CRISPR-Cas9 for selective targeting of somatic mutations in pancreatic cancers.

Somatic mutations are desirable targets for selective elimination of cancer, yet most are found within noncoding regions. We have adapted the CRISPR-Cas9 gene editing tool as a novel, cancer-specific killing strategy by targeting the subset of somatic mutations that create protospacer adjacent motifs (PAMs), which have evolutionally allowed bacterial cells to distinguish between self and non-self DNA for Cas9-induced double strand breaks. Whole genome sequencing (WGS) of paired tumor minus normal (T-N) samples from three pancreatic cancer patients (Panc480, Panc504, and Panc1002) showed an average of 417 somatic PAMs per tumor produced from single base substitutions. Further analyses of 591 paired T-N samples from The International Cancer Genome Consortium found medians of ∼455 somatic PAMs per tumor in pancreatic, ∼2800 in lung, and ∼3200 in esophageal cancer cohorts. Finally, we demonstrated 69-99% selective cell death of three targeted pancreatic cancer cell lines using 4-9 sgRNAs designed using the somatic PAM discovery approach. We also showed no off-target activity from these tumor-specific sgRNAs in either the patient's normal cells or an irrelevant cancer using WGS. This study demonstrates the potential of CRISPR-Cas9 as a novel and selective anti-cancer strategy, and supports the genetic targeting of adult cancers.

TFE3 -Rearranged PEComa/PEComa-like Neoplasms : Report of 25 New Cases Expanding the Clinicopathologic Spectrum and Highlighting its Association With Prior Exposure to Chemotherapy.

Since their original description as a distinctive neoplastic entity, ~50 TFE3 -rearranged perivascular epithelioid cell tumors (PEComas) have been reported. We herein report 25 new TFE3 -rearranged PEComas and review the published literature to further investigate their clinicopathologic spectrum. Notably, 5 of the 25 cases were associated with a prior history of chemotherapy treatment for cancer. This is in keeping with prior reports, based mainly on small case series, with overall 11% of TFE3 -rearranged PEComas being diagnosed postchemotherapy. The median age of our cohort was 38 years. Most neoplasms demonstrated characteristic features such as nested architecture, epithelioid cytology, HMB45 positive, and muscle marker negative immunophenotype. SFPQ was the most common TFE3 fusion partner present in half of the cases, followed by ASPSCR1 and NONO genes. Four of 7 cases in our cohort with meaningful follow-up presented with or developed systemic metastasis, while over half of the reported cases either recurred locally, metastasized, or caused patient death. Follow-up for the remaining cases was limited (median 18.5 months), suggesting that the prognosis may be worse. Size, mitotic activity, and necrosis were correlated with aggressive behavior. There is little evidence that treatment with MTOR inhibitors, which are beneficial against TSC -mutated PEComas, is effective against TFE3 -rearranged PEComas: only one of 6 reported cases demonstrated disease stabilization. As co-expression of melanocytic and muscle markers, a hallmark of conventional TSC -mutated PEComa is uncommon in the spectrum of TFE3 -rearranged PEComa, an alternative terminology may be more appropriate, such as " TFE3 -rearranged PEComa-like neoplasms," highlighting their distinctive morphologic features and therapeutic implications.

High-Grade, Nonsarcomatoid Chromophobe Renal Cell Carcinoma: A Series of 22 Cases With Novel Molecular Features on a Subset.

