ABSTRACT
Background: Phosphodiesterase type 5 inhibitors (PDE5I) are prescribed for erectile dysfunction and pulmonary hypertension. Despite its widespread use, there are only seven cases of drug-induced liver injury (DILI) associated with PDE5I, none associated with vardenafil or avanafil. We report a patient who had taken vardenafil and tadalafil individually for several years without developing symptoms of liver injury. However, after taking vardenafil and tadalafil together on 2 consecutive days, he developed severe cholestasis. Methods: Causality was determined using Roussel Uclaf causality assessment method (RUCAM). Results: The patient is a 72-year-old White man in excellent health who drank 2 units of alcohol, three times/week. Previously, he had used vardenafil for more than 2 years and tadalafil for 3 months as single agent for erectile dysfunction without any complications. He took vardenafil and tadalafil for 2 consecutive days and 5 days later, he developed dyspepsia, loss of appetite, jaundice, and intense itching. Liver tests showed mixed cholestatic/hepatocellular pattern of injury. Histology showed marked cholestasis with minimal inflammation. He remained cholestatic for 5 weeks before a full recovery 2 months later. The patient then resumed vardenafil monotherapy with no recurrent liver dysfunction. RUCAM causality score 7 indicates that the combination of PDE5I is probable cause of liver injury. The similarities among the eight cases of PDE5I DILI include a relatively short latency, cholestatic histological features, and complete recovery. Biochemical pattern of liver injury is variable. Conclusions: PDE5I DILI is a rare event that can result in severe acute liver injury.
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Purpose: Gene fusions involving receptor tyrosine kinases (RTKs) define an important class of genomic alterations with many successful targeted therapies now approved for ALK, ROS1, RET and NTRK gene fusions. Fusions involving the ERBB family of RTKs have been sporadically reported, but their frequency has not yet been comprehensively analyzed and functional characterization is lacking on many types of ERBB fusions. Materials and methods: We analyzed tumor samples submitted to Caris Life Sciences (n=64,354), as well as the TCGA (n=10,967), MSK IMPACT (n=10,945) and AACR GENIE (n=96,324) databases for evidence of EGFR, ERBB2 and ERBB4 gene fusions. We also expressed several novel fusions in cancer cell lines and analyzed their response to EGFR and HER2 tyrosine kinase inhibitors (TKIs). Results: In total, we identified 1,251 ERBB family fusions, representing an incidence of approximately 0.7% across all cancer types. EGFR, ERBB2, and ERBB4 fusions were most frequently found in glioblastoma, breast cancer and ovarian cancer, respectively. We modeled two novel types of EGFR and ERBB2 fusions, one with a tethered kinase domain and the other with a tethered adapter protein. Specifically, we expressed EGFR-ERBB4, EGFR-SHC1, ERBB2-GRB7 and ERBB2-SHC1, in cancer cell lines and demonstrated that they are oncogenic, regulate downstream signaling and are sensitive to small molecule inhibition with EGFR and HER2 TKIs. Conclusions: We found that ERBB fusions are recurrent mutations that occur across multiple cancer types. We also establish that adapter-tethered and kinase-tethered fusions are oncogenic and can be inhibited with EGFR or HER2 inhibitors. We further propose a nomenclature system to categorize these fusions into several functional classes.
ABSTRACT
BACKGROUND: Mifepristone, also known as RU-486, is an anti-progestational steroid with similar chemical structure to anabolic steroids. Given as a single dose in conjunction with misoprostol, mifepristone is used to induce medical abortion. Mifepristone administered chronically at a higher dose is also approved for the management of hypercortisolism. There have been only 2 reported cases of mifepristone associated liver injury, in both cases, in the setting of Cushing syndrome. We report a third patient with Cushing syndrome with mifepristone induced liver injury with unique histological findings that provide insight to the pathophysiology of liver injury in mifepristone and anabolic steroids. CASE PRESENTATION: Patient is a 63-year-old Caucasian female Cushing disease with no prior history of liver disease. She was started on mifepristone and semaglutide. Ninety days after initiating mifepristone, she developed deep jaundice, severe pruritus, fatigue, and nausea. Liver tests revealed a mixed hepatocellular/cholestatic pattern. Viral and autoimmune serologies were negative and there was no biliary dilatation on imaging. Liver biopsy showed severe cholestasis but no bile duct injury. Focal endothelialitis was present within a central venule. Cholestatic symptoms persisted for one month after presentation before slowly subsiding. Four months after stopping mifepristone, the patient's symptoms completely resolved, and liver tests became normal. Compilation of Roussell Uclaf Causality Assessment Method score indicated probable causality. CONCLUSIONS: Mifepristone shares a similar chemical structure as synthetic anabolic/androgenic steroids and there are many similarities in the clinical presentation of liver injury. This case and the 2 other reported cases share similar clinical characteristics. The observation of endothelialitis in our patient may provide a mechanistic link between mifepristone, or anabolic steroids in general, and the development of vascular complications such as peliosis.
