Pedro Mendez - Academia.edu (original) (raw)

Papers by Pedro Mendez

Methods in Molecular Biology, 2022

The chapter was inadvertently published with incorrect figure legends, reference citations, and o... more The chapter was inadvertently published with incorrect figure legends, reference citations, and order of references. These errors have been corrected by updating the correct figure legends, reference citations, and the order of references as seen below:

Molecular Cancer Therapeutics, 2007

C127 Introduction: Trabectedin has recently received positive opinion from EMEA as treatment for ... more C127 Introduction: Trabectedin has recently received positive opinion from EMEA as treatment for advanced soft tissue sarcoma (STS) after failure to anthracyclines and ifosfamide. Objective responses and tumour control have been noted in patients resistant to conventional therapy. Moreover, the emergence of acquired resistance to trabectedin in sensitive patients is generally a late event. In experimental models trabectedin sensitivity correlates with a functional nucleotide excision repair (NER) pathway. Recently it has been described that XPG endonuclease binds to trabectedin adducts in the DNA leading to cell death. The present work characterized the impact of of XPG mRNA and protein expression levels in the tumor samples on the clinical outcome of STS patients treated with trabectedin. Methods: Retrospective analysis of paraffin embedded tumor tissue of STS patients obtained before treatment with trabectedin. A tissue microarray was constructed for analysing XPG protein express...

Methods in Molecular Biology, 2021

A comprehensive study of the cellular components of the immune system demands both deep and broad... more A comprehensive study of the cellular components of the immune system demands both deep and broad immunophenotyping of numerous cell subsets in an effective and practical way. Novel full-spectrum technology reveals the complete emission spectrum of each dye maximizing the amount of information that can be obtained on a single sample regarding conventional flow cytometry and provide an expanded knowledge of biological processes. In this chapter, we describe a 37-color protocol that allows to identify more than 45 different cell populations on whole blood samples of SARS-CoV-2-infected patients.

Nature, 2020

Myeloid malignancies, including acute myeloid leukemia (AML), arise from the expansion of hematop... more Myeloid malignancies, including acute myeloid leukemia (AML), arise from the expansion of hematopoietic stem/progenitor cells which acquire somatic mutations. Bulk molecular profiling suggests step-wise mutation acquisition, where mutant genes with high variant allele frequencies (VAFs) occur early in leukemogenesis and mutations with lower VAFs are thought to be acquired later 1-3. Although bulk sequencing informs leukemia biology and prognostication, it cannot distinguish which mutations occur in the same clone(s), accurately measure clonal complexity, or definitively elucidate mutational order. To delineate the clonal framework of myeloid malignancies, we performed single cell mutational profiling on 146 samples from 123 patients. We found AML is dominated by a small number of clones, which frequently harbor co-occurring mutations in epigenetic regulators. Conversely, mutations in signaling genes often occur more than once in distinct subclones consistent with increasing clonal diversity. We next mapped clonal trajectories for each sample and uncovered mutation combinations that synergized to promote clonal expansion and dominance. Finally, we combined protein expression with mutational analysis to map somatic genotype and clonal architecture with immunophenotype. Our studies of single cell clonal architecture provides novel insights into the pathogenesis of myeloid transformation and how clonal complexity evolves with disease progression.

Cancer Research, 2020

Fusion gene detection has long been a focus of cancer research, when combined with mutations foun... more Fusion gene detection has long been a focus of cancer research, when combined with mutations found in gDNA, can lead to a better understanding of disease progression. One example, BCR-ABL, a marker for CML and AML stem cells, is a target for tyrosine kinase inhibitor (TKI) treatments; however, there are mutations within BCR-ABL that evade TKIs and are selectively resistant to drug therapy. Single-cell technologies are now able to provide information into genomic DNA content, RNA expression, and protein surface markers, unmasked by the heterogeneity found in bulk data. However, multimodal analysis from the same single cell has not been straightforward to implement in a high throughput single-cell workflow. Here, we report the development of chemistries that enable analyses of both targeted genomic DNA and RNA sequencing from the same cell. This workflow relies on the Tapestri platform, which uses a two-step microfluidic droplet system for the analysis of thousands of cells per run. T...

Cancer Research, 2020

Elucidation of biological processes that drive cellular migration, proliferation, stemness and di... more Elucidation of biological processes that drive cellular migration, proliferation, stemness and differentiation have powered our view of tumor initiation and promotion. These avenues of research are reliant on engaging key biomarkers to measure and quantify cell systems. Widely practiced methodologies include metabolomic assays, immunoassays, imaging platforms and genomic tools. Understanding the genomic landscape of cancer has proven valuable for the characterization of molecular events that drive evolution of tumorigenesis and fostering progress in identifying druggable regimens for patient treatment scenarios. For sampling convenience, genomic measurements are typically applied to bulk cell samples. Bulk samples often obscure important rare cell types that define disease progression. Single-cell genomic analysis can highlight rare cells. Such efforts commonly generate one “modal” of information, most often next generation sequencing datasets containing RNA transcriptomic expressio...

