Clinical Implication of KRAS Mutation Variants in Patients With Resected Colon Cancer

Aim: This study evaluated the clinical implication of KRAS proto-oncogene, GTPase (KRAS) mutation variants in patients with resected colon cancer (CC). Patients and Methods: We retrospectively reviewed 482 patients diagnosed with CC who underwent curative surgical resection at Kyungpook National University Chilgok Hospital. The inclusion criteria were: Pathologically diagnosed with primary CC; stage I-III CC according to the 7th edition of American Joint Committee on Cancer staging system; and with available test results for KRAS mutation status. In total, 345 patients met these criteria and were included in this study. Results: Among the 345 patients, 140 (40.6%) exhibited KRAS mutations, with their incidences as follows: 90/140 (64.3%) in exon 2 codon 12, 37/140 (26.4%) in exon 2 codon 13, 1/140 (0.1%) in exon 3 codon 59, 7/140 (5.0%) in exon 3 codon 61, and 5/140 (3.6%) in exon 4 codon 146. KRAS mutation status was not a significant prognostic factor for disease-free survival or overall survival. Although there were no significant differences in survival between patients with exon 2 codon 12 and exon 2 codon 13 mutations, poorer disease-free survival (p=0.085) and overall survival (p=0.005) were seen in those with exon 3 codon 61 mutation than in others. Conclusion: KRAS mutation status was not correlated with survival, but exon 3 codon 61 mutation might be a factor for poor prognosis in patients after resection of CC.

The Unique Genetic and Histological Characteristics of DMBA-Induced Mammary Tumors in an Organoid-Based Carcinogenesis Model

Here, we report a model system using in vitro 7,12-dimethylbenz[a]anthracene (DMBA; 0.6 μM)-treated mammary tissue-derived organoids generated from heterozygous BALB/c-Trp53 knockout mice to induce tumors after injection into the nude mouse subcutis. In parallel, a single oral dose of DMBA (50 mg/kg bodyweight) to the same murine strain induced mammary adenocarcinomas, characterized by biphasic structures differentiated into luminal and myoepithelial lineages and frequent Hras mutations at codon 61. In the present study, the genetic and histological characteristics of DMBA-induced tumors in the organoid-based model were evaluated to validate its similarities to the in vivo study. The organoid-derived tumors were low-grade adenocarcinomas composed of luminal and basal/myoepithelial cells. When the organoid-derived carcinomas were passaged to other nude mice, they partly progressed to squamous cell carcinomas (SCCs). Whole exome sequencing revealed no mutations at Hras codon 61 in the organoid-derived tumors. However, various mutations were detected in other genes such as Tusc3 and Tgfbr2, which have been reported as cancer-associated or homeostatic squamous cell genes. The most common mutational pattern observed in these genes were the G:C to T:A transversions and G:C to A:T transitions, which are not typical of the mutations caused by DMBA treatment. In conclusion, DMBA exhibited carcinogenicity in the both the ex vivo and in vivo mammary carcinogenesis models, albeit with distinct histological and genetical alterations. Further studies are needed to clarify whether organoid-based carcinogenesis models generated following chemical treatment in vitro could be applied to the clarification of the novel mode of action of chemical carcinogenesis.

Combination of ultrasound and molecular testing in malignancy risk estimate of Bethesda category IV thyroid nodules: results from a single-institution prospective study

