Data from A <i>KRAS</i>-Variant in Ovarian Cancer Acts as a Genetic Marker of Cancer Risk
Elena RatnerLingeng LuMarta BoekeRachel BarnettSunitha NallurLena J. ChinCory PelletierRachel BlitzblauRenata TassiTrupti ParanjapePei HuiAndrew K. GodwinHerbert YuHarvey A. RischThomas F. RutherfordPeter E. SchwartzAlessandro D. SantinEllen MatloffDaniel ZeltermanFrank J. SlackJoanne B. Weidhaas
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<div>Abstract<p>Ovarian cancer (OC) is the single most deadly form of women's cancer, typically presenting as an advanced disease at diagnosis in part due to a lack of known risk factors or genetic markers of risk. The <i>KRAS</i> oncogene and altered levels of the microRNA (miRNA) <i>let-7</i> are associated with an increased risk of developing solid tumors. In this study, we investigated a hypothesized association between an increased risk of OC and a variant allele of <i>KRAS</i> at <i>rs61764370</i>, referred to as the <i>KRAS</i>-variant, which disrupts a <i>let-7</i> miRNA binding site in this oncogene. Specimens obtained were tested for the presence of the <i>KRAS</i>-variant from nonselected OC patients in three independent cohorts, two independent ovarian case-control studies, and OC patients with hereditary breast and ovarian cancer syndrome (HBOC) as well as their family members. Our results indicate that the <i>KRAS</i>-variant is associated with more than 25% of nonselected OC cases. Further, we found that it is a marker for a significant increased risk of developing OC, as confirmed by two independent case-control analyses. Lastly, we determined that the <i>KRAS</i>-variant was present in 61% of HBOC patients without <i>BRCA1</i> or <i>BRCA2</i> mutations, previously considered uninformative, as well as in their family members with cancer. Our findings strongly support the hypothesis that the <i>KRAS</i>-variant is a genetic marker for increased risk of developing OC, and they suggest that the <i>KRAS</i>-variant may be a new genetic marker of cancer risk for HBOC families without other known genetic abnormalities. Cancer Res; 70(16); 6509–15. ©2010 AACR.</p></div>e15565 Background: KRAS mutations are common oncogenic events across cancers, but effective RAS-directed therapies are lacking. However, recent studies support use of PD-1 blockade in most subsets of lung cancer with KRAS short variant mutations (KRAS SV ) (PMID: 28039262), and preclinical data supports combined MEK and SHP2 inhibition in KRAS amplified ( KRAS a ) GEA (PMID: 30093730). We sought to explore the landscape of KRAS altered GEA and compare genomic profiles of KRAS-altered and KRAS wild-type (WT) cases for biomarkers of response to targeted therapies and immune checkpoint inhibitors. Methods: 6,667 tissue specimens from patients with advanced GEA were assayed using hybrid capture-based comprehensive genomic profiling. Tumor mutational burden (TMB) was determined on up to 1.1 Mbp of sequenced DNA and microsatellite instability (MSI) was determined on 95 or 114 loci. Descriptive statistics were used to compare among subgroups. Results: KRAS SV and KRAS a were identified in 11% and 5.8% of gastric adenocarcinoma (GA), respectively, and in 7.2% and 17% of esophageal adenocarcinoma (EA), respectively. KRAS a and KRAS SV were nearly mutually exclusive, co-occurring in only 4.4% of KRAS altered cases. ERBB2 alterations were less common in KRAS SV and KRAS a GEA (both 9%) as compared with KRAS WT GEA (19%) (p = 1.9E-16). EGFR a was less common in KRAS SV vs KRAS a GEA (1.9% vs 9.3%, p = 2.6E-8), whereas PIK3CA SV was more common in KRAS SV vs KRAS a (16% vs 5.0%, p = 1.5E-11). Median TMB for all groups was similar; however, KRAS SV GEA had a higher mean TMB (9.7 mut/Mb) as compared to KRAS a (5.1 mut/Mb, p = 5.0E-12) and KRAS WT cases (5.8 mut/Mb, p = 2.2E-07). KRAS codon 12/13 accounted for > 80% of predicted pathogenic mutations. MSI-high was also more prevalent in KRAS SV (11.3%) vs KRAS a (0.9%, p = 4.8E-15) and KRAS WT GEA (2.4%, p = 1.8E-25). MSI-high KRAS SV GEA was associated with older patient age (median 72 years) and with high TMB (median 40.9 mut/mb). Conclusions: GA was enriched for KRAS mutation whereas EA was enriched for KRAS amplification. KRAS WT vs KRAS SV vs KRAS a each presented distinct genomic profiles. KRAS a in the absence of KRAS mutation exists in 11% of GEA and warrants further exploration to inform combination treatment strategies.
