tumor DNA (ctDNA) tested by next-generation sequence (NGS) as a tool to detect BRCA1/2 alterations. Methods: For tissue specimen, genomic DNA from formalin fixed paraffin-embedded (FFPE) tumor specimens or fresh tumor tissues was used for sequence analysis. Genomic DNA (gDNA) from white blood cells was extracted using the QIAamp DNA Mini Kit (Qiagen). For ctDNA, cell-free DNA libraries were prepared using the KAPA Hyper Prep Kit following the manufacturer’s protocol. The captured libraries were loaded onto a NovaSeq 6000 platform (Illumina) for 100bp paired-end sequencing. The testing was performed in the College of American Pathologists (CAP) and Clinical Laboratory Improvement Amendments (CLIA) -certified 3D Medicines Library. Results: A total of 27, 835 patients were tested using tumor tissue during Jan. 1th 2017 to June 1th 2020, including 43% (N = 12089) of non-small cell lung cancer, 19% (N = 5357) of colorectal cancer, 8% (N = 2181) of liver cancer, 6% (N = 1621) of gastric cancer, 5% (N = 1479) of biliary tract cancer, 4% (N = 1084) of kidney cancer, 4% (N = 1045) of pancreas cancer, 3% (N = 689) of breast cancer and 2% (N = 599) of ovarian cancer. Across all tumor types, the known or likely deleterious BRCA1/2 alterations were identified in 2147 (7.7%) patients. Ovarian cancer had the highest frequency of BRCA1/2 alteration (23.4%), followed by endometrial cancer (12.7%) and breast cancer (10.6%). BRCA1/2 alteration was found in 8.8% prostate cancer and 4.2% pancreas cancer respectively. Across all tumor types, the known or likely deleterious gBRCA1/2 alterations were identified in 369 (1.3%) patients. Notably, ovarian cancer had the highest frequency of gBRCA1/2 alteration (13.9%), followed by breast cancer (7%), prostate cancer (4.4%) and endometrial cancer (4.1%). No clear hotspot mutations and mutated codons were spread throughout g or sBRCA1/2 mutations. Additionally, among 15699 patients who suffered ctDNA sequencing, any known or likely deleterious sBRCA1/2 alterations were identified in 358 (2%) patients. Similar to the results of tissue sequencing, ovarian cancer had the highest frequency of sBRCA1/2 alteration (16.67%), followed by endometrial cancer (9.68%), prostate cancer (7.18%) and breast cancer (5.58%) in the blood cohort. Conclusions: BRCA1/2 alterations existed across tumor types and the landscape of g or sBRCA1/2 alterations varied according to cancer type. Furthermore, ctDNA can be used as a potential tool to detect BRCA1/2 alterations.

STUDY OF THE HUMAN PAPILLOMA VIRUS PREVALENCE AND ITS PHYSICAL STATUS IN PATIENS WITH NON-SMALL CELL LUNG CANCER

To date, the role of human papillomavirus (HpV) in the development of lung cancer is still under investigation. The available published data do not provide evidence of the involvement of HpV in lung cancer pathogenesis. In Russia, there are no reports regarding the studies of viral contribution to lung carcinogenesis.The aim of the present study was to examine the presence of high-risk HpV infection in patients with non-small cell lung cancer (NSCLC).Material and Methods. The study included 30 patients with histologically verified stage IIBIIIA NSCLC. Normal and tumor lung tissue samples as well as whole blood samples from each patient were used. dNA was isolated from tissue samples using the QIAamp dNA mini Kit (Qiagen, Germany). Amplisens® reagent kits (Moscow, Russia) were used for dNA isolation and genotyping.Results. The presence of HpV dNA was detected in 3 (10.0 %) patients. HpV dNA was not detected in 27 (90.0 %) patients. HpV-16 genotype was observed in 2 HpV-positive patients, and HpV-33 was observed in 1 patient. A clinically insignificant viral load was found in almost all HpV-positive samples. The assessment of the virus integration in HpV16-positive lung cancer patients revealed mixed and integrated forms of the virus in the tumor tissue of these patients. The 5-year metastasis-free survival rate in all HpV-positive patients was 100 %.Conclusion. Our data showed HpV dNA prevalence rate of 10 % among NSCLC patients in the Russian population. The presence/absence of a viral component for lung cancer patients is a clinically significant parameter.