Subject(s)
Abortion, Induced , Chemical and Drug Induced Liver Injury, Chronic , Chemical and Drug Induced Liver Injury , Cholestasis , Cushing Syndrome , Pregnancy , Female , Humans , Middle Aged , Mifepristone/adverse effects , Cushing Syndrome/chemically induced , Abortion, Induced/methods , Chemical and Drug Induced Liver Injury/etiologyABSTRACT
Background: Reversion mutations in BRCA1/2, resulting in restoration of the open reading frame, have been identified as a mechanism of resistance to platinum-based chemotherapy or PARP inhibition. We sought to explore the incidence of BRCA1/2 reversion mutations in different tumor types. Methods: We retrospectively analyzed molecular profiling results from primary and/or metastatic tumor samples submitted by multiple institutions. The samples underwent DNA and RNA sequencing at a CLIA/CAP-certified clinical lab. Reversion mutations were called only in patients whose available clinical records showed the use of PARP inhibitors or platinum agents prior to tumor profiling. Results: Reversion mutations were identified in 75 of 247,926 samples profiled across all tumor types. Among patients carrying pathogenic or likely pathogenic BRCA1/2 mutations, reversion mutations in BRCA1/2 genes were seen in ovarian cancer (OC) (30/3424), breast cancer (BC) (27/1460), endometrial cancer (4/564), pancreatic cancer (2/340), cholangiocarcinoma (2/178), prostate cancer (5/461), cervical cancer (1/117), cancer of unknown primary (1/244), bladder cancer (1/300), malignant pleural mesothelioma (1/10), and a neuroendocrine tumor of the prostate. We identified 22 reversion mutations in BRCA1 and 8 in BRCA2 in OC. In BC, we detected 6 reversion mutations in BRCA1 and 21 in BRCA2. We compared molecular profile results of 14 high-grade serous ovarian cancers (HGSOC) with reversion mutations against 87 control HGSOC with pathogenic BRCA1/2 mutations without reversion mutations. Tumors with reversion mutations trended to have had lower ER expression (25% vs. 64%, p = 0.024, q = 0.82) and higher KDM6A mutation rate (15% vs. 0, p = 0.016, q = 0.82). Conclusions: We present one of the largest datasets reporting reversion mutations in BRCA1/2 genes across various tumor types. These reversion mutations were rare; this may be because some patients may not have had repeat profiling post-treatment. Repeat tumor profiling at times of treatment resistance can help inform therapy selection in the refractory disease setting.
Subject(s)
Antineoplastic Agents , Retrospective Studies , Female , Humans , Male , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , BRCA1 Protein/genetics , Drug Resistance, Neoplasm , Mutation/genetics , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , Poly(ADP-ribose) Polymerase Inhibitors/therapeutic use , Neoplasms/drug therapy , Neoplasms/genetics , BRCA2 Protein/geneticsABSTRACT
Intrahepatic cholangiocarcinoma (iCCA) is a rare and aggressive malignancy that arises from the intrahepatic biliary tree and is associated with a poor prognosis. Until recently, the treatment landscape of advanced/metastatic iCCA has been limited primarily to chemotherapy. In recent years, the advent of biomarker testing has identified actionable genetic alterations in 40%-50% of patients with iCCA, heralding an era of precision medicine for these patients. Biomarker testing using next-generation sequencing (NGS) has since become increasingly relevant in iCCA; however, several challenges and gaps in standard image-guided liver biopsy and processing have been identified. These include variability in tissue acquisition relating to the imaging modality used for biopsy guidance, the biopsy method used, number of passes, needle choice, specimen preparation methods, the desmoplastic nature of the tumor, as well as the lack of communication among the multidisciplinary team. Recognizing these challenges and the lack of evidence-based guidelines for biomarker testing in iCCA, a multidisciplinary team of experts including interventional oncologists, a gastroenterologist, medical oncologists, and pathologists suggest best practices for optimizing tissue collection and biomarker testing in iCCA.