Chromophobe renal cell carcinoma (ChRCC) is the third most common subtype of renal cell carcinoma and typically exhibits indolent behavior, though a rare subset can exhibit high-grade morphologic features and is associated with a poor prognosis. Although there are limited data on the molecular characteristics of metastatic and sarcomatoid ChRCC, the molecular features of high-grade, nonsarcomatoid ChRCC remain unexplored. Herein, we characterize 22 cases of ChRCC with high-grade, nonsarcomatoid components. High-grade ChRCC frequently demonstrated advanced stage at diagnosis (64% ≥pT3a or N1), with regions of extrarenal extension, nodal metastases, and vascular invasion consisting solely of high-grade ChRCC morphologically. We performed spatially guided panel-based DNA sequencing on 11 cases comparing high-grade and low-grade regions (n = 22 samples). We identified recurring somatic alterations emblematic of ChRCC, including deletions of chromosomes 1, 2, 6, 10, 13, 17, and 21 in 91% (10/11) of cases and recurring mutations in TP53 (81.8%, n = 9/11) and PTEN (36.4%, n = 4/11). Notably, although PTEN and TP53 alterations were found in both high-grade and low-grade regions, private mutations were identified in 3 cases, indicating convergent evolution. Finally, we identified recurring RB1 mutations in 27% (n = 3) of high-grade regions leading to selective protein loss by immunohistochemistry not observed in adjacent low-grade regions. This finding was confirmed in The Cancer Genome Atlas cohort where 2 of 66 cases contained RB1 mutations and demonstrated unequivocal high-grade, nonsarcomatoid morphology. We also detected multiple chromosomal gains confined to the high-grade regions, consistent with imbalanced chromosome duplication. These findings broaden our understanding of the molecular pathogenesis of ChRCC and suggest that subclonal RB1 mutations can drive the evolution to high-grade, nonsarcomatoid ChRCC.

Using Tumor Marker Gene Variants to Improve the Diagnostic Accuracy of DUPAN-2 and Carbohydrate Antigen 19-9 for Pancreatic Cancer.

PURPOSE: Circulating carbohydrate antigen 19-9 (CA19-9) levels reflect FUT3 and FUT2 fucosyltransferase activity. Measuring the related glycan, DUPAN-2, can be useful in individuals unable to synthesize CA19-9. We hypothesized that similar to CA19-9, FUT functional groups determined by variants in FUT3 and FUT2 influence DUPAN-2 levels, and having tumor marker reference ranges for each functional group would improve diagnostic performance. MATERIALS AND METHODS: Using a training/validation study design, FUT2/FUT3 genotypes were determined in 938 individuals from Johns Hopkins Hospital: 607 Cancer of the Pancreas Screening (CAPS) study subjects with unremarkable pancreata and 331 with pancreatic ductal adenocarcinoma (PDAC). Serum DUPAN-2 and CA19-9 levels were measured by immunoassay. RESULTS: In controls, three functional FUT groups were identified with significant differences in DUPAN-2 levels: FUT3-intact, FUT3-null/FUT2-intact, and FUT3-null/FUT2-null. DUPAN-2 training set diagnostic cutoffs for each FUT group yielded higher diagnostic sensitivity in the validation set for patients with stage I/II PDAC than uniform cutoffs (60.4% [95% CI, 50.2 to 70.0] v 39.8% [30.0 to 49.8]), at approximately 99% (96.7 to 99.6) specificity. Combining FUT/CA19-9 and FUT/DUPAN-2 tests yielded 78.4% (72.3 to 83.7) sensitivity for stage I/II PDAC, at 97.7% (95.3 to 99.1) specificity in the combined sets, with higher AUC (stage I/II: 0.960 v 0.935 for CA19-9 + DUPAN-2 without the FUT test; P < .001); for stage I PDAC, sensitivity was 62.0% (49.1 to 73.2; AUC, 0.919 v 0.883; P = .03). CA19-9 levels in FUT3-null/FUT2-null PDAC subjects were higher than in FUT3-null/FUT2-intact subjects (median/IQR; 24.9/57.4 v <1/2.3 U/mL; P = .0044). In a simulated CAPS cohort, AUC precision recall (AUCPR) scores were 0.51 for CA19-9 alone, 0.64 for FUT/CA19-9, 0.73 for CA19-9/DUPAN-2, and 0.84 for FUT/CA19-9/DUPAN-2. CONCLUSION: Using a tumor marker gene test to individualize CA19-9 and DUPAN-2 reference ranges achieves high diagnostic performance for stage I/II pancreatic cancer.