Journal of Clinical Oncology, 2006

17038 Background: STAT5 plays a role in angiogenesis and metastasis. Activation of STAT5 occurs t... more 17038 Background: STAT5 plays a role in angiogenesis and metastasis. Activation of STAT5 occurs through dysregulation of growth factor receptor tyrosine kinases (TK) (including EGFR TK mutations), non-receptor TKs or Janus kinases. Dvl-3, a critical mediator of the Wingless-type (Wnt) signaling pathway, contributes to self-renewal of stem cells. Dicer has a key role in processing microRNAs required for stem cell regulation. We assessed mRNA expression of STAT5, Dvl-3 and Dicer in early-stage NSCLC. Methods: cDNA was derived from paraffin-embedded primary tumors of 47 surgically resected, early-stage NSCLC patients. Real-time quantitative reverse transcriptase PCR was used to measure STAT5, Dvl-3 and Dicer mRNA levels relative to the internal reference gene β-actin. Results: Median expression levels were: STAT5, 0.66 (0.15–6.03); Dvl-3, 3.69 (1.02–25.83); Dicer, 4.53 (1.22–21.96). STAT5 levels for squamous cell carcinoma (SCC) were 0.57 vs 0.89 for non-SCC (P=0.01), while Dicer level...

Blood, 2019

Genomic studies of myeloid malignancies (MM), including acute myeloid leukemia (AML), myeloprolif... more Genomic studies of myeloid malignancies (MM), including acute myeloid leukemia (AML), myeloproliferative neoplasms (MPN) and myelodysplasia (MDS), identified mutations with different allele frequencies. Recent studies of clonal hematopoiesis (CH) discovered a subset of MM disease alleles, while other alleles are only observed in overt MM. These observations suggest an important pathogenetic role for the chronology of mutational acquisition. Although bulk sequencing informs prognostication, it cannot distinguish which mutations occur in the same clone and cannot offer definitive evidence of mutational order. Delineation of clonal architecture at the single cell level is key to understanding how the sequential/parallel acquisition of somatic mutations contributes to myeloid transformation. In order to elucidate the clonal structure of MM, we designed a custom single cell 109 amplicon panel of the most frequently mutated amplicons in 50 MM genes using the Mission Bio Tapestri v2 platfo...

Annals of Oncology, 2012

ABSTRACT Background Response to neoadjuvant chemotherapy has been associated with an improvement ... more ABSTRACT Background Response to neoadjuvant chemotherapy has been associated with an improvement in survival in MIBC. The analysis of genetic markers involved in DNA repair pathways could be useful to predict chemosensitivity to cisplatin-based regimens. BRCA1 plays a central role in DNA repair and cell-cycle checkpoint control. However, BRCA1 function can be modulated by other DNA repair genes. RAP80 acts upstream of BRCA1 and regulates BRCA1 function after DNA damage. AEG-1 can induce BRCA1 expression and cause chemoresistance by activating PI3K/Akt and NF-KB pathways. Methods Paraffin-embedded pre-treatment tumor samples were collected by transurethral resection from 65 p with resectable MIBC stage T2-4N0M0 treated with neoadjuvant cisplatin-based chemotherapy. Gene expression levels of BRCA1, RAP80 and AEG-1 were quantified by real-time quantitative PCR. Expression levels were divided into terciles and correlated with median survival (MS). Results 33 p were treated with cisplatin, methotrexate and vinblastine (CMV) and 32 p with cisplatin and gemcitabine. Chemotherapy was followed by cystectomy in 60 p. Overall MS was not reached and 5-year survival was 51%. 16 p (38%) with low/intermediate BRCA1 levels had a complete pathological response (pR), while only 1 p (5%) with high BRCA1 levels had a complete pR (P = 0.006). MS was 45 months (m) and 5-year survival was 27% in 21 p with high BRCA1 mRNA levels vs 168 m and 59% in 44 p with low/intermediate levels (P = 0.05). No differences in MS were observed according to RAP80 mRNA levels. MS was 50 m in 15 p with high AEG-1 levels, 45 m in 15 p with intermediate levels, and was not reached in 18 p with low levels, although these differences were not statistically significant (P = 0.3). Conclusions Low/intermediate BRCA1 mRNA expression is associated with better pR and longer MS and 5-year survival to cisplatin-based neoadjuvant chemotherapy in p with MIBC. BRCA1 levels can be useful in customizing chemotherapy in these p. Further studies with larger numbers of p are warranted to elucidate the role of other molecular markers, including AEG-1, in this setting. Disclosure All authors have declared no conflicts of interest.