Subject(s)
Bile Duct Neoplasms , Cholangiocarcinoma , Bile Duct Neoplasms/diagnosis , Bile Duct Neoplasms/genetics , Bile Duct Neoplasms/pathology , Bile Ducts, Intrahepatic/pathology , Biomarkers , Cholangiocarcinoma/diagnosis , Cholangiocarcinoma/drug therapy , Cholangiocarcinoma/genetics , Humans , Precision MedicineABSTRACT
PURPOSE: The adoption of precision medicine (PMed) depends on the critical curation of data and interpretation of genomic results. Herein, we sought to study the effect of a coordinated multidisciplinary program to assess results in a community cancer center clinic. METHODS: In a retrospective review from July 2018 to July 2021, we analyzed the implementation of a multidisciplinary PMed program in a tertiary referral community cancer center. Germline genetics test results have been reviewed since 2017. RESULTS: A total of 3,131 tumor samples were analyzed by large panel somatic genomic testing through commercial laboratories during the study period. The number of reviewed cases rose from 661 in the first year to 1,532 in year 3. Additional recommendations beyond what was reported by the commercial laboratory were made in 42.9% of cases. Referrals to the hereditary cancer program for germline testing increased by 32% from the 2017 baseline. Process improvement efforts reduced the rate of DNA quantity nonsufficient for testing to 3.3% compared with a national average of 4.89%. The average time from receipt of orders to issuing of a report of the somatic panel was 15.5 days, compared with 19.1 days for other institutions using the same laboratory. The PMed team has been critical in support of clinical research by assisting in trial procurement and feasibility assessment to the identification of patients for clinical trials. CONCLUSION: The use of somatic genomic testing is increasing at our cancer center. Education and in-depth analysis of the data are valued by cancer physicians. The development and implementation of a PMed program has demonstrated improved physicians' understanding of molecular testing, resulting in improved outcomes for patients.
Subject(s)
Neoplasms , Precision Medicine , Cluster Analysis , Genomics/methods , Humans , Neoplasms/genetics , Retrospective StudiesABSTRACT
Invasive melanoma is the deadliest type of skin cancer, with 101,110 expected cases to be diagnosed in 2021. Recurrent BRAF and NRAS mutations are well documented in melanoma. Biologic implications of gene fusions and the efficacy of therapeutically targeting them remains unknown. Retrospective review of patient samples that underwent next-generation sequencing of the exons of 592 cancer-relevant genes and whole transcriptome sequencing for the detection of gene fusion events and gene expression profiling. Expression of PDL1 and ERK1/2 was assessed by immunohistochemistry (IHC). There were 33 (2.6%) cases with oncogenic fusions (14 novel), involving BRAF, RAF1, PRKCA, TERT, AXL, and FGFR3. MAPK pathway-associated genes were over-expressed in BRAF and RAF1 fusion-positive tumors in absence of other driver alterations. Increased expression in tumors with PRKCA and TERT fusions was concurrent with MAPK pathway alterations. For a subset of samples with available tissue, increased phosphorylation of ERK1/2 was observed in BRAF, RAF1, and PRKCA fusion-positive tumors. Oncogenic gene fusions are associated with transcriptional activation of the MAPK pathway, suggesting they could be therapeutic targets with available inhibitors. Additional analyses to fully characterize the oncogenic effects of these fusions may support biomarker driven clinical trials.
