Publications by authors named "Petr Starostik"

47 Publications

Optimizing COVID-19 testing capabilities and clinical management using pathology informatics.

JAMIA Open 2020 Dec 5;3(4):523-529. Epub 2020 Dec 5.

Department of Pathology, Immunology and Laboratory Medicine, University of Florida, Gainesville, Florida, USA.

Coronavirus disease 2019, first reported in China in late 2019, has quickly spread across the world. The outbreak was declared a pandemic by the World Health Organization on March 11, 2020. Here, we describe our initial efforts at the University of Florida Health for processing of large numbers of tests, streamlining data collection, and reporting data for optimizing testing capabilities and superior clinical management. Specifically, we discuss clinical and pathology informatics workflows and informatics instruments which we designed to meet the unique challenges of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) testing. We hope these results benefit institutions preparing to implement SARS-CoV-2 testing.
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http://dx.doi.org/10.1093/jamiaopen/ooaa055DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7717304PMC
December 2020

A hybrid implementation-effectiveness randomized trial of CYP2D6-guided postoperative pain management.

Genet Med 2021 Apr 8;23(4):621-628. Epub 2021 Jan 8.

Department of Pharmacotherapy and Translation Research, College of Pharmacy, University of Florida, Gainesville, FL, USA.

Purpose: Cytochrome P450 2D6 (CYP2D6) genotype-guided opioid prescribing is limited. The purpose of this type 2 hybrid implementation-effectiveness trial was to evaluate the feasibility of clinically implementing CYP2D6-guided postsurgical pain management and determine that such an approach did not worsen pain control.

Methods: Adults undergoing total joint arthroplasty were randomized 2:1 to genotype-guided or usual pain management. For participants in the genotype-guided arm with a CYP2D6 poor (PM), intermediate (IM), or ultrarapid (UM) metabolizer phenotype, recommendations were to avoid hydrocodone, tramadol, codeine, and oxycodone. The primary endpoints were feasibility metrics and opioid use; pain intensity was a secondary endpoint. Effectiveness outcomes were collected 2 weeks postsurgery.

Results: Of 282 patients approached, 260 (92%) agreed to participate. In the genotype-guided arm, 20% had a high-risk (IM/PM/UM) phenotype, of whom 72% received an alternative opioid versus 0% of usual care participants (p < 0.001). In an exploratory analysis, there was less opioid consumption (200 [104-280] vs. 230 [133-350] morphine milligram equivalents; p = 0.047) and similar pain intensity (2.6 ± 0.8 vs. 2.5 ± 0.7; p = 0.638) in the genotype-guided vs. usual care arm, respectively.

Conclusion: Implementing CYP2D6 to guide postoperative pain management is feasible and may lead to lower opioid use without compromising pain control.
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http://dx.doi.org/10.1038/s41436-020-01050-4DOI Listing
April 2021

Malignant round cell tumor with SS18-POU5F1 fusion: is it a myoepithelial neoplasm, a synovial sarcoma or a new entity?

Histopathology 2020 10 28;77(4):681-684. Epub 2020 Jul 28.

Department of Pathology, Immunology and Laboratory Medicine, University of Florida College of Medicine, Gainesville, FL, USA.

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http://dx.doi.org/10.1111/his.14171DOI Listing
October 2020

How to Transition from Single-Gene Pharmacogenetic Testing to Preemptive Panel-Based Testing: A Tutorial.

Clin Pharmacol Ther 2020 09 6;108(3):557-565. Epub 2020 Jul 6.

Department of Pharmacotherapy and Translational Research, University of Florida College of Pharmacy, Gainesville, Florida, USA.

There have been significant advancements in precision medicine and approaches to medication selection based on pharmacogenetic results. With the availability of direct-to-consumer genetic testing and growing awareness of genetic interindividual variability, patient demand for more precise, individually tailored drug regimens is increasing. The University of Florida (UF) Health Precision Medicine Program (PMP) was established in 2011 to improve integration of genomic data into clinical practice. In the ensuing years, the UF Health PMP has successfully implemented several single-gene tests to optimize the precision of medication prescribing across a variety of clinical settings. Most recently, the UF Health PMP launched a custom-designed pharmacogenetic panel, including pharmacogenes relevant to supportive care medications commonly prescribed to patients undergoing chemotherapy treatment, referred to as "GatorPGx." This tutorial provides guidance and information to institutions on how to transition from the implementation of single-gene pharmacogenetic testing to a preemptive panel-based testing approach. Here, we demonstrate application of the preemptive panel in the setting of an adult solid tumor oncology clinic. Importantly, the information included herein can be applied to other clinical practice settings.
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http://dx.doi.org/10.1002/cpt.1912DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7704727PMC
September 2020

Mapping the Mutation Landscape of Colorectal Cancer.

Am J Med Sci 2019 11 14;358(5):313-314. Epub 2019 Aug 14.

Department of Pathology, Immunology and Laboratory Medicine, College of Medicine, University of Florida, Gainesville, Florida. Electronic address:

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http://dx.doi.org/10.1016/j.amjms.2019.08.004DOI Listing
November 2019

Co-occurring Alterations in the RAS-MAPK Pathway Limit Response to MET Inhibitor Treatment in MET Exon 14 Skipping Mutation-Positive Lung Cancer.

Clin Cancer Res 2020 01 23;26(2):439-449. Epub 2019 Sep 23.

Department of Medicine, University of California, San Francisco, California.