Journal of Clinical Oncology, 2016

11581Background: Targeted therapy has improved outcomes in EGFR mutated and ALK translocated NSCL... more 11581Background: Targeted therapy has improved outcomes in EGFR mutated and ALK translocated NSCLC but options for squamous cell carcinoma and KRAS-mutated tumors remain scarce. Genome re-replicati...

Molecular and Cellular Biology / Genetics, 2019

Biologically annotated specimens such as frozen, fixed and preserved tissues are key sources of c... more Biologically annotated specimens such as frozen, fixed and preserved tissues are key sources of cells for genomic analysis. Bulk NGS using archived solid tumor samples is inadequate to fully characterize somatic variation buried in the landscape of cellular populations. Single-cell targeted DNA sequencing provides an essential solution to elucidate and map genomic variation in such materials. Although the study of frozen, fixed and preserved tissues at the single-cell level is compromised by preservation processes, the isolation of nuclei allows the recovery of suitable gDNA templates. Common tissue disaggregation processes can be complicated by persistence of conglomerated cellular components, ruptured nuclei, and other insoluble extracellular matrices. For challenging samples, we developed a nuclei isolation protocol that demonstrates optimal performance for high-quality targeted DNA sequencing from archived human solid tumor samples. This process begins with physical maceration of ~10-100 mg preserved tissue or 20-100 uM sections, suspension followed by enzymatic treatment, filtration and centrifugal collection. After cell straining, nuclei are ready for counting, staining and sequencing. Fluorescent microscopy using membrane, cytoplasmic and nuclear stains reveal highly purified intact nuclei, recovering at least 500K nuclei from 30-50 mg of tissue containing greater than 70% nucleated cells by HE 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2532.

Molecular and Cellular Biology / Genetics, 2019

Journal of Clinical Oncology, 2015

e13516 Background: Targeted lung cancer therapy has undoubtedly made a difference to the treatmen... more e13516 Background: Targeted lung cancer therapy has undoubtedly made a difference to the treatment of EGFR mutation and ALK translocation carriers. However, targeted therapies for other subgroups like squamous cell carcinoma are still scarce. Re-replication of the genome could initiate gene amplification and cause chromosomal translocation and loss, contributing to tumor progression. It has been shown that cell cycle checkpoints and DNA damage response are activated when re-replication is induced. Cell cycle checkpoints, mediated by CHK1 and 2, are essential to prevent re-replication and maintain genomic integrity. Specific CHK1 inhibitors such as LY2603618 have been shown to delay tumor growth when given in combination with pemetrexed in NSCLC xenograft models. Methods: We selected a panel of NSCLC adenocarcinoma and squamous cell carcinoma cell lines representing different genetic backgrounds with TP53, KRAS and EGFR mutations. In addition, six PC9-derived, TKI resistant cell lines were included (PC9-ER...

Journal of Clinical Oncology, 2004

7148 Background: BRCA1 plays a central role in DNA repair, and decreased BRCA1 mRNA expression in... more 7148 Background: BRCA1 plays a central role in DNA repair, and decreased BRCA1 mRNA expression in the human breast cancer HCC1937 cell line caused cisplatin hypersensitivity, but its role in NSCLC has never been examined. HIF-1α overexpression activates the transcription of genes involved in angiogenesis and chemoresistance. We have examined the potential correlation and predictive value of BRCA1 and HIF-1α mRNA expression in surgically resected tumors of gemcitabine/cisplatin-treated NSCLC patients (p). METHODS Resected specimens were available from 57 stage IIB-IIIA-IIIB NSCLC p underwent surgery between September 1998 and December 2002 after receiving gemcitabine/platinum chemotherapy. Total RNA was recovered from formalin-fixed, paraffin-embedded surgical specimens. BRCA1 and HIF-1α mRNA expression was quantified with real-time PCR Results: Median overall survival (MOS) was 37.8 months (m) for all p, 51.9 m for p who underwent lobectomy, and 25.8 m for p who underwent pneumonectomy. Significant correlations were found between BRCA1 and HIF-1α mRNA expression (P<0.0001). 15 p in the bottom quartile of BRCA1 mRNA levels had a decreased risk of death compared to 12 p in the top quartile (P=0.008). MOS was 51.97 m for p in the bottom quartile, 12.7 m for p in the top quartile (P=0.03), and 25.8 m for 40 p in the top three quartiles (P=0.04). Lobectomy was performed more often in p in the bottom quartile (P=0.01). No significant differences were found according to HIF-1α mRNA levels Conclusions: BRCA1 expression should be assessed for predicting cisplatin resistance and tailoring chemotherapy in NSCLC. No significant financial relationships to disclose.

