KRAS Copy Research Articles

Abstract A092: Determinants of sensitivity to BI KRASmulti inhibitor using high-throughput in-vitro drug screens

Abstract BI 3706674 is a novel small molecule inhibitor targeting KRAS in its inactive (GDP-bound) state and is currently undergoing IND enabling studies. Here, we assess the sensitivity of BI 3706674 in a large panel of cancer cell lines with the aim to identify biomarkers predictive of patient response. We employ two complementary, high-throughput cell viability screening setups: the pooled PRISM (Profiling Relative Inhibition Simultaneously in Mixtures) platform (868 cancer cell lines representing 80 tumor types) and a well-based custom screen performed in collaboration with Horizon Discovery (292 cell lines representing 32 tumor types). Based on correlation with publicly available data of over 1464 drug sensitivity profiles, the top 10 correlated drugs (ranked by Pearson correlation) are exclusively MEK inhibitors. This analysis shows that compounds with high similarity in their sensitivity profile to our KRAS inhibitor target proteins in the MAPK pathway. Correlation of drug sensitivity data with CRISPR gene dependency data for KRAS showed that drug selectivity of BI 3706674 is specific for KRAS (Pearson R = -0.49) compared to other members of the RAS family, such as HRAS or NRAS (R=0.03 & R=0.16). BI 3706674 shows sensitivity across a wide range of KRAS alleles (KRAS wild-type (wt) copy number amplification, G12V, G12C, G13D, G12D, G12A, Q61H) with the highest sensitivity for cell lines with KRAS wt amplifications (relative copy number of > 10) followed by cell lines with a KRAS G12V and G12C mutant alleles. Efficacy was observed in 8/9 cell lines with KRAS wt relative copy number of > 10 (sensitivity threshold of 1- AUC = 0.25) in the PRISM screen, emphasizing the utility of KRAS copy number as a predictive biomarker for drug response. Furthermore, KRAS copy number amplifications and KRAS expression are highly correlated features across cell lines (Pearson R = 0.72, P=2.2e-16), indicating that both KRAS copy number amplification and KRAS expression could serve as sensitivity biomarkers for BI 3706674. In this study, we show that BI 3706674 is a potent and selective KRAS inhibitor and that KRAS copy number alterations represent a highly predictive biomarker. Conclusively, high-throughput drug screens are powerful tools to define and further refine biomarkers and study drug mechanism of actions. Citation Format: Fiorella Schischlik, Antonio Tedeschi, Dorothea Rudolph, Daniel Gerlach, Birgit Wilding, Matthias Treu, Julian Fuchs, Lorenz Herdeis, Joachim Broeker, Tobias Wunberg, Andrew S. Boghossian, Matthew G. Rees, Melissa M. Ronan, Jennifer A. Roth, Darryl McConnell, Mark Pearson, Norbert Kraut, Christian Haslinger, Jesse Lipp. Determinants of sensitivity to BI KRASmulti inhibitor using high-throughput in-vitro drug screens [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A092.

The copy number and mutational landscape of recurrent ovarian high-grade serous carcinoma

The drivers of recurrence and resistance in ovarian high grade serous carcinoma remain unclear. We investigate the acquisition of resistance by collecting tumour biopsies from a cohort of 276 women with relapsed ovarian high grade serous carcinoma in the BriTROC-1 study. Panel sequencing shows close concordance between diagnosis and relapse, with only four discordant cases. There is also very strong concordance in copy number between diagnosis and relapse, with no significant difference in purity, ploidy or focal somatic copy number alterations, even when stratified by platinum sensitivity or prior chemotherapy lines. Copy number signatures are strongly correlated with immune cell infiltration, whilst diagnosis samples from patients with primary platinum resistance have increased rates of CCNE1 and KRAS amplification and copy number signature 1 exposure. Our data show that the ovarian high grade serous carcinoma genome is remarkably stable between diagnosis and relapse and acquired chemotherapy resistance does not select for common copy number drivers.

Abstract B032: KRAS copy number variation and mutant allele fractions predict in vitro response of PDX-derived human pancreatic cancer cell lines to KRASG12D inhibitor MRTX1133