Purpose Malignancy prediction in indeterminate thyroid nodules is still challenging. We prospectively evaluated whether the combination of ultrasound (US) risk stratification and molecular testing improves the assessment of malignancy risk in Bethesda Category IV thyroid nodules. Methods Ninety-one consecutively diagnosed Bethesda Category IV thyroid nodules were prospectively evaluated before surgery by both ACR- and EU-TIRADS US risk-stratification systems and by a further US-guided fine-needle aspiration cytology (FNAC) for the following molecular testing: BRAFV600E, N-RAS codons 12/13, N-RAS codon 61, H-RAS codons 12/13, H-RAS codon 61, K-RAS codons 12/13, and K-RAS codon 61 point-mutations, as well as PAX8/PPARγ, RET/PC1, and RET/PTC 3 rearrangements. Results At histology, 37% of nodules were malignant. No significant association was found between malignancy and either EU- or ACR-TIRADS. In total, 58 somatic mutations were identified, including 3 BRAFV600E (5%), 5 N-RAS 12/13 (9%), 13 N-RAS 61 (22%), 7 H-RAS 12/13 (12%), 11 H-RAS 61 (19%), 6 K-RAS 12/13 (10%), 8 K-RAS 61 (14%) mutations and 2 RET/PTC1 (4%), 0 RET/PTC 3 (0%), 3 PAX8/PPARγ (5%) rearrangements. At least one somatic mutation was found in 28% and 44% of benign and malignant nodules, respectively, although malignancy was not statistically associated with the outcome of the mutational test. However, the combination of ACR-, but not EU-, TIRADS with the presence of at least one somatic mutation, was significantly associated with malignant histology (P = 0.03). Conclusion US risk stratification and FNAC molecular testing may synergistically contribute to improve malignancy risk estimate of Bethesda category IV thyroid nodules.

Impact of KRAS alterations in localized pancreatic cancer (PC).

431 Background: Patients (pts) with localized PC do not routinely undergo comprehensive genomic profiling (CGP) unless they develop recurrent or metastatic disease. KRAS is the most frequently mutated gene in PC, however, the impact of different KRAS mutations in localized PC has not been well characterized. We interrogated our genomic database to analyze the KRAS status in PC pts who presented with localized disease at diagnosis (Dx). Methods: We identified PC pts at our institution who underwent CGP utilizing the Foundation One CDx assay and had localized disease at initial Dx; these pts were categorized into resectable/borderline resectable PC (LPC) and locally advanced PC (LAPC). All pts with LPC and LAPC underwent neoadjuvant chemotherapy and chemoradiation prior to possible surgery (all intended therapy - AIT). Tissue from metastatic sites was used for CGP in pts who developed recurrent/metastatic disease before or after completion of AIT. The primary tumor was used for CGP in pts who completed AIT without subsequent relapse or in the absence of adequate metastatic tissue. Effect of each gene on response and survival outcomes was estimated using proportional odds and Cox regression analysis, respectively, adjusting for stage. Results: 75 pts were identified, median age at Dx was 65 years, 59% were male, 65% had a primary tumor in the pancreatic head. 38 (86%) pts with LPC completed AIT compared to 21 (68%) pts with LAPC (p<0.001). KRAS mutation was detected in 95% (71/75) of pts– 94% (67/71) in codon 12 and 6% (4/71) in codon 61. The various KRAS mutations and their association with completion of AIT is summarized in the table. The likelihood of completing AIT did not differ based on KRAS wildtype (WT) vs mutated status (p =1.00), the mutated codon (codon 12 vs. codon 61; p =1.00) or the individual KRAS point mutations (p = 0.7); however, all patients with G12A (N= 1), G12C (N=1), G12L (N=1) and G12R (N=11) mutations completed AIT. KRAS status (mutated vs. WT) and the individual KRAS mutations were not associated with overall survival (OS) after adjusting for stage (p= 0.13 and p = 0.26 respectively). Median OS for patients with LPC and LAPC, was 39 months (mos) and 29 mos respectively. Conclusions: KRAS status and individual KRAS mutations did not have an impact on completing AIT or mOS; however, these findings need to be interpreted with caution due to the inherent biases involved in such analyses. The clinical significance and functional relevance of KRAS G12A, G12C, G12L and G12R mutations, though relatively rare, needs further characterization as well as mechanistic elucidation. [Table: see text]

RAS status in circulating-tumor DNA (ctDNA) and outcomes during rechallenge treatments with anti-EGFR antibodies in metastatic colorectal cancer (mCRC).