Scientific reports, Jan 22, 2017

Sequencing key cancer-driver genes using formalin-fixed, paraffin-embedded (FFPE) cancer tissues ... more Sequencing key cancer-driver genes using formalin-fixed, paraffin-embedded (FFPE) cancer tissues is becoming the standard for identifying the best treatment regimen. However, about 25% of all samples are rejected for genetic analyses for reasons that include too little tissue to extract enough high quality DNA. One way to overcome this is to do whole-genome amplification (WGA) in clinical samples, but only limited studies have tested different WGA methods in FFPE cancer specimens using targeted next-generation sequencing (NGS). We therefore tested the two most commonly used WGA methods, multiple displacement amplification (MDA-Qiagen REPLI-g kit) and the hybrid or modified PCR-based method (Sigma/Rubicon Genomics Inc. GenomePlex kit) in FFPE normal and tumor tissue specimens. For the normalized copy number analysis, the FFPE process caused none or very minimal bias. Variations in copy number were minimal in samples amplified using the GenomePlex kit, but they were statistically sign...

PloS one, 2017

The development of next-generation sequencing (NGS) technology allows to sequence whole exomes or... more The development of next-generation sequencing (NGS) technology allows to sequence whole exomes or genome. However, data analysis is still the biggest bottleneck for its wide implementation. Most laboratories still depend on manual procedures for data handling and analyses, which translates into a delay and decreased efficiency in the delivery of NGS results to doctors and patients. Thus, there is high demand for developing an automatic and an easy-to-use NGS data analyses system. We developed comprehensive, automatic genetic analyses controller named Mobile Genome Express (MGE) that works in smartphones or other mobile devices. MGE can handle all the steps for genetic analyses, such as: sample information submission, sequencing run quality check from the sequencer, secured data transfer and results review. We sequenced an Actrometrix control DNA containing multiple proven human mutations using a targeted sequencing panel, and the whole analysis was managed by MGE, and its data revie...

Cancer Research, 2016

Malignant pleural mesothelioma (MPM) is a rare malignancy with a highly unfavorable prognosis. A ... more Malignant pleural mesothelioma (MPM) is a rare malignancy with a highly unfavorable prognosis. A strong link has been established between increased risk for MPM and exposure to asbestos or erionite. As asbestos had widely been used in different industries, the incidence of MPM in the United States is expected to steadily rise and peak with about 70,000 new MPM cases over the next 20 years. Median survival has ranged from 10-17 months. But the underlying genetic mechanism is not fully understood. Moreover, genetic alterations and causes for multiple primary cancer development including MPM are unknown. We used whole exome sequencing to identify somatic mutations in a patient with MPM and two additional primary cancers who had no evidence of venous, arterial, lymphovascular, or perineural invasion indicating dissemination of a primary lung cancer to the pleura. The development of multiple primary malignancies including a rare MPM led us to search for underlying genetic alterations. In...

International journal of oncology, Jan 11, 2016

Next-generation sequencing (NGS) is becoming a standard for genetic analyses of clinical samples.... more Next-generation sequencing (NGS) is becoming a standard for genetic analyses of clinical samples. DNAs retrieved from formalin-fixed, paraffin-embedded (FFPE) tissue specimens are commonly degraded, and specimens such as core biopsies are sometimes too small to obtain enough DNA for NGS applications. Thus, it is important to measure both the DNA quantity and quality accurately from clinical samples. However, there is no standard method for DNA quantity and quality analyses for NGS library preparation. We tested four different methods (PicoGreen, Qubit® fluorometry, TaqMan and SYBR-Green-based qPCR assay) and compared each to RNase P TaqMan as a reference control. We found that SYBR-Green-based qPCR assay provides a consistent and accurate DNA quantification while keeping its cost relatively low and the throughput high. We designed a dual-probe SYBR-Green qPCR assay for DNA quantity and quality assessment for targeted NGS library preparation. This assay provides a Dscore (degradation...

Cancer Research, 2016

The emergence of next-generation sequencing (NGS) has changed the paradigm for genetic and genomi... more The emergence of next-generation sequencing (NGS) has changed the paradigm for genetic and genomic studies in many medical and life science fields. Despite its growing popularity and importance in many life science applications, several factors such as complicated sample preparation, high cost, and time-consuming data analyses may prevent NGS applications from being more widely used in clinical and research settings. Therefore, it is crucial that the current methods are improved or newly developed to become faster, more robust, and accurate in NGS applications. Targeted sequencing, focusing on small but important gene sets or genetic regions, is a very powerful approach to screen disease-related key genes. Reductions in cost and experimental time as well as availability of targeted sequencing are fueling the use of NGS for many genetic applications. We have developed a new, simple, and robust sample preparation method called ‘NextDay Seq,’ enabling us to obtain targeted deep sequenc...

