Search

Background Tumor mutational burden (TMB) measurements aid in identifying patients who are likely to benefit from immunotherapy; however, there is empirical variability across panel assays and factors contributing to this variability have not been comprehensively investigated. Identifying sources of variability can help facilitate comparability across different panel assays, which may aid in broader adoption of panel assays and development of clinical applications. Materials and methods Twenty-nine tumor samples and 10 human-derived cell lines were processed and distributed to 16 laboratories; each used their own bioinformatics pipelines to calculate TMB and compare to whole exome results. Additionally, theoretical positive percent agreement (PPA) and negative percent agreement (NPA) of TMB were estimated. The impact of filtering pathogenic and germline variants on TMB estimates was assessed. Calibration curves specific to each panel assay were developed to facilitate translation of panel TMB values to whole exome sequencing (WES) TMB values. Results Panel sizes >667 Kb are necessary to maintain adequate PPA and NPA for calling TMB high versus TMB low across the range of cut-offs used in practice. Failure to filter out pathogenic variants when estimating panel TMB resulted in overestimating TMB relative to WES for all assays. Filtering out potential germline variants at >0% population minor allele frequency resulted in the strongest correlation to WES TMB. Application of a calibration approach derived from The Cancer Genome Atlas data, tailored to each panel assay, reduced the spread of panel TMB values around the WES TMB as reflected in lower root mean squared error (RMSE) for 26/29 (90%) of the clinical samples. Conclusions Estimation of TMB varies across different panels, with panel size, gene content, and bioinformatics pipelines contributing to empirical variability. Statistical calibration can achieve more consistent results across panels and allows for comparison of TMB values across various panel assays. To promote reproducibility and comparability across assays, a software tool was developed and made publicly available.

Over the past decade, several distinct novel renal epithelial neoplasms driven by underlying tuberous sclerosis comples ( TSC)/ mammalian target of rapamycin (MTOR) pathway mutations have been described. We report herein two distinctive TSC2 -mutated renal cell carcinomas which do not fit any previously described entity. The two renal carcinomas occurred in young patients (ages 10 and 31 y), and were characterized by highly permeative growth within the kidney with metastases to perirenal lymph nodes. The neoplastic cells were predominantly large, multinucleated giant cells having variably eosinophilic to xanthomatous cytoplasm with basophilic stippling and frequent vacuolization. While the discohesive nature of the neoplastic cells, xanthomatous cytoplasm, immunoreactivity for histiocytic markers and minimal immunoreactivity for conventional epithelial markers raised the possibility of a histiocytic neoplasm, multifocal immunoreactivity for cytokeratin 20 helped establish their epithelial nature. Despite the aggressive growth pattern of these neoplasms and lymph node metastases, mitotic figures were rare and Ki-67 indices were low (1%). One patient with follow-up shows no evidence of disease seven years after nephrectomy with no adjuvant therapy. Next-generation sequencing demonstrated TSC2 mutations in each case. By immunohistochemistry, downstream markers of mTOR pathway activation S6K1, 4EBP1, and glycoprotein nonmetastatic melanoma protein B were all highly expressed in these neoplasms, suggesting mTOR pathway activation as the neoplastic driver. While the cytokeratin 20 immunoreactivity and focal basophilic cytoplasmic stippling suggest a relationship to eosinophilic solid and cystic renal cell carcinoma, and cytoplasmic vacuolization suggests a relationship to eosinophilic vacuolated tumor, these neoplasms appear to be distinctive given their permeative growth patterns and predominant xanthomatous giant cell morphology. Addition of cytokeratin 20 to a panel of epithelial markers helps avoid misdiagnosis in such cases.

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. Electronic supplementary material The online version of this article (10.1186/s12885-019-5864-1) contains supplementary material, which is available to authorized users.

Fields of forensics, transplantation, and paternity rely on human identity testing. Currently, this is accomplished through amplification of microsatellites followed by capillary electrophoresis. An alternative and theoretically better approach uses multiple single-nucleotide polymorphisms located within a small region of DNA, a method we initially developed using HLA-A and called haplotype counting. Herein, we validated seven additional polymorphic loci, sequenced a total of 45 individuals from three of the 1000 Genomes populations (15 from each), and determined the number of haplotypes, heterozygosity, and polymorphic information content for each locus. In addition, we developed a multiplex PCR that amplifies five of these loci simultaneously. Using this strategy with a small cohort of leukemic patients who underwent allogeneic bone marrow transplantation, we first attempted to define a threshold (0.26% recipient) by examining seven patients who tested all donor and did not relapse. Although this initial threshold will need to be confirmed in a larger cohort, we detected increased recipient DNA above this threshold 90 to 145 days earlier than microsatellite positivity, and 127 to 142 days before clinical relapse in four of eight patients (50%). Haplotype counting using these novel loci may be useful for ultrasensitive detection in fields such as bone marrow transplantation, solid organ transplant rejection, patient identification, and forensics.

