10 results on '"Fitarelli-Kiehl M"'
Search Results
2. Sensitive Detection of Minimal Residual Disease in Patients Treated for Early-Stage Breast Cancer.
- Author
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Parsons HA, Rhoades J, Reed SC, Gydush G, Ram P, Exman P, Xiong K, Lo CC, Li T, Fleharty M, Kirkner GJ, Rotem D, Cohen O, Yu F, Fitarelli-Kiehl M, Leong KW, Hughes ME, Rosenberg SM, Collins LC, Miller KD, Blumenstiel B, Trippa L, Cibulskis C, Neuberg DS, DeFelice M, Freeman SS, Lennon NJ, Wagle N, Ha G, Stover DG, Choudhury AD, Getz G, Winer EP, Meyerson M, Lin NU, Krop I, Love JC, Makrigiorgos GM, Partridge AH, Mayer EL, Golub TR, and Adalsteinsson VA
- Subjects
- Adult, Breast Neoplasms blood, Breast Neoplasms genetics, Breast Neoplasms therapy, Circulating Tumor DNA blood, Combined Modality Therapy, Female, Follow-Up Studies, Humans, Neoplasm Recurrence, Local blood, Neoplasm Recurrence, Local genetics, Neoplasm Recurrence, Local therapy, Neoplasm, Residual blood, Neoplasm, Residual genetics, Neoplasm, Residual therapy, Prognosis, Prospective Studies, Retrospective Studies, Survival Rate, Breast Neoplasms pathology, Circulating Tumor DNA genetics, Estrogen Receptor alpha metabolism, Neoplasm Recurrence, Local pathology, Neoplasm, Residual pathology
- Abstract
Purpose: Existing cell-free DNA (cfDNA) methods lack the sensitivity needed for detecting minimal residual disease (MRD) following therapy. We developed a test for tracking hundreds of patient-specific mutations to detect MRD with a 1,000-fold lower error rate than conventional sequencing., Experimental Design: We compared the sensitivity of our approach to digital droplet PCR (ddPCR) in a dilution series, then retrospectively identified two cohorts of patients who had undergone prospective plasma sampling and clinical data collection: 16 patients with ER+/HER2- metastatic breast cancer (MBC) sampled within 6 months following metastatic diagnosis and 142 patients with stage 0 to III breast cancer who received curative-intent treatment with most sampled at surgery and 1 year postoperative. We performed whole-exome sequencing of tumors and designed individualized MRD tests, which we applied to serial cfDNA samples., Results: Our approach was 100-fold more sensitive than ddPCR when tracking 488 mutations, but most patients had fewer identifiable tumor mutations to track in cfDNA (median = 57; range = 2-346). Clinical sensitivity was 81% ( n = 13/16) in newly diagnosed MBC, 23% ( n = 7/30) at postoperative and 19% ( n = 6/32) at 1 year in early-stage disease, and highest in patients with the most tumor mutations available to track. MRD detection at 1 year was strongly associated with distant recurrence [HR = 20.8; 95% confidence interval, 7.3-58.9]. Median lead time from first positive sample to recurrence was 18.9 months (range = 3.4-39.2 months)., Conclusions: Tracking large numbers of individualized tumor mutations in cfDNA can improve MRD detection, but its sensitivity is driven by the number of tumor mutations available to track., (©2020 American Association for Cancer Research.)
- Published
- 2020
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3. Denaturation-Enhanced Droplet Digital PCR for Liquid Biopsies.