Methods in Molecular Biology, 2022

The chapter was inadvertently published with incorrect figure legends, reference citations, and o... more The chapter was inadvertently published with incorrect figure legends, reference citations, and order of references. These errors have been corrected by updating the correct figure legends, reference citations, and the order of references as seen below:

Molecular Cancer Therapeutics, 2007

C127 Introduction: Trabectedin has recently received positive opinion from EMEA as treatment for ... more C127 Introduction: Trabectedin has recently received positive opinion from EMEA as treatment for advanced soft tissue sarcoma (STS) after failure to anthracyclines and ifosfamide. Objective responses and tumour control have been noted in patients resistant to conventional therapy. Moreover, the emergence of acquired resistance to trabectedin in sensitive patients is generally a late event. In experimental models trabectedin sensitivity correlates with a functional nucleotide excision repair (NER) pathway. Recently it has been described that XPG endonuclease binds to trabectedin adducts in the DNA leading to cell death. The present work characterized the impact of of XPG mRNA and protein expression levels in the tumor samples on the clinical outcome of STS patients treated with trabectedin. Methods: Retrospective analysis of paraffin embedded tumor tissue of STS patients obtained before treatment with trabectedin. A tissue microarray was constructed for analysing XPG protein express...

Methods in Molecular Biology, 2021

A comprehensive study of the cellular components of the immune system demands both deep and broad... more A comprehensive study of the cellular components of the immune system demands both deep and broad immunophenotyping of numerous cell subsets in an effective and practical way. Novel full-spectrum technology reveals the complete emission spectrum of each dye maximizing the amount of information that can be obtained on a single sample regarding conventional flow cytometry and provide an expanded knowledge of biological processes. In this chapter, we describe a 37-color protocol that allows to identify more than 45 different cell populations on whole blood samples of SARS-CoV-2-infected patients.

Nature, 2020

Myeloid malignancies, including acute myeloid leukemia (AML), arise from the expansion of hematop... more Myeloid malignancies, including acute myeloid leukemia (AML), arise from the expansion of hematopoietic stem/progenitor cells which acquire somatic mutations. Bulk molecular profiling suggests step-wise mutation acquisition, where mutant genes with high variant allele frequencies (VAFs) occur early in leukemogenesis and mutations with lower VAFs are thought to be acquired later 1-3. Although bulk sequencing informs leukemia biology and prognostication, it cannot distinguish which mutations occur in the same clone(s), accurately measure clonal complexity, or definitively elucidate mutational order. To delineate the clonal framework of myeloid malignancies, we performed single cell mutational profiling on 146 samples from 123 patients. We found AML is dominated by a small number of clones, which frequently harbor co-occurring mutations in epigenetic regulators. Conversely, mutations in signaling genes often occur more than once in distinct subclones consistent with increasing clonal diversity. We next mapped clonal trajectories for each sample and uncovered mutation combinations that synergized to promote clonal expansion and dominance. Finally, we combined protein expression with mutational analysis to map somatic genotype and clonal architecture with immunophenotype. Our studies of single cell clonal architecture provides novel insights into the pathogenesis of myeloid transformation and how clonal complexity evolves with disease progression.

Cancer Research, 2020

Fusion gene detection has long been a focus of cancer research, when combined with mutations foun... more Fusion gene detection has long been a focus of cancer research, when combined with mutations found in gDNA, can lead to a better understanding of disease progression. One example, BCR-ABL, a marker for CML and AML stem cells, is a target for tyrosine kinase inhibitor (TKI) treatments; however, there are mutations within BCR-ABL that evade TKIs and are selectively resistant to drug therapy. Single-cell technologies are now able to provide information into genomic DNA content, RNA expression, and protein surface markers, unmasked by the heterogeneity found in bulk data. However, multimodal analysis from the same single cell has not been straightforward to implement in a high throughput single-cell workflow. Here, we report the development of chemistries that enable analyses of both targeted genomic DNA and RNA sequencing from the same cell. This workflow relies on the Tapestri platform, which uses a two-step microfluidic droplet system for the analysis of thousands of cells per run. T...

Cancer Research, 2020

Elucidation of biological processes that drive cellular migration, proliferation, stemness and di... more Elucidation of biological processes that drive cellular migration, proliferation, stemness and differentiation have powered our view of tumor initiation and promotion. These avenues of research are reliant on engaging key biomarkers to measure and quantify cell systems. Widely practiced methodologies include metabolomic assays, immunoassays, imaging platforms and genomic tools. Understanding the genomic landscape of cancer has proven valuable for the characterization of molecular events that drive evolution of tumorigenesis and fostering progress in identifying druggable regimens for patient treatment scenarios. For sampling convenience, genomic measurements are typically applied to bulk cell samples. Bulk samples often obscure important rare cell types that define disease progression. Single-cell genomic analysis can highlight rare cells. Such efforts commonly generate one “modal” of information, most often next generation sequencing datasets containing RNA transcriptomic expressio...