Purpose: Although patients with advanced-stage non-small cell lung cancers (NSCLC) harboring exon 14 skipping mutations (ex14) often benefit from MET tyrosine kinase inhibitor (TKI) treatment, clinical benefit is limited by primary and acquired drug resistance. The molecular basis for this resistance remains incompletely understood.

Experimental Design: Targeted sequencing analysis was performed on cell-free circulating tumor DNA obtained from 289 patients with advanced-stage ex14-mutated NSCLC.

Results: Prominent co-occurring RAS-MAPK pathway gene alterations (e.g., in ) were detected in NSCLCs with ex14 skipping alterations as compared with -mutated NSCLCs. There was an association between decreased MET TKI treatment response and RAS-MAPK pathway co-occurring alterations. In a preclinical model expressing a canonical ex14 mutation, KRAS overexpression or NF1 downregulation hyperactivated MAPK signaling to promote MET TKI resistance. This resistance was overcome by cotreatment with crizotinib and the MEK inhibitor trametinib.

Conclusions: Our study provides a genomic landscape of co-occurring alterations in advanced-stage ex14-mutated NSCLC and suggests a potential combination therapy strategy targeting MAPK pathway signaling to enhance clinical outcomes.
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http://dx.doi.org/10.1158/1078-0432.CCR-19-1667DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6980768PMC
January 2020

SMARCA4-Deficient Thoracic Sarcoma: A Case Report and Review of Literature.

Int J Surg Pathol 2020 Feb 5;28(1):102-108. Epub 2019 Aug 5.

University of Florida, Gainesville, FL, USA.

SMARCA4-deficient thoracic sarcoma (SMARCA4-DTS) is a recently described entity with a poor prognosis that is defined by certain genetic alterations in the BAF chromatin remodeling complex, specifically and . We present a case of a SMARCA4-DTS in a 59 year-old male with a heavy smoking history who was found to have an unexpected right upper lobe lung mass on routine chest radiograph after a visit to his primary care physician. This led to a biopsy with a diagnosis of poorly differentiated carcinoma at an outside institution. The patient was subsequently seen at our facility for surgical intervention. The right upper lobectomy contained a 7.2-cm poorly differentiated malignancy with slightly discohesive cells arranged in sheets and nests, abundant geographic necrosis, and with many areas showing rhabdoid morphology. The tumor was focally reactive for CK7, AE1/3, Cam5.2, and SALL4 and showed scattered reactivity for CD34 and SOX2. There was complete loss of reactivity for both SMARCA4 and SMARCA2. The histology and immunophenotype were all consistent with the diagnosis of a SMARCA4-DTS. Next-generation sequencing showed a frameshift mutation in the gene and no abnormality with the gene. Interestingly, this tumor was confined to the pulmonary parenchyma with no invasion of the visceral pleura nor the mediastinum and with no clinically apparent metastases at the time of presentation. This case is presented to add to the cohort of cases described to date and to discuss the immunohistochemical and molecular findings with regard to .
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http://dx.doi.org/10.1177/1066896919865944DOI Listing
February 2020

Gastric Plexiform Fibromyxoma: A Great Mimic of Gastrointestinal Stromal Tumor (GIST) and Diagnostic Pitfalls.

J Surg Res 2019 07 26;239:76-82. Epub 2019 Feb 26.

Department of Pathology and Laboratory Medicine, Cedars-Sinai Medical Center, Los Angeles, California.

Through a multicenter study, we collected seven cases of gastric plexiform fibromyxoma including four females and three males, 21 to 79 y old (46.1 ± 10.1). All cases showed a unilocular lesion measuring 0.3 to 17 cm (5.3 ± 2.4), arising from antrum (5/7) or body (2/7). Six of the seven cases had intraoperative frozen sections and/or endoscopic ultrasound fine needle aspiration (EUS-FNA), and all of them were preoperatively or intraoperatively diagnosed as gastrointestinal stromal tumor (GIST). EUS-FNA material showed markedly elongated spindle cells with streaming oval to elongated nuclei with rounded ends. Histologically, the tumors exhibited a plexiform growth pattern and were composed of a rich myxoid stroma and cytologically bland uniform spindle cells without mitotic figures, with the exception of one case which displayed nuclear pleomorphism and increased mitosis. Immunostains showed the tumor cells to be focally positive for SMA (6/6), focally and weakly positive for desmin (3/6) and caldesmon (2/3), negative for CD117 (0/7), CD34 (0/7), DOG1 (0/4), and S100 (0/5). No mutations were identified on Next-Generation Sequencing test, and no loss of SDHB immunoreactivity was identified in the tumor with nuclear pleomorphism. One case was treated with Gleevec because of the initial diagnosis of GIST. All patients had a follow-up for up to 11 y, with no tumor recurrence or metastasis reported. Our results suggest that gastric plexiform fibromyxoma is rare and may be underrecognized and misinterpreted as GIST during intraoperative frozen section or preoperative EUS-FNA diagnosis without immunostains leading to inappropriate treatment.
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http://dx.doi.org/10.1016/j.jss.2019.01.062DOI Listing
July 2019

CYP2D6-guided opioid therapy improves pain control in CYP2D6 intermediate and poor metabolizers: a pragmatic clinical trial.

Genet Med 2019 08 23;21(8):1842-1850. Epub 2019 Jan 23.

Department of Pharmacotherapy and Translational Research, College of Pharmacy, University of Florida, Gainesville, FL, USA.

