Kipp, Benjamin R.

Abstract Background: KRAS gene mutations are present in approximately 40% of colorectal adenocarcinomas and predict for nonresponse to the anti-epidermal growth factor receptor antibodies used in routine clinical practice. The most common activating mutations in this malignancy occur in codons 12 and 13 of exon 2. Determination of KRAS mutational status from tumor samples is an essential tool for managing patients with colorectal cancer (CRC) but requires tissue obtained by invasive percutaneous or surgical biopsies. Recent advances allow for the isolation and analysis of cell-free DNA (cfDNA) from the peripheral blood. A highly sensitive multiplex technology that uses low amounts of input DNA is needed to establish cfDNA as a liquid biopsy to personalize care. Methods: Digital droplet PCR (ddPCR) is a particularly sensitive method for detecting rare and low copy targets. Related work led to 7 individual assays, and to companion 4-plex and 3-plex assays, to accommodate the 7 common KRAS mutations and internal controls. A single 7-plex assay is favored to maximize efficiency and minimize cost. We optimized the RainDrop Digital PCR System to collectively detect the G12/G13 KRAS mutations, given that documentation of a mutation (not the specific mutation) is the factor of clinical relevance. Commercially available cell lines and genomic DNA aliquots were used as reference standards and for determinations of sensitivity and limit of blank (LOB). Initial clinical validation samples included tumor DNA derived from FFPE tissue (n = 10), cfDNA from healthy donors (n = 10), and cfDNA from metastatic CRC patients with tumors of known KRAS mutation status (n = 4). All assays were run in triplicate to confirm reproducibility. Results: Custom Taqman MGB probes were selected for KRAS wild type (WT), G12A, G12C, G12D, G12R, G12S, G12V, and G13D to allow for testing in a single reaction. LOB was 9 per 1×10(6) droplets on the basis of WT controls, including cfDNA samples from healthy donors. Evaluation of cell line DNA, genomic DNA aliquots, and FFPE samples demonstrates that the assay detects mutant KRAS with 100% accuracy and 0.1% mutant DNA in a total input of 10 ng WT/mutant DNA. With the clinical samples, concordant KRAS findings between cfDNA and tissue were obtained for two patients with WT tumors and one patient with a KRAS G12S mutant tumor. cfDNA analysis did not detect the G12V mutation identified in the colon primary of the other patient. Evaluation of additional clinical samples is in progress. Conclusions: Our 7-plex G12/G13 KRAS assay has excellent accuracy and is able to detect the KRAS mutations of interest at low (0.1%) mutation frequencies. Additional studies are being performed on prospectively collected plasma samples from patients with metastatic CRC of known KRAS mutation status. Large scale correlation between tumor mutation status and cfDNA findings will be performed to validate our assay for clinical implementation. Citation Format: Rajeswari Avula, Benjamin R. Kipp, Jesse S. Voss, Keegan E. Haselkorn, W. Edward Highsmith, Kevin C. Halling, Jeremy C. Jones, Steven R. Alberts, Michael B. Campion, Cassandra J. Nelson, Jin Jen, Eric D. Wieben, Julie M. Cunningham, Minetta C. Liu. Optimized 7-Plex cell-free DNA assay for clinically relevant KRAS mutations in colorectal cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 505.

Ciliated muconodular papillary tumors (CMPTs) are rare peripheral lung lesions, characterized by papillary architecture and ciliated columnar cells admixed with mucinous cells and basal cells. They often have prominent surrounding intra-alveolar mucin, which can lead to diagnostic confusion with mucinous adenocarcinoma. Recognition of the ciliated component is the key to diagnosis of CMPT. The literature contains few reported cases to date, all occurring in East-Asian patients. Although follow-up data are limited, CMPT seems to be an indolent tumor with very good prognosis, leading some to question whether it is a reactive or hamartomatous lesion. However, a very recent molecular study has identified BRAF (40%) and EGFR (30%) alterations in CMPT, supporting a truly neoplastic process. Here for the first time, we report 4 cases of morphologically typical CMPT in western patients, occurring in 1 man (60 y) and 3 women (71 to 83 y). Interestingly, 1 case occurred in background of pronounced small airway disease with necrotizing bronchiolitis and multiple carcinoid tumorlets. We further analyzed 1 tumor using a 50 gene next-generation sequencing oncology panel that identified 2 pathogenic mutations (BRAF V600E and AKT1 E17K). Our study is the first to describe that CMPT can occur in western (non-Asian) patients. Our data confirm BRAF V600E mutation as a probable driver in a subset of these tumors, along with AKT1 mutation, which further supports that CMPT are indolent pulmonary neoplasms.

Abstract Many somatic mutations have been detected in pancreatic ductal adenocarcinoma (PDAC), leading to the identification of some key drivers of disease progression, but the involvement of large genomic rearrangements has often been overlooked. In this study, we performed mate pair sequencing (MPseq) on genomic DNA from 24 PDAC tumors, including 15 laser-captured microdissected PDAC and 9 patient-derived xenografts, to identify genome-wide rearrangements. Large genomic rearrangements with intragenic breakpoints altering key regulatory genes involved in PDAC progression were detected in all tumors. SMAD4, ZNF521, and FHIT were among the most frequently hit genes. Conversely, commonly reported genes with copy number gains, including MYC and GATA6, were frequently observed in the absence of direct intragenic breakpoints, suggesting a requirement for sustaining oncogenic function during PDAC progression. Integration of data from MPseq, exome sequencing, and transcriptome analysis of primary PDAC cases identified limited overlap in genes affected by both rearrangements and point mutations. However, significant overlap was observed in major PDAC-associated signaling pathways, with all PDAC exhibiting reduced SMAD4 expression, reduced SMAD-dependent TGFβ signaling, and increased WNT and Hedgehog signaling. The frequent loss of SMAD4 and FHIT due to genomic rearrangements strongly implicates these genes as key drivers of PDAC, thus highlighting the strengths of an integrated genomic and transcriptomic approach for identifying mechanisms underlying disease initiation and progression. Cancer Res; 76(3); 749–61. ©2015 AACR.