Journal of Clinical Oncology, 2006

17038 Background: STAT5 plays a role in angiogenesis and metastasis. Activation of STAT5 occurs t... more 17038 Background: STAT5 plays a role in angiogenesis and metastasis. Activation of STAT5 occurs through dysregulation of growth factor receptor tyrosine kinases (TK) (including EGFR TK mutations), non-receptor TKs or Janus kinases. Dvl-3, a critical mediator of the Wingless-type (Wnt) signaling pathway, contributes to self-renewal of stem cells. Dicer has a key role in processing microRNAs required for stem cell regulation. We assessed mRNA expression of STAT5, Dvl-3 and Dicer in early-stage NSCLC. Methods: cDNA was derived from paraffin-embedded primary tumors of 47 surgically resected, early-stage NSCLC patients. Real-time quantitative reverse transcriptase PCR was used to measure STAT5, Dvl-3 and Dicer mRNA levels relative to the internal reference gene β-actin. Results: Median expression levels were: STAT5, 0.66 (0.15–6.03); Dvl-3, 3.69 (1.02–25.83); Dicer, 4.53 (1.22–21.96). STAT5 levels for squamous cell carcinoma (SCC) were 0.57 vs 0.89 for non-SCC (P=0.01), while Dicer level...

Blood, 2019

Genomic studies of myeloid malignancies (MM), including acute myeloid leukemia (AML), myeloprolif... more Genomic studies of myeloid malignancies (MM), including acute myeloid leukemia (AML), myeloproliferative neoplasms (MPN) and myelodysplasia (MDS), identified mutations with different allele frequencies. Recent studies of clonal hematopoiesis (CH) discovered a subset of MM disease alleles, while other alleles are only observed in overt MM. These observations suggest an important pathogenetic role for the chronology of mutational acquisition. Although bulk sequencing informs prognostication, it cannot distinguish which mutations occur in the same clone and cannot offer definitive evidence of mutational order. Delineation of clonal architecture at the single cell level is key to understanding how the sequential/parallel acquisition of somatic mutations contributes to myeloid transformation. In order to elucidate the clonal structure of MM, we designed a custom single cell 109 amplicon panel of the most frequently mutated amplicons in 50 MM genes using the Mission Bio Tapestri v2 platfo...

Annals of Oncology, 2012

ABSTRACT Background Response to neoadjuvant chemotherapy has been associated with an improvement ... more ABSTRACT Background Response to neoadjuvant chemotherapy has been associated with an improvement in survival in MIBC. The analysis of genetic markers involved in DNA repair pathways could be useful to predict chemosensitivity to cisplatin-based regimens. BRCA1 plays a central role in DNA repair and cell-cycle checkpoint control. However, BRCA1 function can be modulated by other DNA repair genes. RAP80 acts upstream of BRCA1 and regulates BRCA1 function after DNA damage. AEG-1 can induce BRCA1 expression and cause chemoresistance by activating PI3K/Akt and NF-KB pathways. Methods Paraffin-embedded pre-treatment tumor samples were collected by transurethral resection from 65 p with resectable MIBC stage T2-4N0M0 treated with neoadjuvant cisplatin-based chemotherapy. Gene expression levels of BRCA1, RAP80 and AEG-1 were quantified by real-time quantitative PCR. Expression levels were divided into terciles and correlated with median survival (MS). Results 33 p were treated with cisplatin, methotrexate and vinblastine (CMV) and 32 p with cisplatin and gemcitabine. Chemotherapy was followed by cystectomy in 60 p. Overall MS was not reached and 5-year survival was 51%. 16 p (38%) with low/intermediate BRCA1 levels had a complete pathological response (pR), while only 1 p (5%) with high BRCA1 levels had a complete pR (P = 0.006). MS was 45 months (m) and 5-year survival was 27% in 21 p with high BRCA1 mRNA levels vs 168 m and 59% in 44 p with low/intermediate levels (P = 0.05). No differences in MS were observed according to RAP80 mRNA levels. MS was 50 m in 15 p with high AEG-1 levels, 45 m in 15 p with intermediate levels, and was not reached in 18 p with low levels, although these differences were not statistically significant (P = 0.3). Conclusions Low/intermediate BRCA1 mRNA expression is associated with better pR and longer MS and 5-year survival to cisplatin-based neoadjuvant chemotherapy in p with MIBC. BRCA1 levels can be useful in customizing chemotherapy in these p. Further studies with larger numbers of p are warranted to elucidate the role of other molecular markers, including AEG-1, in this setting. Disclosure All authors have declared no conflicts of interest.