Abstract Introduction: KRASG12D is the most common KRAS mutation in pancreatic ductal adenocarcinoma (PDAC). It is well known that activating point mutations in one allele are sufficient for tumor growth in patients harboring KRASG12D mutations and it is challenging to target this mutation with effective KRASG12D inhibitors. Driver gene amplification is a known mechanism of acquired resistance to therapy in pancreatic cancers. Although other genetic features, such as KRAS gene copy number variations (CNV) and mutant allele fractions (MAF), have been observed, their influence on response to treatment remains unclear. Current discovery revealed that MRTX1133 is a potent, non-covalent and high-affinity small molecule that selectively targets KRASG12D. Therefore, with the efforts underway to develop effective therapy response using the KRASG12D-targeted drug, MRTX1133, it is paramount to improve our understanding regarding the role of KRAS CNV/MAF in PDAC. Methods: Using quantitative digital PCR (DPCR), we first measured the absolute copy number of KRAS wild type (WT), and mutant type (MT) and we then calculated MAF by formula: MAF = MT allele numbers/(MT+WT allele numbers) in DNA from various KRASG12D mutant PDAC cell lines and PDX-derived primary cell lines. 3D viability and cell signaling assays determined sensitivity to MRTX1133 in established and PDX-derived primary cell lines in vitro. We also assessed the representative KRAS gene CNV of those cell lines by TaqMan real time PCR. DPCR was also utilized to determine the MAF in PDX-derived tissues. Results: We found a trend in response to MRTX1133 in KRASG12D cell lines based on their MAF and representative KRAS gene CNV. Resistant, and sensitive KRASG12D cell lines have average MAF of 75.23%, and 52.81% respectively. Most of the resistant cells have higher KRAS gene copy number gain (6-8) compared to sensitive cells (3-4). Interestingly, our data suggests that higher MAF (>53%) and gene copy numbers (>4) may predict a resistant phenotype in KRASG12D cells to MRTX1133. Mechanistically, MRTX1133 inhibits ERK1/2 signaling and increases apoptosis in sensitive cells. Representative sensitive and resistant cell lines are further evaluated for any changes in baseline and MRTX1133-induced MAF and copy number alterations by DPCR and TaqMan real time PCR. However, the results indicated that there are no significant differences in MAF and KRAS CNV in the sensitive and resistant cells on pre and post treatment of MRTX1133. We further extended our study by evaluating the MAF targeting KRASG12D in various PDX-derived tissues by quantitative DPCR. With exception to a few cases, most of the analyzed PDX tissues contained balanced MAF (~50%), which may show sensitivity to MRTX1133 according to our findings. Conclusions: Our current observations indicate 1) KRAS CN gain and MAF increase may appear to play major roles in the progression of a resistant phenotype 2) KRAS CNV and MAF status may contribute to criteria to select PDAC patients who are most likely to benefit from therapy with MRTX1133. Citation Format: Bhaswati Sarcar, Alexandra Tassielli, Liang Wang, Ruifan Dai, Meagan D. Read, Francisca Beato, Dae W. Kim, Pamela J. Hodul, Jennifer B. Permuth, Eric B. Haura, Mokenge P. Malafa, Jason B. Fleming. KRAS copy number variation and mutant allele fractions predict in vitro response of PDX-derived human pancreatic cancer cell lines to KRASG12D inhibitor MRTX1133 [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr B032.

Molecular markers as predictors of response to perioperative chemotherapy in locally advanced gastric cancer

Introduction. Perioperative FLOT chemotherapy has improved prognosis in patients with locally advanced resectable gastric cancer (GC). However, in 80 % of cases, the tumor is resistant to the therapy, resulting in unnecessary toxicity and delayed surgical treatment.Aim. Evaluation of clinico-morphological patterns of microsatellite instability, HER2 gene amplification, changes in gene copy number and their relationship with the response to perioperative FLOT chemotherapy in patients with locally advanced resectable GC.Materials and methods. The retrospective study included 185 patients. All tumor samples were assessed for HER2 and microsatellite instability status. Among all cases there were 45 patients with locally advanced T2–4N1–2 M0 GC, who underwent a total or subtotal gastrectomy with D2 lymphadenectomy and perioperative chemotherapy with FLOT. Microsatellite instability detection was performed using fragment analysis, HER2 gene amplification testing – fluorescent in situ hybridization. Also 19 patients were tested for copy number changes of the FGFR1, FGFR2, KRAS, MET, EGFR, CCND1, MYC genes using Multiplex ligation-dependent probe amplification. The endpoints were progression-free survival and objective response rate.Results. Microsatellite instability was detected in 4.8 % (9/185) of GC cases. Microsatellite instability was associated with advanced age (p = 0.005), low grade of differentiation (p = 0.011), presence of tumor-infiltrating lymphocytes (p = 0.0004), and high preoperative CA 72–4 levels (p = 0.025). Prevalence of HER2 amplification was 7.5 % (14/185). It was associated with low grade of differentiation (p = 0.048) and metastasis in regional lymph nodes (p = 0.037). PFS in patients with HER2-positive (HER2 – human epidermal growth factor receptor 2) GC treated with perioperative FLOT chemotherapy (4/45) was significantly lower than in patients with HER2-negative GC: the median was 156 and 317 days, respectively (hazard ratio 0.49; 95 % confidence interval 0.16–1.47; p = 0.0006). There was no correlation between the presence of the alteration and ORR (p = 1.0). Progression-free survival in GC patients with KRAS amplification (3/19) was significantly lower comparing with patients without it: the median was 98 and 327 days, respectively (hazard ratio 0.29; 95 % confidence interval 0.07–1.19; p <0.0001). There was no association between an increase in KRAS copy number and objective response rate (p = 1.0). For microsatellite instability and other studied markers no statistically significant correlation with progression-free survival and objective response rate was found (p >0.05).Conclusion. The presence of HER2 and KRAS amplification have been shown as promising predictive markers of the treatment failure in patients treated with perioperative FLOT chemotherapy for locally advanced resectable GC.

