Publications by authors named "James R Eshleman"

169 Publications

Utility of Targeted Next-Generation Sequencing Assay to Detect 1p/19q Co-Deletion in Formalin-fixed Paraffin-embedded Glioma Specimens.

Hum Pathol 2022 May 10. Epub 2022 May 10.

Johns Hopkins Genomics; Department of Pathology, Johns Hopkins University School of Medicine; Cytogenetics Laboratory, Johns Hopkins University Hospital, Baltimore, MD 21205. Electronic address:

Molecular classification of brain neoplasms is important for diagnosis, prognosis, and treatment outcome of histologically similar tumors. Oligodendroglioma is a glioma subtype characterized by 1p/19q co-deletion and IDH1/IDH2 mutations, which predicts a good prognosis, responsiveness to therapy and an improved overall survival compared to other adult gliomas. In a routine clinical setting, 1p/19q co-deletion is detected by interphase-FISH and SNP microarray, and somatic mutations are detected by targeted next generation sequencing (NGS). The aim of this proof-of-principle study was to investigate the feasibility of using targeted NGS to simultaneously detect both 1p/19q co-deletion and somatic mutations. Among two hundred forty-seven consecutive patients with formalin-fixed paraffin-embedded brain tumors with various subtypes, NGS revealed 1p/19q co-deletion in twenty-six oligodendrogliomas and an IDH-wildtype astrocytoma, and partial loss across chromosomes 1p and 19q/whole-arm loss of 1p or 19q/copy neutral loss of heterozygosity in eleven non-oligodendrogliomas. For this 247 brain-tumor cohort, the overall sensitivity, specificity, and accuracy of detecting 1p/19q co-deletion by NGS in oligodendrogliomas were 96.2%, 99.6%, and 99.2%, respectively. The oligodendroglioma cohort had more mutations in IDH1/IDH2, CIC, FUBP1, and TERT, and fewer mutations in ATRX and TP53 than the non-oligodendroglioma cohort. This proof-of-concept study demonstrated that targeted NGS can simultaneously detect both 1p/19q co-deletion and somatic mutations, which can provide a more comprehensive genetic profiling for patients with gliomas using a single assay in a clinical setting.
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http://dx.doi.org/10.1016/j.humpath.2022.05.001DOI Listing
May 2022

Artificial intelligence-assisted serial analysis of clinical cancer genomics data identifies changing treatment recommendations and therapeutic targets.

Clin Cancer Res 2022 Mar 21. Epub 2022 Mar 21.

Johns Hopkins Medical Institutions, Baltimore, MD, United States.

Purpose: Given the pace of predictive biomarker and targeted therapy development, it is unknown if repeat annotation of the same next-generation sequencing data can identify additional clinically actionable targets that could be therapeutically leveraged. In this study, we sought to determine the predictive yield of serial re-analysis of clinical tumor sequencing data.

Experimental Design: Using artificial intelligence (AI)-assisted variant annotation, we retrospectively re-analyzed sequencing data from 2,219 cancer patients from a single academic medical center at 3-month intervals totaling 9 months in 2020. The yield of serial re-analysis was assessed by the proportion of patients with improved strength of therapeutic recommendations.

Results: 1,775 patients (80%) had {greater than or equal to}1 potentially clinically actionable mutation at baseline, including 243 (11%) patients who had an alteration targeted by an FDA-approved drug for their cancer type. By month nine, the latter increased to 458 (21%) patients mainly due to a single pan-cancer agent directed against tumors with high tumor mutation burden. Within this timeframe, 67 new therapies became available and 45 were no longer available. Variant pathogenicity classifications also changed leading to changes in treatment recommendations for 124 patients (6%).

Conclusions: Serial re-annotation of tumor sequencing data improved the strength of treatment recommendations (based on level of evidence) in a mixed cancer cohort and showed substantial changes in available therapies and variant classifications. These results suggest a role for repeat analysis of tumor sequencing data in clinical practice, which can be streamlined with AI support.
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http://dx.doi.org/10.1158/1078-0432.CCR-21-4061DOI Listing
March 2022

Functional CDKN2A assay identifies frequent deleterious alleles misclassified as variants of uncertain significance.

Elife 2022 01 10;11. Epub 2022 Jan 10.

The Sol Goldman Pancreatic Cancer Research Center, Department of Pathology, Johns Hopkins University, Baltimore, United States.

Pathogenic germline variants are associated with an increased risk of pancreatic ductal adenocarcinoma (PDAC). variants of uncertain significance (VUSs) are reported in up to 4.3% of patients with PDAC and result in significant uncertainty for patients and their family members as an unknown fraction are functionally deleterious, and therefore, likely pathogenic. Functional characterization of VUSs is needed to reclassify variants and inform clinical management. Twenty-nine germline VUSs previously reported in patients with PDAC or in ClinVar were evaluated using a validated in vitro cell proliferation assay. Twelve of the 29 VUSs were functionally deleterious (11 VUSs) or potentially functionally deleterious (1 VUS) and were reclassified as likely pathogenic variants. Thus, over 40% of VUSs identified in patients with PDAC are functionally deleterious and likely pathogenic. When incorporating VUSs found to be functionally deleterious, and reclassified as likely pathogenic, the prevalence of pathogenic/likely pathogenic in patients with PDAC reported in the published literature is increased to up to 4.1% of patients, depending on family history. Therefore, VUSs may play a significant, unappreciated role in risk of pancreatic cancer. These findings have significant implications for the counselling and care of patients and their relatives.
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http://dx.doi.org/10.7554/eLife.71137DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8824478PMC
January 2022

A Cost-Effective and Non-Invasive pfeRNA-Based Test Differentiates Benign and Suspicious Pulmonary Nodules from Malignant Ones.

Noncoding RNA 2021 Dec 16;7(4). Epub 2021 Dec 16.

The Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins School of Medicine, 1650 Orleans Street, Baltimore, MD 21287, USA.

The ability to differentiate between benign, suspicious, and malignant pulmonary nodules is imperative for definitive intervention in patients with early stage lung cancers. Here, we report that plasma protein functional effector sncRNAs (pfeRNAs) serve as non-invasive biomarkers for determining both the existence and the nature of pulmonary nodules in a three-stage study that included the healthy group, patients with benign pulmonary nodules, patients with suspicious nodules, and patients with malignant nodules. Following the standards required for a clinical laboratory improvement amendments (CLIA)-compliant laboratory-developed test (LDT), we identified a pfeRNA classifier containing 8 pfeRNAs in 108 biospecimens from 60 patients by sncRNA deep sequencing, deduced prediction rules using a separate training cohort of 198 plasma specimens, and then applied the prediction rules to another 230 plasma specimens in an independent validation cohort. The pfeRNA classifier could (1) differentiate patients with or without pulmonary nodules with an average sensitivity and specificity of 96.2% and 97.35% and (2) differentiate malignant versus benign pulmonary nodules with an average sensitivity and specificity of 77.1% and 74.25%. Our biomarkers are cost-effective, non-invasive, sensitive, and specific, and the qPCR-based method provides the possibility for automatic testing of robotic applications.
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http://dx.doi.org/10.3390/ncrna7040080DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8709422PMC
December 2021

Validation of Long Mononucleotide Repeat Markers for Detection of Microsatellite Instability.