Purpose: CYP2D6 bioactivates codeine and tramadol, with intermediate and poor metabolizers (IMs and PMs) expected to have impaired analgesia. This pragmatic proof-of-concept trial tested the effects of CYP2D6-guided opioid prescribing on pain control.

Methods: Participants with chronic pain (94% on an opioid) from seven clinics were enrolled into CYP2D6-guided (n = 235) or usual care (n = 135) arms using a cluster design. CYP2D6 phenotypes were assigned based on genotype and CYP2D6 inhibitor use, with recommendations for opioid prescribing made in the CYP2D6-guided arm. Pain was assessed at baseline and 3 months using PROMIS measures.

Results: On stepwise multiple linear regression, the primary outcome of composite pain intensity (composite of current pain and worst and average pain in the past week) among IM/PMs initially prescribed tramadol/codeine (n = 45) had greater improvement in the CYP2D6-guided versus usual care arm (-1.01 ± 1.59 vs. -0.40 ± 1.20; adj P = 0.016); 24% of CYP2D6-guided versus 0% of usual care participants reported ≥30% (clinically meaningful) reduction in the composite outcome. In contrast, among normal metabolizers prescribed tramadol or codeine at baseline, there was no difference in the change in composite pain intensity at 3 months between CYP2D6-guided (-0.61 ± 1.39) and usual care (-0.54 ± 1.69) groups (adj P = 0.540).

Conclusion: These data support the potential benefits of CYP2D6-guided pain management.
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http://dx.doi.org/10.1038/s41436-018-0431-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6650382PMC
August 2019

Clinical implementation of rapid CYP2C19 genotyping to guide antiplatelet therapy after percutaneous coronary intervention.

J Transl Med 2018 04 11;16(1):92. Epub 2018 Apr 11.

Division of Cardiology, Department of Medicine, University of Florida, Jacksonville, FL, USA.

Background: The CYP2C19 nonfunctional genotype reduces clopidogrel effectiveness after percutaneous coronary intervention (PCI). Following clinical implementation of CYP2C19 genotyping at University Florida (UF) Health Shands Hospital in 2012, where genotype results are available approximately 3 days after PCI, testing was expanded to UF Health Jacksonville in 2016 utilizing a rapid genotyping approach. We describe metrics with this latter implementation.

Methods: Patients at UF Health Jacksonville undergoing left heart catheterization with intent to undergo PCI were targeted for genotyping using the Spartan RX™ system. Testing metrics and provider acceptance of testing and response to genotype results were examined, as was antiplatelet therapy over the 6 months following genotyping.

Results: In the first year, 931 patients, including 392/505 (78%) total patients undergoing PCI, were genotyped. The median genotype test turnaround time was 96 min. Genotype results were available for 388 (99%) PCI patients prior to discharge. Of 336 genotyped PCI patients alive at discharge and not enrolled in an antiplatelet therapy trial, 1/6 (17%) poor metabolizers (PMs, with two nonfunctional alleles), 38/93 (41%) intermediate metabolizers (IMs, with one nonfunctional allele), and 119/237 (50%) patients without a nonfunctional allele were prescribed clopidogrel (p = 0.110). Clopidogrel use was higher among non-ACS versus ACS patients (78.6% vs. 42.2%, p < 0.001). Six months later, among patients with follow-up data, clopidogrel was prescribed in 0/4 (0%) PMs, 33/65 (51%) IMs, and 115/182 (63%) patients without a nonfunctional allele (p = 0.008 across groups; p = 0.020 for PMs versus those without a nonfunctional allele).

Conclusion: These data demonstrate that rapid genotyping is clinically feasible at a high volume cardiac catheterization facility and allows informed chronic antiplatelet prescribing, with lower clopidogrel use in PMs at 6 months. Trial registration ClinicalTrials.gov Identifier: NCT02724319; registered March 31, 2016; https://www.clinicaltrials.gov/ct2/show/NCT02724319?term=angiolillo&rank=7.
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http://dx.doi.org/10.1186/s12967-018-1469-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5896099PMC
April 2018

Personalized Dosing of Dichloroacetate Using GSTZ1 Clinical Genotyping Assay.

Genet Test Mol Biomarkers 2018 Apr 19;22(4):266-269. Epub 2018 Mar 19.

4 Department of Pathology, College of Medicine, University of Florida , Gainesville, Florida.

Aims: Dichloroacetate (DCA) represents the first targeted therapy for pyruvate dehydrogenase complex deficiency; it is metabolized by glutathione transferase zeta1 (GSTZ1). Variation in the GSTZ1 haplotype is the principal variable influencing DCA kinetics and dynamics in humans. We aimed to develop a sensitive and rapid clinical genetic screening test for determining GSTZ1 haplotype status in individuals who would be treated with DCA, and then apply the test for the investigation of the plasma pharmacokinetics (PK) of DCA as a function of GSTZ1 haplotype.

Materials And Methods: DNA samples from 45 healthy volunteer study participants were genotyped for three functional GSTZ1 single nucleotide polymorphisms (rs7975, rs7972, and rs1046428) by TaqMan. Prior studies showed that subjects with at least one EGT haplotype (EGT carrier) metabolized DCA faster than EGT noncarriers. The clinical genetic test for GSTZ1 was developed and validated at our CLIA-certified Clinical Laboratory. Four fast metabolizer EGT carriers and four slow metabolizer EGT noncarriers were selected to complete a standard PK study. Each participant received a single oral dose of 25 mg/kg of DCA (IND 028625) for 5 days.