166 Background: We have evaluated rechallenge treatment with irinotecan plus cetuximab (JACCRO CC-08, n = 36) or panitumumab (JACCRO CC-09, n = 25) in patients (pts) with KRAS wild-type mCRC [Tsuji A, WCGC 2018], and the primary endpoint of PFS rate at 3 months was met in both trials. RAS status in ctDNA may potentially predict responders of the rechallenge treatment in mCRC resistant to anti-EGFR antibody [Cremolini C, JAMA Oncol 2018]. Methods: A post-hoc biomarker study was performed to investigate an association between RAS status in ctDNA and clinical outcomes in the JACCRO phase II trials comprised mCRC pts who achieved a clinical benefit from 1st-line anti-EGFR antibody-based therapy, then had a disease progression at 2nd-line treatment. RAS status in ctDNA was analyzed at the time points of baseline, 8 weeks, and progression using OncoBEAM RAS CRC Kit for specific KRAS/ NRAS mutations, using cut-off value defined as the number of beads with amplified-mutant molecules specifically set per each codon. Results: Sixteen pts (median 67.5-y old, 50% of PS0, 25% of right-sided primary, cet/pani: 4/12) were enrolled in this study, with response rate of 0% and disease control rate (DCR) of 62.5%. RAS mutations (mt) were found at the baseline in 6 out of 16 pts (all left-sided pts with KRAS codon 12, codon 61 and/or NRAS codon 61 mt simultaneously). Pts without RAS mt at baseline experienced longer PFS in 1st-line treatment (11.5 vs. 9.0 m). The DCR was 33% in pts with RAS mt in ctDNA, while it was 80% in pts without RAS mt at baseline. Pts with RAS mt at baseline had significantly shorter PFS and OS than pts without RAS mt [median PFS 2.3 vs 4.7 m, HR 6.2 (95%CI 1.6-30.5), p = 0.013; median OS 3.8 vs. 16.0 m, HR 12.4 (95%CI 2.7-87.7), p = 0.0028]. Six of 10 pts without RAS mt at baseline acquired RAS mt ( KRAS/ NRAS codon 12 or 61) in ctDNA at progression, however there was no difference in survival between pts with/without RAS mt at progression. Conclusions: Our study demonstrated that RAS status in ctDNA using OncoBEAM RAS CRC Kit predicts survival of rechallenge treatment with anti-EGFR antibody in mCRC pts. These data can support the application of RAS monitoring into clinical practice. Clinical trial information: UMIN000015914/UMIN000015916.

Molecular biomarkers and histological parameters impact on survival and response to first- line systemic therapy of metastatic colorectal cancer patients

Background Histological parameters of primary tumour and nodal metastases are prognostic factors for survival of operable colorectal (CRC) patients, but not predictive for response rate of systemic therapy. KRAS mutations in codons 12 and 13 were first recognized as a predictive factor for resistance to anti-EGFR monoclonal antibodies. Not all patients with wild-type KRAS (wtKRAS) respond to anti-EGFR antibody treatment. Additional mechanisms of resistance may activate mutations of the other main EGFR effectors pathway, such as other mutations in RAS gene, mutations in P13K and PTEN expression. Patients and methods In the prospective study prognostic and predictive impact of histological parameters of primary tumour, KRAS and BRAF mutations on overall survival (OS) and objective response (OR) rate of metastatic CRC (mCRC) patients treated with 1st line systemic therapy were analysed. We additionally retrospectively analysed other mutations in RAS genes and their impact on survival and time to progression. Results From November 2010 to December 2012, we enrolled 154 patients in the study, 95 men and 59 women. Mutations in KRAS gene and V600E BRAF gene were found in 42% and in 3% of patients, respectively. Median OS of the patients with T1, T2 and T3 tumour was 65.4 months (95% CI, 55.7–75.6) while in patients with T4 tumour, lymphangiosis, vascular and perineural invasion it has not been reached yet. Median OS of the patients with G1, G2 and G3 of tumour differentiation was 65.6 (95% CI, 53.7–77.5) and 25.3 months (95% CI, 16.6–34.1), respectively. Median OS of the patients with stage N0, N1 and N2 was 65.6 (95% CI, 56.4–74.8) and 58.0 months (95% CI, 21.9–94.2), respectively. Median OS of wtKRAS and mutated KRAS patients was 56.5 (95% CI, 48.2–64.9) and 58 months (95% CI, 52.6–63.4), respectively. Median OS of mutated codon 12 and codon 13 patients was 57 (95% CI, 50.9–64.4) and 44 months (95% CI, 40.1–48.4), respectively. Median OS of wtBRAF and of mutated BRAF patients was 59.2 (95% CI, 52.5–65.9) and 27.6 months (95% CI, 12.6–42.5), respectively. wtKRAS significantly affected the response to the first systemic therapy (p = 0.028), while other parameters did not affected it, p= 0.07. In 14 patients (17%), additional mutations in NRAS gene, codon 61 and codon 146 were found. Median OS of wtNRAS, codon 61 and 146 patients was 67.1 months (50.3–67.6) while median OS of mutated NRAS patients has not been reached yet (p = 0.072). Median time to progression of wtNRAS, codon 61 and 146 patients was 11.7 months (10.4–14.5) while median time to progression of mutated NRAS patients was 7.9 months (6.1–11.0), (p = 0.025). Conclusions Mutated BRAF, N2 and G3 of primary tumour were poor prognostic factors for OS in mCRC patients. wtKRAS significantly affected the response to the first line systemic therapy. Histological parameters included in the analysis and mutated BRAF did not affect significantly the efficacy of 1st line systemic therapy in mCRC patients.