J Mol Diagn 2022 02 2;24(2):144-157. Epub 2021 Dec 2.

The Sol Goldman Pancreatic Cancer Research Center, Department of Pathology, The Johns Hopkins University School of Medicine, Baltimore, Maryland; Department of Oncology, The Johns Hopkins University School of Medicine, Baltimore, Maryland. Electronic address:

Mismatch repair deficiency (dMMR) predicts response to immune checkpoint inhibitor therapy in solid tumors. Long mononucleotide repeat (LMR) markers may improve the interpretation of microsatellite instability (MSI) assays. Our cohorts included mismatch repair (MMR) proficient and dMMR colorectal cancer (CRC) samples, MMR proficient and dMMR endometrial cancer (EC) samples, dMMR prostate cancer samples, MSI-high (MSI-H) samples of other cancer types, and MSI-low (MSI-L) samples of various cancer types. MMR status was determined by immunohistochemical staining and/or MSI Analysis System Version 1.2 (V1.2). The sensitivity and specificity of the LMR MSI panel for dMMR detection were both 100% in CRC. The sensitivity values of the MSI V1.2 and LMR MSI panels in EC were 88% and 98%, respectively, and the specificity values were both 100%. The sensitivity of the LMR panel was 75% in dMMR prostate cancer detected by immunohistochemistry. The 22 samples of other cancer types that were previously classified as MSI-H were also classified as MSI-H using the LMR MSI panel. For the 12 samples that were previously classified as MSI-L, 1 sample was classified as microsatellite stable using the LMR MSI panel, 8 as MSI-L, and 3 as MSI-H. The LMR MSI panel showed high concordance to the MSI V1.2 panel in CRC and greater sensitivity in EC. The LMR MSI panel improves dMMR detection in noncolorectal cancers.
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http://dx.doi.org/10.1016/j.jmoldx.2021.10.011DOI Listing
February 2022

Adult Granulosa Cell Tumor With Sarcomatous Transformation: A Case Study With Emphasis on Molecular Alterations.

Int J Gynecol Pathol 2021 Dec 3. Epub 2021 Dec 3.

Departments of Pathology (W.D., V.P., J.R.E., R.V., B.M.R., D.X.) Oncology (J.H., C.F.M., S.L.W., J.R.E., D.X.) Gynecology and Obstetrics (S.L.W., R.V., B.M.R., D.X.), The Johns Hopkins Medical Institutions, Baltimore, Maryland.

Adult granulosa cells tumors (AGCTs) are typically low-grade indolent tumors. On rare occasions, they undergo high-grade/sarcomatous transformation and behave aggressively. This transformation is postulated to occur as the result of acquired genetic alterations, some of which may be eligible for targeted therapy. Here we report a rare case of AGCT with sarcomatous transformation that harbored distinct molecular alterations from those typically seen with AGCTs supporting a molecularly driven approach to these malignancies. The patient is a 56-yr-old G3P3 woman with a history of multiple recurrences of ovarian AGCT for which the first diagnosis was made at the age of 25 when she was evaluated for infertility. The ovarian tumor displayed typical features of AGCT with low-grade, bland morphology. The first extraovarian spread of tumor involving the cul-de-sac was reported at the age of 39. After that, recurrences occurred every 2 to 3 yr with involvement of multiple anatomic sites and repeated surgical resections. At the age of 55 she developed a symptomatic recurrence in the pelvis and underwent resection of an isolated lesion (specimen 1) to no gross residual disease. Within 4 wk of resection she developed significant pelvic pain and imaging showed recurrence of the mass. Therefore, in 5 mo after the initial resection she underwent repeat excision of the lesion (specimen 2) and associated bowel. The sections from specimen 1 showed a biphasic morphology: a low-grade component with morphology and immunophenotype consistent with a typical AGCT and a high-grade spindle cell component with features consistent with a high-grade sarcoma. Specimen 2 featured a pure high-grade sarcoma characterized by coagulative tumor cell necrosis, readily recognizable mitoses, highly atypical cells with vesicular nuclei and prominent nucleoli. SF-1 positivity and the presence of FOXL2 C134W mutation in the sarcomatous component support the notion of transformation of typical AGCT. While detected TERT promoter C228T mutation may play a role in this process, we further identified genetic alterations affecting PI3K/AKT/mTOR pathway, including mutations in PIK3CA, PIK3R1, AKT1, and NF2, which may also contribute to tumor progression/transformation. These findings provide rationale for molecular/pathway-based targeted therapy for patients with advanced AGCT.
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http://dx.doi.org/10.1097/PGP.0000000000000845DOI Listing
December 2021

Double PIK3CA Alterations and Parallel Evolution in Colorectal Cancers.

Am J Clin Pathol 2022 Feb;157(2):244-251

Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, MD, USA.

Objectives: To demonstrate clinicopathologic features and evaluate the clonality of double PIK3CA alterations in colorectal cancers (CRCs).

Methods: Clonality was examined in 13 CRCs with double PIK3CA alterations (1.7% of CRCs or 9.6% of PIK3CA-mutated CRCs). Multiregional analyses were performed to confirm subclonal PIK3CA alterations.

Results: PIK3CA alterations were detected within exon 9 (51%), exon 20 (23%), exon 1 (15%), and exon 7 (6.0%). CRCs with exon 7 alterations showed a significantly higher incidence of double PIK3CA alterations. Most double PIK3CA alterations consisted of a hotpsot alteration and an uncommon alteration; they were often clonal and present within a single tumor population. Multiregional analyses of CRCs with predicted subclonal double-alterations revealed multiclonal CRCs with divergent PIK3CA variant status originating from a common APC- and KRAS-mutated founder lineage of adenoma.

Conclusions: The findings supported multiclonal CRCs resulting from parallel evolution during the progression from adenoma to adenocarcinoma within the mitogen-activated protein kinase pathway, as previously demonstrated, or the mammalian target of rapamycin pathway. Further studies are warranted to elucidate clinical significance and potential targeted therapy for CRC patients with double PIK3CA alterations and impacts on clinical decision-making in patients with multiclonal CRCs harboring divergent PIK3CA mutational status.
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http://dx.doi.org/10.1093/ajcp/aqab119DOI Listing
February 2022

CloneRetriever: An Automated Algorithm to Identify Clonal B and T Cell Gene Rearrangements by Next-Generation Sequencing for the Diagnosis of Lymphoid Malignancies.

Clin Chem 2021 Nov;67(11):1524-1533

Department of Pathology, Johns Hopkins School of Medicine, Baltimore, MD.

Background: Clonal immunoglobulin and T-cell receptor rearrangements serve as tumor-specific markers that have become mainstays of the diagnosis and monitoring of lymphoid malignancy. Next-generation sequencing (NGS) techniques targeting these loci have been successfully applied to lymphoblastic leukemia and multiple myeloma for minimal residual disease detection. However, adoption of NGS for primary diagnosis remains limited.

Methods: We addressed the bioinformatics challenges associated with immune cell sequencing and clone detection by designing a novel web tool, CloneRetriever (CR), which uses machine-learning principles to generate clone classification schemes that are customizable, and can be applied to large datasets. CR has 2 applications-a "validation" mode to derive a clonality classifier, and a "live" mode to screen for clones by applying a validated and/or customized classifier. In this study, CR-generated multiple classifiers using 2 datasets comprising 106 annotated patient samples. A custom classifier was then applied to 36 unannotated samples.