- Author
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Fitarelli-Kiehl M, Yu F, Ashtaputre R, Leong KW, Ladas I, Supplee J, Paweletz C, Mitra D, Schoenfeld JD, Parangi S, and Makrigiorgos GM
- Subjects
- Cell-Free Nucleic Acids chemistry, Cell-Free Nucleic Acids genetics, DNA Repair, ErbB Receptors genetics, Humans, Male, Mutation, Neoplasms blood, Proto-Oncogene Proteins B-raf genetics, Workflow, Liquid Biopsy, Neoplasms genetics, Nucleic Acid Denaturation genetics, Polymerase Chain Reaction methods
- Abstract
Background: Although interest in droplet-digital PCR technology (ddPCR) for cell-free circulating DNA (cfDNA) analysis is burgeoning, the technology is compromised by subsampling errors and the few clinical targets that can be analyzed from limited input DNA. The paucity of starting material acts as a "glass ceiling" in liquid biopsies because, irrespective how analytically sensitive ddPCR techniques are, detection limits cannot be improved past DNA input limitations., Methods: We applied denaturation-enhanced ddPCR (dddPCR) using fragmented genomic DNA (gDNA) with defined mutations. We then tested dddPCR on cfDNA from volunteers and patients with cancer for commonly-used mutations. gDNA and cfDNA were tested with and without end repair before denaturation and digital PCR., Results: By applying complete denaturation of double-stranded DNA before ddPCR droplet formation the number of positive droplets increased. dddPCR using gDNA resulted in a 1.9-2.0-fold increase in data-positive droplets, whereas dddPCR applied on highly-fragmented cfDNA resulted in a 1.6-1.7-fold increase. End repair of cfDNA before denaturation enabled cfDNA to display a 1.9-2.0-fold increase in data-positive signals, similar to gDNA. Doubling of data-positive droplets doubled the number of potential ddPCR assays that could be conducted from a given DNA input and improved ddPCR precision for cfDNA mutation detection., Conclusions: dddPCR is a simple and useful modification in ddPCR that enables extraction of more information from low-input clinical samples with minor change in protocols. It should be applicable to all ddPCR platforms for mutation detection and, potentially, for gene copy-number analysis in cancer and prenatal screening., (© 2018 American Association for Clinical Chemistry.)
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- 2018
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4. Enhanced detection of microsatellite instability using pre-PCR elimination of wild-type DNA homo-polymers in tissue and liquid biopsies.
- Author
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Ladas I, Yu F, Leong KW, Fitarelli-Kiehl M, Song C, Ashtaputre R, Kulke M, Mamon H, and Makrigiorgos GM
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- Artifacts, Cell Line, Tumor, Circulating Tumor DNA blood, Colonic Neoplasms blood, Colonic Neoplasms pathology, DNA chemistry, Electrophoresis, Capillary, Humans, INDEL Mutation, Liquid Biopsy, Oligonucleotide Probes, Biopsy, Colonic Neoplasms genetics, Microsatellite Instability, Polymerase Chain Reaction
- Abstract
Detection of microsatellite-instability in colonoscopy-obtained polyps, as well as in plasma-circulating DNA, is frequently confounded by sensitivity issues due to co-existing excessive amounts of wild-type DNA. While also an issue for point mutations, this is particularly problematic for microsatellite changes, due to the high false-positive artifacts generated by polymerase slippage (stutter-bands). Here, we describe a nuclease-based approach, NaME-PrO, that uses overlapping oligonucleotides to eliminate unaltered micro-satellites at the genomic DNA level, prior to PCR. By appropriate design of the overlapping oligonucleotides, NaME-PrO eliminates WT alleles in long single-base homopolymers ranging from 10 to 27 nucleotides in length, while sparing targets containing variable-length indels at any position within the homopolymer. We evaluated 5 MSI targets individually or simultaneously, NR27, NR21, NR24, BAT25 and BAT26 using DNA from cell-lines, biopsies and circulating-DNA from colorectal cancer patients. NaME-PrO enriched altered microsatellites and detected alterations down to 0.01% allelic-frequency using high-resolution-melting, improving detection sensitivity by 500-1000-fold relative to current HRM approaches. Capillary-electrophoresis also demonstrated enhanced sensitivity and enrichment of indels 1-16 bases long. We anticipate application of this highly-multiplex-able method either with standard 5-plex reactions in conjunction with HRM/capillary electrophoresis or massively-parallel-sequencing-based detection of MSI on numerous targets for sensitive MSI-detection.
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- 2018
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5. Multiplexed Elimination of Wild-Type DNA and High-Resolution Melting Prior to Targeted Resequencing of Liquid Biopsies.