KRAS/NRAS/BRAF genotyping in patients with mestastatic colorectal cancer.

718 Background: mCRC is the 2º cause of death in the world. Development of targeted therapies has increased the survival. The efficacy of these drugs (such Bevacizumab and monoclonal antibodies against EGFR) depends on the use of genetic biomarkers such as KRAS and NRAS. The BRAF status acts as prognostic factor. The objective of this study was to perform a mutational analysis of KRAS/NRAS/BRAF in a cohort of 326 Spanish patients, and correlate the findings with clinical factors. Methods: We analyzed KRAS by chip-array analysis (Infiniti System, USA), NRAS using pyrosequencing on a Pyromark Q96MD instrument (Qiagen, USA) and BRAF mutations (in 80 of these samples) by means of Infiniti System (AutoGenomics, USA). Results: KRAS mutations 129/326 patients had KRAS mutation (31.2% in codon 12 and 6.7% in codon 13). The median age was 68.9, 55% male, 66.4% had left-sided tumor, 83% histologically grade 2 tumor. The pattern of spread was liver (51.1%), lung (33.3%) and peritoneum (11.6%). There were 6 types of mutation in codon 12 (p.Gly12Asp, p.Gly12Cys, p.Gly12Val, p.Gly12Arg, p.Gly12Ser, p.Gly12Ala) and 1 type in codon 13 (p.Gly13Asp). We apply a logistic model (age&sex corrected) to location, and we found association between KRAS wild type and left location, OR 1.73 [CI:1.03-2.9] p = 0.035 NRAS mutations: 13 patients had NRAS mutation (3.98%), 8 male, 92% had left-sided, 46% liver metastasis and 53.9% lung metastasis. Type of NRAS mutation: 1.6% in exon 2 (1.2% in codon 12 and 0.4% in codon 13) and 1.8% in exon 3 (0.2% in codon 59 and 1.6% in codon 61). In exon 2 we found 2 types of changes, 1 in codon 12 (p.Gly12Asp) and 1 in codon 13 (p.Gly13Arg). In exon 3 we found 5 types of changes, 1 in codon 59 (p.Ala59Thr) and 4 in codon 61 (p.Gln61Glu; p.Gln61Leu; p.Gln61Arg; p.Gln61His). BRAF mutation: 9/80 patients harboured mutations, all of them at the residue V600E. Conclusions: In our cohort 39.5% had mutations in KRAS, 3.98% in NRAS and 11% in BRAF. KRAS mutation is associated to right-sided location.