Results: The EGT haplotype carrier group demonstrated significantly faster metabolism of DCA and higher rates of plasma DCA clearance after 5 days of drug exposure compared with EGT noncarriers (p = 0.04).

Conclusions: These preliminary data establish the validity and practicality of our rapid genotyping/haplotyping procedure for genetic-based DCA dosing to mitigate or prevent adverse effects in patients treated chronically with this drug.
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http://dx.doi.org/10.1089/gtmb.2017.0261DOI Listing
April 2018

Design and rational for the precision medicine guided treatment for cancer pain pragmatic clinical trial.

Contemp Clin Trials 2018 05 10;68:7-13. Epub 2018 Mar 10.

Department of Pharmacotherapy and Translational Research, College of Pharmacy, University of Florida, Gainesville, FL, USA; Center for Pharmacogenomics, University of Florida, Gainesville, FL, USA; Clinical and Translational Science Institute, University of Florida, Gainesville, FL, USA. Electronic address:

Introduction: Pain is one of the most burdensome symptoms associated with cancer and its treatment, and opioids are the cornerstone of pain management. Opioid therapy is empirically selected, and patients often require adjustments in therapy to effectively alleviate pain or ameliorate adverse drug effects that interfere with quality of life. There are data suggesting CYP2D6 genotype may contribute to inter-patient variability in response to opioids through its effects on opioid metabolism. Therefore, we aim to determine if CYP2D6 genotype-guided opioid prescribing results in greater reductions in pain and symptom severity and interference with daily living compared to a conventional prescribing approach in patients with cancer.

Methods: Patients with solid tumors with metastasis and a self-reported pain score ≥ 4/10 are eligible for enrollment and randomized to a genotype-guided or conventional pain management strategy. For patients in the genotype-guided arm, CYP2D6 genotype information is integrated into opioid prescribing decisions. Patients are asked to complete questionnaires regarding their pain, symptoms, and quality of life at baseline and 2, 4, 6, and 8 weeks after enrollment. The primary endpoint is differential change in pain severity by treatment strategy (genotype-guided versus conventional pain management). Secondary endpoints include change in pain and symptom interference with daily living.

Conclusion: Pharmacogenetic-guided opioid selection for cancer pain management has potential clinical utility, but current evidence is limited to retrospective and observational studies. Precision Medicine Guided Treatment for Cancer Pain is a pragmatic clinical trial that seeks to determine the utility of CYP2D6 genotype-guided opioid prescribing in patients with cancer.
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http://dx.doi.org/10.1016/j.cct.2018.03.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5899651PMC
May 2018

Multisite Investigation of Outcomes With Implementation of CYP2C19 Genotype-Guided Antiplatelet Therapy After Percutaneous Coronary Intervention.

JACC Cardiovasc Interv 2018 01 1;11(2):181-191. Epub 2017 Nov 1.

Department of Medicine, University of Maryland, Baltimore, Maryland.

Objectives: This multicenter pragmatic investigation assessed outcomes following clinical implementation of CYP2C19 genotype-guided antiplatelet therapy after percutaneous coronary intervention (PCI).

Background: CYP2C19 loss-of-function alleles impair clopidogrel effectiveness after PCI.

Methods: After clinical genotyping, each institution recommended alternative antiplatelet therapy (prasugrel, ticagrelor) in PCI patients with a loss-of-function allele. Major adverse cardiovascular events (defined as myocardial infarction, stroke, or death) within 12 months of PCI were compared between patients with a loss-of-function allele prescribed clopidogrel versus alternative therapy. Risk was also compared between patients without a loss-of-function allele and loss-of-function allele carriers prescribed alternative therapy. Cox regression was performed, adjusting for group differences with inverse probability of treatment weights.

Results: Among 1,815 patients, 572 (31.5%) had a loss-of-function allele. The risk for major adverse cardiovascular events was significantly higher in patients with a loss-of-function allele prescribed clopidogrel versus alternative therapy (23.4 vs. 8.7 per 100 patient-years; adjusted hazard ratio: 2.26; 95% confidence interval: 1.18 to 4.32; p = 0.013). Similar results were observed among 1,210 patients with acute coronary syndromes at the time of PCI (adjusted hazard ratio: 2.87; 95% confidence interval: 1.35 to 6.09; p = 0.013). There was no difference in major adverse cardiovascular events between patients without a loss-of-function allele and loss-of-function allele carriers prescribed alternative therapy (adjusted hazard ratio: 1.14; 95% confidence interval: 0.69 to 1.88; p = 0.60).

Conclusions: These data from real-world observations demonstrate a higher risk for cardiovascular events in patients with a CYP2C19 loss-of-function allele if clopidogrel versus alternative therapy is prescribed. A future randomized study of genotype-guided antiplatelet therapy may be of value.
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http://dx.doi.org/10.1016/j.jcin.2017.07.022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5775044PMC
January 2018

Institutional profile: University of Florida Health Personalized Medicine Program.

Pharmacogenomics 2017 Apr 27;18(5):421-426. Epub 2017 Mar 27.

Department of Pharmacotherapy & Translational Research, College of Pharmacy, University of Florida, Gainesville, FL 32610, USA.