ABSTRACT
Selective IgM deficiency (SIgMD) and isolated collagenous gastritis are two independent rare disorders. Our purpose is to report the 1st case of SIgMD and isolated collagenous gastritis and collagenous gastritis that has transitioned to EBV + gastric adenocarcinoma. Gastric biopsy tissue was analyzed by EBV-related encoded RNA in situ hybridization assay. Subsets of CD4, CD8, T follicular helper cells (TFH), and members of the "regulatory lymphocytes club" were measured with multiple panels of monoclonal antibodies and isotype controls by multicolor flow cytometry. The patient was diagnosed with SIgMD (extremely low serum IgM 9 mg/dl and normal IgG and IgA and exclusion of secondary causes of low IgM). Soon after SIgMD diagnosis, the patient developed collagenous gastritis and, 8 years later, developed gastric adenocarcinoma that was positive for EBV. An extensive immunological analysis revealed reduced naïve CD4 and CD8 effector memory T cells and increased naïve and central memory CD8 T cells. Among the circulating follicular helper T cells (cTFH), TFH1 and TFH2 were increased whereas TFH17 was decreased. CD4 Treg cells and TFR cells were increased, whereas Breg and CD8 Treg were comparable to control. In conclusion, SIgMD may be associated with isolated collagenous gastritis, and collagenous gastritis may transition to EBV + gastric adenocarcinoma. A role of regulatory lymphocytes in gastric cancer is discussed.
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BACKGROUND: Complementary and alternative medicine use among Americans is prevalent. Originating in India, Ayurvedic medicine use in the United States has grown 57% since 2002. CAM accounts for a significant proportion of drug induced liver injury in India and China, but there have been only three reports of drug induced liver injury from Ayurvedic medications in the U.S. We report three cases of suspected Ayurvedic medication associated liver injury seen at a Southern California community hospital and review literature of Ayurvedic medication induced liver injury. CASE PRESENTATIONS: Three patients presented with acute hepatocellular injury and jaundice after taking Ayurvedic supplements for 90-120 days. First patient took Giloy Kwath consisting solely of Tinospora cordifolia. Second patient took Manjishthadi Kwatham and Aragwadhi Kwatham, which contained 52 and 10 individual plant extracts, respectively. Third patient took Kanchnar Guggulu, containing 10 individual plant extracts. Aminotransferase activities decreased 50% in < 30 days and all 3 patients made a full recovery. Roussel Uclaf Causality Assessment Method (RUCAM) scores were 7-8, indicating probable causality. These products all contained ingredients in other Ayurvedic and traditional Chinese medicines with previously reported associations with drug induced liver injury. CONCLUSIONS: These patients highlight the risk of drug induced liver injury from Ayurvedic medications and the complexity of determining causality. There is a need for a platform like LiverTox.gov to catalog Ayurvedic ingredients causing liver damage.
Subject(s)
Chemical and Drug Induced Liver Injury/etiology , Medicine, Ayurvedic/adverse effects , Adult , Aged , Female , Humans , Middle AgedABSTRACT
BACKGROUND: At presentation, 21% to 49% of patients with adrenocortical cancer have metastases. Standard chemotherapy has a 23% response rate. We assessed whether next generation sequencing could elucidate additional treatment options in refractory adrenocortical cancer. METHODS: Retrospective analysis using a commercial, 592-gene DNA-based panel was performed of next generation sequencing data from 94 adrenocortical cancer tumors profiled for clinical care. We compared our data to the adrenocortical cancer database of The Cancer Genome Atlas containing survival data. We evaluated mutations, indels, amplifications, tumor mutation burden, microsatellite instability, and programmed death-ligand 1 protein expression. RESULTS: Our cohort included 54 primary neoplasms and 40 metastatic lesions. The most frequently mutated genes were TP53 (36%) and CTNNB1 (19%). Low prevalence mutations were noted in 37 genes including DNA damage repair genes in 15 samples. High tumor mutation burden was seen in 3 patients, and programmed death-ligand 1 was positive in 12. Potential targets to Food and Drug Administration-approved drugs were seen in 16% of cases. CONCLUSION: DNA sequencing panel tests may identify therapeutic options for some patients with adrenocortical cancer. TP53 and mutations were associated with an adverse outcome. An expanded repertoire of drugs and, perhaps, more expansive multi-omic sequencing are needed to advance the treatment of adrenocortical cancer.