Development of a novel biomarker for the early detection of colorectal cancer

Colorectal cancer (CRC) is one of the most common cancer and a leading cause of cancer related deaths both in men and in women worldwide including US. A positive family history is strongly correlated with an increased risk of CRC diagnosis during lifetime. However, CRC is an indolent disease in its early stages and usually becomes symptomatic when it progresses to more advanced stages. Conventional screening methods have several limitations and currently, there is no screening procedure available to detect CRC at a very early stage. Under normal physiological condition, the epithelial lining of the gut gets breached by various factors including food, that creates local wound. The surviving epithelial cells around the wound lose their differentiated structure and undergoes epithelial mesenchymal transition (EMT) and migrate and proliferate to cover the injured area. Once the wound is covered, the cells start differentiating back into their normal cell type and become a part of the epithelium. If the cells fail to stop proliferating following wound closure by overriding the switch to differentiating that can lead to colonic polyp, or tumor, and if not detected in a timely manner than, it leads ultimately to CRC development. Previously, we reported that β-catenin; an adherence junction protein gets phosphorylated by Janus kinase 3 (Jak3), a non-receptor tyrosine kinase, where Jak3 directly phosphorylates three tyrosine residues, viz. Tyr30, Tyr64, and Tyr86 in the N-terminal domain (NTD) of β-catenin. Now, our data show that human biopsies from colonic polyps not only associate with EMT, but also with only those mutation in β-catenin where Jak3, fail to phosphorylate β-catenin thereby making the cells override the switch and undergo uncontrolled proliferation and polyp formation. Based on this mechanistic understanding, the present study demonstrates the development of probe that detects the transition of colonic cells from proliferative stage to a differentiated (non-proliferative) stage that can diagnose CRC at the pre-polyp stage. The probe can not only play an important role in the early detection and/or treatment of patients with colorectal cancer but also increase life expectancy post detection. For this study, we collectedde-identified and discarded specimen from normal and/or polyps (n=40 cases; 20 normal and 20 polyps) through our collaboration with clinicians from local hospitals. Briefly, snap-frozen tissues were used to isolate DNA with the QIAamp DNA mini kit (Qiagen) as reported before by our group. All DNA samples were quantified using UV spectrophotometer and analyzed through agarose gel electrophoresis. The DNA sample was used as template in PCR reactions using the β-catenin-probes to amplify the regions encompassing Y30, Y64, and Y86 followed by sequencing of PCR samples. This was followed by histological analysis of colonic polyp sections from each patient stained with H&E and images were taken with phase contrast microscope. Our results show strongly correlation between mutations and histological analysis indicating the aforementioned detection of intactness of EMT switch as potential novel biomarker for the early detection of colorectal cancer.

Mitochondrial nicotinamide adenine dinucleotide hydride dehydrogenase (NADH) subunit 4 (MTND4) polymorphisms and their association with male infertility