Results: The optimal classifier for clonality required clonal dominance ≥4.5× above background, read representation ≥8% of all reads, and technical replicate agreement. Depending on the dataset and analysis step, the optimal algorithm yielded sensitivities of 81%-90%, specificities of 97%-100%, areas under the curve of 91%-94%, positive predictive values of 92-100%, and negative predictive values of 88%-98%. Customization of the algorithms yielded 95%-100% concordance with gold-standard clonality determination, including rescue of indeterminate samples. Application to a set of unknowns showed concordance rates of 83%-96%.

Conclusions: CR is an out-of-the-box ready and user-friendly software designed to identify clonal rearrangements in large NGS datasets for the diagnosis of lymphoid malignancies.
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http://dx.doi.org/10.1093/clinchem/hvab141DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8965457PMC
November 2021

Heat Shock Protein 90 Inhibitor Effects on Pancreatic Cancer Cell Cultures.

Pancreas 2021 04;50(4):625-632

Departments of Pathology and Oncology, Johns Hopkins University School of Medicine, Baltimore, MD.

Objectives: Pancreatic ductal adenocarcinoma is one of the deadliest cancers for which few curative therapies are available to date. Heat shock protein 90 (Hsp90) inhibitors have shown activity against numerous cancers in vitro; therefore, we tested whether they could be used to target pancreatic ductal adenocarcinoma.

Methods: Inhibitors of Hsp90 ATPase activity were applied on low-passage pancreatic cell line cultures (Panc10.05, Panc215, A6L) in a dose-response manner, and the inhibitor in vitro effect on cell growth was evaluated. Seven of novel Hsp90 inhibitors based on resorcinol fragment and 5 commercially available Hsp90 inhibitors (17-AAG, AT-13387, AUY-922, ganetespib, and rifabutin) as well as control compound triptolide were tested yielding IC50 values in 2- and 3-dimensional assays.

Results: The novel Hsp90 inhibitors exhibited strong effects on all 3 tested pancreatic cell line cultures (Panc10.05, Panc215, A6L) reaching the IC50 of 300 to 600 nM in 2- and 3-dimensional assays.

Conclusions: Novel Hsp90 inhibitors can be developed as antipancreatic cancer agents. Their chemical structures are simpler, and they are likely to exhibit lower side effects than the much more complex inhibitors used as controls.
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http://dx.doi.org/10.1097/MPA.0000000000001807DOI Listing
April 2021

IDH1 and IDH2 Mutations in Colorectal Cancers.

Am J Clin Pathol 2021 Oct;156(5):777-786

Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, MD, USA.

Objectives: To elucidate clinicopathologic and molecular characteristics of IDH1 and IDH2 (IDH1/2) mutations in colorectal cancers (CRCs).

Methods: We evaluated IDH1/2 mutations in 1,623 CRCs using a next-generation sequencing assay.

Results: IDH1/2 mutations, predominantly IDH1 p.R132C, were detected in 15 (0.9%) CRCs and in 5 (3.0%) of 167 BRAF p.V600E-mutated CRCs. Three IDH1/2-mutated CRCs were associated with inflammatory bowel disease. They were significantly associated with old age, mucinous or signet ring cell adenocarcinoma, and high-grade histomorphology. Concordance of variant allele frequency between IDH1/2 mutants and other trunk drivers in CRCs and presence of IDH1/2 mutation in the adenoma and early adenocarcinoma indicated IDH1/2 mutations could be trunk drivers suitable for targeted therapy.

Conclusions: IDH1/2 mutations in CRCs were uncommon but enriched in BRAF p.V600E-mutated CRCs and perhaps colitis-associated CRCs. Further studies on IDH1/2-mutated CRCs are needed to clarify their clinicopathologic features and implications for targeted therapy.
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http://dx.doi.org/10.1093/ajcp/aqab023DOI Listing
October 2021

Nivolumab plus ipilimumab, with or without enzalutamide, in AR-V7-expressing metastatic castration-resistant prostate cancer: A phase-2 nonrandomized clinical trial.

Prostate 2021 05 26;81(6):326-338. Epub 2021 Feb 26.

Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

Background: AR-V7-positive metastatic prostate cancer is a lethal phenotype with few treatment options and poor survival.

Methods: The two-cohort nonrandomized Phase 2 study of combined immune checkpoint blockade for AR-V7-expressing metastatic castration-resistant prostate cancer (STARVE-PC) evaluated nivolumab (3 mg/kg) plus ipilimumab (1 mg/kg), without (Cohort 1) or with (Cohort 2) the anti-androgen enzalutamide. Co-primary endpoints were safety and prostate-specific antigen (PSA) response rate. Secondary endpoints included time-to-PSA-progression-free survival (PSA-PFS), time-to-clinical/radiographic-PFS, objective response rate (ORR), PFS lasting greater than 24 weeks, and overall survival (OS).

Results: Thirty patients were treated with ipilimumab plus nivolumab (N = 15, Cohort 1, previously reported), or ipilimumab plus nivolumab and enzalutamide (N = 15, Cohort 2) in patients previously progressing on enzalutamide monotherapy. PSA response rate was 2/15 (13%) in cohort 1 and 0/15 in cohort 2, ORR was 2/8 (25%) in Cohort 1 and 0/9 in Cohort 2 in those with measureable disease, median PSA-PFS was 3.0 (95% confidence interval [CI]: 2.1-NR) in cohort 1 and 2.7 (95% CI: 2.1-5.9) months in cohort 2, and median PFS was 3.7 (95% CI: 2.8-7.5) in cohort 1 and 2.9 (95% CI: 1.3-5.8) months in cohort 2. Three of 15 patients in cohort 1 (20%, 95% CI: 7.1%-45.2%) and 4/15 patients (26.7%, 95% CI: 10.5%-52.4%) in cohort 2 achieved a durable PFS lasting greater than 24 weeks. Median OS was 8.2 (95% CI: 5.5-10.4) in cohort 1 and 14.2 (95% CI: 8.5-NA) months in cohort 2. Efficacy results were not statistically different between cohorts. Grade-3/4 adverse events occurred in 7/15 cohort 1 patients (46%) and 8/15 cohort 2 patients (53%). Combined cohort (N = 30) baseline alkaline phosphatase and cytokine analysis suggested improved OS for patients with lower alkaline phosphatase (hazards ratio [HR], 0.30; 95% CI: 0.11-0.82), lower circulating interleukin-7 (IL-7) (HR, 0.24; 95% Cl: 0.06-0.93) and IL-6 (HR, 0.13; 95% Cl: 0.03-0.52) levels, and higher circulating IL-17 (HR, 4.53; 95% CI: 1.47-13.93) levels. There was a trend towards improved outcomes in men with low sPD-L1 serum levels.

Conclusion: Nivolumab plus ipilimumab demonstrated only modest activity in patients with AR-V7-expressing prostate cancer, and was not sufficient to justify further exploration in unselected patients. Stratification by baseline alkaline phosphatase and cytokines (IL-6, -7, and -17) may be prognostic for outcomes to immunotherapy.
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http://dx.doi.org/10.1002/pros.24110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8018565PMC
May 2021

Pancreatic cancer pathology viewed in the light of evolution.