Subject(s)
Adrenal Cortex Neoplasms/genetics , Antineoplastic Agents/therapeutic use , Biomarkers, Tumor/genetics , Adolescent , Adrenal Cortex/pathology , Adrenal Cortex Neoplasms/drug therapy , Adrenal Cortex Neoplasms/pathology , Adult , Aged , Antineoplastic Agents/pharmacology , Biomarkers, Tumor/antagonists & inhibitors , Child , DNA Repair/genetics , Datasets as Topic , Drug Resistance, Neoplasm/drug effects , Drug Resistance, Neoplasm/genetics , Female , Gene Amplification , Genomics , High-Throughput Nucleotide Sequencing , Humans , Male , Middle Aged , Mutation , Precision Medicine/methods , Retrospective Studies , Sequence Analysis, DNA , Tumor Suppressor Protein p53/genetics , Young Adult , beta Catenin/geneticsSubject(s)
Enterocolitis/diagnostic imaging , Inflammatory Bowel Diseases/diagnostic imaging , Lymphocytes , Phlebitis/diagnostic imaging , Aged , Enterocolitis/complications , Enterocolitis/surgery , Humans , Inflammatory Bowel Diseases/complications , Inflammatory Bowel Diseases/surgery , Male , Phlebitis/complications , Phlebitis/surgeryABSTRACT
Advances in immuno-oncology over the last several years have led to FDA approvals of novel agents. As our understanding of immune response and its checkpoints has evolved, further advances have been made in treatment for several cancer types. To predict a response to immunotherapy, the initial biomarkers used were expression of the PD-1 receptor and PD-L1, as assessed by immunohistochemistry. More recently, predictive biomarkers have included microsatellite instability, DNA mismatch repair, and tumor mutational burden. Although these markers may be clinically relevant in predicting an immunotherapy response, cancer immunotherapy fails some patients. Improved understanding of the human immune system is necessary, as is a careful evaluation of the methods used to predict and assess response to immuno-oncology treatments. With the application of therapeutic immune-modulating agents, more comprehensive assays, and associated bioinformatics tools to accurately assess the tumor microenvironment, we may better predict responses to immuno-oncology agents and the ever-increasing complexity of their clinical use.
Subject(s)
Antineoplastic Agents, Immunological/therapeutic use , B7-H1 Antigen/antagonists & inhibitors , Biomarkers, Tumor/immunology , Immunotherapy/methods , Neoplasms/drug therapy , Neoplasms/immunology , Programmed Cell Death 1 Receptor/antagonists & inhibitors , B7-H1 Antigen/immunology , B7-H1 Antigen/metabolism , Biomarkers, Tumor/genetics , Biomarkers, Tumor/metabolism , Humans , Microsatellite Instability , Mutation , Neoplasms/metabolism , Neoplasms/pathology , Programmed Cell Death 1 Receptor/immunology , Programmed Cell Death 1 Receptor/metabolismABSTRACT
The Sialyl Lewis A antigen, or CA 19-9, is the prototype serum biomarker for adenocarcinoma of the pancreas. Despite extensive clinical study of CA 19-9 in gastrointestinal malignancies, surprisingly little is known concerning the specific cell types that express this marker during development, tissue regeneration and neoplasia. SOX9 is a transcription factor that plays a key role in these processes in foregut tissues. We report the biochemistry and tissue expression of the GCTM-5 antigen, a pancreatic cancer marker related to, but distinct from, CA19-9. This antigen, defined by two monoclonal antibodies recognising separate epitopes on a large glycoconjugate protein complex, is co-expressed with SOX9 by foregut ductal progenitors in the developing human liver and pancreas, and in pancreatic adenocarcinoma. These progenitors are distinct from cell populations identified by DCLK1, LGR5, or canonical markers of liver and pancreatic progenitor cells. Co-expression of this antigen complex and SOX9 also characterises the ductal metaplasia of submucosal glands that occurs during the development of Barrett's oesophagus. The GCTM-5 antigen complex can be detected in the sera of patients with pancreatic adenocarcinoma. The GCTM-5 epitope shows a much more restricted pattern of expression in the normal adult pancreas relative to CA19-9. Our findings will aid in the identification, characterisation, and monitoring of ductal progenitor cells during development and progression of pancreatic adenocarcinoma in man.