PurposeThe purpose of the present study was to determine the relationship between infertility and the polymorphisms of mitochondrial NADH dehydrogenase subunit 4 (MTND4) by spermatozoa analysis in fertile and subfertile men.MethodsSamples were divided into 68 subfertile men (case group) and 44 fertile men (control group). After semen analysis, samples were purified. The whole genome was extracted using a QIAamp DNA Mini Kit and the mitochondrial DNA was amplified by using the REPLI-g Mitochondrial DNA Kit. Polymerase chain reaction (PCR) was used to amplify the MT-ND4 gene. Then, samples were purified and sequenced using the Sanger method.ResultsTwenty-five single-nucleotide polymorphisms (SNPs) were identified in the MTND4 gene. The genotype frequencies of the study population showed a statistically significant association between rs2853495 G>A (Gly320Gly) and male infertility (P = 0.0351). Similarly, the allele frequency test showed that rs2853495 G>A (Gly320Gly) and rs869096886 A>G (Leu164Leu) were significantly associated with male infertility (adjusted OR = 2.616, 95% CI = 1.374–4.983, P = 0.002; adjusted OR = 2.237, 95% CI = 1.245–4.017, P = 0.007, respectively).ConclusionIn conclusion, our findings suggested that male infertility was correlated with rs2853495 and rs869096886 SNPs in MTND4.

Quality of metagenomic DNA extracted for molecular identification of microorganisms from CSF samples of patients with suspected cerebrospinal meningitis in northern Nigeria

Background: Following an increase in the practice of starting antimicrobial therapy prior to clinical sample collection, the ability to confirm pathogenic microorganisms of bacterial meningitis has decreased by approximately 30%. Culture results may be false negative when fastidious or culture-resistant bacteria are involved or when patient samples are obtained after antimicrobial therapy has started. Molecular diagnosis using PCR can be performed directly on clinical samples after metagenomic DNA (mDNA) extraction not requiring live organisms for a positive result. The specific objectives of this study are to perform mDNA extraction directly from cerebrospinal fluids (CSF) using appropriate spin column method, and to determine the quality of the mDNA elute.Methodology: Cerebrospinal fluid specimens were collected from 210 patients with suspected acute cerebrospinal meningitis (CSM) in the Federal Capital Territory and some States in Northern Nigeria during the 2017 and 2018 outbreak seasons. Metagenomic DNA was extracted from approximately 200µL of CSF specimens using the Qiagen QIAamp(R) DNA Mini kit specific for bacterial agents only. DNA quality check was performed on all DNA elutes using fluorometric, spectrophotometric and agarose gel electrophoresis methods.Results: Of the 210 CSF samples analyzed microbiologically, Gram reaction was positive in 94 cases (44.8 %) but only 17 (8.1 %) were culture positive for two of the three major bacterial causes of meningitis. One hundred and eighty (85.7%) samples had DNA concentrations ≥ 0.005 ng/µL, 55 (30.6 %) of these had DNA purity (A260/A280) of ≥ 1.7, 103 (57.2%) had purity value between 1.0 - 1.69, 14 (7.8%) had value of 0.57 - 0.99, and 8 (4.4%) failed purity evaluation with value of 0.00 at A260/A280.Conclusion: The essence of mDNA extraction is multipurpose. A multiplex PCR can be performed on the extracted mDNA to interrogate the presence of microbial pathogens of interest using specific primers and probes (when applicable). Quality mDNA from CSF samples will ensure successful qPCR results for rapid and accurate detection of bacterial pathogens in meningitis. This will eliminate the challenges associated with traditional culture methods.&#x0D; Keywords: Meningitis, CSF, DNA Quality Check, Fluorometry

Association between polymorphisms in catechol-O-methyl transferase, opioid receptor Mu 1 and serotonin receptor genes with postoperative pain following root canal treatment.