Cancer Metastasis Rev 2021 09 8;40(3):661-674. Epub 2021 Feb 8.

Sol Goldman Pancreatic Cancer Research Center, Department of Pathology, The Johns Hopkins University School of Medicine, Carnegie 415, 600 North Wolfe Street, Baltimore, MD, 21287, USA.

One way to understand ductal adenocarcinoma of the pancreas (pancreatic cancer) is to view it as unimaginably large numbers of evolving living organisms interacting with their environment. This "evolutionary view" creates both expected and surprising perspectives in all stages of neoplastic progression. Advances in the field will require greater attention to this critical evolutionary prospective.
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http://dx.doi.org/10.1007/s10555-020-09953-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8556193PMC
September 2021

Val16A SOD2 Polymorphism Promotes Epithelial-Mesenchymal Transition Antagonized by Muscadine Grape Skin Extract in Prostate Cancer Cells.

Antioxidants (Basel) 2021 Feb 1;10(2). Epub 2021 Feb 1.

Center for Cancer Research and Therapeutic Development and Department of Biological Sciences, Clark Atlanta University, Atlanta, GA 30314, USA.

Epithelial-mesenchymal transition (EMT), a key event in cancer metastasis, allows polarized epithelial cells to assume mesenchymal morphologies, enhancing invasiveness and migration, and can be induced by reactive oxygen species (ROS). Val16A (Ala) SOD2 polymorphism has been associated with increased prostate cancer (PCa) risk. We hypothesized that SOD2 Ala single nucleotide polymorphism (SNP) may promote EMT. We analyzed SOD2 expression and genotype in various prostate cell lines. Stable overexpression of Ala-SOD2 or Val-SOD2 allele was performed in Lymph Node Carcinoma of the Prostate (LNCaP) cells followed by analysis of intracellular ROS and EMT marker protein expression. Treatments were performed with muscadine grape skin extract (MSKE) antioxidant, with or without addition of HO to provide further oxidative stress. Furthermore, MTS cell proliferation, cell migration, and apoptosis assays were completed. The results showed that SOD2 expression did not correlate with tumor aggressiveness nor SOD2 genotype. We demonstrated that the Ala-SOD2 allele was associated with marked induction of EMT indicated by higher Snail and vimentin, lower E-cadherin, and increased cell migration, when compared to Val-SOD2 allele or Neo control cells. Ala-SOD2 SNP cells exhibited increased levels of total ROS and superoxide and were more sensitive to co-treatment with HO and MSKE, which led to reduced cell growth and increased apoptosis. Additionally, MSKE inhibited Ala-SOD2 SNP-mediated EMT. Our data indicates that treatment with a combination of HO-generative drugs, such as certain chemotherapeutics and antioxidants such as MSKE that targets superoxide, hold promising therapeutic potential to halt PCa progression in the future.
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http://dx.doi.org/10.3390/antiox10020213DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7912849PMC
February 2021

Tumor Frameshift Mutation Proportion Predicts Response to Immunotherapy in Mismatch Repair-Deficient Prostate Cancer.

Oncologist 2021 02 3;26(2):e270-e278. Epub 2020 Dec 3.

The Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

Background: Genomic biomarkers that predict response to anti-PD1 therapy in prostate cancer are needed. Frameshift mutations are predicted to generate more neoantigens than missense mutations; therefore, we hypothesized that the number or proportion of tumor frameshift mutations would correlate with response to anti-PD1 therapy in prostate cancer.

Methods: To enrich for response to anti-PD1 therapy, we assembled a multicenter cohort of 65 men with mismatch repair-deficient (dMMR) prostate cancer. Patient characteristics and outcomes were determined by retrospective chart review. Clinical somatic DNA sequencing was used to determine tumor mutational burden (TMB), frameshift mutation burden, and frameshift mutation proportion (FSP), which were correlated to outcomes on anti-PD1 treatment. We subsequently used data from a clinical trial of pembrolizumab in patients with nonprostatic dMMR cancers of various histologies as a biomarker validation cohort.

Results: Nineteen of 65 patients with dMMR metastatic castration-resistant prostate cancer were treated with anti-PD1 therapy. The PSA response rate was 65%, and the median progression-free survival (PFS) was 24 (95% confidence interval 16-54) weeks. Tumor FSP, more than overall TMB, correlated most strongly with prolonged PFS and overall survival (OS) on anti-PD1 treatment and with density of CD8+ tumor-infiltrating lymphocytes. High FSP similarly identified patients with longer PFS as well as OS on anti-PD1 therapy in a validation cohort.

Conclusion: Tumor FSP correlated with prolonged efficacy of anti-PD1 treatment among patients with dMMR cancers and may represent a new biomarker of immune checkpoint inhibitor sensitivity.

Implications For Practice: Given the modest efficacy of immune checkpoint inhibition (ICI) in unselected patients with advanced prostate cancer, biomarkers of ICI sensitivity are needed. To facilitate biomarker discovery, a cohort of patients with DNA mismatch repair-deficient (dMMR) prostate cancer was assembled, as these patients are enriched for responses to ICI. A high response rate to anti-PD1 therapy in these patients was observed; however, these responses were not durable in most patients. Notably, tumor frameshift mutation proportion (FSP) was identified as a novel biomarker that was associated with prolonged response to anti-PD1 therapy in this cohort. This finding was validated in a separate cohort of patients with nonprostatic dMMR cancers of various primary histologies. This works suggests that FSP predicts response to anti-PD1 therapy in dMMR cancers, which should be validated prospectively in larger independent cohorts.
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http://dx.doi.org/10.1002/onco.13601DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7873327PMC
February 2021

Tumor Frameshift Mutation Proportion Predicts Response to Immunotherapy in Mismatch Repair-Deficient Prostate Cancer.

Oncologist 2021 02 3;26(2):e270-e278. Epub 2020 Dec 3.

The Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

Background: Genomic biomarkers that predict response to anti-PD1 therapy in prostate cancer are needed. Frameshift mutations are predicted to generate more neoantigens than missense mutations; therefore, we hypothesized that the number or proportion of tumor frameshift mutations would correlate with response to anti-PD1 therapy in prostate cancer.

Methods: To enrich for response to anti-PD1 therapy, we assembled a multicenter cohort of 65 men with mismatch repair-deficient (dMMR) prostate cancer. Patient characteristics and outcomes were determined by retrospective chart review. Clinical somatic DNA sequencing was used to determine tumor mutational burden (TMB), frameshift mutation burden, and frameshift mutation proportion (FSP), which were correlated to outcomes on anti-PD1 treatment. We subsequently used data from a clinical trial of pembrolizumab in patients with nonprostatic dMMR cancers of various histologies as a biomarker validation cohort.

Results: Nineteen of 65 patients with dMMR metastatic castration-resistant prostate cancer were treated with anti-PD1 therapy. The PSA response rate was 65%, and the median progression-free survival (PFS) was 24 (95% confidence interval 16-54) weeks. Tumor FSP, more than overall TMB, correlated most strongly with prolonged PFS and overall survival (OS) on anti-PD1 treatment and with density of CD8+ tumor-infiltrating lymphocytes. High FSP similarly identified patients with longer PFS as well as OS on anti-PD1 therapy in a validation cohort.