The University of Florida (UF) Health Personalized Medicine Program launched in 2012 with CYP2C19 genotyping for clopidogrel response at UF Health Shands Hospital. We have since expanded CYP2C19 genotyping to UF Health Jacksonville and established the infrastructure at UF Health to support clinical implementation for five additional gene-drug pairs: TPMT-thiopurines, IFNL3 (IL28B)-PEG IFN-α-based regimens, CYP2D6-opioids, CYP2D6/CYP2C19-antidepressants and CYP2C19-proton pump inhibitors. We are contributing to the evidence based on outcomes with genotype-guided therapy through pragmatic studies of our clinical implementations. In addition, we have developed a broad array of educational programs for providers, trainees and students that incorporate personal genotype evaluation to enhance participant learning.
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http://dx.doi.org/10.2217/pgs-2017-0028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5558508PMC
April 2017

Spontaneous Remission in an Older Patient with Relapsed ITD Mutant AML.

Case Rep Hematol 2016 29;2016:1259759. Epub 2016 Dec 29.

Leukemia Service, Department of Medicine, Roswell Park Cancer Institute, Buffalo, NY, USA.

Spontaneous remission (SR) of acute myeloid leukemia (AML) is a very rare phenomenon. AML characterized by internal tandem duplication ( ITD) is typically associated with an aggressive clinical course with rapid progression, relapse, and short overall survival in the absence of transplantation. We report here the first case of SR of ITD mutant AML in the literature. Our patient was an elderly woman with relapsed and ITD mutant AML whose disease underwent SR for a brief duration without precipitating cause. We review the potential immune mechanisms underlying SR in AML and discuss the implications for novel immunotherapeutic approaches for mutant AML.
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http://dx.doi.org/10.1155/2016/1259759DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5227128PMC
December 2016

Clinical mutation assay of tumors: new developments.

Authors:
Petr Starostik

Anticancer Drugs 2017 01;28(1):1-10

Department of Pathology, Immunology, and Laboratory Medicine, University of Florida College of Medicine, Gainesville, Florida, USA.

Mutation detection in tumors started with classical cytogenetics as the method of choice more than 50 years ago. Karyotyping proved to be sensitive enough to detect deletions or duplications of large chromosome segments, and translocations. Over time, new techniques were developed to detect mutations that are much smaller in scope. The availability of Sanger sequencing and the invention of the PCR improved the discriminatory power of mutation detection to just one base change in the genomic DNA sequence. Techniques derived from PCR (allele-specific PCR, qPCR) and improved or modified sequencing methods (capillary electrophoresis, pyrosequencing) considerably increased the efficiency and sample throughput of mutation detection assays. With the advent of massive parallel sequencing [also called next-generation sequencing (NGS)] in the past decade, a major shift to even higher sample throughput and a significant decrease in cost per sequenced base occurred. The application of the new technology provided a whole slew of novel biomarkers and potential therapy targets to improve diagnosis and treatment. It even led to changes in cancer classification as new information on the mutation profile of tumors became available that characterizes some disease entities better than morphology. NGS, which usually interrogates multiple genes at once and is a prime example of a multianalyte assay, started to replace older single analyte assays focused on analysis of one target, one gene. However, the transition to these extremely complex NGS-based assays is associated with multiple challenges. There are issues with adequate tissue source of nucleic acids, sequencing library preparation, bioinformatics, government regulations and oversight, reimbursement, and electronic medical records that need to be resolved to successfully implement the new technology in a clinical laboratory.
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http://dx.doi.org/10.1097/CAD.0000000000000427DOI Listing
January 2017

Routine Clinical Mutation Profiling of Non-Small Cell Lung Cancer Using Next-Generation Sequencing.

Arch Pathol Lab Med 2015 Jul;139(7):913-21

From the Department of Pathology and Laboratory Medicine, Roswell Park Cancer Institute, Buffalo, New York. Dr Deeb is now with the University Hospitals Case Medical Center, Cleveland, Ohio; Mr Risch is now with Life Technologies, Grand Island, New York; and Dr Starostik is now with the Department of Pathology, Immunology and Laboratory Medicine, College of Medicine University of Florida, Gainesville.

Context: The availability of massive, parallel-sequencing technologies makes possible efficient, simultaneous detection of driver and druggable mutations in cancer.

Objective: To develop an amplicon-based, next-generation sequencing, mutation-detection assay for lung cancer using the 454 GS Junior (Roche Applied Science, Indianapolis, Indiana) platform.

Design: Fusion primers incorporating target sequence, 454 adaptors, and multiplex identifiers were designed to generate 35 amplicons (median length 246 base pairs) covering 8.9 kilobases of mutational hotspots in AKT1, BRAF, EGFR, ERBB2, HRAS, KRAS, NRAS, PIK3CA, and MAP2K1 genes and all exons of the PTEN gene.

Results: The assay was validated on 23 formalin-fixed, paraffin-embedded lung cancer specimens. A minimum number of reads was consistently achieved with overall median read depth of 529× per amplicon. In total, 25 point mutations and 4 insertions/deletions (indels) with a frequency of 5.5% to 93.1% mutant alleles were detected. All EGFR, ERBB2, KRAS, PIK3CA, KRAS, and PTEN mutations, as detected by next-generation sequencing, were confirmed by pyrosequencing, with the exception of 3 point mutations in a tumor sample with low mutant-allele burden (below the pyrosequencing limit of detection).

Conclusions: The GS Junior-based, targeted, resequencing assay for a focused set of non-small cell lung cancer driver genes allows for quick and sensitive detection of point mutations and indels for the most relevant therapeutic genes in this type of cancer.
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http://dx.doi.org/10.5858/arpa.2014-0095-OADOI Listing
July 2015

Deletion and deletion/insertion mutations in the juxtamembrane domain of the FLT3 gene in adult acute myeloid leukemia.