Subject(s)
Adenocarcinoma/metabolism , Antibodies, Neoplasm/chemistry , CA-19-9 Antigen/metabolism , Fetus/metabolism , Neoplasm Proteins/metabolism , Neoplastic Stem Cells/metabolism , Pancreas/metabolism , Pancreatic Neoplasms/metabolism , SOX9 Transcription Factor/metabolism , Adenocarcinoma/pathology , Cell Line , Fetus/pathology , Gene Expression Regulation, Neoplastic , Humans , Neoplastic Stem Cells/pathology , Pancreas/embryology , Pancreas/pathology , Pancreatic Neoplasms/pathologySubject(s)
Duodenal Neoplasms , Trastuzumab , Humans , Immunotherapy, Adoptive , Leucovorin , Neoadjuvant Therapy , Receptor, ErbB-2ABSTRACT
Only a single case report exists in the literature of hepatic adenocarcinoma expressing InhibinA in a young woman, in which the authors proposed it to be a rare variant of intrahepatic cholangiocarcinoma (iCCA). We present novel molecular and histologic findings in our series of three cases occurring in young women and show these tumors may mimic well-differentiated neuroendocrine tumors (NET). Immunohistochemical (IHC) profiling was performed along with a next-generation sequencing (NGS) 47-gene solid tumor panel, and cytogenomic profiling via single-nucleotide polypmorphism microarray. IHC for inhibinA, chromogranin A (ChrA), and synaptophysin (Syn) was surveyed in liver tumors and in fetal liver. Two of the three patients recurred with metastatic disease with two confirmed deaths. Histological patterns present in the tumors included solid, trabecular, organoid, microcystic, and blastemal-like. IHC was positive for cytokeratin 7 in 3/3, cytokeratin 19 in 3/3, inhibinA in 3/3, ChrA in 3/3, Syn in 3/3, Sox9 in 2/3 and HepPar1 in 0/3. NGS was negative for pathogenic mutations. Recurrent cytogenomic abnormalities included gain of 17q, and loss of 6q. InhibinA was strong and diffusely expressed in 0/10 (0%) iCCA, 0/15 (0%) hepatocellular carcinomas (HCC), in the biliary component of 1/4 (25%) combined HCC-iCCA, 0/4 hepatoblastomas, 1/8 (13%) metastatic NET, and in 1/8 fetal liver tissues. We propose a classification of "cholangioblastic variant of intrahepatic cholangiocarcinoma" and molecular pathogenesis for this rare malignancy. Accurate identification on core biopsy is crucial for clinical management as it may mimic neuroendocrine neoplasms.
Subject(s)
Adenocarcinoma/diagnosis , Bile Duct Neoplasms/diagnosis , Biomarkers, Tumor/analysis , Biomarkers, Tumor/genetics , Cholangiocarcinoma/diagnosis , Cytogenetic Analysis , Inhibins/analysis , Liver Neoplasms/diagnosis , Molecular Diagnostic Techniques , Neuroendocrine Tumors/diagnosis , Adenocarcinoma/chemistry , Adenocarcinoma/genetics , Adenocarcinoma/secondary , Adolescent , Adult , Bile Duct Neoplasms/chemistry , Bile Duct Neoplasms/genetics , Bile Duct Neoplasms/pathology , Cholangiocarcinoma/chemistry , Cholangiocarcinoma/genetics , Cholangiocarcinoma/pathology , Diagnosis, Differential , Fatal Outcome , Female , Gene Expression Profiling , Genetic Predisposition to Disease , Humans , Immunohistochemistry , Liver Neoplasms/chemistry , Liver Neoplasms/genetics , Liver Neoplasms/pathology , Neuroendocrine Tumors/chemistry , Neuroendocrine Tumors/genetics , Neuroendocrine Tumors/pathology , Phenotype , Polymorphism, Single Nucleotide , Predictive Value of Tests , Terminology as Topic , Treatment OutcomeABSTRACT