Conclusion: Tumor FSP correlated with prolonged efficacy of anti-PD1 treatment among patients with dMMR cancers and may represent a new biomarker of immune checkpoint inhibitor sensitivity.

Implications For Practice: Given the modest efficacy of immune checkpoint inhibition (ICI) in unselected patients with advanced prostate cancer, biomarkers of ICI sensitivity are needed. To facilitate biomarker discovery, a cohort of patients with DNA mismatch repair-deficient (dMMR) prostate cancer was assembled, as these patients are enriched for responses to ICI. A high response rate to anti-PD1 therapy in these patients was observed; however, these responses were not durable in most patients. Notably, tumor frameshift mutation proportion (FSP) was identified as a novel biomarker that was associated with prolonged response to anti-PD1 therapy in this cohort. This finding was validated in a separate cohort of patients with nonprostatic dMMR cancers of various primary histologies. This works suggests that FSP predicts response to anti-PD1 therapy in dMMR cancers, which should be validated prospectively in larger independent cohorts.
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http://dx.doi.org/10.1002/onco.13601DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7873327PMC
February 2021

PIN-like ductal carcinoma of the prostate has frequent activating RAS/RAF mutations.

Histopathology 2021 Jan 24;78(2):327-333. Epub 2020 Sep 24.

Department of Pathology, Johns Hopkins School of Medicine, Baltimore, MD, USA.

Aims: Prostatic intraepithelial neoplasia-like (PIN-like) ductal carcinoma is a rare tumour characterised by often cystically dilated glands architecturally resembling high-grade PIN, but lacking basal cells. These tumours are frequently accompanied by grade group 1 acinar cancer and behave relatively indolently. In contrast, conventional ductal adenocarcinoma of the prostate is an aggressive variant comparable to grade group 4 acinar cancer. Here, we used targeted next-generation sequencing to molecularly profile PIN-like ductal carcinoma cases at radical prostatectomy.

Methods And Results: Five PIN-like ductal carcinoma samples at radical prostatectomy with sufficient tumour tissue available were analysed for genomic alterations by targeted next-generation sequencing using the Johns Hopkins University (JHU) solid tumour panel. DNA was captured using SureSelect for 640 genes and sequenced on the Illumina HiSeq platform. Three of five (60%) of the PIN-like ductal carcinomas showed activating mutations in the RAS/RAF pathways, which are extraordinarily rare in conventional primary prostate carcinoma (<3% of cases), including an activating hot-spot BRAF mutation (p.K601E), an activating hot-spot mutation in HRAS (p.Q61K) and an in-frame activating deletion in BRAF (p.T488_Q493delinsK). An additional two cases lacked BRAF or HRAS mutations, but harboured in-frame insertions of uncertain significance in MAP2K4 and MAP3K6. One case had sufficient acinar tumour for sequencing, and showed a similar molecular profile as the concurrent PIN-like ductal carcinoma, suggesting a clonal relationship between the two components.

Conclusions: PIN-like ductal carcinoma represents a molecularly unique tumour, enriched for potentially targetable oncogenic driver mutations in the RAS/RAF/MAPK pathway. This molecular profile contrasts with that of conventional ductal adenocarcinoma, which is typically enriched for pathogenic mutations in the mismatch repair (MMR) and homologous recombination (HR) DNA repair pathways.
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http://dx.doi.org/10.1111/his.14224DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7775281PMC
January 2021

The genetics of ductal adenocarcinoma of the pancreas in the year 2020: dramatic progress, but far to go.

Mod Pathol 2020 12 23;33(12):2544-2563. Epub 2020 Jul 23.

Department of Pathology, The Sol Goldman Pancreatic Cancer Research Center, The Johns Hopkins University School of Medicine, Baltimore, MD, USA.

The publication of the "Pan-Cancer Atlas" by the Pan-Cancer Analysis of Whole Genomes Consortium, a partnership formed by The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC), provides a wonderful opportunity to reflect on where we stand in our understanding of the genetics of pancreatic cancer, as well as on the opportunities to translate this understanding to patient care. From germline variants that predispose to the development of pancreatic cancer, to somatic mutations that are therapeutically targetable, genetics is now providing hope, where there once was no hope, for those diagnosed with pancreatic cancer.
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http://dx.doi.org/10.1038/s41379-020-0629-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8375585PMC
December 2020

Molecular characterization of organoids derived from pancreatic intraductal papillary mucinous neoplasms.

J Pathol 2020 11 19;252(3):252-262. Epub 2020 Sep 19.

Department of Pathology, The Sol Goldman Pancreatic Cancer Research Center, The Johns Hopkins University School of Medicine, Baltimore, MD, USA.

Intraductal papillary mucinous neoplasms (IPMNs) are commonly identified non-invasive cyst-forming pancreatic neoplasms with the potential to progress into invasive pancreatic adenocarcinoma. There are few in vitro models with which to study the biology of IPMNs and their progression to invasive carcinoma. Therefore, we generated a living biobank of organoids from seven normal pancreatic ducts and ten IPMNs. We characterized eight IPMN organoid samples using whole genome sequencing and characterized five IPMN organoids and seven normal pancreatic duct organoids using transcriptome sequencing. We identified an average of 11,344 somatic mutations in the genomes of organoids derived from IPMNs, with one sample harboring 61,537 somatic mutations enriched for T→C transitions and T→A transversions. Recurrent coding somatic mutations were identified in 15 genes, including KRAS, GNAS, RNF43, PHF3, and RBM10. The most frequently mutated genes were KRAS, GNAS, and RNF43, with somatic mutations identified in six (75%), four (50%), and three (37.5%) IPMN organoid samples, respectively. On average, we identified 36 structural variants in IPMN derived organoids, and none had an unstable phenotype (> 200 structural variants). Transcriptome sequencing identified 28 genes differentially expressed between normal pancreatic duct organoid and IPMN organoid samples. The most significantly upregulated and downregulated genes were CLDN18 and FOXA1. Immunohistochemical analysis of FOXA1 expression in 112 IPMNs, 113 mucinous cystic neoplasms, and 145 pancreatic ductal adenocarcinomas demonstrated statistically significant loss of expression in low-grade IPMNs (p < 0.0016), mucinous cystic neoplasms (p < 0.0001), and pancreatic ductal adenocarcinoma of any histologic grade (p < 0.0001) compared to normal pancreatic ducts. These data indicate that FOXA1 loss of expression occurs early in pancreatic tumorigenesis. Our study highlights the utility of organoid culture to study the genetics and biology of normal pancreatic duct and IPMNs. © 2020 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
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http://dx.doi.org/10.1002/path.5515DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8162794PMC
November 2020

and Methylation and Somatic Mutations as Precision Medicine Biomarkers for Diagnosis and Prognosis of High-grade Serous Ovarian Cancer.

Cancer Prev Res (Phila) 2020 09 24;13(9):783-794. Epub 2020 Jun 24.

Otolaryngology Department, Head and Neck Cancer Research Division, The Johns Hopkins University, School of Medicine, Baltimore, Maryland.