- Author
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Ladas I, Fitarelli-Kiehl M, Song C, Adalsteinsson VA, Parsons HA, Lin NU, Wagle N, and Makrigiorgos GM
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- Cell Line, Tumor, DNA blood, DNA chemistry, Exome, Humans, Multiplex Polymerase Chain Reaction methods, Mutation, Neoplasms blood, Nucleic Acid Denaturation, DNA genetics, DNA Mutational Analysis methods, Neoplasms genetics, Proto-Oncogene Proteins p21(ras) genetics
- Abstract
Background: The use of clinical samples and circulating cell-free DNA (cfDNA) collected from liquid biopsies for diagnostic and prognostic applications in cancer is burgeoning, and improved methods that reduce the influence of excess wild-type (WT) portion of the sample are desirable. Here we present enrichment of mutation-containing sequences using enzymatic degradation of WT DNA. Mutation enrichment is combined with high-resolution melting (HRM) performed in multiplexed closed-tube reactions as a rapid, cost-effective screening tool before targeted resequencing., Methods: We developed a homogeneous, closed-tube approach to use a double-stranded DNA-specific nuclease for degradation of WT DNA at multiple targets simultaneously. The No Denaturation Nuclease-assisted Minor Allele Enrichment with Probe Overlap (ND-NaME-PrO) uses WT oligonucleotides overlapping both strands on putative DNA targets. Under conditions of partial denaturation (DNA breathing), the oligonucleotide probes enhance double-stranded DNA-specific nuclease digestion at the selected targets, with high preference toward WT over mutant DNA. To validate ND-NaME-PrO, we used multiplexed HRM, digital PCR, and MiSeq targeted resequencing of mutated genomic DNA and cfDNA., Results: Serial dilution of KRAS mutation-containing DNA shows mutation enrichment by 10- to 120-fold and detection of allelic fractions down to 0.01%. Multiplexed ND-NaME-PrO combined with multiplexed PCR-HRM showed mutation scanning of 10-20 DNA amplicons simultaneously. ND-NaME-PrO applied on cfDNA from clinical samples enables mutation enrichment and HRM scanning over 10 DNA targets. cfDNA mutations were enriched up to approximately 100-fold (average approximately 25-fold) and identified via targeted resequencing., Conclusions: Closed-tube homogeneous ND-NaME-PrO combined with multiplexed HRM is a convenient approach to efficiently enrich for mutations on multiple DNA targets and to enable prescreening before targeted resequencing., (© 2017 American Association for Clinical Chemistry.)
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- 2017
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6. Methylation-sensitive enrichment of minor DNA alleles using a double-strand DNA-specific nuclease.
- Author
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Liu Y, Song C, Ladas I, Fitarelli-Kiehl M, and Makrigiorgos GM
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- Alleles, DNA blood, Humans, Lung Neoplasms blood, Oligonucleotide Probes, Real-Time Polymerase Chain Reaction, Sequence Analysis, DNA, Sulfites, DNA Methylation, Deoxyribonucleases, Nucleic Acid Amplification Techniques methods
- Abstract
Aberrant methylation changes, often present in a minor allelic fraction in clinical samples such as plasma-circulating DNA (cfDNA), are potentially powerful prognostic and predictive biomarkers in human disease including cancer. We report on a novel, highly-multiplexed approach to facilitate analysis of clinically useful methylation changes in minor DNA populations. Methylation Specific Nuclease-assisted Minor-allele Enrichment (MS-NaME) employs a double-strand-specific DNA nuclease (DSN) to remove excess DNA with normal methylation patterns. The technique utilizes oligonucleotide-probes that direct DSN activity to multiple targets in bisulfite-treated DNA, simultaneously. Oligonucleotide probes targeting unmethylated sequences generate local double stranded regions resulting to digestion of unmethylated targets, and leaving methylated targets intact; and vice versa. Subsequent amplification of the targeted regions results in enrichment of the targeted methylated or unmethylated minority-epigenetic-alleles. We validate MS-NaME by demonstrating enrichment of RARb2, ATM, MGMT and GSTP1 promoters in multiplexed MS-NaME reactions (177-plex) using dilutions of methylated/unmethylated DNA and in DNA from clinical lung cancer samples and matched normal tissue. MS-NaME is a highly scalable single-step approach performed at the genomic DNA level in solution that combines with most downstream detection technologies including Sanger sequencing, methylation-sensitive-high-resolution melting (MS-HRM) and methylation-specific-Taqman-based-digital-PCR (digital Methylight) to boost detection of low-level aberrant methylation-changes., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)
- Published
- 2017
- Full Text
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7. Comparison of multiple genotyping methods for the identification of the cancer predisposing founder mutation p.R337H in TP53.