To evaluate the effect of single nucleotide polymorphisms in the COMT, OPRM1, 5HT1A, 5HT2A and 5HTR3B genes on the intensity of postoperative pain following root canal treatment. Ninety-five patients with mandibular and maxillary molar teeth diagnosed with symptomatic apical periodontitis and a level of preoperative pain greater than 50 on a 100mm visual analogue scale (VAS) were included. Salivary DNA was collected from the participants and stored in Eppendorf tubes at -80°C. Preoperative percussion pain values were recorded before the root canal treatment procedures. After completion of root canal treatment, the participants were given instructions to record their postoperative pain intensity levels at 24, 48 and 72h, 5days and 1week after treatment, using the VAS. A second visit for the patients after seven days was planned to record their intensity levels of percussion pain on VAS. The percussion test was performed by tapping on the occlusal surface of the tooth with a blunt instrument. A QIAamp DNA Mini Kit was used to isolate DNA from saliva, and SNP Genotyping Analysis software version 1 was used to analyse the genotypes by calculating FAM and HEX signals. The Kruskal-Wallis and Mann-Whitney U-tests were used to evaluate pain intensity values amongst the genotypes, alleles, haplotypes and allele combinations. Nominal data (gender, intake and tooth number) were analysed using a Chi-square test. Bonferroni correction was performed. Thus, the significance level was set at 1.6% (P=0.016), 2.5% (P=0.025) and 1.25% (P=0.0125) for genotype, allele and haplotype comparisons, respectively. There was no significant difference amongst the genotypes and alleles in terms of pre- and postoperative pain intensity. There was no significant difference amongst the haplotypes formed for the COMT gene in terms of pain intensity. Additionally, there was no significant association between the allelic combination formed for 5HT1A+5HT2A genes and the intensity of postoperative pain. The findings indicate that none of the evaluated SNPs for COMT, OPRM1, 5HT1A, 5HT2A and 5HTR3B genes were associated with the intensity of postoperative pain.

Effect of TOP2A and ERCC1 gene polymorphisms on the efficacy and toxicity of cisplatin and etoposide-based chemotherapy in small cell lung cancer patients.

IntroductionThe main treatment regimen for small cell lung cancer (SCLC) involves platinum-based chemotherapy (cisplatin or carboplatin) and etoposide. Single nucleotide polymorphisms (SNPs) in TOP2A and ERCC1 genes were tested as prognostic and predictive factors in non-small cell lung cancer (NSCLC). There are limited data about the clinical relevance of these genetic alterations in SCLC. We undertook this retrospective study to determine the influence of SNPs in TOP2A (rs34300454; rs13695; rs11540720) and ERCC1 (rs11615; rs3212986) genes on the efficiency and toxicity of chemotherapy with platinum and etoposide in SCLC Caucasian patients.Material and methodsThe studied group included 103 Caucasian SCLC patients (65 male, 38 female, median age 65 ±7.5 years). Detailed clinical-demographical data were collected and response to treatment was monitored. DNA was isolated from peripheral blood leukocytes using QIAamp DNA Mini Kit. Single nucleotide polymorphisms were analyzed using TaqMan hydrolyzing probes in real-time PCR technique on an Eco Illumina device.ResultsPatients with C/C genotype in rs13695 of the TOP2A gene had significantly lower risk of neutropenia during chemotherapy than C/T heterozygous patients (p = 0.02, χ² = 5.51, OR = 2.676, 95% CI: 1.165–6.143). Patients harbouring homozygous C/C genotype in rs3212986 of the ERCC1 gene had significantly higher risk of anaemia during chemotherapy, than heterozygous C/A patients (p = 0.045, χ² = 4.01, OR = 0.417, 95% CI: 0.175–0.991). Furthermore, heterozygous G/A genotype in rs11615 of the ERCC1 gene was associated with significant shortening of OS (9 vs. 12 months) compared to homozygous A/A genotype (p = 0.01, χ² = 6.31, HR = 1.657, 95% CI: 1.0710–2.5633).ConclusionsSNPs in ERCC1 and TOP2 genes may be associated with the toxicities and survival of SCLC patients treated with cisplatin and etoposide.