Molecular alterations that contribute to long-term (LT) and short-term (ST) survival in ovarian high-grade serous carcinoma (HGSC) may be used as precision medicine biomarkers. DNA promoter methylation is an early event in tumorigenesis, which can be detected in blood and urine, making it a feasible companion biomarker to somatic mutations for early detection and targeted treatment workflows. We compared the methylation profile in 12 HGSC tissue samples to 30 fallopian tube epithelium samples, using the Infinium Human Methylation 450K Array. We also used 450K methylation arrays to compare methylation among HGSCs long-term survivors (more than 5 years) and short-term survivors (less than 3 years). We verified the array results using bisulfite sequencing and methylation-specific PCR (qMSP). in another cohort of HGSC patient samples ( = 35). Immunoblot and clonogenic assays after pharmacologic unmasking show that and promoter methylation downregulates mRNA expression levels in ovarian cancer cells. We then used qMSP in paired tissue, ascites, plasma/serum, vaginal swabs, and urine from a third cohort of patients with HGSC cancer ( = 85) to test the clinical potential of and in precision medicine workflows. We also performed next-generation exome sequencing of 50 frequently mutated in human cancer genes, using the Ion AmpliSeqCancer Hotspot Panel, to show that the somatic mutation profile found in tissue and plasma can be quantified in paired urine samples from patients with HGSC. Our results suggest that HIST1H2BB and MAGI2 have growth-suppressing roles and can be used as HGSC precision medicine biomarkers.
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http://dx.doi.org/10.1158/1940-6207.CAPR-19-0412DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8082233PMC
September 2020

Clonal Origin Evaluated by Trunk and Branching Drivers and Prevalence of Mutations in Multiple Lung Tumor Nodules.

Mol Diagn Ther 2020 08;24(4):461-472

Department of Pathology, Johns Hopkins University School of Medicine, Carnegie 469, Pathology, 600 North Wolfe Street, Baltimore, MD, 21287, USA.

Introduction: Differentiation between intrapulmonary metastasis (IPM) and multiple primary lung cancers (MPLC) in patients with synchronous or metachronous lung tumor nodules is critical but challenging.

Objective: We proposed an algorithm to evaluate clonal origin based on trunk (initiating) versus branching drivers and the prevalence of mutations in lung adenocarcinomas.

Methods: Driver mutations were examined using next-generation sequencing in five trunk driver genes (BRAF, EGFR, ERBB2, KRAS, and NRAS) and three branching driver genes (ATK1, PIK3CA, and TP53).

Results: Mutational profiling supported same clonality and likely same clonality, respectively, in 39 and 14 of 66 pairs of specimens with known identical clonal origin. Discordance of TP53 mutations (branching drivers) was observed in three pairs. Subsequent analyses of 30 pairs of synchronous or metachronous lung tumor nodules revealed different clonality and likely different clonality in 17 and 2 pairs, respectively, including three pairs with similar histomorphology; same clonality and likely same clonality in three and five pairs, respectively, including two pairs with different histomorphology; and inconclusive or noninformative results in three pairs.

Conclusion: While discordance of trunk drivers indicated MPLC in patients with synchronous or metachronous lung tumor nodules, discordance of branching drivers did not exclude IPM. Concordance of uncommon drivers supported IPM, whereas concordance of common drivers did not exclude MPLC. Additional recommendations from official organizations are needed to guide applications of molecular markers in defining clonality of multiple lung tumor nodules.
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http://dx.doi.org/10.1007/s40291-020-00471-wDOI Listing
August 2020

IDH1 and IDH2 mutations in lung adenocarcinomas: Evidences of subclonal evolution.

Cancer Med 2020 06 25;9(12):4386-4394. Epub 2020 Apr 25.

Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, MD, USA.

Background: Selective IDH1 and IDH2 inhibitors have been approved for targeted therapy of acute myeloid leukemia. Clinical trials for solid tumors with IDH1 and IDH2 (IDH1/2) mutations are ongoing. Reports of IDH1/2-mutated non-small cell lung cancers (NSCLCs), however, are limited.

Methods: We evaluated IDH1/2 mutations in 1,924 NSCLC specimens (92% adenocarcinoma) using a next-generation sequencing assay.

Results: Retrospective quality assessments identified false detection of IDH1 c.395G>A (p.R132H) resulting from cytosine deamination (C:G→T:A) artifact in one specimen. IDH1/2 mutations were detected in 9 (0.5%) adenocarcinomas taken by fine-needle aspiration (n = 3), thoracentesis (n = 2) or core biopsy (n = 4). All nine adenocarcinomas showed high-grade features. Extensive clear cell change, however, was not observed. High expression (50% or greater) of PD-L1 was observed in two of five specimens examined. IDH1/2 mutations were associated with old age, smoking history, and coexisting KRAS mutation. Lower than expected variant allele frequency of IDH1/2 mutants and coexistence of IDH1/2 mutations with known trunk drivers in the BRAF, EGFR, and KRAS genes suggest they could be branching drivers leading to subclonal evolution in lung adenocarcinomas. Multiregional analysis of an adenocarcinoma harboring two IDH2 mutations revealed parallel evolution originating from a KRAS-mutated lineage, further supporting subclonal evolution promoted by IDH1/2 mutations.

Conclusions: IDH1/2 mutations in NSCLCs are uncommon. They occur in adenocarcinomas with high-grade features and may be branching drivers leading to subclonal evolution. Accumulation of more IDH1/2-mutated NSCLCs is needed to clarify their clinicopathological characteristics and implications for targeted therapy.
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http://dx.doi.org/10.1002/cam4.3058DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7300411PMC
June 2020

Detection of Circulating Tumor DNA in Patients with Pancreatic Cancer Using Digital Next-Generation Sequencing.

J Mol Diagn 2020 06 20;22(6):748-756. Epub 2020 Mar 20.

Department of Pathology, The Sol Goldman Pancreatic Cancer Research Center, Johns Hopkins Medical Institutions, Baltimore, Maryland; Department of Oncology, The Sol Goldman Pancreatic Cancer Research Center, Johns Hopkins Medical Institutions, Baltimore, Maryland; Department of Medicine, The Sol Goldman Pancreatic Cancer Research Center, Johns Hopkins Medical Institutions, Baltimore, Maryland. Electronic address:

Circulating tumor DNA (ctDNA) measurements can be used to estimate tumor burden, but avoiding false-positive results is challenging. Herein, digital next-generation sequencing (NGS) is evaluated as a ctDNA detection method. Plasma KRAS and GNAS hotspot mutation levels were measured in 140 subjects, including 67 with pancreatic ductal adenocarcinoma and 73 healthy and disease controls. To limit chemical modifications of DNA that yield false-positive mutation calls, plasma DNA was enzymatically pretreated, after which DNA was aliquoted for digital detection of mutations (up to 384 aliquots/sample) by PCR and NGS. A digital NGS score of two SDs above the mean in controls was considered positive. Thirty-seven percent of patients with pancreatic cancer, including 31% of patients with stages I/II disease, had positive KRAS codon 12 ctDNA scores; only one patient had a positive GNAS mutation score. Two disease control patients had positive ctDNA scores. Low-normal-range digital NGS scores at mutation hotspots were found at similar levels in healthy and disease controls, usually at sites of cytosine deamination, and were likely the result of chemical modification of plasma DNA and NGS error rather than true mutations. Digital NGS detects mutated ctDNA in patients with pancreatic cancer with similar yield to other methods. Detection of low-level, true-positive ctDNA is limited by frequent low-level detection of false-positive mutation calls in plasma DNA from controls.
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http://dx.doi.org/10.1016/j.jmoldx.2020.02.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7338889PMC
June 2020

Multiclonal colorectal cancers with divergent histomorphological features and RAS mutations: one cancer or separate cancers?