Microsatellite Instability
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Abstract Allelic imbalance is reported to be a frequent event in cancers and a common feature associated with oncogenes such as KRAS and BRAF. However the functional and therapeutic consequences of such imbalances are poorly understood. Mutations in the KRAS oncogene are one of the most prevalent events in human cancers and heterozygous KRAS mutations are well-described functionally and are thus viewed as sufficient for tumorigenesis. Recent evidence shows high incidence of KRAS gene dosage changes in human cancers, including loss of the normal wild-type allele of KRAS. However, there is still much debate over the function of wild-type KRAS in tumour initiation, progression and therapy response. Using advanced genetically engineered mouse models of colorectal cancer (VillinCRE Apcfl/fl; LSL-KrasG12D/+ (AK), LSL-KrasG12D/+; Trp53fl/fl; Rosa26N1iCD/+ (KPN)) we investigated the functional impact of wild-type Kras in oncogenic Kras driven tumour initiation (AK), progression and metastasis (KPN) in vivo. Mechanistically, wild-type Kras restrains oncogenic Kras signalling and significantly affects the efficiency of the oncogenic Kras induced transformation and response to therapy. We demonstrate a suppressive role for wild-type Kras during tumorigenesis and highlight the critical impact of wild-type Kras upon therapeutic response and tumour progression in Kras mutant CRC. Wild-type KRAS-deficient colorectal tumours are characterized by increased MAPK signalling and transcriptional activation of MAPK regulators. Importantly, loss of wild-type Kras in oncogenic KRAS-driven aggressive KPN tumours significantly alter tumour progression and liver metastasis, showing increased immune infiltration and WNT signalling. In addition, loss of wild-type Kras modulates response to therapeutic intervention and sensitizes wild-type Kras deficient tumours to MEK1/2 inhibition. This study demonstrates a suppressive role for wild-type Kras during tumour initiation and highlights the critical impact of wild-type Kras upon therapeutic response to MAPK and tumour progression in KRAS mutant CRC. Citation Format: Arafath K. Najumudeen, Sigrid K. Fey, Andrew D. Campbell, Owen J. Sansom. KRAS allelic imbalance drives an epithelial MAPK-dependent tumor initiation program that is inefficient in provoking metastasis in colorectal cancer in vivo [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 A004.
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e19324 Background: KRAS G12C mutations are present in 15% of non-small cell lung cancer (NSCLC) and have recently been shown to confer sensitivity to KRAS(G12C) inhibitors. This study aims to assess the clinical features and outcomes with KRAS G12C mutant NSCLC in a real-world setting. Methods: Patients enrolled in an Australian prospective cohort study, Thoracic Malignancies Cohort (TMC), between July 2012 to October 2019 with metastatic or recurrent non-squamous NSCLC, with available KRAS test results, and without EGFR, ALK, or ROS1 gene aberrations, were selected. Data was extracted from TMC and patient records. Clinicopathologic features, treatment and overall survival was compared for KRAS wildtype ( KRAS WT ) and KRAS mutated ( KRAS mut ) patients, and between KRAS G12C ( KRAS G12C ) and other ( KRAS other ) mutations. Results: Of 1386 patients with non squamous NSCLC, 1040 were excluded for: non metastatic or recurrent (526); KRAS not tested (356); ALK, EGFR or ROS1 positive (154); duplicate (4). Of 346 patients analysed, 202 (58%) were KRAS WT and 144 (42%) were KRAS mut , of whom 65 (45%) were KRAS G12C . 100% of pts with KRAS G12C were smokers, compared to 92% of KRAS other and 83% of KRAS WT . The prevalence of brain metastases over entire follow-up period was similar between KRAS mut and KRAS WT (33% vs 40%, p = 0.17), and KRAS G12C and KRAS other (40% vs 41%, p = 0.74). Likewise, there was no difference in the proportion of patients receiving one or multiple lines of systemic therapy. Overall survival (OS) was also similar between KRAS mut and KRAS WT (p = 0.54), and KRAS G12C and KRAS other (p = 0.39). Conclusions: In this real-world prospective cohort, patients had comparable clinical features regardless of having a KRAS mut , KRAS G12C or KRAS other mutation, or being KRAS WT . Treatment and survival were also similar between groups. While not prognostic, KRAS G12C may be an important predictive biomarker as promising KRAS G12C covalent inhibitors continue to be developed.