Single Nucleotide Polymorphisms of Pfdhfr and Pfdhps Genes: Implications for Malaria Prophylactic Strategies in Maiduguri, Northeast Nigeria

Background. The success of Intermittent Preventive Treatment in Pregnancy (IPTp), Intermittent Preventive Treatment in Infancy (IPTi), and Seasonal Malaria Chemoprevention (SMC) depends on sulfadoxine-pyrimethamine (SP) efficacy. Objective. The study determined Single Nucleotide Polymorphisms (SNPs) of Plasmodium falciparum dihydrofolate reductase (Pfdhfr) and dihydropteroate synthase (Pfdhps) in Maiduguri, Northeast Nigeria. Materials and Methods. Giemsa-stained blood smears, capillary blood, and dried blood spot samples were collected from 63 subjects with uncomplicated malaria in Maiduguri between May and October 2018. Plasmodium species was determined and parasite density (PD) was estimated using the smears. Genomic DNA (gDNA) of P. falciparum was extracted from the dried blood spot samples using QIAamp DNA Mini Kit. The gDNA was subjected to nested PCR followed by restriction fragment length polymorphism (RFLP) to determine SNPs at Pfdhfr codons N51I, C59R, and S108N and Pfdhps codons S436A/F, A437G, and K540E. Results. The subjects’ mean age ± standard deviation was 23.6 ± 8.7 (2.0–67.0) years with a geometric mean PD of 8,948 (2,100–13,400) asexual parasites/µl blood. SNPs prevalence at any of the six Pfdhfr and Pfdhps codons was 85.7% (54/63); the prevalence was higher ( p &lt; 0.05 ) in Pfdhfr (82.5%; 52/63) than Pfdhps (58.7%; 37/63). Pfdhfr allele 108N (82.5%; 52/63) was the highest ( p &lt; 0.05 ) mutant when compared with alleles 51I (60.3%; 38/63) and 59R (66.7%; 42/63). Triple Pfdhfr mutation was observed in 60.3% (38/63) of the isolates and was higher ( p &lt; 0.05 ) among female subjects and SP recipients. Prevalence of Pfdhps allele 436A (28.6%; 18/63) was similar ( p &gt; 0.05 ) to allele 437G (34.9%; 22/63), with double mutation recorded in 4.8% (3/63). K540E mutation was not observed. Conclusion. Pfdhfr and Pfdhps mutations observed in Maiduguri are suggestive of SP resistance level, and this could constitute a setback to malaria prophylactic strategies in the region if unchecked. Thus, there is a need to investigate the clinical efficacy of SP.

Comparison of HPV detection rate in formalin-fixed paraffin-embedded tissues of head and neck carcinoma using two DNA extraction kits and three amplification methods.

The potential problems of DNA extraction from formalin-fixed paraffin-embedded (FFPE) tissue samples and amplification efficiency of Human papilloma virus (HPV) may occur in the molecular studies of head and neck squamous carcinoma (HNSCC). The aim of this study was to compare HPV detection rate in FFPE tissues of oral, oropharyngeal, hypopharyngeal, and laryngeal cancers using two silica-based extraction kits and three amplification methods. A total of 50 FFPE specimens from HNSCC tissues were analyzed. The quality and quantity of the extracted DNA were tested by spectrophotometry. HPV DNA was detected using a single polymerase chain reaction (PCR), a nested PCR, and a Real-time PCR kit. Statistically significantly higher DNA quality and quantity was observed using the QIAamp DNA FFPE Tissue Kit than when using the QIAamp DNA Mini Kit. There was not HPV amplification in any of the 50 FFPE samples using the single PCR and Real-time PCR kits, whereas HPV DNA was detected in 22% of samples using nested PCR. Comparing results of the three different methods showed that HPV DNA was detected only with nested PCR. The results presented imply that nested PCR is the most appropriate method for the detection of HPV DNA in FFPE samples, along with adequate DNA extraction methods.