Hum Pathol 2020 04 12;98:120-128. Epub 2020 Mar 12.

Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, MD, 21205, USA; Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, MD, 21205, USA.

Detection of coexisting mutations within the same signal transduction pathway, which are expected to be mutually exclusive, raises a concern of laboratory errors. We have previously confirmed the presence of different RAS (KRAS and NRAS) mutations in the adenoma and/or adenocarcinoma subpopulations of colorectal cancers (CRCs). In this study, multiregional analyses by next-generation sequencing were conducted to elucidate the mechanisms underlying multiple RAS mutations seen in 5 CRC specimens. Multiregional analyses were initially conducted in a single tissue block originally submitted for mutational profiling. In 2 specimens, mutational status of the APC gene was not identical, indicating collisional adenoma and adenocarcinoma. In 3 specimens, the same APC mutation was present in different subpopulations with divergent RAS mutations, indicating a common clonal origin. Subsequent comprehensive multiregional analyses of additional adenoma and adenocarcinoma components revealed multiclonal CRCs with divergent histomorphological features and RAS mutations originating from a common APC-mutated founder lineage of adenoma, but from different RAS-mutated founder lineages of adenocarcinoma. These findings are consistent with the stepwise model of colorectal tumorigenesis along with parallel evolution, which affects RAS genes within the mitogen-activated protein kinase pathway and occurs during the progression from adenomas to adenocarcinomas. Evaluation of tumor subpopulations with divergent histomorphological features by pathologists may help identify multiclonal CRCs. Further studies are warranted to evaluate the incidence of multiclonality in CRCs and its impact on clinical outcomes. Perhaps, multiclonal CRCs originating from the same APC-mutated founder lineage of adenoma but from different RAS-mutated founder lineages of adenocarcinomas should be defined and managed as separate CRCs.
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http://dx.doi.org/10.1016/j.humpath.2020.03.002DOI Listing
April 2020

Gene Variants That Affect Levels of Circulating Tumor Markers Increase Identification of Patients With Pancreatic Cancer.

Clin Gastroenterol Hepatol 2020 05 30;18(5):1161-1169.e5. Epub 2019 Oct 30.

Department of Pathology, The Sol Goldman Pancreatic Cancer Research Center, Johns Hopkins Medical Institutions, Baltimore, Maryland; Department of Oncology, The Sol Goldman Pancreatic Cancer Research Center, Johns Hopkins Medical Institutions, Baltimore, Maryland; Department of Medicine, The Sol Goldman Pancreatic Cancer Research Center, Johns Hopkins Medical Institutions, Baltimore, Maryland. Electronic address:

Background & Aims: Levels of carcinoembryonic antigen (CEA), carbohydrate antigen 19-9 (CA19-9), and cancer antigen 125 (CA-125) in blood are used as markers to determine the response of patients with cancer to therapy, but are not used to identify patients with pancreatic cancer.

Methods: We obtained blood samples from 504 patients undergoing pancreatic surveillance from 2002 through 2018 who did not develop pancreatic cancer and measured levels of the tumor markers CA19-9, CEA, CA-125, and thrombospondin-2. Single-nucleotide polymorphisms (SNPs) in FUT3, FUT2, ABO, and GAL3ST2 that have been associated with levels of tumor markers were used to establish SNP-defined ranges for each tumor marker. We also tested the association between additional SNPs (in FUT6, MUC16, B3GNT3, FAM3B, and THBS2) with levels of tumor markers. To calculate the diagnostic specificity of each SNP-defined range, we assigned the patients under surveillance into training and validation sets. After determining the SNP-defined ranges, we determined the sensitivity of SNP-adjusted tests for the tumor markers, measuring levels in blood samples from 245 patients who underwent resection for pancreatic ductal adenocarcinoma (PDAC) from 2010 through 2017.

Results: A level of CA19-9 that identified patients with PDAC with 99% specificity had 52.7% sensitivity. When we set the cut-off levels of CA19-9 based on each SNP, the test for CA19-9 identified patients with PDAC with 60.8% sensitivity and 98.8% specificity. Among patients with FUT3 alleles that encode a functional protein, levels of CA19-9 greater than the SNP-determined cut-off values identified 66.4% of patients with PDAC, with 99.3% specificity. In the validation set, levels of CEA varied among patients with vs without SNP in FUT2, by blood group, and among smokers vs nonsmokers; levels of CA-125 varied among patients with vs without the SNP in GAL3ST2. The use of the SNPs to define the ranges of CEA and CA-125 did not significantly increase the diagnostic accuracy of the assays for these proteins. Combining data on levels of CA19-9 and CEA, CA19-9 and CA-125, or CA19-9 and thrombospondin-2 increased the sensitivity of detection of PDAC, but slightly reduced specificity.

Conclusions: Including information on SNPs associated with levels of CA19-9, CEA, and CA-125 can improve the diagnostic accuracy of assays for these tumor markers in the identification of patients with PDAC. Clinicaltrials.gov no: NCT02000089.
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http://dx.doi.org/10.1016/j.cgh.2019.10.036DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7166164PMC
May 2020

Tetraploid Partial Hydatidiform Moles: Molecular Genotyping and Determination of Parental Contributions.

J Mol Diagn 2020 01 24;22(1):90-100. Epub 2019 Oct 24.

Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland. Electronic address:

DNA genotyping studies have established that most partial hydatidiform moles (PHMs) are diandric dispermic triploid conceptions. Rare triandric tetraploid PHMs have been described, but genotyping cannot determine the manner in which three paternal chromosome complements are derived (one sperm with triplication, two sperm with one duplication, three different sperm, or one diploid and one haploid sperm). In a large prospective analysis of potentially molar products of conception, five tetraploid PHMs were encountered among 235 PHMs. Single-nucleotide polymorphism (SNP) arrays were used to define different paternal chromosomal contributions. Short tandem repeat analysis of the five tetraploid PHMs established that these contained three paternal and one maternal chromosome complements. In each case, the corresponding SNP array found five tracts with segmented absence of the central tract across approximately 25% of the genome. Meiotic crossovers could be observed directly in the chromosomes via the total number of starts and stops of regions of loss of heterozygosity. The findings are consistent with each conceptus having three different paternal contributions and one maternal contribution. These findings suggest that tetraploid PHMs arise when three different sperm fertilize a single, normal ovum. SNP array is useful to determine the parental contributions in triploid/tetraploid conceptuses. It also allows for direct visualization of meiotic crossover frequency and sites in these conceptions, providing insight into their biology.
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http://dx.doi.org/10.1016/j.jmoldx.2019.09.006DOI Listing
January 2020

Biphenotypic Differentiation of Pancreatic Cancer in 3-Dimensional Culture.

Pancreas 2019 10;48(9):1225-1231

From the Department of Pathology, Johns Hopkins University School of Medicine, The Sol Goldman Pancreatic Cancer Research Center.