Somatic mutation detection and KRAS amplification in testicular germ cell tumors.

Testicular Germ Cell Tumors (TGCT) are the most common cancer among young adult men. The TGCT histopathology is diverse, and the frequency of genomic alterations, along with their prognostic role, remains largely unexplored. Herein, we evaluate the mutation profile of a 15-driver gene panel and copy number variation of KRAS in a large series of TGCT from a single reference cancer center. A cohort of 97 patients with TGCT, diagnosed at the Barretos Cancer Hospital, was evaluated. Real-time PCR was used to assess copy number variation (CNV) of the KRAS gene in 51 cases, and the mutation analysis was performed using the TruSight Tumor 15 (Illumina) panel (TST15) in 65 patients. Univariate analysis was used to compare sample categories in relation to mutational frequencies. Survival analysis was conducted by the Kaplan-Meier method and log-rank test. KRAS copy number gain was a very frequent event (80.4%) in TGCT and presented a worse prognosis compared with the group with no KRAS copy gain (10y-OS, 90% vs. 81.5%, p = 0.048). Among the 65 TGCT cases, different variants were identified in 11 of 15 genes of the panel, and the TP53 gene was the most recurrently mutated driver gene (27.7%). Variants were also detected in genes such as KIT, KRAS, PDGFRA, EGFR, BRAF, RET, NRAS, PIK3CA, MET, and ERBB2, with some of them potentially targetable. Although larger studies incorporating collaborative networks may shed the light on the molecular landscape of TGCT, our findings unveal the potential of actionable variants in clinical management for applying targeted therapies.

Peripheral and Portal Venous KRAS ctDNA Detection as Independent Prognostic Markers of Early Tumor Recurrence in Pancreatic Ductal Adenocarcinoma.

KRAS circulating tumor DNA (ctDNA) has shown biomarker potential for pancreatic ductal adenocarcinoma (PDAC) but has not been applied in clinical routine yet. We aim to improve clinical applicability of ctDNA detection in PDAC and to study the impact of blood-draw site and time point on the detectability and prognostic role of KRAS mutations. 221 blood samples from 108 PDAC patients (65 curative, 43 palliative) were analyzed. Baseline peripheral and tumor-draining portal venous (PV), postoperative, and follow-up blood were analyzed and correlated with prognosis. Significantly higher KRAS mutant detection rates and copy numbers were observed in palliative compared to curative patients baseline blood (58.1% vs 24.6%; P = 0.002; and P < 0.001). Significantly higher KRAS mutant copies were found in PV blood compared to baseline (P < 0.05) samples. KRAS detection in pre- and postoperative and PV blood were significantly associated with shorter recurrence-free survival (all P < 0.015) and identified as independent prognostic markers. KRAS ctDNA status was also an independent unfavorable prognostic factor for shorter overall survival in both palliative and curative cohorts (hazard ratio [HR] 4.9, P = 0.011; HR 6.9, P = 0.008). KRAS ctDNA detection is an independent adverse prognostic marker in curative and palliative PDAC patients-at all sites of blood draw and a strong follow-up marker. The most substantial prognostic impact was seen for PV blood, which could be an effective novel tool for identifying prognostic borderline patients-guiding future decision-making on neoadjuvant treatment despite anatomical resectability. In addition, higher PV mutant copy numbers contribute to an improved technical feasibility.

KRAS Pathway Alterations in Malignant Pleural Mesothelioma: An Underestimated Player.

Simple SummaryMalignant pleural mesothelioma (MPM) is a rare, incurable cancer. KRAS pathway alterations are frequent in human MPM but have been likely underestimated by next generation sequencing studies.Malignant pleural mesothelioma (MPM) is a rare, incurable cancer of the mesothelial cells lining the lungs and the chest wall that is mainly caused by asbestos inhalation. The molecular mechanisms of mesothelial carcinogenesis are still unclear despite comprehensive studies of the mutational landscape of MPM, and the most frequently mutated genes BAP1, NF2, CDKN2A, TP53, and TSC1 cannot cause MPM in mice in a standalone fashion. Although KRAS pathway alterations were sporadically detected in older studies employing targeted sequencing, they have been largely undetected by next generation sequencing. We recently identified KRAS mutations and copy number alterations in a significant proportion of MPM patients. Here, we review and analyze multiple human datasets and the published literature to show that, in addition to KRAS, multiple other genes of the KRAS pathway are perturbed in a significant proportion of patients with MPM.