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74 Background: KRAS mutation is rare ( < 5%) in gastroesophageal cancer (GEC). However, the incidence of KRAS gene amplification (amp+), consequent protein levels, and prognostic and/or therapeutic implications are unknown. Methods: 410 GEC samples and 30 cell lines were assessed for KRAS gene copy number (GCN) by fluorescence in situ hybridization (FISH) (n = 90), Kras expression by selected reaction monitoring mass spectrometry (Kras-SRM-MS) (n = 393), and Kras-SRM level evaluated for correlation with KRAS amp+ status (n = 73). Survival analysis was performed comparing KRAS amp+ versus non-amp+ patients. When possible, concurrent 315 gene next-generation sequencing was also performed. Four KRAS-amplified xenograft lines (CAT-2,12,14,15) were established from malignant effusions. Tumorigenic activity of KRAS amp+ lines (CAT lines, MKN-1) were assessed using MTT and soft agar assays in vitro and subcutaneous xenograft models, compared to non-amp+ lines. Inhibitory assays were performed using KRAS siRNA and CRIPSR, and commercial inhibitors targeting downstream effectors MEK and/or PIK3CA. Results: KRAS FISH revealed clustered gene amp+ in 28.9% (26/90); these patients had worse prognosis than non-amp+ patients. GCN significantly correlated with Kras expression. All KRAS amp+ cell lines significantly overexpressed Kras protein and were tumorigenic in xenograft subcutaneous models. KRAS siRNA and KRAS CRISPR of KRAS amp+ cell lines demonstrated inhibition in MTT viability and soft agar assays, compared to appropriate controls, and demonstrated significant and durable xenograft growth reduction. Conversely, inhibition using MEK and/or PI3K inhibitors demonstrated only transient growth reduction in vivo. Conclusions: KRAS gene amp+ was observed in a large subset (26%) of GEC patients, which correlated with extreme expression by mass spectrometry. Established xenograft lines serve as models to investigate therapeutic strategies for KRAS amp+ patients. Inhibition using MEK/PIK3CA inhibitors provided transient benefit for KRAS amp+ tumors while durable inhibition was observed with Kras protein knockdown, suggesting potential benefit from novel siRNA therapeutics currently in development.
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Abstract Background : KRAS is the most frequently mutated oncogene in cancer, however efforts to develop targeted therapies have been largely unsuccessful. Recently, two small-molecule inhibitors, AMG 510 and MRTX849, have shown promising activity in KRAS G12C-mutant solid tumors. The current study aims to assess the molecular profile of KRAS G12C in colorectal (CRC) and non-small-cell lung cancer (NSCLC) tested in a clinical certified laboratory. Methods : CRC and NSCLC samples submitted for KRAS testing between 2017 and 2019 were reviewed. CRC samples were tested for KRAS and NRAS by pyrosequencing, while NSCLC samples were submitted to next generation sequencing of KRAS, NRAS, EGFR, and BRAF. Results : The dataset comprised 4,897 CRC and 4,686 NSCLC samples. Among CRC samples, KRAS was mutated in 2,354 (48.1%). Most frequent codon 12 mutations were G12D in 731 samples (15.2%) and G12V in 462 (9.6%), followed by G12C in 167 (3.4%). KRAS mutations were more frequent in females than males (p=0.003), however this difference was exclusive of non-G12C mutants (p<0.001). KRAS mutation frequency was lower in the South and North regions (p=0.003), but again KRAS G12C did not differ significantly (p=0.80). In NSCLC, KRAS mutations were found in 1,004 samples (21.4%). As opposed to CRC samples, G12C was the most common mutation in KRAS, in 346 cases (7.4%). The frequency of KRAS G12C was higher in the South and Southeast regions (p=0.012), and lower in patients younger than 50 years (p<0.001). KRAS G12C mutations were largely mutually exclusive with other driver mutations; only 11 NSCLC (3.2%) and 3 CRC (1.8%) cases had relevant co-mutations. Conclusions : KRAS G12C presents in frequencies higher than several other driver mutations, represent a large volume of patients in absolute numbers. KRAS testing should be considered in all CRC and NSCLC patients, independently of clinical or demographic characteristics.