"Oncocytoid Renal Cell Carcinomas After Neuroblastoma" Represent TSC -mutated Eosinophilic Solid and Cystic Renal Cell Carcinomas : Association With Prior Childhood Malignancy and Multifocality With Therapeutic Implications.

The concept of oncocytoid renal cell carcinoma in patients who have survived neuroblastoma as a distinct biologic entity has been controversial since its original description in 1999. This is in part because similar oncocytoid renal cell carcinomas have been described in association with other pediatric cancers, and also because other renal cell carcinoma subtypes (such as MiT family translocation renal cell carcinoma) have been described in children who have survived neuroblastoma. We identified an index case of a child who survived medulloblastoma and developed multifocal bilateral oncocytoid renal cell carcinomas with morphology and immunophenotype compatible with eosinophilic solid and cystic renal cell carcinoma (ESC RCC) and demonstrated that both neoplasms harbored distinctive mutations in the TSC1/TSC2 genes. Remarkably, the child's remaining bilateral multifocal renal neoplasms completely responded to MTOR inhibitor therapy without need for further surgery. To confirm our hypothesis that oncocytoid renal cell carcinomas after childhood cancer represent ESC RCC, we obtained formalin-fixed paraffin-embedded tissue blocks from 2 previously published cases of oncocytoid renal cell carcinoma after neuroblastoma, confirmed that the morphology and immunophenotype was consistent with ESC RCC, and demonstrated that both cases harbored somatic TSC gene mutations. Both expressed markers previously associated with neoplasms harboring TSC gene mutations, glycoprotein nonmetastatic B, and cathepsin K. Of note, one of these patients had 2 ESC RCC which harbored distinctive TSC2 mutations, while the background kidney of the other patient had multiple small cysts lined by similar oncocytoid cells which showed loss of TSC2 protein. We then reviewed 3 of 4 cases from the original 1999 report of oncocytoid renal cell carcinomas after neuroblastoma, found that all 3 demonstrated morphology (including basophilic cytoplasmic stippling) that is characteristic of ESC RCC, showed that all 3 overexpressed glycoprotein nonmetastatic B, and showed that both cases with adequate material demonstrated loss of TSC2 protein and expressed cytokeratin 20 and cathepsin K by immunohistochemistry. In summary, "oncocytoid renal cell carcinomas after neuroblastoma" represent ESC RCC which are often multifocal in patients who have survived childhood cancer, likely representing an incompletely characterized tumor predisposition syndrome. MTOR-targeted therapy represents an effective therapeutic option for such patients to preserve functional nephrons.

Colorectal cancer in patients of advanced age is associated with increased incidence of BRAF p.V600E mutation and mismatch repair deficiency.

Introduction: The highest incidence of colorectal cancer (CRC) is in patients diagnosed at 80 years or older highlighting a need for understanding the clinical and molecular features of these tumors. Methods. In this retrospective cohort study, 544 CRCs underwent next generation sequencing and mismatch repair (MMR) evaluation. Molecular and clinical features were compared between 251 patients with traditional-onset CRC (50-69 years at diagnosis) and 60 with late-onset CRC (>80 years at diagnosis). Results: Late-onset CRC showed a significantly higher rate of right-sided tumors (82% vs 35%), MMR deficiency (35% vs. 8%) and BRAF p.V600E mutations (35% vs. 8%) and a significantly lower rate of stage IV disease (15% vs 28%) and APC mutations (52% vs. 78%). Association of these features with advanced age was supported by stratifying patients into 6 age groups (<40, 40-49, 50-59, 60-69, 70-79 and >80 years). However, the age-related rise in MMR deficient (dMMR) CRC was only seen in the female patients with an incidence of 48% (vs. 10% in the male patient) in the >80y group. In addition, BRAF p.V600E was significantly enriched in MMR deficient CRC of advanced age (67% in late-onset CRC). Categorizing CRC by mutational profiling, late-onset CRC revealed a significantly higher rate of dMMR/BRAF + APC - (18% vs. 2.0%), dMMR/BRAF - APC - (8.3% vs. 1.2%) and MMR proficient (pMMR)/BRAF + APC - (12% vs. 4.0%) as compared to traditional-onset CRC. Discussion: In summary, there was a higher rate of dMMR and BRAF p.V600E in late-onset CRC, independently or in combination. The higher incidence of dMMR in late-onset CRC in females is most likely predominantly driven by BRAF p.V600E induced hypermethylation. Prospective studies with treatment plans designed specifically for these older patients are warranted to improve their outcomes.