Analysis of molecular characterization: Pancreatic ductal adenocarcinoma with hepatic metastases.

4129 Background: Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal solid tumors. Distant metastasis is the leading cause of cancer death. Approximately 50% of PDAC patients are discovered to have distant metastases, mainly in the liver. Chemotherapy is the main recommended treatment for metastatic PDACs, with a median survival time of only 4-6 months and the 5-year survival of 1%. Therefore, deciphering the molecular mechanisms of metastatic PDAC is urgently for exploring effective therapeutic targets. Methods: 93 normal-paired samples from patients with PDAC were analyzed using hybridization capture-based next generation sequencing. Somatic and germline mutations were identified including 500 genes associated with tumor development. Sequencing data were analyzed to call tumor specific single nucleotide variants (SNV), small insertions and deletions (InDels), copy number alterations (CNA) and gene fusions and rearrangements. Results: A total 93 patients with PDAC were analyzed, including hepatic metastases (15/93), lymph node metastases (1/93), colon metastases (1/93), pelvic metastases (1/93), and no distant metastasis (75/93). In PDAC patients without distant metastasis, the most frequent mutated gene was KRAS (72%), followed by TP53 (60%), CDKN2A (21%), SMAD4 (17%), ARID1A (11%), RNF43 (9%), and RBM10 (8%). In PDAC patients with hepatic metastases, we found that the most frequent one was KRAS (100%), followed by TP53 (87%), CDKN2A (40%), SMAD4 (40%), RNF43 (13%), and RBM10 (6%). Furthermore, four PDAC patients occurred KRAS copy number amplification, including 2 cases of hepatic metastases. And a hepatic metastases PDAC patient had ERBB2 and MET copy number amplification. We also found that seven no-metastasis patients carried pathogenic or likely pathogenic germline variants, including BRIP1, PALB2, RECQL, ATM, RAD50, FANCM and BRCA1. Conclusions: High frequency mutation genes in PDAC patients with hepatic metastases were highly consistent with those of no-metastasis patients. KRAS and TP53 gene co-mutations were extensively mutated in hepatic metastases patients. Our results revealed the molecular mechanisms driving PDAC hepatic metastases should be considered in further study.

Circulating KRAS variant-specific shedding and association with survival in patients with metastatic pancreatic ductal adenocarcinoma (mPDAC) receiving chemoimmunotherapy.

2548 Background: Circulating tumor DNA (ctDNA) is increasingly used as a prognostic marker with high ctDNA shedding associated with poor survival. Gene-, but not variant-specific, differences in ctDNA shedding have been reported. Tumor burden, mitotic rate, and cell death rate have been proposed as contributors to ctDNA shedding. Here we investigate associations of ctDNA shedding for the two most common mPDAC KRAS variants, G12D and G12V, with tumor burden, mitotic score, and overall survival (OS). Methods: Pretreatment (baseline) ctDNA was analyzed by droplet digital PCR for 86 (including 44 G12D, 30 G12V) patients with mPDAC receiving front-line chemoimmunotherapy in a randomized open-label Phase II study (NCT03214250). Baseline tumor burden in total, within the pancreas, and distally, was assessed by sum of RECIST target lesion diameters. Tumor tissue variant allele fraction (tVAF) and mitotic score (geometric mean expression of 65 mitosis-associated genes) were calculated from DNA and RNA sequencing. Results: ctKRAS shedding (dichotomized at median mutant copies/mL) was associated with OS for G12D bearing tumors (p = 0.03) but not G12V (p = 0.17, log-rank test). To identify variant-specific features of shedding, we examined the Spearman correlation for total tumor burden and ctKRAS shedding; G12D but not G12V shedding was correlated with tumor burden (p = 0.01 and p = 0.22 respectively). However, the higher tVAF in G12V compared to G12D tumors (p = 0.048, Mann-Whitney test) could result from differences in purity, ploidy, and KRAS copy number. Thus, we used tVAF as a scalar to calculate an adjusted tumor burden which was significantly correlated with both G12D and G12V ctDNA shedding (p = 0.004 and 0.02, respectively). When a patient’s distal vs. pancreatic lesions were analyzed separately, pancreatic tumor burden was not correlated with G12D or G12V shedding (p = 0.10 and 0.33, respectively) but distal burden was correlated with both (p = 0.001 and 0.02, respectively). While there was no difference by KRAS variant for the correlation between adjusted tumor burden and shedding, these results do suggest that, in patients with mPDAC, distal rather than primary tumor burden may drive ctDNA shedding. Finally, tumor mitotic rate was combined with adjusted total tumor burden in a linear regression model; both were significant for predicting G12D shedding (p = 0.007 and p &lt; 0.0001, respectively) but not for G12V (p = 0.045 and p = 0.16, respectively). Conclusions: These data suggest that ctDNA shedding and survival associations may be KRAS variant-specific in mPDAC. Tumor mitotic score and location of tumors may explain some variant-specific differences in shedding. As clinical ctDNA tests become more widely used, further investigation of variant-specific shedding in KRAS and other genes may be key for proper interpretation of ctDNA tests.