Objective: Pancreatic ductal adenocarcinoma (PDAC) is the third most common cause of cancer death in the United States. Improved characterized models of PDAC are needed for drug screening.

Methods: We grew 4 established pancreatic cancer cell lines in hanging drop cultures to produce spheroids. We also grew organoids from explanted xenografted PDAC and surgically resected primary PDAC. We performed transmission and scanning electron microscopy and compared findings with those of the normal pancreatic duct. We also performed single-cell cloning to determine the potential options for differentiation.

Results: Spheroids contained tight junctions and desmosomes but lacked zymogen granules, as expected. The former features were present in normal pancreatic duct but absent from PDAC cell lines grown in standard 2-dimensional culture. Spheroids functionally excluded macromolecules in whole mounts. Cells on the surface of PDAC spheroids were carpeted by microvilli except for rare cells with prominent stereocilia. Carpets of microvilli were also seen in low passage organoids produced from xenografts and surgically resected human PDAC, in addition to normal human pancreatic duct. We performed single-cell cloning and resulting spheroids produced both cell phenotypes at the same approximate ratios as those from bulk cultures.

Conclusions: Pancreatic cancer spheroids/organoids are capable of biphenotypic differentiation.
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http://dx.doi.org/10.1097/MPA.0000000000001390DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6791773PMC
October 2019

A pilot study of prostate-specific membrane antigen (PSMA) dynamics in men undergoing treatment for advanced prostate cancer.

Prostate 2019 10 30;79(14):1597-1603. Epub 2019 Jul 30.

Department of Radiology and Radiological Science, Johns Hopkins University, Baltimore, Maryland.

Background: Prostate-specific membrane antigen (PSMA) is a rational target for noninvasive detection of recurrent prostate cancer (PCa) and for therapy of metastatic castration-resistant prostate cancer (mCRPC) with PSMA-targeted agents. Here we conducted serial measurements of PSMA expression on circulating tumor cells (CTCs) to evaluate patterns of longitudinal PSMA dynamics over the course of multiple sequential therapies.

Methods: A retrospective investigation of men with mCRPC undergoing evaluation at medical oncology clinics at our institution assessed the dynamics of PSMA expression in the context of different systemic treatments administered sequentially. Eligibility included patients who began systemic therapies with androgen receptor (AR)-directed agents or taxane agents for whom peripheral blood samples were tested for CTC mRNA of AR splice variant-7 (AR-V7), prostate-specific antigen (PSA), and PSMA (with >2 CTC + results) in a CLIA-accredited laboratory.

Results: From August 2015 to November 2017, we identified 96 eligible men. Fifteen had greater than or equal to 2 sequential therapies and evaluable CTC samples, greater than or equal to 1 expressing PSMA (PSMA+). Among the 15 patients included in this analysis, a total of 54 PSMA status evaluations were performed in the context of 48 therapies during a median follow-up of 18 months. At baseline, PSMA signal was detected ("positive") in 11 of 15 (73.3%) patients, while for 4 of 15 (26.7%) patients PSMA signal was undetectable ("negative"). In all but two patients, the baseline collection corresponded with a change in treatment. On the second assessment, PSMA increases were detected in all 4/4 (100%) PSMA-negative patients and 8 of 11 (72.7%) PSMA-positive patients. PSMA significantly decreased in a patient treated with Lu-PSMA-617. Serum PSA declines were seen in 7 of 8 (88%) of the treatment periods where PSMA decreased.

Conclusions: PSMA expression in CTCs is a dynamic marker. PSMA transcript declines appear to be associated with concurrent decreases in serum PSA. Sequential CTC sampling could provide a noninvasive response assessment to systemic treatment for mCRPC.
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http://dx.doi.org/10.1002/pros.23883DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6818502PMC
October 2019

Clinical mutational profiling and categorization of BRAF mutations in melanomas using next generation sequencing.

BMC Cancer 2019 Jul 5;19(1):665. Epub 2019 Jul 5.

Department of Pathology, Johns Hopkins University School of Medicine, Johns Hopkins University School of Medicine, 1812 Ashland Ave, Suite 200, Baltimore, MD, 21205, USA.

Background: Analysis of melanomas for actionable mutations has become the standard of care. Recently, a classification scheme has been proposed that categorizes BRAF mutations based on their mechanisms for activation of the MAPK pathway.

Methods: In this analysis BRAF, KIT, NRAS, and PIK3CA mutations were examined by next generation sequencing (NGS) in 446 melanomas in a clinical diagnostic setting. KRAS and HRAS were also analyzed to elucidate coexisting BRAF and RAS mutations. BRAF mutations were categorized into class-1 (kinase-activated, codon 600), class-2 (kinase-activated, non-codon 600) and class-3 (kinase-impaired), based on the newly proposed classification scheme.

Results: NGS demonstrated high analytic sensitivity. Among 355 mutations detected, variant allele frequencies were 2-5% in 21 (5.9%) mutations and 2-10% in 47 (13%) mutations. Mutations were detected in BRAF (42%), NRAS (25%), KIT (4.9%) and PIK3CA (2.7%). The incidence of class-1, class-2 and class-3 mutations were 33% (26% p.V600E and 6.1% p.V600K), 3.1 and 4.9% respectively. With a broader reportable range of NGS, class-1, class-2 and class-3 mutations accounted for 77, 7.4 and 12% of all BRAF mutations. Class-3 mutations, commonly affecting codons 594, 466 and 467, showed a higher incidence of coexisting RAS mutations, consistent with their RAS-dependent signaling. Significant association with old age and primary tumors of head/neck/upper back suggest chronic solar damage as a contributing factor for melanomas harboring BRAF p.V600K or class-3 mutations.

Conclusion: This study categorizes the range, frequency, coexisting driver mutations and clinical characteristics of the three classes of BRAF mutations in a large cohort of melanomas in a clinical diagnostic setting. Further prospective studies are warranted to elucidate the clinical outcomes and benefits of newly developed targeted therapy in melanoma patients carrying each class of BRAF mutation.
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http://dx.doi.org/10.1186/s12885-019-5864-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6612071PMC
July 2019

Clinical Validation of Discordant Trunk Driver Mutations in Paired Primary and Metastatic Lung Cancer Specimens.

Am J Clin Pathol 2019 10;152(5):570-581

Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, MD.

Objectives: To propose an operating procedure for validation of discordant trunk driver mutations.

Methods: Concordance of trunk drivers was examined by next-generation sequencing in 15 patients with two to three metastatic lung cancers and 32 paired primary and metastatic lung cancers.

Results: Tissue identity was confirmed by genotyping 17 single-nucleotide polymorphisms within the panel. All except three pairs showed concordant trunk drivers. Quality assessment conducted in three primary and metastatic pairs with discordant trunk drivers indicates metastasis from a synchronous or remote lung primary in two patients. Review of literature revealed high discordant rates of EGFR and KRAS mutations, especially when Sanger sequencing was applied to examine primary and lymph node metastatic tumors.

Conclusions: Trunk driver mutations are highly concordant in primary and metastatic tumors. Discordance of trunk drivers, once confirmed, may suggest a second primary cancer. Guidelines are recommended to establish standard operating procedures for validation of discordant trunk drivers.
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http://dx.doi.org/10.1093/ajcp/aqz077DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6779251PMC
October 2019
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