Faculty Bibliography

Screening for therapeutic targets is standard of care in the management of advanced non-small cell lung cancer. However, most molecular assays utilize tumor tissue, which may not always be available. "Liquid biopsies" are plasma-based next generation sequencing (NGS) assays that use circulating tumor DNA to identify relevant targets. To compare the sensitivity, specificity, and accuracy of a plasma-based NGS assay to solid-tumor-based NGS we retrospectively analyzed sequencing results of 100 sequential patients with lung adenocarcinoma at our institution who had received concurrent testing with both a solid-tissue-based NGS assay and a commercially available plasma-based NGS assay. Patients represented both new diagnoses (79%) and disease progression on treatment (21%); the majority (83%) had stage IV disease. Tissue-NGS identified 74 clinically relevant mutations, including 52 therapeutic targets, a sensitivity of 94.8%, while plasma-NGS identified 41 clinically relevant mutations, a sensitivity of 52.6% (p < 0.001). Tissue-NGS showed significantly higher sensitivity and accuracy across multiple patient subgroups, both in newly diagnosed and treated patients, as well as in metastatic and nonmetastatic disease. Discrepant cases involved hotspot mutations and actionable fusions including those in EGFR, ALK, and NTRK1. In summary, tissue-NGS detects significantly more clinically relevant alterations and therapeutic targets compared to plasma-NGS, suggesting that tissue-NGS should be the preferred method for molecular testing of lung adenocarcinoma when tissue is available. Plasma-NGS can still play an important role when tissue testing is not possible. However, given its low sensitivity, a negative result should be confirmed with a tissue-based assay.

Background: Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in the genes encoding polycystin 1 and polycystin 2 (PKD1 and PKD2, respectively), leading to florid cystic change of the renal parenchyma. The incidence of carcinoma associated with ADPKD remains unclear although there are studies to suggest that the incidence may be higher.
Design(s): We queried our department pathology database for surgical specimens with ADPKD from 1990 to 2020. We evaluated these cases for the presence of associated malignant or benign neoplasia, as well as pathological and clinical parameters.
Result(s): The majority of the surgical specimens are kidney explants with a clinical diagnosis of ADPKD and the status of end stage kidney diseases. All specimens showed radiological, gross and microscopic features of ADPKD. Eight of 33 ADPKD patients with kidney resection specimens examined contained a malignant neoplasm, including 2 patients with bilateral malignancy. The types of renal cell carcinoma (RCC) associated with the following types: four cases of clear cell RCC, two cases of papillary RCC, type 2, two cases of unclassified high grade RCC, one case of unclassified low grade, as well as one case of TFE3 translocated RCC. Associated carcinomas ranged in size from less than 1 cm to 12 cm. One case with a concurrent oncocytoma and several cases with associated papillary adenoma were also reported.
Conclusion(s): In this cohort, a wide distribution of renal cell carcinoma subtypes were observed, with clear cell RCC being the most common type. The incidence of associated malignancy (24%) is higher than previously reported by Jilg et al. 2013 (5%), possibly due to differences in patient management or patient populations between the institutions. This case series highlights the high occurrence of carcinoma in APKD nephrectomies suggesting a clinical risk of malignancy in patients with ADPKD. Additionally this case series reports the first case of a TFE3 translocated renal cell carcinoma arising synchronously with a contralateral clear cell renal cell carcinoma in a patient with ADPKD. The heterogeneity of renal carcinoma subtypes within the group (and within contralateral kidneys in one patient with bilateral involvement) suggests that stimuli for tumorigenesis arise at the kidney microenvironment level rather than on the basis of gene mutation alone. Accrual of an expanded cohort of patients is planned to enable confirmation of differences between carcinomas arising in the setting of ADPKD versus those arising in end stage renal disease due to other causes, and in the sporadic setting. Furthermore a role for molecular studies is suggested to evaluate if any of the ADPKD causing mutations (PKD1, PKD2, or other) is associated with the development of carcinoma

CONTEXT.—:An increasing number of molecular laboratories are implementing next-generation sequencing platforms to identify clinically actionable and relevant genomic alterations for precision oncology. OBJECTIVE.—:To describe the validation studies as per New York State-Department of Health (NYS-DOH) guidelines for the Oncomine Comprehensive Panel v2, which was originally tailored to the National Cancer Institute Molecular Analysis for Therapy Choice (NCI-MATCH) trial. DESIGN.—:Accuracy, precision, and reproducibility were investigated by using 130 DNA and 18 RNA samples from cytology cell blocks; formalin-fixed, paraffin-embedded tissues; and frozen samples. Analytic sensitivity and specificity were tested by using ATCC and HapMap cell lines. RESULTS.—:High accuracy and precision/reproducibility were observed for single nucleotide variants and insertion/deletions. We also share our experience in the detection of gene fusions and copy number alterations from an amplicon-based sequencing platform. After sequencing analysis, variant annotation and report generation were performed by using the institutional knowledgebase. CONCLUSIONS.—:This study serves as an example for validating a comprehensive targeted next-generation sequencing assay with both DNASeq and RNASeq components for NYS-DOH.