KRAS gene status in gastric signet-ring cell carcinoma patients and acts as biomarker of MEK inhibitor.

Signet-ring cell carcinoma (SRCC) is a specific subtype of stomach cancer with unique epidemiology. Here, we sought to explore the role of KRAS in SRCC. KRAS status was studied both in The Cancer Genome Atlas (TCGA) and internal cohorts. Immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) were performed in formalin-fixed and paraffin-embedded (FFPE) samples. We explored patients' survival and clinicopathological characteristics in terms of KRAS mutation and expression. We also explored KRAS status and drug response curve of MEK/mTOR inhibitors in SRCC cell lines. Patients with KRAS mutations and copy number variation (CNV) showed higher mRNA level compared to non-mutant cases (P=0.003 and P<0.001). In internal cohort, 15 samples harbored KRAS mutations. Survival analysis showed that these patients had significantly lower overall survival (OS) (P=0.048). We further analyzed 75 patients with sufficient FFPE samples. Eight patients showed KRAS mutations and one patient showed KRAS amplification. The median OS was 12.5 months for patients with KRAS mutation, and 19.5 months for patients without KRAS mutation (P=0.005). Positive expression of KRAS as shown by IHC was detected in majority of SRCC samples, which was higher than our intestinal cohort (28% vs. 12.6%, P=0.033). We further explored the correlation between KRAS status and drug sensitivity in 4 SRCC cell lines. SNU601 and SNU668, which harbored KRAS mutation, were hypersensitive to MEK and mTOR inhibitors than KRAS wide type cell lines KATO-III and NUGC-4. Our findings demonstrate that KRAS gene plays an important role in SRCC and reveals therapeutic potential of targeting tumors by inhibiting MEK and mTOR pathways.

Digital next-generation sequencing of cell-free DNA for pancreatic cancer.

Background and AimThe clinical applicability of digital next‐generation sequencing (dNGS), which eliminates polymerase chain reaction (PCR) and sequencing error‐derived noise by using molecular barcodes (MBs), has not been fully evaluated. We evaluated the utility of dNGS of cell‐free DNA (cfDNA) in liquid biopsies obtained from patients with pancreatic cancer.MethodsFifty‐eight patients with pancreatic cancer undergoing endoscopic ultrasound‐guided fine‐needle aspiration (EUS‐FNA) were included. Samples were subjected to sequencing of 50 cancer‐related genes using next‐generation sequencing (NGS). The results were used as reference gene alterations. NGS of cfDNA from plasma was performed for patients with a mutant allele frequency (MAF) >1% and an absolute mutant number > 10 copies/plasma mL in KRAS or GNAS by digital PCR. Sequence readings with and without MBs were compared with reference to EUS‐FNA‐derived gene alterations.ResultsThe concordance rate between dNGS of cfDNA and EUS‐FNA‐derived gene alterations was higher with than without MBs (p = 0.039), and MAF cut‐off values in dNGS could be decreased to 0.2%. dNGS using MBs eliminated PCR and sequencing error by 74% and 68% for TP53 and all genes, respectively. Overall, dNGS detected mutations in KRAS (45%) and TP53 (26%) and copy number alterations in CCND2, CCND3, CDK4, FGFR1, and MYC, which are targets of molecular‐targeted drugs.ConclusionsdNGS of cfDNA using MBs is useful for accurate detection of gene alterations even with low levels of MAFs. These results may be used to inform the development of diagnostics and therapeutics that can improve the prognosis of pancreatic cancer.

Prognostic and predictive effect of KRAS gene copy number and mutation status in early stage non-small cell lung cancer patients.