- Author
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Fitarelli-Kiehl M, Macedo GS, Schlatter RP, Koehler-Santos P, Matte Uda S, Ashton-Prolla P, and Giacomazzi J
- Abstract
Germline mutations in the TP53 gene are associated with Li-Fraumeni and Li-Fraumeni-Like Syndromes, characterized by increased predisposition to early-onset cancers. In Brazil, the prevalence of the TP53-p.R337H germline mutation is exceedingly high in the general population and in cancer-affected patients, probably as result of a founder effect. Several genotyping methods are used for the molecular diagnosis of LFS/LFL, however Sanger sequencing is still considered the gold standard. We compared performance, cost and turnaround time of Sanger sequencing, PCR-RFLP, TaqMan-PCR and HRM in the p.R337H genotyping. The performance was determined by analysis of 95 genomic DNA samples and results were 100% concordant for all methods. Sequencing was the most expensive method followed by TaqMan-PCR, PCR-RFLP and HRM. The overall cost of HRM increased with the prevalence of positive samples, since confirmatory sequencing must be performed when a sample shows an abnormal melting profile, but remained lower than all other methods when the mutation prevalence was less than 2.5%. Sequencing had the highest throughput and the longest turnaround time, while TaqMan-PCR showed the lowest turnaround and hands-on times. All methodologies studied are suitable for the detection of p.R337H and the choice will depend on the application and clinical scenario.
- Published
- 2016
- Full Text
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8. The breast cancer immunophenotype of TP53-p.R337H carriers is different from that observed among other pathogenic TP53 mutation carriers.
- Author
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Fitarelli-Kiehl M, Giacomazzi J, Santos-Silva P, Graudenz MS, Palmero EI, Michelli RA, Achatz MI, de Toledo Osório CA, de Faria Ferraz VE, Picanço CG, and Ashton-Prolla P
- Subjects
- Adult, Aged, Breast Neoplasms etiology, Female, Genetic Predisposition to Disease, Humans, Immunophenotyping, Middle Aged, Receptor, ErbB-2 analysis, Retrospective Studies, Breast Neoplasms genetics, Genes, p53, Germ-Line Mutation, Heterozygote
- Abstract
Germline TP53 mutations are associated with Li-Fraumeni syndrome, an autosomal dominant disorder characterized by a predisposition to multiple early-onset cancers including breast cancer (BC), the most prevalent tumor among women. The majority of germline TP53 mutations are clustered within the DNA-binding domain of the gene, disrupting the structure and function of the protein. A specific germline mutation in the tetramerization domain of p53, p.R337H, was reported at a high frequency in Southern and Southeastern Brazil. This mutation appears to result in a more subtle defect in the protein, which becomes functionally deficient only under particular conditions. Recent studies show that the BC phenotype in TP53 mutation carriers is often HER2 positive (63-83%). Considering that the immunophenotype of BC among p.R337H carriers has not been reported, we reviewed immunohistochemistry data of 66 p.R337H carriers in comparison with 12 patients with other non-functional TP53 germline mutation. Although 75% of carriers of these mutations showed significant HER2 overexpression (3+), corroborating previous studies, only 22.7% of p.R337H patients had BC overexpressing HER2. These results reinforce the notion that different germline mutations in TP53 may predispose to BC via different mechanisms.
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- 2015
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9. Expression of cancer stem cell markers in basal and penta-negative breast carcinomas--a study of a series of triple-negative tumors.