Applications of precision oncology strategies rely on accurate tumor genotyping from clinically available specimens. Fine needle aspirations (FNA) are frequently obtained in cancer management and often represent the only source of tumor tissues for patients with metastatic or locally advanced diseases. However, FNAs obtained from pancreas ductal adenocarcinoma (PDAC) are often limited in cellularity and/or tumor cell purity, precluding accurate tumor genotyping in many cases. Digital PCR (dPCR) is a technology with exceptional sensitivity and low DNA template requirement, characteristics that are necessary for analyzing PDAC FNA samples. In the current study, we sought to evaluate dPCR as a mutation analysis tool for pancreas FNA specimens. To this end, we analyzed alterations in the KRAS gene in pancreas FNAs using dPCR. The sensitivity of dPCR mutation analysis was first determined using serial dilution cell spiking studies. Single-cell laser-microdissection (LMD) was then utilized to identify the minimal number of tumor cells needed for mutation detection. Lastly, dPCR mutation analysis was performed on 44 pancreas FNAs (34 formalin-fixed paraffin-embedded (FFPE) and 10 fresh (non-fixed)), including samples highly limited in cellularity (100 cells) and tumor cell purity (1%). We found dPCR to detect mutations with allele frequencies as low as 0.17%. Additionally, a single tumor cell could be detected within an abundance of normal cells. Using clinical FNA samples, dPCR mutation analysis was successful in all preoperative FNA biopsies tested, and its accuracy was confirmed via comparison with resected tumor specimens. Moreover, dPCR revealed additional KRAS mutations representing minor subclones within a tumor that were not detected by the current clinical gold standard method of Sanger sequencing. In conclusion, dPCR performs sensitive and accurate mutation analysis in pancreas FNAs, detecting not only the dominant mutation subtype, but also the additional rare mutation subtypes representing tumor heterogeneity.
Subject(s)
Carcinoma, Pancreatic Ductal/genetics , DNA Mutational Analysis/methods , Mutation , Pancreas/metabolism , Pancreatic Neoplasms/genetics , Polymerase Chain Reaction/methods , Proto-Oncogene Proteins p21(ras)/genetics , Biopsy, Fine-Needle , Carcinoma, Pancreatic Ductal/metabolism , Carcinoma, Pancreatic Ductal/pathology , Cell Line, Tumor , Genotype , Humans , Pancreas/pathology , Pancreatic Neoplasms/metabolism , Pancreatic Neoplasms/pathology , Sensitivity and SpecificityABSTRACT
Determining gastrointestinal tract dysplasia level is clinically important but can be difficult, and given this challenge, we investigated colonic and esophageal dysplastic progression using digital image analysis (IA). Whole slide images were obtained for colonic normal mucosa (NCM), hyperplastic polyps (HP), conventional tubular adenomas (TA), and adenomas with high-grade dysplasia (HGD), and esophageal intestinal metaplasia negative for dysplasia (IM), indefinite for dysplasia (IFD), low-grade dysplasia (LGD), and HGD. Characteristic nuclei were circumscribed, and parameters discriminating groups included nuclear circumference (µm), area (µm(2)), and 15 positive pixel count (PPC) algorithm IA measurements. In colon polyps and esophageal lesions, average nuclear area and circumference ranged 30-108.6 µm(2) and 27.5-48.9 µm, respectively. Differences for average nuclear area and circumference met statistical significance (p < 0.05) between diagnostic groups in the esophagus and colon, except for IM versus IFD nuclear area. Pixel intensity (brightness) separated lesions within both groups with statistical significance except for colonic TAs versus HPs and esophageal LGD versus IM. HGD nuclei in both groups demonstrated more pixel staining heterogeneity than other lesions. Hierarchical clustering and principal component analysis demonstrated that lesions with similar diagnoses tended to cluster together on a low- to high-grade spectrum. Our results confirm that quantitative IA is an effective adjunct reflecting dysplasia in colon polyps and Barrett esophagus lesions. Nuclear area, circumference, and PPC algorithm findings distinguished lesions in a statistically significant manner. This suggests utility for future studies on similar methods, which may provide an adjunctive ancillary technique for pathologists and enhance patient care.