Leuk Res Rep 2014 16;3(2):86-9. Epub 2014 Oct 16.

Molecular Diagnostics Laboratory, Department of Pathology and Laboratory Medicine, Roswell Park Cancer Institute, Buffalo, NY 14263, United States.

In contrast to FLT3 ITD mutations, in-frame deletions in the FLT3 gene have rarely been described in adult acute leukemia. We report two cases of AML with uncommon in-frame mutations in the juxtamembrane domain of the FLT3 gene: a 3-bp (c.1770_1774delCTACGinsGT; p.F590_V592delinsLF) deletion/insertion and a 12-bp (c.1780_1791delTTCAGAGAATAT; p.F594_Y597del) deletion. We verified by sequencing that the reading frame of the FLT3 gene was preserved and by cDNA analysis that the mRNA of the mutant allele was expressed in both cases. Given the recent development of FLT3 inhibitors, our findings may be of therapeutic value for AML patients harboring similar FLT3 mutations.
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http://dx.doi.org/10.1016/j.lrr.2013.09.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4220013PMC
November 2014

Phase II trial of clofarabine and daunorubicin as induction therapy for acute myeloid leukemia patients greater than or equal to 60 years of age.

Leuk Res 2013 Nov 15;37(11):1468-71. Epub 2013 Aug 15.

Leukemia Section, Department of Medicine, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263, United States.

We designed a phase II study evaluating the upfront combination of clofarabine and daunorubicin in acute myeloid leukemia (AML) patients≥60 years old. The median age of the 21 patients was 69 (range 60-85) years. Fourteen patients (67%) had unfavorable risk features. The principal toxicities were grade ≥3 infections and prolonged myelosuppression. Three (14%) deaths occurred from infectious complications. Six (28.6%) patients achieved complete remission including three (21.4%) of 14 patients with unfavorable AML. The median disease-free survival was 6.8 months and the median overall survival was 11.2 months.
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http://dx.doi.org/10.1016/j.leukres.2013.07.036DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3818466PMC
November 2013

Primary intraplacental gestational choriocarcinoma: histologic and genetic analyses.

Int J Gynecol Pathol 2013 Jan;32(1):71-5

Department of Pathology and Laboratory Medicine, Roswell Park Cancer Institute, Buffalo, NY, USA.

Gestational choriocarcinoma is a highly malignant form of gestational trophoblastic tumors. Placental involvement of the tumor is exceedingly rare. We describe a case of intraplacental choriocarcinoma in a full-term placenta. Microscopically, there were sheets of highly pleomorphic trophoblasts invading the placenta, leaving isolated chorionic villi within the tumor. We utilized molecular genetic methods to determine the genotype of the tumor and compared it with that of the placenta. Short tandem repeat polymorphism analysis was performed on tumor and placental DNA macrodissected from unstained slides of formalin-fixed, paraffin-embedded tissue. The assay included polymerase chain reaction reactions of 10 polymorphic genetic loci. Comparing the polymorphic genetic markers of the tumor with the placenta revealed a complete match in the genotype of all loci examined. The results suggest that the choriocarcinoma originated from the current placenta sharing exactly the same genotype of multiple polymorphic markers. It also excludes a nongestational choriocarcinoma, which is of germ cell origin.
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http://dx.doi.org/10.1097/PGP.0b013e3182566552DOI Listing
January 2013

Development and characterization of a novel human Waldenström macroglobulinemia cell line: RPCI-WM1, Roswell Park Cancer Institute - Waldenström Macroglobulinemia 1.

Leuk Lymphoma 2013 Feb 27;54(2):387-96. Epub 2012 Aug 27.

Department of Hematology and Oncology, Mayo Clinic, Jacksonville, FL 32224, USA.

Understanding the biology of Waldenström macroglobulinemia is hindered by a lack of preclinical models. We report a novel cell line, RPCI-WM1, from a patient treated for WM. The cell line secretes human immunoglobulin M (h-IgM) with κ-light chain restriction identical to the primary tumor. The cell line has a modal chromosomal number of 46 and harbors chromosomal changes such as deletion of 6q21, monoallelic deletion of 9p21 (CDKN2A), 13q14 (RB1) and 18q21 (BCL-2), with a consistent amplification of 14q32 (immunoglobulin heavy chain; IgH) identical to its founding tumor sample. The clonal relationship is confirmed by identical CDR3 length and single nucleotide polymorphisms as well as a matching IgH sequence of the cell line and founding tumor. Both also harbor a heterozygous, non-synonymous mutation at amino acid 265 in the MYD88 gene (L265P). The cell line expresses most of the cell surface markers present on the parent cells. Overall, RPCI-WM1 represents a valuable model to study Waldenström macroglobulinemia.
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http://dx.doi.org/10.3109/10428194.2012.713481DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4406272PMC
February 2013

Monoallelic and biallelic deletions of 13q14.3 in chronic lymphocytic leukemia: FISH vs miRNA RT-qPCR detection.

Am J Clin Pathol 2012 Apr;137(4):641-6

Molecular Diagnostics Laboratory, Roswell Park Cancer Institute, Buffalo, NY 14263, USA.