CRISPR-Cas9 for selective targeting of somatic mutations in pancreatic cancers.

Somatic mutations are desirable targets for selective elimination of cancer, yet most are found within the noncoding regions. We propose a novel, cancer-specific killing approach using CRISPR-Cas9 which exploits the requirement of a protospacer adjacent motif (PAM) for Cas9 activity. Through whole genome sequencing (WGS) of paired tumor minus normal (T-N) samples from three pancreatic cancer patients (Panc480, Panc504, and Panc1002), we identified an average of 417 somatic PAMs per tumor produced from single base substitutions. We analyzed 591 paired T-N samples from The International Cancer Genome Consortium and discovered medians of ~455 somatic PAMs per tumor in pancreatic, ~2800 in lung, and ~3200 in esophageal cancer cohorts. Finally, we demonstrated >80% selective cell death of two targeted pancreatic cancer cell lines in co-cultures using 4-9 sgRNAs, targeting noncoding regions, designed from the somatic PAM discovery approach. We also showed no off-target activity from these tumor-specific sgRNAs through WGS.

Morphology-guided transcriptomic analysis of human pancreatic cancer organoids reveals microenvironmental signals that enhance invasion.

Pancreatic ductal adenocarcinoma (PDAC) frequently presents with metastasis, but the molecular programs in human PDAC cells that drive invasion are not well understood. Using an experimental pipeline enabling PDAC organoid isolation and collection based on invasive phenotype, we assessed the transcriptomic programs associated with invasion in our organoid model. We identified differentially expressed genes in invasive organoids compared with matched noninvasive organoids from the same patients, and we confirmed that the encoded proteins were enhanced in organoid invasive protrusions. We identified 3 distinct transcriptomic groups in invasive organoids, 2 of which correlated directly with the morphological invasion patterns and were characterized by distinct upregulated pathways. Leveraging publicly available single-cell RNA-sequencing data, we mapped our transcriptomic groups onto human PDAC tissue samples, highlighting differences in the tumor microenvironment between transcriptomic groups and suggesting that non-neoplastic cells in the tumor microenvironment can modulate tumor cell invasion. To further address this possibility, we performed computational ligand-receptor analysis and validated the impact of multiple ligands (TGF-ß1, IL-6, CXCL12, MMP9) on invasion and gene expression in an independent cohort of fresh human PDAC organoids. Our results identify molecular programs driving morphologically defined invasion patterns and highlight the tumor microenvironment as a potential modulator of these programs.

Aberrant p53 Expression in Gastric Biopsies and Resection Specimens Following Neoadjuvant Chemoradiation: A Diagnostic Pitfall.