AimsFibrolamellar carcinoma is characterized by a recurrent DNAJB1–PRKACA chimeric transcript. The functional properties of the fusion are unknown, but are believed to include PRKACA up‐regulation. PRKCA is a subunit of protein kinase A. The downstream targets of protein kinase A are unknown, but may include interactions with fibroblast growth factor receptor (FGFR) pathways. In addition, inhibitors for FGFR proteins have been developed recently.Methods and resultsNineteen histologically confirmed fibrolamellar carcinomas were studied. All showed the characteristic DNAJB1–PRKACA transcript by reverse transcription–polymerase chain reaction (RT–PCR). Immunohistochemistry for FGFR1 was negative in 19 of 19 cases using a monoclonal antibody, while a polyclonal antibody showed no expression (n = 11) or weak and focal expression (n = 8). RNAin‐situ hybridization was 2+ in two cases, 1+ in four cases and negative in four cases. FGFR1 fluorescence in‐situ hybridization (FISH) revealed polysomy of chromosome 8 in 17 of 19 cases. Break‐apart FISH for FGFR2 was negative for rearrangements in 12 of 12 informative cases.ConclusionsFibrolamellar carcinomas show polysomy of chromosome 8 and the FGFR1 locus, and only modest mRNA expression and weak or absent expression at the protein level. FGFR2 rearrangement was not detected. These data reduce the likelihood that FGFR inhibitors will be effective in the treatment of most fibrolamellar carcinomas.

Circulating tumor cells (CTCs) in blood are associated with poor survival of patients with breast, prostate, or colon cancer. We hypothesized that CTCs are associated with poor survival of patients with cholangiocarcinoma (CCA). Eighty‐eight patients with CCA were prospectively enrolled at Mayo Clinic Rochester between June 2010 and September 2014. The CellSearch system by Veridex was used for detection of CTCs in peripheral blood. Associations between CTC, patient and tumor characteristics, and survival were examined using the Cox's proportional hazards model. Fifteen patients (17%) were positive for CTC ≥2 and 8 patients (9%) for CTC ≥5. CTCs were associated with tumor extent. CTC ≥2 (hazard ratio [HR]: 2.5; 95% confidence interval [CI]: 1.1‐5.4; P = 0.02) and CTC ≥5 (HR, 4.1; 95% CI: 1.4‐10.8; P = 0.01) were both independent predictors of survival. In subgroup analyses, CTC ≥2 (HR, 8.2; 95% CI: 1.8‐57.5; P < 0.01) and CTC ≥5 (HR, 7.7; 95% CI: 1.4‐42.9; P = 0.02) were both associated with shorter survival among patients with metastasis. There was a trend toward association of CTC ≥5 with shorter survival in patients with nonmetastatic CCA (HR, 4.3; 95% CI: 1.0‐13.8; P = 0.06). CTC ≥2 (HR, 10.5; 95% CI: 2.2‐40.1; P < 0.01) and CTC ≥5 (HR, 10.2; 95% CI: 1.5‐42.3; P = 0.02) were both associated with shorter survival among patients with perihilar/distal CCA. CTC ≥5 was associated with shorter survival of patients with intrahepatic CCA (HR, 4.2; 95% CI: 1.1‐14.1; P = 0.04). Conclusion: CTCs were associated with more‐aggressive tumor characteristics and independently associated with survival in patients with CCA. Assessment of CTCs may be useful for identifying CCA patients at risk of early mortality. (Hepatology 2016;63:148–158)

Purpose Distinguishing independent primary tumors from intrapulmonary metastases in non–small-cell carcinoma remains a clinical dilemma with significant clinical implications. Using next-generation DNA sequencing, we developed a chromosomal rearrangement–based approach to differentiate multiple primary tumors from metastasis. Methods Tumor specimens from patients with known independent primary tumors and metastatic lesions were used for lineage test development, which was then applied to multifocal tumors. Laser capture microdissection was performed separately for each tumor. Genomic DNA was isolated using direct in situ whole-genome amplification methodology, and next-generation sequencing was performed using an Illumina mate-pair library protocol. Sequence reads were mapped to the human genome, and primers spanning the fusion junctions were used for validation polymerase chain reaction. Results A total of 41 tumor samples were sequenced (33 adenocarcinomas [ADs] and eight squamous cell carcinomas [SQCCs]), with a range of three to 276 breakpoints per tumor identified. Lung tumors predicted to be independent primary tumors based on different histologic subtype did not share any genomic rearrangements. In patients with lung primary tumors and paired distant metastases, shared rearrangements were identified in all tumor pairs, emphasizing the patient specificity of identified breakpoints. Multifocal AD and SQCC samples were reviewed independently by two pulmonary pathologists. Concordance between histology and genomic data occurred in the majority of samples. Discrepant tumor samples were resolved by genome sequencing. Conclusion A diagnostic lineage test based on genomic rearrangements from mate-pair sequencing demonstrates promise for distinguishing independent primary from metastatic disease in lung cancer.