Deletion of 13q14.3 (del(13q)) is the most common cytogenetic abnormality in chronic lymphocytic leukemia (CLL) and implies a favorable prognosis. We explored the feasibility of detecting del(13q) by real-time quantitative polymerase chain reaction (PCR) for miR-15a and miR-16-1, whose loci are located in the deleted region. We analyzed 23 cases of B-CLL with monoallelic (10 cases) or biallelic del(13q) (5 cases) and used trisomy 12-positive CLL samples (n = 8) as control samples. As expected, miR-15a was expressed at significantly lower levels in monoallelic del(13qx1) samples compared with trisomy 12 control samples (P = .001). Biallelic del(13q) (del(13qx2)) samples showed further reduction of miR-15a levels compared with monoallelic del(13q) (del(13qx1)) (P = .009). In contrast, miR-16-1 expression levels were generally much lower and variable, with the highest levels detected in del(13qx1). Analyzed retrospectively, miR-15a levels differ among the del(13q) groups. However, only del(13qx2) miR-15a levels are reduced enough to determine the allelic status of an individual sample prospectively by real-time quantitative PCR.
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http://dx.doi.org/10.1309/AJCPP31FSSRQTTAQDOI Listing
April 2012

ERBB2 juxtamembrane domain (trastuzumab binding site) gene mutation is a rare event in invasive breast cancers overexpressing the ERBB2 gene.

Mod Pathol 2011 Aug 15;24(8):1055-9. Epub 2011 Apr 15.

Department of Pathology, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263, USA.

The recent development of targeted therapies using monoclonal antibodies has added new dimensions to breast cancer treatment. Trastuzumab has been added to the regimens that contain chemotherapeutic agents, which has improved the clinical outcomes of patients in both the adjuvant and metastatic settings. However, trastuzumab resistance, both de novo and acquired, continues to be problematic. There have been scattered studies reporting ERBB2 gene mutation, but nothing is currently known about the ERBB2 binding site mutations. In the current study, we examined the ERBB2 juxtamembrane domain trastuzumab binding site for mutations in invasive breast cancers overexpressing ERBB2. Pure tumor cells of 54 breast cancer patients were procured using laser capture microdissection. Two polymerase chain reaction primer pairs were designed to amplify the trastuzumab binding site sequence. The polymerase chain reaction product was sequenced. Standard clinicopathological data were recorded. For the 54 patients, there was one (2%) case that showed missense point mutation in exon 17 (H559A). There were nine patients treated with trastuzumab in the metastatic setting, none of which had gene mutation. Therefore, we conclude that ERBB2 juxtamembrane domain (trastuzumab binding site) gene mutation is a rare event in breast cancer. Although it is unclear whether this substitution would result in trastuzumab target therapy resistance, this would not account for the relatively high frequency of this resistance encountered clinically.
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http://dx.doi.org/10.1038/modpathol.2011.64DOI Listing
August 2011

Hypoxia-inducible factor-1α protein expression is associated with poor survival in normal karyotype adult acute myeloid leukemia.

Leuk Res 2011 May 21;35(5):579-84. Epub 2010 Dec 21.

Department of Pathology and Laboratory Medicine, Roswell Park Cancer Institute, Buffalo, NY, USA.

We examined the predictive impact of HIF-1α protein expression on clinical outcome of 84 normal karyotype acute myeloid leukemia (NK-AML) patients (median age 66.5 years) at our institute. Thirty percent of NK-AML cells expressed cytoplasmic HIF-1α. In univariate analysis, low HIF-1α (≤ 5%, n = 66) was associated with improved event-free survival (p = 0.0453, HR = 0.22). Multivariate analysis incorporating age, complete remission, FLT3-ITD mutation, and marrow blast percentage demonstrated that HIF-1α was independently associated with poorer overall and event-free survival. HIF-1α expression correlated with VEGF-C but not VEGF-A, marrow angiogenesis, FLT3 ITD or NPM1 mutations. These results support HIF-1α as an outcome marker for NK-AML.
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http://dx.doi.org/10.1016/j.leukres.2010.10.020DOI Listing
May 2011

Genomic, immunophenotypic, and NPM1/FLT3 mutational studies on 17 patients with normal karyotype acute myeloid leukemia (AML) followed by aberrant karyotype AML at relapse.

Cancer Genet Cytogenet 2010 Oct;202(2):101-7

Leukemia Service, Department of Medicine, Roswell Park Cancer Institute, Buffalo, NY 14263, USA.

Normal karyotype (NK) is the most common cytogenetic group in acute myeloid leukemia (AML) diagnosis; however, up to 50% of these patients at relapse will have aberrant karyotype (AK) AML. To determine the etiology of relapsed AK AML cells, we evaluated cytogenetic, immunophenotypic, and molecular results of 17 patients with diagnostic NK AML and relapsed AK AML at our institute. AK AML karyotype was diverse, involving no favorable and largely (8 of 17) complex cytogenetics. Despite clear cytogenetic differences, immunophenotype and NPM1/FLT3 gene mutation status did not change between presentation and relapse in 83% (10 of 12) and 94% (15 of 16) cases, respectively. High-resolution array-based comparative genomic hybridization (aCGH) performed via paired aCGH on NK AML and AK AML samples from the same patient confirmed cytogenetic aberrations only in the relapse sample. Analysis of 16 additional diagnostic NK AML samples revealed no evidence of submicroscopic aberrations undetected by conventional cytogenetics in any case. These results favor evolution of NK AML leukemia cells with acquisition of novel genetic changes as the most common etiology of AK AML relapse as opposed to secondary leukemogenesis. Additional studies are needed to confirm whether AK AML cells represent selection of rare preexisting clones below aCGH detection and to further characterize the molecular lesions found at time of AK AML relapse.
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http://dx.doi.org/10.1016/j.cancergencyto.2010.07.117DOI Listing
October 2010

Multiparameter flow cytometry for the diagnosis and monitoring of small GPI-deficient cellular populations.