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.

Table S1. 474 specimens from 457 tumors of 455 patients with melanoma. Table S2. Patients stratified according to gender and age. Table S3. KIT mutations in 22 melanomas. Table S4. Variant allele frequency (VAF) detected by next generation sequencing. (DOCX 22 kb)

Eosinophilic solid and cystic (ESC) renal cell carcinoma (RCC) has recently been described as a potentially new subtype of RCC based upon morphologic and immunohistochemical features. These neoplasms typically demonstrate solid and cystic architecture, and the neoplastic cells contain voluminous eosinophilic cytoplasm with granular cytoplasmic stippling. There is frequently focal immunoreactivity for cytokeratin 20. Although the initial cases all occurred in adult females and had benign outcome, we recently expanded the proposed spectrum of this neoplasm to include pediatric cases, multifocal neoplasms, and a case with hematogenous metastasis. ESC has been postulated to be analogous to a subtype of RCC consistently identified in tuberous sclerosis complex patients, and while previous work has demonstrated loss of heterozygosity at the TSC1 locus and copy number gains at TSC2 in ESC RCC, these genes have not been sequenced in ESC RCC. Using capture-based and amplicon-based next-generation sequencing, we now demonstrate the consistent presence of either TSC1 or TSC2 gene mutations in pediatric ESC RCC (8/9 cases) and adult ESC RCC (6/6 cases). These included a metastatic ESC RCC which had a complete response to mTOR targeted therapy. We also found these mutations in some neoplasms with variant morphology and thus potentially expand the spectrum of ESC RCC. These include one of our adult cases which demonstrated dominant "type 2" papillary RCC morphology and 2 of 3 previously unclassified pediatric RCC with features of ESC RCC minus granular cytoplasmic stippling. We also demonstrate TSC mutations in a case of so-called "oncocytoid RCC after neuroblastoma" with identical morphology and immunoprofile, providing a molecular link between the latter and ESC RCC. In summary, ESC RCC consistently harbors actionable TSC1 or TSC2 mutations, which are infrequently seen in established subtypes of RCC. These findings support TSC1/2 mutation as a molecular marker of ESC RCC, and suggest expansion of the clinicopathologic spectrum to include neoplasms with papillary architecture, occasional cases lacking well-developed granular cytoplasmic stippling, and a subset of RCC with oncocytic features in patients who have survived neuroblastoma.

Fields of forensics, transplantation, and paternity rely on human identity testing. Currently, this is accomplished through amplification of microsatellites followed by capillary electrophoresis. An alternative and theoretically better approach uses multiple single-nucleotide polymorphisms located within a small region of DNA, a method we initially developed using HLA-A and called haplotype counting. Herein, we validated seven additional polymorphic loci, sequenced a total of 45 individuals from three of the 1000 Genomes populations (15 from each), and determined the number of haplotypes, heterozygosity, and polymorphic information content for each locus. In addition, we developed a multiplex PCR that amplifies five of these loci simultaneously. Using this strategy with a small cohort of leukemic patients who underwent allogeneic bone marrow transplantation, we first attempted to define a threshold (0.26% recipient) by examining seven patients who tested all donor and did not relapse. Although this initial threshold will need to be confirmed in a larger cohort, we detected increased recipient DNA above this threshold 90 to 145 days earlier than microsatellite positivity, and 127 to 142 days before clinical relapse in four of eight patients (50%). Haplotype counting using these novel loci may be useful for ultrasensitive detection in fields such as bone marrow transplantation, solid organ transplant rejection, patient identification, and forensics.

Phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) mutations and amplification are detected in 1% of primary lung adenocarcinomas (ADCs) and in 38% of primary lung squamous cell carcinomas. Alterations of PIK3CA in metastatic non-small cell lung carcinoma (NSCLC), however, are still not fully understood. This study investigated PIK3CA alterations in metastatic ADCs and correlated the findings with those for other commonly tested molecular abnormalities via fine-needle aspiration (FNA) and small-core biopsy materials.This study identified 115 FNA cases of metastatic lung ADC with standard lung cancer panel analysis by targeted next-generation sequencing and fluorescence in situ hybridization at the Johns Hopkins Medical Institute over a 12-month period. The panel included mutational analysis of PIK3CA, AKT, BRAF, EGFR, ERBB2, KRAS, and NRAS genes and tests of rearrangements for ALK and ROS1 genes.A PIK3CA mutation was detected in 7 of 115 cases of metastatic ADC (6.1%). The majority of the mutations were located in exon 9 or exon 20; however, a mutation in exon 1 was seen in 1 case. Furthermore, p.V344G in exon 4 was detected in 2 cases. Among cases with PIK3CA mutations, 4 had coexisting EGFR mutations, whereas 2 had a coexisting BRAF or KRAS mutation.Several common mutations as well as a novel mutation in the PIK3CA gene were observed in metastatic NSCLC (particularly ADC). The unique role, however, of PIK3CA mutations in metastatic NSCLC and the clinical implications need to be further investigated. Cancer Cytopathol 2016;124:485-92. © 2016 American Cancer Society.