Gastric mucosal biopsies and resections from patients treated with neoadjuvant radiation and/or chemotherapy are frequently encountered. These samples may show histologic features related to therapy including inflammation, ulceration, and epithelial atypia. In some cases, epithelial atypia may be marked, prompting the use of adjunct p53 immunohistochemistry. We examined p53 expression by immunohistochemistry in gastric mucosa following therapy.We evaluated the histology and p53 immunohistochemical expression in gastric mucosa from 57 resections and 3 mucosal biopsies, from 60 patients treated with radiation and/or chemotherapy for gastroesophageal carcinoma (n = 33) or pancreatic carcinoma (n = 27).We identified histomorphologic features of therapy-related epithelial changes in 50 of 60 cases (83%). Abnormal p53 expression was present at least focally in nearly half the cases (27 of 60 cases; 45%), all of which showed morphologic evidence of therapy-related epithelial changes. Neuroendocrine cell micronests were present in 37 of 60 cases (62%). Next-generation sequencing (NGS) of foci with therapy-related epithelial changes showing abnormal p53 expression and carcinoma from the same patient was attempted and yielded results in 1 patient. Interestingly, differing TP53 alterations in the patient's adenocarcinoma and in a histologically benign esophageal submucosal gland with therapy-related epithelial changes and abnormal p53 expression were identified.Our results demonstrate that abnormal p53 expression is relatively common in gastric mucosal samples following radiation and/or chemotherapy and suggest that p53 expression should be avoided when distinguishing therapy-related changes from dysplasia or carcinoma. Furthermore, our NGS results raise interesting biological questions, which may warrant further investigation.

Optimizing Insertion and Deletion Detection Using Next-Generation Sequencing in the Clinical Laboratory.

Detection of insertions and deletions (InDels) by short-read next-generation sequencing (NGS) technology can be challenging because of frequent misaligned reads. A systematic analysis of short InDels (1 to 30 bases) and fms-related receptor tyrosine kinase 3 (FLT3) internal tandem duplications (ITDs; 6 to 183 bases) from 46 clinical cases of solid or hematologic malignancy processed with a clinical NGS assay identified misaligned reads in every case, ranging from 3% to 100% of reads with the InDel showing mismapped bases. Mismaps also increased with InDel size. As a consequence, the clinical NGS bioinformatics pipeline undercalled the variant allele frequency by 1% to 84%, incorrectly called simultaneous single-base substitutions along with InDels, or did not report an FLT3 ITD that had been detected by capillary electrophoresis. To improve the ability of the pipeline to better detect and quantify InDels, we utilized a software program called Assembly-Based ReAligner (ABRA2) to more accurately remap reads. ABRA2 was able to correct 41% to 100% of the reads with mismapped bases and led to absolute increases in the variant allele frequency from 1% to 61% along with correction of all of the single-base substitutions except for two cases. ABRA2 could also detect multiple FLT3 ITD clones except for one 183-base ITD. Our analysis has found that ABRA2 performs well on short InDels as well as FLT3 ITDs that are <100 bases.

Artificial Intelligence-Assisted Serial Analysis of Clinical Cancer Genomics Data Identifies Changing Treatment Recommendations and Therapeutic Targets.

PURPOSE: Given the pace of predictive biomarker and targeted therapy development, it is unknown whether 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 reanalysis of clinical tumor sequencing data. EXPERIMENTAL DESIGN: Using artificial intelligence (AI)-assisted variant annotation, we retrospectively reanalyzed sequencing data from 2,219 patients with cancer from a single academic medical center at 3-month intervals totaling 9 months in 2020. The yield of serial reanalysis was assessed by the proportion of patients with improved strength of therapeutic recommendations. RESULTS: A total of 1,775 patients (80%) had ≥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 9, 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 reannotation 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.

Adult Wilms Tumor: Genetic Evidence of Origin of a Subset of Cases From Metanephric Adenoma.

The genetics of nephroblastoma (Wilms tumor) occurring in adults is largely unknown, as studies have largely been limited to isolated case reports. We, therefore, studied 14 adult Wilms tumors for genetic alterations, using expanded targeted sequencing on 11 cases. The patients ranged from 17 to 46 years of age (mean and median, 31 y), and there were 8 males and 6 females. Five Wilms tumors harbored BRAF V600E mutations. All of these had better-differentiated areas identical to metanephric adenoma, as has previously been described. In 3 such cases, microdissection studies revealed that the BRAF V600E mutation was present in both the metanephric adenoma and Wilms tumor areas; however, additional genetic alterations (including TERT promoter mutations in 2 cases, ASLX1/ATR mutations in 1 other case) were limited to the Wilms tumor component. These findings suggest that the Wilms tumor developed from the metanephric adenoma. Other adult Wilms tumors harbored genetic alterations previously reported in the more common pediatric Wilms tumors, including WT1 mutations (2 cases), ASLX1 mutations (3 additional cases), NSD2 mutation (1 additional case), and 11p loss (3 cases). In summary, a significant subset of adult Wilms tumors (specifically those of epithelial type with differentiated areas) harbor targetable BRAF V600E mutations and appear to arise from metanephric adenomas as a consequence of additional acquired genetic alterations. Other adult Wilms tumors often harbor genetic alterations found in their more common pediatric counterparts, suggesting at least some similarities in their pathogenesis.