Cytometry B Clin Cytom 2010 Sep 7;78(5):348-56. Epub 2010 Jun 7.

Department of Medicine, Roswell Park Cancer Institute, Buffalo, New York, USA.

Background: Glycosyl-phosphatidylinositol (GPI)-negative blood cells are diagnostic for Paroxysmal Nocturnal Hemoglobinuria (PNH). Marrow failure states are often associated with GPI-negative cell populations. Quantification of small clonal populations of GPI-negative cells influences clinical decisions to administer immunosuppressive therapy in marrow failure states (aplastic anemia or myelodysplastic syndrome) and to monitor minimal residual disease after allogeneic blood or marrow transplantation (BMT). We studied the reliability of high-resolution flow cytometry markers operating at the limits of detection.

Methods: We performed serial quantification of the PNH clone size in 38 samples using multiparameter flow cytometry. Granulocytes, monocytes, and RBCs were gated using forward and side scatter as well as lineage-specific markers. The GPI-linked markers fluorescent aerolysin (FLAER), CD55, and CD59 were comparatively evaluated. We also evaluated CD16 on granulocytes and CD14 on monocytes. The sensitivity of detection by each marker was further defined by serial dilution experiments on a flow-sorted sample. Two patients had quantification of their GPI-negative clones before and after allogeneic BMT.

Results: FLAER was the most discriminant marker and allowed identification of 0.1% of GPI-negative cells despite other markers having superior signal-to-noise characteristics. CD14 and CD16 were inferior to CD55 at lower concentrations and in clinical application.

Conclusions: Multiparameter flow cytometry permits quantification of small GPI-negative clones with a sensitivity limit of about 0.1%. The single most reliable marker to monitor small granulocyte or monocyte PNH clones is FLAER, especially in conditions such as myelodysplastic syndromes or BMT, when traditional GPI-linked surface marker expression can be significantly altered. © 2010 International Clinical Cytometry Society.
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http://dx.doi.org/10.1002/cyto.b.20519DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2937167PMC
September 2010

Acquirement of rituximab resistance in lymphoma cell lines is associated with both global CD20 gene and protein down-regulation regulated at the pretranscriptional and posttranscriptional levels.

Clin Cancer Res 2008 Mar;14(5):1561-70

Department of Medicine, Roswell Park Cancer Institute, Buffalo, New York 14263, USA.

Acquirement of resistance to rituximab has been observed in lymphoma patients. To define mechanisms associated with rituximab resistance, we developed various rituximab-resistant cell lines (RRCL) and studied changes in CD20 expression/structure, lipid raft domain (LRD) reorganization, calcium mobilization, antibody-dependent cellular cytotoxicity, and complement-mediated cytotoxicity (CMC) between parental and RRCL. Significant changes in surface CD20 antigen expression were shown in RRCL. Decreased calcium mobilization and redistribution of CD20 into LRD were found in RRCL. Western blotting identified a unique 35 kDa protein band in RRCL, which was not seen in parental cells and was secondary to an increase in surface and cytoplasmic expression of IgM light chains. CD20 gene expression was decreased in RRCL. In vitro exposure to PS341 increased CD20 expression in RRCL and minimally improved the sensitivity to rituximab-associated CMC. Our data strongly suggest that the acquisition of rituximab resistance is associated with global gene and protein down-regulation of the CD20 antigen affecting LRD organization and downstream signaling. CD20 expression seems to be regulated at the pretranscriptional and posttranscriptional levels. Proteasome inhibition partially reversed rituximab resistance, suggesting the existence of additional mediators of rituximab resistance. Future research is geared to identify drugs and/or biological agents that are effective against RRCL.
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http://dx.doi.org/10.1158/1078-0432.CCR-07-1254DOI Listing
March 2008

Increased frequency of uridine diphosphate glucuronosyltransferase 1A1 7/7 in patients experiencing severe irinotecan-induced toxicities.

Clin Colorectal Cancer 2007 Jul;6(8):583-7

Department of Medicine, Roswell Park Cancer Institute, Buffalo, NY 14263, USA.

Background: Uridine diphosphate glucuronosyltransferase (UGT) 1A1 7/7 polymorphism has been linked with an increased risk of irinotecan-induced severe toxicities. We evaluated UGT1A1 polymorphism in patients developing grade 3/4 toxicity after initiation of irinotecan to determine the frequency of this polymorphism in this population.

Patients And Methods: Twenty patients with grade 3/4 irinotecan-induced toxicity underwent UGT1A1 genotyping in an exploratory study. The frequency of UGT1A1 7/7 and the pattern of toxicity associated with this polymorphism were described.

Results: Forty percent of patients with grade 3/4 toxicities had a UGT1A 7/7 polymorphism (vs. 10% in general population). Six of 7 patients requiring hospitalization, 7 of 10 patients with grade 4 neutropenia, and 3 of 3 patients with grade 4 diarrhea, had UGT1A1 7/7 polymorphism.

Conclusion: Uridine diphosphate glucuronosyltransferase 1A1 7/7 is prevalent in patients with irinotecan grade 3/4 toxicity, especially in patients with treatment-related hospitalizations and grade 4 toxicities. Our data support the need for more prospective studies that evaluate the predictive value of UGT1A1 as well as UGT1A1-based dosing in patients receiving irinotecan.
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http://dx.doi.org/10.3816/CCC.2007.n.026DOI Listing
July 2007