BackgroundIn the current analysis, we characterize the prognostic significance of KRAS mutations with concomitant copy number aberrations (CNA) in early stage non-small cell lung cancer (NSCLC), and evaluate the ability to predict survival benefit from adjuvant chemotherapy.MethodsClinical and genomic data from the LACE (Lung Adjuvant Cisplatin Evaluation)-Bio consortium was utilized. CNAs were categorized as Gain (CN ≥2) or Neutral (Neut)/Loss; KRAS status was defined as wild type (WT) or mutant (MUT). The following groups were compared in all patients and the adenocarcinoma subgroup, and were correlated to survival endpoints using a Cox proportional hazards model: WT + Neut/Loss (reference), WT + Gain, MUT + Gain and MUT + Neut/Loss. A treatment-by-variable interaction was added to evaluate predictive effect.ResultsOf the 946 (399 adenocarcinoma) NSCLC patients, 41 [30] had MUT + Gain, 145 [99] MUT + Neut/Loss, 125 [16] WT + Gain, and 635 [254] WT + Neut/Loss. A non-significant trend towards worse lung cancer-specific survival (LCSS; HR =1.34; 95% CI, 0.83–2.17, P=0.232), DFS (HR =1.34; 95% CI, 0.86–2.09, P=0.202) and OS (HR =1.59; 95% CI, 0.99–2.54, P=0.055) was seen in KRAS MUT + Gain patients relative to KRAS WT + Neut/Loss patients. A negative prognostic effect of KRAS MUT + Neut/Loss was observed for LCSS (HR =1.32; 95% CI, 1.01–1.71, P=0.038) relative to KRAS WT + Neut/Loss on univariable analysis, but to a lesser extent after adjusting for covariates (HR =1.28; 95% CI, 0.97–1.68, P=0.078). KRAS MUT + Gain was associated with a greater beneficial effect of chemotherapy on DFS compared to KRAS WT + Neut/Loss patients (rHR =0.33; 95% CI, 0.11–0.99, P=0.048), with a non-significant trend also seen for LCSS (rHR =0.41; 95% CI, 0.13–1.33, P=0.138) and OS (rHR =0.40; 95% CI, 0.13–1.26, P=0.116) in the adenocarcinoma subgroup.ConclusionsA small prognostic effect of KRAS mutation was identified for LCSS, and a trend towards worse LCSS, DFS and OS was noted for KRAS MUT + Gain. A potential predictive effect of concomitant KRAS mutation and copy number gain was observed for DFS in adenocarcinoma patients. These results could be driven by the small number of patients and require validation.

A Protocol for Cancer-Related Mutation Detection on Exosomal DNA in Clinical Application.

BackgroundRecently, some genomic mutations in exosomal DNA have been found to be related to disease progress and clinical outcomes of patients in several cancers. Unfortunately, the methods for exosome isolation and exosomal DNA analysis are still lack of relevant research to ensure their optimal performance and the comparability. Here we aim to establish a protocol for cancer-related mutation detection on exosomal DNA in clinical application.MethodsTaking KRAS mutation in pancreatic cancer as an example, we tested whether the types of blood samples, the potential factors in the courses of exosome isolation and exosomal DNA preparation, as well as the detail in mutation detection by droplet digital PCR (ddPCR) could influence the exosomal DNA analysis.ResultsWe found that the concentration of exosomal DNA from serum was higher than that from plasma, whereas the mutant allele fraction (MAF) of KRAS in serum-derived exosomal DNA was obviously lower. The membrane-based method for exosome isolation showed no evident difference in both exosomal DNA yield and KRAS MAF from the classical ultracentrifugation method. DNase I pretreatment on exosomes could remove the wild-type DNA outside of exosomes and increase the KRAS MAF. PBS might interfere with the effect of DNase I and should not be recommended as resuspension buffer for exosomes if the subsequent experiments would be done with exosomal DNA. Besides, the denaturation of exosomal DNA before droplet generation during ddPCR could effectively improve the total KRAS copy number and mutation-positive droplet number.ConclusionThis study provides some methodological evidences for the selection of the optimal experimental conditions in exosomal DNA analysis. We also suggest a protocol for mutation detection on exosomal DNA, which might be suitable for the clinical testing and could be helpful to the comparison of results from different laboratories.

Abstract B04: High-level expression of oncogenic KRAS is required to transform LKB1 mutant tissue in vivo

Abstract Although activating mutations in the GTPase KRAS are oncogenic and implicated in various malignancies, mutations in KRAS alone are inefficient to transform epithelia. In lung adenocarcinoma, KRAS mutations occur at high frequency and co-segregate with loss of the LKB1 tumor suppressor, a master serine/threonine kinase that phosphorylates and activates multiple AMPK family kinases to control energy homeostasis, cell polarity, and cell:matrix signaling. Despite our current knowledge of LKB1 biology, the complexity of the signaling pathway has precluded investigations into the precise mechanism by which alteration of this kinase contributes to oncogenic transformation and tumor progression in vivo. In the present study, we introduced mutations in Kras and Lkb1 in the genetically tractable organism Drosophila melanogaster. We show that low-level expression of oncogenic Kras (KrasLow) promotes the survival of Lkb1-mutant tissue, eventually leading to a G1 arrest, and concomitant overgrowth of the surrounding tumor microenvironment. Alternatively, high-level expression of oncogenic Kras (KrasHigh) promotes the neoplastic transformation of Lkb1-mutant tissue, leading to autonomous growth and increased cell cycle progression, invasion of mutant tissue into the brain, and the survival and growth of explanted tumor tissue in a wild-type Drosophila host. Interestingly, phosphorylation of the canonical Lkb1 target AMPK is maintained in KrasHigh/Lkb1 neoplastic tissue, but not in KrasLow/Lkb1 tissue. Finally, we analyzed TCGA Pan Lung Cancer data and show that high-level, but not low-level, expression of oncogenic KRAS results in worse overall survival in the context of LKB1 loss. Taken together, these data suggest that increasing levels of oncogenic KRAS, perhaps through mutant KRAS copy gains, are a driving event in the malignant transformation of LKB1-mutant tissue, and that the rewired AMPK signaling axis may offer a potential therapeutic target for patients with this subset of mutations. Citation Format: Briana B. Rackley, Chang-Soo Seong, Melissa Gilbert-Ross. High-level expression of oncogenic KRAS is required to transform LKB1 mutant tissue in vivo [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr B04.