Double PIK3CA Alterations and Parallel Evolution in Colorectal Cancers.

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.

Solid Organ Transplantation Is Associated with an Increased Rate of Mismatch Repair Deficiency and PIK3CA Mutations in Colorectal Cancer.

Solid organ transplants are associated with a modestly increased risk of colorectal cancers (CRC). However, the molecular profile of these cancers has not been described. We hypothesized that transplant-related immunosuppression may promote development of more immunogenic tumors as suggested by a high tumor mutation burden or mismatch repair deficiency. We performed an electronic medical record search for patients seen in the Johns Hopkins University Health System (JHHS) between 2017 and 2022 who developed CRC following solid organ transplantation. A comparator cohort of patients treated for CRC at JHHS with molecular profiling data was also identified. In this case, 29 patients were identified that developed post-transplant CRC (renal transplant, n = 18; liver transplant, n = 8; kidney-liver transplantation, n = 3). Compared to the JHHS general population CRC cohort, patients who developed post-transplant CRC had a higher rate of mismatch repair deficiency (41% versus 12%, p-value = 0.0038), and elevated tumor mutation burden (median of 22 mut/Mb versus 3.5 mut/Mb, p-value = 0.033) (range 3.52-53.65). Post-transplant tumors were enriched for PIK3CA mutations (43% versus 24%, p-value = 0.042). Post-Transplant CRCs are associated with clinical and molecular features of immune sensitivity, supporting a potential role for impaired immune surveillance in shaping the landscape of CRCs. These results may help inform the management of patients with post-transplant CRC.

Analytical Validation of SOD2 Genotyping.

Manganese superoxide dismutase-2 (SOD2) plays a crucial role in cells' protection against mitochondrial oxidative damage. A genetic polymorphism in the mitochondrial targeting sequence of the SOD2 gene has been implicated in various diseases, including prostate cancer. Paller et al. have shown an increase in prostate-specific antigen (PSA) doubling time in patients with the Ala/Ala (wildtype) genotype when treated with pomegranate/grape extract antioxidants. We developed and validated a pyrosequencing assay that detects the common germline SOD2 SNP (rs_4880) with the aim of identifying men with castrate-resistant prostate cancer eligible for an antioxidant therapy clinical trial. We first selected 37 samples from the 1000 genomes study with known genotypes determined using Illumina-based sequencing and confirmed them by Sanger sequencing. In a blinded design, we then performed the new pyrosequencing assay on these samples and assigned genotypes. Genotypes for all 37 samples (13 homozygous Ala, 12 heterozygous Ala/Val, and 12 homozygous Val) were all concordant by pyrosequencing. The pyrosequencing assay has been live since May 2018 and has proven to be robust and accurate.

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

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.

Concomitance of a novel RMDN2-ALK fusion and an EML4-ALK fusion in a lung adenocarcinoma.