Background: Intraductal carcinoma of the prostate (IDC-P) is considered a harbinger of aggressive disease and is associated with poor clinical prognosis. It is believed to represent retrograde spread of high grade invasive carcinoma into the prostatic ductular system, although there is minimal molecular evidence to support this theory. We aimed to further study the molecular evolution of IDC-P and how it relates to invasive carcinoma and tumor metastases.
Design(s): Radical prostatectomy specimens where the dominant tumor nodule had an abundant IDC-P component (>20%) were identified. Cases were reviewed by GU pathologists to annotate areas of IDC-P and high grade (> Grade Group 3) invasive carcinoma (HGIC) in ten selected cases. Five cases also had lymph node metastases. DNA was extracted from each macrodissected area and subjected to copy number alteration (CNA) analysis using the OncoScan Assay (Affymetrix). CNA was performed using the Oncoscan Nexus Express software v3.0. FISH was utilized to validate particular key findings with centromeric probes for chromosomes 8, 10, and 12.
Result(s): Similar 8p and 10q losses were seen in both the IDC-P and high grade invasive carcinoma in all 10 cases, and 8q gains were also seen in both areas in 7 cases. In 3 of 10 cases, the IDC-P component showed copy number gains spanning the entire genome in the regions without chromosomal losses, and in one of these cases, the HGIC area showed similar whole genome gains. In addition, those non-amplified regions of chromosomal loss are common to the HGIC and IDC-P components, including both deletion and uniparental disomy. In 2 of 5 cases, the lymph node metastases also showed a copy number profile that was more similar to IDC-P than it was to HGIC, including one case with whole genome gain. Whole genome gains were further validated by FISH, which showed a mixed population of cells containing a range of 2-5 copies of each of the selected chromosomes.
Conclusion(s): IDC-P and invasive high grade carcinoma are clonally related, as evidenced by similar focal chromosomal gains and losses. Whole genome gains are seen in IDC-P, and share the same loss pattern with HGIC. This suggests a clonal evolution from invasive carcinoma to IDC-P in some cases. The copy number alteration pattern of the lymph node metastases is sometimes more similar to IDC-P than it is to HGIC, suggesting that the tumor clones present within IDC-P have metastatic potential

Background: Radical prostatectomy (RP) specimens often harbor multiple prostate cancer (PCa) nodules that are spatially separated. These tumor nodules can be molecularly heterogeneous. The current practice is to separate the dominant tumor nodule histologically, usually the one with highest Gleason score and/or largest size, and assign individual Grade Groups. There is no current recommendation to use ancillary studies to further differentiate theses nodules molecularly. Current assays that interrogate biopsies and RP assume that only one tumor is present in the tissue sample. The aim of this study was to establish the frequency of molecularly heterogeneous tumors within one spatially distinct nodule in RP specimens.
Design(s): 269 RP specimens - Grade Groups 1 to 5 - collected prospectively from three participating institutions of the Early Detection Research Network (EDRN) trial were reviewed. Dual ERG/SPINK1 IHC was performed on all tumor foci. The nodule with the highest Grade Group score was defined as dominant. In case of multiple nodules with the same Gleason score, the largest one was considered as the dominant. A "collision tumor" was defined as having two molecular subtypes within one spatially distinct nodule. The presence of cribriform pattern and intraductal carcinoma (IDC-P), both features associated with unfavorable outcome were also recorded.
Result(s): IHC slides from 260 RP cases were available for analysis. 25 collision tumors (9.6%) were identified (Table), of which, 48% included nodules with different grade groups. The most common collision being between the ERG+/SPINK1- and ERG-/SPINK1-(64%) (Fig. 1). The frequency of cribriform morphology and IDC-P when applicable was 15% and 12%, respectively. Finally, we found ERG+ benign-appearing glands in 8% of total cases (Fig. 2), which were present close to ERG+ tumors in only 15% of these cases. (Table presented)
Conclusion(s): Collison tumors of two molecular subclasses of prostate cancer are not uncommon, affecting the final Grade Group in about half of these cases. Ancillary studies - e.g. dual I