KRAS mutations by digital PCR in circulating tumor cells isolated from the mesenteric vein are associated with residual disease and overall survival in resected colorectal cancer patients.

In colorectal cancer (CRC), circulating tumor cells (CTCs) are released into the mesenteric veins (MV). We chose to determine whether KRAS mutations detected in CTCs from blood obtained at the time of surgery could be a marker of survival. From 52 surgically resected CRC patients who later relapsed, samples of tumor tissue, normal tissue, and blood from the peripheral vein (PV) and MV were obtained from each patient at the time of surgery. KRAS mutations were assessed by Sanger sequencing and digital PCR (DGPCR) in tissue samples and by DGPCR in CTCs. Mutant KRAS copy number was assessed in CTCs. Results were correlated with overall survival (OS). Sanger sequencing detected KRAS mutations in ten tumor samples (19.2%), while DGPCR detected mutations in 30 (58%). Mutations were detected in CTCs in 21 MV samples (40.4%) and 18 PV samples (34.6%). Patients with G13D mutations in CTCs from the MV had shorter OS than those with G12D mutations (28.1 vs 54.6months; p = 0.025). Patients with a high mutant KRAS copy number in CTCs had shorter OS than those with a low mutant KRAS copy number (MV: 20.5 vs 43.7months; p = 0.002; PV: 15.1 vs 38.2months; p = 0.027). DGPCR is more efficient than Sanger sequencing for detecting KRAS mutations. KRAS G13D mutations and high mutant KRAS copy number are associated with shorter OS. The analysis of KRAS mutations in CTCs from blood obtained at the time of surgery can identify patients with a higher risk of relapse.

Analysis of circulating cell-free DNA identifies KRAS copy number gain and mutation as a novel prognostic marker in Pancreatic cancer

Serial biopsy of pancreatic ductal adenocarcinoma (PDAC), to chart tumour evolution presents a significant challenge. We examined the utility of circulating free DNA (cfDNA) as a minimally invasive approach across a cohort of 55 treatment-naïve patients with PDAC; 31 with metastatic and 24 with locally advanced disease. Somatic mutations in cfDNA were detected using next generation sequencing in 15/24 (62.5%) and 27/31 (87%) of patients with locally advanced and metastatic disease, respectively. Copy number changes were detected in cfDNA of 10 patients of whom 7 exhibited gain of chromosome 12p harbouring KRAS as well as a canonical KRAS codon 12 mutation. In multivariable Cox Regression analysis, we show for the first time that patients with KRAS copy number gain and KRAS mutation have significantly worse outcomes, suggesting that this may be linked to PDAC progression. The simple cfDNA assay we describe will enable determination of the presence of KRAS copy number gain and KRAS mutations in larger studies and clinical trials.

Abstract 3660: Can the presence of fusion genes in circulating cell-free DNA serve as potential biomarkers for Ewing's sarcoma

Abstract Background: Circulating cell-free DNA (cfDNA) is fragmented DNA derived from tumors that circulates in the blood of patients. With liquid biopsy, cfDNA has the potential to be used to monitor tumor burden in real time. Most reports have described that tumor-specific alterations in cfDNA are mutations, although little has been reported for the detection of tumor specific gene fusions. The objective of this study was to identify whether cfDNA can be used to detect tumor specific gene fusions and whether this would reflect the tumor burden of patients with Ewing's sarcoma. Patients and Methods: Multiplex long-range genomic PCR was performed to detect patient-specific genomic breakpoints in eight patients. Probes for digital PCR were designed based on the genomic breakpoint of each patient. cfDNA was extracted from the serum of patients, which was drawn at multiple time-points (pretreatment, completion of the treatment, and/or relapse). cfDNA quantification was performed by digital PCR (BioRad Qx-200). Results: Gene fusions in the cfDNA were detected in six (75%) of eight patients with high specificity. The relative gene fusion copy ratio (gene fusion copy number/wild KRAS copy number) was higher in the patients with M1b (extrapulmonary metastases at diagnosis) compared to the patients with M1a (pulmonary metastases at diagnosis) or M0 (no metastasis), although no significant relationship was observed between the relative gene fusion copy ratio and chemosensitivity. In each patient, the relative gene fusion copy ratio was the highest in pretreatment samples and decreased as the treatment progressed. Patients with relapse displayed an elevated relative gene fusion copy ratio in advance of clinical manifestation or elevation of lactase dehydrogenase. Conclusion: The amount of cfDNA in the serum strongly correlated with the treatment stage and would be useful as an early biomarker for Ewing's sarcoma patients. Citation Format: Shintaro Iwata, Hajime Kageyama, Tsukasa Yonemoto, Makiko Itami. Can the presence of fusion genes in circulating cell-free DNA serve as potential biomarkers for Ewing's sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3660.