Subject(s)
Barrett Esophagus/pathology , Esophageal Neoplasms/pathology , Hyperplasia/pathology , Intestinal Neoplasms/pathology , Intestines/pathology , Metaplasia/pathology , Adenocarcinoma/pathology , Adult , Aged , Barrett Esophagus/diagnosis , Colon/pathology , Colonic Polyps/pathology , Esophageal Neoplasms/diagnosis , Humans , Middle AgedABSTRACT
Cancer somatic genetic evolution is a direct contributor to heterogeneity at the clonal and molecular level in colorectal adenocarcinoma (COAD). We sought to determine the extent to which genetic evolution may be detected in COAD in routinely obtained single clinical specimens and establish clinical significance with regard to clinicopathologic and outcome data. One hundred and twenty three cases of routinely collected mismatch repair proficient COAD were sequenced on the Illumina Truseq Amplicon assay. Measures of intratumoral heterogeneity and the preferential timing of mutational events were assessed and compared to clinicopathologic data. Survival subanalysis was performed on 55 patients. Patient age (p = 0.013) and specimen percent tumor (p = 0.033) was associated with clonal diversity, and biopsy (p = 0.044) and metastasis (p = 0.044) returned fewer mutations per case. APC and TP53 mutations preferentially occurred early while alterations in FBXW7, FLT3, SMAD4, GNAS and PTEN preferentially occurred as late events. Temporal heterogeneity was evident in KRAS and PIK3CA mutations. Hierarchical clustering revealed a TP53 mutant subtype and a MAPK-PIK3CA subtype with differing patterns of late mutational events. Survival subanalysis showed a decreased median progression free survival for the MAPK-PIK3CA subtype (8 months vs. 13 months; univariate logrank p = 0.0380, cox model p= 0.018). Neoadjuvant therapy associated mutations were found for ERBB2 (p = 0.0481) and FBXW7 (p = 0.015). Our data indicate novel molecular subtypes of mismatch repair proficient COAD display differing patterns of genetic evolution which correlate with clinical outcomes. Furthermore, we report treatment acquired and/or selected mutations in ERBB2 and FBXW7.
Subject(s)
Adenocarcinoma/genetics , Adenocarcinoma/pathology , Colorectal Neoplasms/genetics , Colorectal Neoplasms/pathology , DNA Mismatch Repair , Cell Cycle Proteins/genetics , Cluster Analysis , Evolution, Molecular , F-Box Proteins/genetics , F-Box-WD Repeat-Containing Protein 7 , Female , Gene Frequency , Humans , Male , Middle Aged , Mutation , Neoplasm Grading , Neoplasm Staging , Prognosis , Receptor, ErbB-2/genetics , Ubiquitin-Protein Ligases/geneticsABSTRACT
BACKGROUND: GATA3 as a diagnostic marker of metastatic breast carcinoma in cytology specimens has not been fully established. METHODS: Metastatic breast carcinoma was assessed for GATA3, mammaglobin, and GCDFP-15 immunohistochemistry on cell blocks. GATA3 was scored by intensity (0, negative; 1, weakly positive; 2, moderately positive; 3, strongly positive), and area (0-100%). Mammaglobin (MMG) and GCDFP-15 staining was scored qualitatively (positive vs. negative). Results were correlated with specimen type (fine-needle aspiration vs. body fluid), breast prognostic markers estrogen receptor (ER), progesterone receptor (PR), Her-2/Neu (Her2), and Ki67, and with each other. Statistical significance was determined by chi-squared test and ANOVA for numerical variables. Alpha was set as 0.05. A total of 40 CB specimens containing metastatic breast carcinoma were studied. RESULTS: GATA3 was positive in 32 (80%) cases. All ER-positive cases (n = 25) were positive for GATA3. Conversely, all GATA3-negative cases (n = 8) were triple-negative breast cancers. On qualitative univariate analysis, GATA3 was statistically associated with ER (P = 0.0001), and PR (P = 0.0468). GATA3 intensity was statistically associated with ER (P ≤ 0.0001), PR (P = 0.0157), Her2 (P = 0.0256), and cancer category (P = 0.0127). GATA3 staining was statistically associated with ER (P ≤ 0.0001), PR (P = 0.0160), Her2 (P = 0.0451), and cancer category (P = 0.0002). MMG and GCDFP-15 were directly compared to GATA3 in 35 samples. The sensitivity was 86% for GATA3, 26% for MMG, and 14% for GCDFP-15. CONCLUSION: GATA3 is a more sensitive diagnostic marker of metastatic breast carcinoma in CB samples than MMG and GCDFP-15.