Journal of Clinical Oncology, 2016

11581Background: Targeted therapy has improved outcomes in EGFR mutated and ALK translocated NSCL... more 11581Background: Targeted therapy has improved outcomes in EGFR mutated and ALK translocated NSCLC but options for squamous cell carcinoma and KRAS-mutated tumors remain scarce. Genome re-replicati...

Molecular and Cellular Biology / Genetics, 2019

Biologically annotated specimens such as frozen, fixed and preserved tissues are key sources of c... more Biologically annotated specimens such as frozen, fixed and preserved tissues are key sources of cells for genomic analysis. Bulk NGS using archived solid tumor samples is inadequate to fully characterize somatic variation buried in the landscape of cellular populations. Single-cell targeted DNA sequencing provides an essential solution to elucidate and map genomic variation in such materials. Although the study of frozen, fixed and preserved tissues at the single-cell level is compromised by preservation processes, the isolation of nuclei allows the recovery of suitable gDNA templates. Common tissue disaggregation processes can be complicated by persistence of conglomerated cellular components, ruptured nuclei, and other insoluble extracellular matrices. For challenging samples, we developed a nuclei isolation protocol that demonstrates optimal performance for high-quality targeted DNA sequencing from archived human solid tumor samples. This process begins with physical maceration of ~10-100 mg preserved tissue or 20-100 uM sections, suspension followed by enzymatic treatment, filtration and centrifugal collection. After cell straining, nuclei are ready for counting, staining and sequencing. Fluorescent microscopy using membrane, cytoplasmic and nuclear stains reveal highly purified intact nuclei, recovering at least 500K nuclei from 30-50 mg of tissue containing greater than 70% nucleated cells by HE 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2532.

Molecular and Cellular Biology / Genetics, 2019

Journal of Clinical Oncology, 2015

e13516 Background: Targeted lung cancer therapy has undoubtedly made a difference to the treatmen... more e13516 Background: Targeted lung cancer therapy has undoubtedly made a difference to the treatment of EGFR mutation and ALK translocation carriers. However, targeted therapies for other subgroups like squamous cell carcinoma are still scarce. Re-replication of the genome could initiate gene amplification and cause chromosomal translocation and loss, contributing to tumor progression. It has been shown that cell cycle checkpoints and DNA damage response are activated when re-replication is induced. Cell cycle checkpoints, mediated by CHK1 and 2, are essential to prevent re-replication and maintain genomic integrity. Specific CHK1 inhibitors such as LY2603618 have been shown to delay tumor growth when given in combination with pemetrexed in NSCLC xenograft models. Methods: We selected a panel of NSCLC adenocarcinoma and squamous cell carcinoma cell lines representing different genetic backgrounds with TP53, KRAS and EGFR mutations. In addition, six PC9-derived, TKI resistant cell lines were included (PC9-ER...

Journal of Clinical Oncology, 2004

7148 Background: BRCA1 plays a central role in DNA repair, and decreased BRCA1 mRNA expression in... more 7148 Background: BRCA1 plays a central role in DNA repair, and decreased BRCA1 mRNA expression in the human breast cancer HCC1937 cell line caused cisplatin hypersensitivity, but its role in NSCLC has never been examined. HIF-1α overexpression activates the transcription of genes involved in angiogenesis and chemoresistance. We have examined the potential correlation and predictive value of BRCA1 and HIF-1α mRNA expression in surgically resected tumors of gemcitabine/cisplatin-treated NSCLC patients (p). METHODS Resected specimens were available from 57 stage IIB-IIIA-IIIB NSCLC p underwent surgery between September 1998 and December 2002 after receiving gemcitabine/platinum chemotherapy. Total RNA was recovered from formalin-fixed, paraffin-embedded surgical specimens. BRCA1 and HIF-1α mRNA expression was quantified with real-time PCR Results: Median overall survival (MOS) was 37.8 months (m) for all p, 51.9 m for p who underwent lobectomy, and 25.8 m for p who underwent pneumonectomy. Significant correlations were found between BRCA1 and HIF-1α mRNA expression (P<0.0001). 15 p in the bottom quartile of BRCA1 mRNA levels had a decreased risk of death compared to 12 p in the top quartile (P=0.008). MOS was 51.97 m for p in the bottom quartile, 12.7 m for p in the top quartile (P=0.03), and 25.8 m for 40 p in the top three quartiles (P=0.04). Lobectomy was performed more often in p in the bottom quartile (P=0.01). No significant differences were found according to HIF-1α mRNA levels Conclusions: BRCA1 expression should be assessed for predicting cisplatin resistance and tailoring chemotherapy in NSCLC. No significant financial relationships to disclose.