Integrated Molecular Characterization of Testicular Germ Cell Tumors

SUMMARYWe studied 137 primary testicular germ cell tumors (TGCTs) using high-dimensional assays of genomic, epigenomic, transcriptomic, and proteomic features. These tumors exhibited high aneuploidy and a paucity of somatic mutations. Somatic mutation of only three genes achieved significance—KIT, KRAS, and NRAS—exclusively in samples with seminoma components. Integrated analyses identified distinct molecular patterns that characterized the major recognized histologic subtypes of TGCT: seminoma, embryonal carcinoma, yolk sac tumor, and teratoma. Striking differences in global DNA methylation and microRNA expression between histology subtypes highlight a likely role of epigenomic processes in determining histologic fates in TGCTs. We also identified a subset of pure seminomas defined by KIT mutations, increased immune infiltration, globally demethylated DNA, and decreased KRAS copy number. We report potential biomarkers for risk stratification, such as miRNA specifically expressed in teratoma, and others with molecular diagnostic potential, such as CpH (CpA/CpC/CpT) methylation identifying embryonal carcinomas.

Mutant KRAS Status Is Associated with Increased KRAS Copy Number Imbalance: a Potential Mechanism of Molecular Heterogeneity.

Mutation rates determined by allele-specific PCR can be highly different in KRAS exon 2 mutant colorectal carcinoma (CRC) samples suggesting intratumoural heterogeneity. To address the effect of KRAS gene copy number on the relative mutant allele frequency the KRAS locus was individually quantified following FISH analysis in 36 cases. We observed, that mutant KRAS status was associated with an elevated KRAS locus number (2.36±0.42 vs 2.63±0.75; p=0.037) reflecting an increased aneuploidy status but no true amplification of the locus. In parallel, KRAS locus copy numbers showed significant intercellular variability (1-16 copies/cell nucleus) within individual tumours also indicating to a dynamic intratumoural oscillation of the mutant allele copy number. In conclusion, aneusomy is a common feature of KRAS mutant CRC and KRAS copy number variations may have an impact on the relative mutant allele frequency detected by allele specific PCR/sequencing), potentially leading to subclonal diversity and influencing tumour behaviour.

Mutant Kras copy number defines metabolic reprogramming and therapeutic susceptibilities.

SummaryThe RAS/MAPK-signalling pathway is frequently deregulated in non-small cell lung cancer (NSCLC), often through KRAS activating mutations1-3. A single endogenous mutant Kras allele is sufficient to promote lung tumour formation in mice but malignant progression requires additional genetic alterations4-7. We recently showed that advanced lung tumours from KrasG12D/+;p53-null mice frequently exhibit KrasG12D allelic enrichment (KrasG12D/Kraswild-type>1)7, implying that mutant Kras copy gains are positively selected during progression. Through a comprehensive analysis of mutant Kras homozygous and heterozygous MEFs and lung cancer cells we now show that these genotypes are phenotypically distinct. In particular, KrasG12D/G12D cells exhibit a glycolytic switch coupled to increased channelling of glucose-derived metabolites into the TCA cycle and glutathione biosynthesis, resulting in enhanced glutathione-mediated detoxification. This metabolic rewiring is recapitulated in mutant KRAS homozygous NSCLC cells and in vivo, in spontaneous advanced murine lung tumours (which display a high frequency of KrasG12D copy gain), but not in the corresponding early tumours (KrasG12D heterozygous). Finally, we demonstrate that mutant Kras copy gain creates unique metabolic dependences that can be exploited to selectively target these aggressive mutant Kras tumours. Our data demonstrate that mutant Kras lung tumours are not a single disease but rather a heterogeneous group comprised of two classes of tumours with distinct metabolic profiles, prognosis and therapeutic susceptibility, which can be discriminated based on their relative mutant allelic content. We also provide the first in vivo evidence of metabolic rewiring during lung cancer malignant progression.