BACKGROUND: Diffuse large B cell lymphoma (DLBCL) is the most common non-Hodgkin lymphoma. While most relapses occur within 2 years, a small proportion of patients present with late relapse (LR) after 5 years. As there are very few studies addressing the pathobiology of LR-DLBCL, the aim of this study is to further characterize the clinical, pathologic and molecular features of these neoplasms. METHODS: A retrospective analysis of all patients with DLBCL treated at Johns Hopkins Hospital between 1984 and 2013 was performed. Patients with low-grade lymphoma at any time-point were excluded. Disease-free intervals (DFI) of 5 years or greater were designated as LR. Five paired diagnostic (D) and relapse (R) samples were available for further studies. DNA was extracted from formalin fixed paraffin embedded tissue. IGH gene rearrangement status was determined by PCR. SNP microarray was performed, and copy number variations (CNV) were defined as loss or gain of signal over at least 2 megabases. Targeted next generation sequencing (NGS) using a cancer hotspot panel was also performed. Variant calls were generated using Torrent variant caller and a laboratory-developed analysis pipeline. RESULTS: One hundred thirty-three patients with relapsed DLBCL were identified. Forty-three (32.3%) patients were diagnosed in the pre-rituximab era. One hundred fourteen (85.7%) patients had early relapse (ER) with 99 (74.4%) patients recurring within 2 years. Nineteen (14.3%) patients had LR (mean 7.9 years; median 7.3 years; up to 15.6 years). There were no significant differences in age at diagnosis, race, staging marrow status, or overall survival (OS) in ER versus LR patients. Extra-nodal presentation at diagnosis (89.5% vs. 65.8%; p = 0.04) and extra-nodal-only disease over time (73.7% vs. 48.2%; p = 0.04) were more common in LR cases. Both groups had similar rates of recurring at a different site from the original disease (79.3% vs. 89.5%; p = 0.30). Table 1. Molecular profile of paired D and R DLBCL Patient IGH clonality comparison (D vs. R) Clonal Heterogeneity (D / R) Total CNVs (D / R) Shared CNVs Unique CNVs (% of D / % of R) 1 Same + / + 24 / 21 15 37.5 / 28.6 2 Same + / + 15 / 32 11 26.7 / 65.6 3 Same + / + 32 / 15 7 78.1 / 53.3 4 2 in D / 1 persists in R + / + 8 / 20 1 87.5 / 95.0 5 Different - / + 4 / 5 0 100 / 100 The average DFI was 7.1 years in the 5 LR patients selected for additional studies. IGH gene rearrangement analysis demonstrated identical D and R IGH clones in 3 cases (Table 1). Patient 4 showed 2 rearranged alleles at D with only 1 persisting at R. Patient 5 had lymphomas with unique IGH rearrangements. SNP microarray data demonstrated the presence of clonal heterogeneity in all but 1 sample (4 of 5 at D; 5 of 5 at R). Among the 4 patients with clonally related IGH gene rearrangements, there was only partial overlap in CNVs (approximately 40% on average) between the D and R lymphomas. The average CNVs was similar in the D and R samples (16.6 vs. 18.6 respectively; p = 0.75). Chromosomes 2, 3, 6, 9, and 17 were frequently altered, and CNVs involving the BCL-6, CDKN2A, TP53, and MYC loci were also commonly seen; but there was no systematic difference between the CNVs identified at D and R. NGS showed a variety of mutations, but no consistent pattern of mutations acquired at R. There was a nonsense mutation in exon 2 of CDKN2A in the R sample in patient 1, and both D and R samples showed the same copy-neutral loss-of-heterozygosity of 9p encompassing the CDKN2A gene. In addition, missense mutations of TP53 were detected in patients 4 (only at R) and 5 (only at D). CONCLUSIONS: This study demonstrates that LR-DLBCL is an uncommon phenomenon with most cases representing recurrence of the original disease. LR patients have similar OS as ER patients, and the only clinical factors segregating LR from ER are higher rates of extra-nodal presentation and extra-nodal-only sites of disease. Although most paired D and R cases share IGH clones, there is clear evidence of clonal heterogeneity with clonal evolution over time. This suggests that DLBCL may contain minor subclones not susceptible to chemotherapy, which persist subclinically acquiring additional mutations over time eventually generating clinically-evident relapse. In rare cases, the late “relapse” may occur as an unrelated lymphoma that arises spontaneously or secondary to the mutagenic effects of chemotherapy. The precise mechanism of this long latency is yet unclear, and requires further investigation. Disclosures Borowitz: Becton Dickinson Biosciences: Research Funding.