- Author
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Uchôa Dde M, Graudenz MS, Callegari-Jacques SM, Hartmann CR, Ferreira BP, Fitarelli-Kiehl M, and Edelweiss MI
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- Adult, CD24 Antigen biosynthesis, ErbB Receptors biosynthesis, Female, Humans, Hyaluronan Receptors biosynthesis, Immunohistochemistry, Kaplan-Meier Estimate, Keratin-5 biosynthesis, Keratin-6 biosynthesis, Middle Aged, Neoplasm Grading, Neoplasms, Basal Cell mortality, Neoplasms, Basal Cell pathology, Tissue Array Analysis, Triple Negative Breast Neoplasms metabolism, Triple Negative Breast Neoplasms mortality, Biomarkers, Tumor analysis, Neoplastic Stem Cells pathology, Triple Negative Breast Neoplasms pathology
- Abstract
Purpose: Breast cancer is a heterogeneous disease. Immunohistochemistry has given rise to triple-negative carcinoma (TNC). Concomitantly, biological origins of neoplasia and its heterogeneity has been strongly debated in cancer stem cells (CSC) theme. This study investigates the prevalence of basal (BCC) and penta-negative carcinomas (5NC) in TNC and establishes associations with CSC (CD44CD24)., Materials and Methods: 94 TNC were tested for CK5/6, HER1, CD44 and CD24, evaluated by a simple immunohistochemistry score and correlated with clinicopathological and survival data., Results: BCC had higher tumor grades than 5NC (p=0.004). CD44 negativity (p=0.007) and CD44(-)CD24(+) phenotype (p=0.013) were associated with less vascular invasion amongst TNC. CD44 expression was associated with BCC (p=0.007). CD44(-)CD24(-/low) phenotype was associated with 5NC. None of the variables were associated with clinical outcome., Conclusion: BCC and 5NC are closely related tumor subtypes. CD44(-)CD24(-/low) phenotype was associated with 5NC and CD44(-)CD24(+) phenotype was associated with vascular invasion. These results require histogenetic confirmation in larger studies., (Copyright © 2014 Elsevier GmbH. All rights reserved.)
- Published
- 2014
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10. A TP53 founder mutation, p.R337H, is associated with phyllodes breast tumors in Brazil.
- Author
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Giacomazzi J, Koehler-Santos P, Palmero EI, Graudenz MS, Rivero LF, Lima E, Pütten AC, Hainaut P, Camey SA, Michelli RD, Neto CS, Fitarelli-Kiehl M, Geyer G, Meurer L, Geiger A, Azevedo MB, da Silva VD, and Ashton-Prolla P
- Subjects
- Adolescent, Adult, Aged, Aged, 80 and over, Brazil, Female, Haplotypes, Humans, Middle Aged, Breast Neoplasms genetics, Genes, p53, Germ-Line Mutation, Phyllodes Tumor genetics
- Abstract
A few studies have reported phyllodes tumors (PT) of the breast with germline TP53 mutations. Given this potential association and the high frequency of the TP53 p.R337H in southern and southeastern Brazil, the aim of this study was to assess whether p.R337H occurs among women diagnosed with such rare tumors in this region. Benign, borderline, and malignant breast PT were retrieved from eight pathology laboratories, and DNA was extracted from tumor tissue to perform p.R337H analysis. Overall, 128 cases classified as benign, 7 as borderline, and 13 as malignant PT were included in the study. The TP53 p.R337H mutation was identified in tumor cells of eight (5.4 %) cases. Analysis of DNA from non-tumoral tissue was possible in two of these, and both were p.R337H carriers in the germline. In addition, haplotype analysis was done in these two p.R337H carriers showing the presence of the founder haplotype previously reported in Brazilian mutation-positive individuals. Mutation frequency was significantly higher among malignant (3 of 13; 23 %) compared to benign tumors (5 of 128; 3.4 %) (p = 0.004). Mean age at PT diagnosis was not significantly different between mutation carriers and non-carriers. However, when subgroups where analyzed, the difference in age at diagnosis of carriers versus non-carriers within the group of benign tumors reached borderline significance. Our findings reinforce previous evidence that TP53 mutations have an important role in the development of both benign and malignant PT of the breast.
- Published
- 2013
- Full Text
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