The possible role of genetic testing in the early identification of patients at increased risk of developing anthracycline-induced cardiotoxicity

Abstract Background In the last years prognosis of cancer patients (pts) has been improved, but the develop of cardiotoxicity (CTX) in pts with low CV risk treated with cumulative doses of Anthracycline (ANT) considered safe, led to investigate the possible role of the genetic profile in the onset of CTX. Purpose To study the role of some single nucleotide polymorphisms (SNPs) as predictive genetic markers of individual susceptibility to CTX induced by anticancer treatment. Methods We have enrolled women with non-metastatic breast cancer who had to start a therapy with ANT. The presence of a known heart disease, a previous mediastinal irradiation and a previous treatment with ANT, were the main exclusion criteria. All pts underwent complete cardiological evaluation (ECG, echo) before the beginning of the therapy (T0), after each ANT cycle and every 3 months up to 1 year of follow-up after the end of treatment. During each visit, we performed the determination of troponin I (TnI) and NT-proBNP. The genetic profile of each pts was also investigated, analyzing 6 SNPs belonging to 3 different genes (two for each genes) coding for enzymes or enzyme systems involved in the metabolism of ANT. DNA extraction from blood samples was performed using the QIAamp DNA Mini kit (QIAGEN). The DNA extracted was genotyped (by performing TaqMan SNP Genotyping assays) identifying 3 possible variants for each SNPs: homozygosity for the protective and “at risk” variant and heterozygosity. Results 179 pts finished the follow-up and from the analysis of the trend of biomarkers we found that 53 pts (30%) showed a significant increase in TnI (&amp;gt;0.04ng/mL) or less than this cut-off but persistently &amp;gt;0.015ng/mL in at least 2 measurements (“TnI+ group”), in the remaining 126 pts the TnI remained non-measurable (“TnI− group”); 76 pts (43%) showed NT-proBNP values ≥125 ng/L in at least two consecutive determinations (“NT-proBNP + group”),in the remaining 57% of pts this increase was not detected (“NT-proBNP - group”). Comparing “TnI+ group” to “TnI− group” we observed that only the genotyping of the SNPs rs1149222 (G/T) belonging to the ABCB4 gene is distributed differently in the two groups, in particular the homozygosity for the “at risk” variant (G/G) was present in 13% of the pts of the “TnI+ group” vs 5% in the “TnI− group” (p 0.06). The results of the comparison of “NT-proBNP+ group” vs “NT-proBNP− group”, showed that the genotyping of the SNPs rs6759892 (G/T) belonging to the UGT1A6 gene was distributed differently, the homozygosity for the “at risk” variant (G/G) was present in 28% of the “NT-proBNP+ group” vs 17% in the “NT-proBNP− group” (p 0.07). Conclusion Together with the baseline clinical evaluation, genetic markers could contribute to the early identification of pts at high risk of developing CTX especially following treatment with ANT and they could support future therapeutic decisions and the planning of taylored strategies for the prevention of the CTX development. Funding Acknowledgement Type of funding source: None

Isolation and Characterization of Infectious Vibrio Species from the Whiteleg Shrimp: Litopenaeus vannamei

Aquaculture shrimp farming has serious problems with diseases caused by viruses and bacteria, which lead to severe economic loss. The genus Vibrio is a group of facultative anaerobic microbes, most frequently found in aquatic environments and marine hosts. The Gram-negative genus Vibrio is one of the most important classes of bacterial pathogens in aquaculture systems and is a key cause of high mortality. The isolation, identification, and molecular characterization of Vibrio spp are studied from infected shrimp Litopenaeus vannamei. The microorganisms were isolated from the tissues of a white gut infected shrimp collected from diseased aquaculture ponds. After subculturing, microbial isolates were obtained using TCBS agar plates for vibrio selection, and the biochemical key was developed. Meanwhile, 11 isolates of bacteria belonging to the Vibrionaceae family have been identified in this study. For these, six Vibrio species-related isolates are responsible for vibriosis in the shrimp. These included Vibrio harveyi, Vibrio campbellii, Vibrio parahaemolyticus, Vibrio owensii, Vibrio rotiferianus, and Vibrio alginolyticus. The extraction of DNA was carried out with a QIAamp DNA Mini Kit (QIAGEN). The extracted DNA was analyzed using an electrophoresis of 1 % agarose gel, the consistency and quantity of Vibrio spp's gDNA. NanoDropTM 2000 spectrophotometer (Thermo Scientific) was used for determination. In addition, the Agilent 2100 Bioanalyzer (Agilent Technologies) has been used to verify the gDNA libraries' validity, purity, concentration, and scale. In addition, Vibrio spp, a phylogenetic tree focused on maximum likelihood and bootstrapping, was performed using the MEGA version to examine the evolutionary phylogenetic relationship between the strains. This study's main objective would be useful in integrating strain variation in predicting microbiology and microbial risk assessment and may provide scientific guidelines for major Vibrio strains that may be responsible for vibriosis infection in the management of shrimp aquaculture disease.