Scientific reports, Jan 22, 2017

Sequencing key cancer-driver genes using formalin-fixed, paraffin-embedded (FFPE) cancer tissues ... more Sequencing key cancer-driver genes using formalin-fixed, paraffin-embedded (FFPE) cancer tissues is becoming the standard for identifying the best treatment regimen. However, about 25% of all samples are rejected for genetic analyses for reasons that include too little tissue to extract enough high quality DNA. One way to overcome this is to do whole-genome amplification (WGA) in clinical samples, but only limited studies have tested different WGA methods in FFPE cancer specimens using targeted next-generation sequencing (NGS). We therefore tested the two most commonly used WGA methods, multiple displacement amplification (MDA-Qiagen REPLI-g kit) and the hybrid or modified PCR-based method (Sigma/Rubicon Genomics Inc. GenomePlex kit) in FFPE normal and tumor tissue specimens. For the normalized copy number analysis, the FFPE process caused none or very minimal bias. Variations in copy number were minimal in samples amplified using the GenomePlex kit, but they were statistically sign...

PloS one, 2017

The development of next-generation sequencing (NGS) technology allows to sequence whole exomes or... more The development of next-generation sequencing (NGS) technology allows to sequence whole exomes or genome. However, data analysis is still the biggest bottleneck for its wide implementation. Most laboratories still depend on manual procedures for data handling and analyses, which translates into a delay and decreased efficiency in the delivery of NGS results to doctors and patients. Thus, there is high demand for developing an automatic and an easy-to-use NGS data analyses system. We developed comprehensive, automatic genetic analyses controller named Mobile Genome Express (MGE) that works in smartphones or other mobile devices. MGE can handle all the steps for genetic analyses, such as: sample information submission, sequencing run quality check from the sequencer, secured data transfer and results review. We sequenced an Actrometrix control DNA containing multiple proven human mutations using a targeted sequencing panel, and the whole analysis was managed by MGE, and its data revie...

Cancer Research, 2016

Malignant pleural mesothelioma (MPM) is a rare malignancy with a highly unfavorable prognosis. A ... more Malignant pleural mesothelioma (MPM) is a rare malignancy with a highly unfavorable prognosis. A strong link has been established between increased risk for MPM and exposure to asbestos or erionite. As asbestos had widely been used in different industries, the incidence of MPM in the United States is expected to steadily rise and peak with about 70,000 new MPM cases over the next 20 years. Median survival has ranged from 10-17 months. But the underlying genetic mechanism is not fully understood. Moreover, genetic alterations and causes for multiple primary cancer development including MPM are unknown. We used whole exome sequencing to identify somatic mutations in a patient with MPM and two additional primary cancers who had no evidence of venous, arterial, lymphovascular, or perineural invasion indicating dissemination of a primary lung cancer to the pleura. The development of multiple primary malignancies including a rare MPM led us to search for underlying genetic alterations. In...

International journal of oncology, Jan 11, 2016

Next-generation sequencing (NGS) is becoming a standard for genetic analyses of clinical samples.... more Next-generation sequencing (NGS) is becoming a standard for genetic analyses of clinical samples. DNAs retrieved from formalin-fixed, paraffin-embedded (FFPE) tissue specimens are commonly degraded, and specimens such as core biopsies are sometimes too small to obtain enough DNA for NGS applications. Thus, it is important to measure both the DNA quantity and quality accurately from clinical samples. However, there is no standard method for DNA quantity and quality analyses for NGS library preparation. We tested four different methods (PicoGreen, Qubit® fluorometry, TaqMan and SYBR-Green-based qPCR assay) and compared each to RNase P TaqMan as a reference control. We found that SYBR-Green-based qPCR assay provides a consistent and accurate DNA quantification while keeping its cost relatively low and the throughput high. We designed a dual-probe SYBR-Green qPCR assay for DNA quantity and quality assessment for targeted NGS library preparation. This assay provides a Dscore (degradation...

Cancer Research, 2016

The emergence of next-generation sequencing (NGS) has changed the paradigm for genetic and genomi... more The emergence of next-generation sequencing (NGS) has changed the paradigm for genetic and genomic studies in many medical and life science fields. Despite its growing popularity and importance in many life science applications, several factors such as complicated sample preparation, high cost, and time-consuming data analyses may prevent NGS applications from being more widely used in clinical and research settings. Therefore, it is crucial that the current methods are improved or newly developed to become faster, more robust, and accurate in NGS applications. Targeted sequencing, focusing on small but important gene sets or genetic regions, is a very powerful approach to screen disease-related key genes. Reductions in cost and experimental time as well as availability of targeted sequencing are fueling the use of NGS for many genetic applications. We have developed a new, simple, and robust sample preparation method called ‘NextDay Seq,’ enabling us to obtain targeted deep sequenc...