Background: Clear cell papillary renal cell carcinoma (ccpRCC) is a relatively new entity and it has been described as an indolent renal neoplasm. ccpRCC shares features of clear cell RCC (ccRCC) and papillary RCC (pRCC) morphologically and immunohistochemically, even though it carries a very different prognostic potential. Epigenetic alterations play a significant role in the development and progression of human tumors. A large scale sequencing efforts demonstrated that hypermethylation in RCC tumors is associated with poor prognosis and may dictate treatment options. However, ccpRCC has not been included and further molecular elucidation is to be done. In this study, we attempt to investigate the methylation patterns in ccpRCC and test if differential patterns can be correlated with histologic subtypes and known biological behaviors.
Design(s): Nephrectomy specimens from our institution were reviewed and 8 cases from 2017-2019 were selected and confirmed as ccpRCC by three pathologists. Tumors were microdissected and DNA from FFPE was extracted and profiled using the Illumina MethylationEPIC array. Methylation data were analyzed with the R Bioconductor package minfi, including quality control, data normalization and differentially methylated CpG site analysis. Subsequent filtering was performed using a p-value cutoff = 0.01 and a minimum mean difference of the Beta-value of 0.1. Clustering was performed using tSNE analysis. Copy numbers were analyzed using conumee package. The methylation data were compared to ccRCC and pRCC from publicly available TCGA dataset.
Result(s): All 8 cases passed QC metrics on bisulfite conversion, hybridization and signal intensity confirming that the DNA quality was optimal for methylation study. ccpRCC clustered very tightly together illustrating that they represented a homogeneous group of tumors. When this cluster was compared to the TCGA dataset, ccpRCC was found to be located in between ccRCC and pRCC which might explain the characteristic features of this tumor subtype (figure 1). (Figure presented)
Conclusion(s): 1. DNA methylation is a useful molecular hallmark of many cancers including renal cancers and is increasingly utilized in diagnosis, prognostication, and clinical trials (epigenetic therapy). 2. Morphologically and immunohistochemically confirmed ccpRCC forms a tight cluster validating the use of methylation assay for future studies. 3. Promotor and pathway specific methylation patterns will be further studied which can distinguish the indolent clinical behavior

Background: The NCCN currently recommends microsatellite instability (MSI) or mismatch repair (MMR) immunohistochemistry (IHC) for patients with endometrial cancer (EC) as a screen for Lynch Syndrome and criteria for immune checkpoint inhibitor therapy. Recently, MSI testing has become available through next generation sequencing (NGS). We sought to compare the concordance of MMR IHC and MSI status obtained via NGS in EC.
Design(s): Patients with newly diagnosed primary EC were prospectively consented to an IRB-approved protocol. ECs were subjected to MMR IHC and targeted NGS with a panel covering over 400 cancer-related genes. MSIsensor was used to bioinformatically infer the MSI status, with an MSIsensor score >=10 deemed MSI-high. Tumor cell content was inferred bioinformatically using ABSOLUTE. Concordance between IHC and sequencing results was defined using Cohen's Kappa.
Result(s): 175 ECs were included (116 endometrioid, 17 serous, 11 carcinosarcoma, 13 mixed, 6 clear cell, 7 dedifferentiated, 5 other). 50 (29%) were considered MMRd based on the loss of at least one MMR protein by IHC, of which 30 (60%) ECs were classified as MSI-high by MSIsensor (Table). Of the 125 ECs with retained MMR expression, the vast majority (123; 98%) was molecularly concordant and was not MSI-high (Table). The overall agreement between MMRd and MSI-high by MSIsensor was 153/175 (87%), with a Kappa of 0.749 (good agreement). Of the 20 MMRd classified as non-MSI-high, 9 were MSI indeterminate and 11 microsatellite stable. Of these 11 discrepant ECs (i.e. MMRd and microsatellite stable), 4 displayed loss of MLH1 and PMS2 expression, 1 PMS2 loss and 6 MSH6 loss of expression (Table). The tumor purity was significantly lower in the 20 MMRd non-MSI-high ECs (median 26%, range 19-60%) compared to the 30 MMRd MSI-high concordant ECs (median 49%, range 20-95%; p<0.001). (Table presented)
Conclusion(s): Our findings revealed a good agreement between MMR IHC and MSI status inferred from NGS for EC. Tumor purity may falsely decrease the degree of MSI in EC. However, in addition to MSI status, multi-gene sequencing assays provide information on specific somatic/ germline mutations, copy number alterations and tumor mutational burden in a single assay