The anaplastic lymphoma kinase (ALK) fusions/rearrangements in non-small cell lung cancer (NSCLC) act as oncogenic driver mutations. ALK tyrosine kinase inhibitors have anti-tumor activities in ALK-positive NSCLC. Although the EML4-ALK fusion is common in NSCLC, concomitance of an additional ALK fusion together with an EML4-ALK fusion is not common. Here, we present a lung adenocarcinoma with two ALK fusions, a novel RMDN2-ALK fusion accompanied by an EML4-ALK fusion, detected by a targeted next generation sequencing assay. The genomic translocation breakpoints of the RMDN2-ALK fusion were mapped to intron 2 for RMDN2 and exon 15 for ALK, and EML4-ALK breakpoints were mapped to intron 13 for EML4 and intron 19 for ALK. ALK break-apart FISH detected multiple ALK rearrangements, a gene fusion panel (NanoString) test confirmed the EML4-ALK fusion, and RNA-sequencing revealed two ALK fusions. The RMDN2 gene locates at the short arm of chromosome 2 between ALK and EML4 genes. The intact ALK kinase domain fused to RMDN2. Genome-wide copy number variants were found in multiple chromosome arms and the short arm of chromosome 2, suggestive of complex rearrangements. Further detailed analyses of breakpoints and copy number variants may shed light on mechanisms of their formation and pathogenesis in lung malignancies.

IDH1 and IDH2 Mutations in Colorectal Cancers.

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.

Assessment of RAS Dependency for BRAF Alterations Using Cancer Genomic Databases.

Importance: Understanding RAS dependency and mechanisms of RAS activation in non-V600 BRAF variant cancers has important clinical implications. This is the first study to date to systematically assess RAS dependency of BRAF alterations with real-world cancer genomic databases. Objective: To evaluate RAS dependency of individual BRAF alterations through alteration coexistence analysis using cancer genomic databases. Design and Setting: A cross-sectional data analysis of 119 538 nonredundant cancer samples using cancer genomics databases including GENIE (Genomics Evidence Neoplasia Information Exchange) and databases in cBioPortal including TCGA (The Cancer Genome Atlas) (accessed March 24, 2020), in addition to 2745 cancer samples from Mayo Clinic Genomics Laboratory (January 1, 2015, to July 1, 2020). Frequencies and odds ratios of coexisting alterations of RAS (KRAS, NRAS and HRAS) and RAS regulatory genes (NF1, PTPN11 and CBL) were calculated for individual BRAF alterations, and compared according to the current BRAF alteration classification; cancer type specificity of coexisting alterations of RAS or RAS regulatory genes was also evaluated. Main Outcomes and Measures: Primary outcome measurement is enrichment of RAS (KRAS, NRAS and HRAS) alterations in BRAF variant cancers. Secondary outcome measurement is enrichment of RAS regulatory gene (NF1, PTPN11, and CBL) in BRAF variant cancers. Results: A total of 2745 cancer samples from 2708 patients (female/male ratio: 1.0) tested by Mayo Clinic Genomics Laboratory and 119 538 patients (female/male ratio: 1.1) from GENIE and cBioPortal database were included in the study. In 119 538 nonredundant cancer samples, class 1 BRAF alterations and BRAF fusions were found to be mutually exclusive to alterations of RAS or RAS regulatory genes (odds ratio range 0.03-0.13 and 0.03-0.73 respectively), confirming their RAS independency. Both class 2 and class 3 BRAF alterations show variable and overlapping levels of enriched RAS alterations (odds ratio range: 0.03-5.9 and 0.63-2.52 respectively), suggesting heterogeneity in RAS dependency and a need to revisit BRAF alteration classification. For RAS-dependent BRAF alterations, the coexisting alterations also involve RAS regulatory genes by enrichment analysis (for example, S467L shows an odds ratio of 8.26 for NF1, 9.87 for PTPN11, and 15.23 for CBL) and occur in a variety of cancer types with some coalterations showing cancer type specificity (for example, HRAS variations account for 46.7% of all coexisting RAS alterations in BRAF variant bladder cancers, but 0% in non-small cell lung cancers). Variant-level assessment shows that BRAF alterations involving the same codon may differ in RAS dependency. In addition, RAS dependency of previously unclassified BRAF alterations could be assessed. Conclusions and Relevance: Current BRAF alteration classification based on in vitro assays does not accurately predict RAS dependency in vivo for non-V600 BRAF alterations. RAS-dependent BRAF variant cancers with different mechanisms of RAS activation suggest the need for different treatment strategies.