LOSS OF HETEROZYGOSITY IN THE BRCA1 AND BRCA2 LOCUS IN BREAST CANCER

One of the factors of variability of malignant neoplasms is the loss of heterozygosity (LOH). The biological meaning of LOH, in relation to carcinogenesis, is associated with the inactivation of heterozygous loci of pathogenetically significant genes. Thus, the aim of this work was to study BRCA1/2 LOH in breast tumors.Material and Methods. The study included 122 patients with stage IIAIIIC breast cancer. DNA was isolated from 122 biopsy samples of tumor tissue using the QIAamp DNA mini Kit (Qiagen, Germany). To assess the status of LOH, microarray analysis was performed on high-density DNA chips from Affymetrix CytoScanTM HD Array. To process the results of microchipping, we used the Chromosome Analysis Suite 3.3 program (Affymetrix, USA).Results. The loss of heterozygosity in the BRCA1 gene was found to be associated with response to NAC. It was shown that in 59 patients LOH in the BRCA1gene was associated with an objective response to treatment (p=0.005). The presence of LOH in the studied genes was associated with a favorable prognosis. The 5-year non-metastatic survival rates were 75 % and 100 % in patients with LOH in the BRCA1 and BRCA2 genes, respectively (log-rank test: p=0.003 and p=0.05, respectively).Conclusion. The phenomenon of LOH in the BRCA1/2 genes was shown to be associated with response to NACT. BRCA1/2. Further studies are needed to evaluate the frequency of BRCA1/2 LOH after NAC for choosing and changing treatment tactics.

Optimization of isothermal amplification method for Mycobacteriumtuberculosisdetection and visualization method for fieldwork

Background/aim Tuberculosis is still one of the most contagious diseases around the world. Key factors of tuberculosis control are rapid diagnostic, efficient treatment, and prevention of contamination by surveillance and monitoring. However, culture is the gold standard method for laboratory diagnosis of tuberculosis; the results are several weeks to obtain. In order to prevent contamination of tuberculosis, diagnosis must be made in short time and treatment should be started as soon as possible. The aim of this study is to optimize the loop-mediated isothermal amplification (LAMP) method, which provides a much faster and more sensitive result than the polymerase chain reaction (PCR) method and allows the replication of target nucleic acid sequences under isothermal conditions without the need for laboratory infrastructure.Materials and methods Sputum samples were homogenized with 5% trypsin solution in CaCl2 to obtain DNA. DNA was purified using QIAGEN QIAamp DNA mini kit. LAMP primers were design using Primer explorer V5 program according to IS6110 gene of Mycobacterium tuberculosis. NEB Bst 3.0 DNA polymerase kit was used for LAMP reactions. Besides, LAMP reactions were compared with TaqMan based RT-PCR method using NEB’s Taq polymerase kit. Finally, for visualization of LAMP products, lateral flow dipsticks that produced by Milenia Biotec, colorimetric 2X LAMP master mix that produced by NEB and 2% w/v agarose gel electrophoresis methods were used.ResultsOptimum amplification temperature for LAMP was found to be 71.4 °C. The detection limit of the method was 102 CFU/mL and sensitivity was determined 100% compared to five different Mycobacterium species. Conclusion The current study indicated that the LAMP-LFD and colorimetric LAMP protocol optimized with sputum samples can be reliable used as a rapid, sensitive and specific assay in the diagnosis of tuberculosis in the field.