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70 Background: KRAS mutations are common oncogenic events across cancers, but effective RAS-directed therapies are lacking. However, recent studies support use of PD-1 blockade in most subsets of lung cancer with KRAS short variant mutations (KRAS SV ) (PMID: 28039262), and preclinical data supports combined MEK and SHP2 inhibition in KRAS amplified ( KRAS a ) GEA (PMID: 30093730). We sought to explore the landscape of KRAS altered GEA and compare genomic profiles of KRAS-altered and KRAS wild-type (WT) cases for biomarkers of response to targeted therapies and immune checkpoint inhibitors. Methods: 6,667 tissue specimens from patients with advanced GEA were assayed using hybrid capture-based comprehensive genomic profiling. Tumor mutational burden (TMB) was determined on up to 1.1 Mbp of sequenced DNA and microsatellite instability (MSI) was determined on 95 or 114 loci. Descriptive statistics were used to compare among subgroups. Results: KRAS SV and KRAS a were identified in 11% and 5.8% of gastric adenocarcinoma (GA), respectively, and in 7.2% and 17% of esophageal adenocarcinoma (EA), respectively. KRAS a and KRAS SV were nearly mutually exclusive, co-occurring in only 4.4% of KRAS altered cases. ERBB2 alterations were less common in KRAS SV and KRAS a GEA (both 9%) as compared with KRAS WT GEA (19%) (p = 1.9E-16). EGFR a was less common in KRAS SV versus KRAS a GEA (1.9% vs. 9.3%, p = 2.6E-8), whereas PIK3CA SV was more common in KRAS SV versus KRAS a (16% vs 5.0%, p = 1.5E-11). Median TMB for all groups was similar; however, KRAS SV GEA had a higher mean TMB (9.7 mut/Mb) as compared to KRAS a (5.1 mut/Mb, p = 5.0E-12) and KRAS WT cases (5.8 mut/Mb, p = 2.2E-07). KRAS codon 12/13 accounted for > 80% of predicted pathogenic mutations. MSI-high was also more prevalent in KRAS SV (11.4%) versus KRAS a (0.9%, p = 4.8E-15) and KRAS WT GEA (3.0%, p = 1.8E-25). MSI-high KRAS SV GEA was associated with older patient age (median 72 years) and with high TMB (median 40.9 mut/mb). Conclusions: GA was enriched for KRAS mutation whereas EA was enriched for KRAS amplification. KRAS WT versus KRAS SV versus KRAS a each presented distinct genomic profiles. KRAS a in the absence of KRAS mutation exists in 11% of GEA and warrants further exploration to inform combination treatment strategies.
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KRAS遺伝子変異は非小細胞肺癌を含むヒトの癌で頻度の高いがん遺伝子変異の一つである．発見から30年以上のKRAS変異陽性癌の治療法開発にもかかわらず，臨床的有用性を示す薬物は得られず，創薬不能な標的とされてきた．理由として，KRASとGTPの親和性は高く結合阻害は困難，KRASの下流シグナルや膜結合に必要な翻訳後修飾はいくつも平行しており，単一の経路や修飾反応の阻害では他の活性化が起こる，KRAS変異陽性癌は必ずしもKRASに生死が依存していないことなどが考えられる．2013年にGDP結合KRASに低分子化合物がはまるポケットが見出され，G12C変異KRASに限定的ながら，KRASを不活性なGDP結合型に非可逆的に固定する化合物が報告された．この発見に基づき，ソトラシブやアダグラシブなどのG12C特異的阻害剤が開発され，前者は2021年に米国で，2次治療以降のKRASG12C変異陽性非小細胞肺癌に対し迅速承認された．今後，G12C以外の直接阻害剤，G12C阻害剤との併用療法，耐性獲得後の対策，有効な患者選択のためのバイオマーカーなどについて，さらなる研究開発が待たれる．
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Abstract Background KRAS is the most frequently mutated oncogene in cancer, however efforts to develop targeted therapies have been largely unsuccessful. Recently, two small-molecule inhibitors, AMG 510 and MRTX849, have shown promising activity in KRAS G12C-mutant solid tumors. The current study aims to assess the molecular profile of KRAS G12C in colorectal (CRC) and non-small-cell lung cancer (NSCLC) tested in a clinical certified laboratory. Methods CRC and NSCLC samples submitted for KRAS testing between 2017 and 2019 were reviewed. CRC samples were tested for KRAS and NRAS by pyrosequencing, while NSCLC samples were submitted to next generation sequencing of KRAS , NRAS , EGFR , and BRAF . Results The dataset comprised 4897 CRC and 4686 NSCLC samples. Among CRC samples, KRAS was mutated in 2354 (48.1%). Most frequent codon 12 mutations were G12D in 731 samples (14.9%) and G12V in 522 (10.7%), followed by G12C in 167 (3.4%). KRAS mutations were more frequent in females than males ( p = 0.003), however this difference was exclusive of non-G12C mutants ( p < 0.001). KRAS mutation frequency was lower in the South and North regions ( p = 0.003), but again KRAS G12C did not differ significantly ( p = 0.80). In NSCLC, KRAS mutations were found in 1004 samples (21.4%). As opposed to CRC samples, G12C was the most common mutation in KRAS , in 346 cases (7.4%). The frequency of KRAS G12C was higher in the South and Southeast regions ( p = 0.012), and lower in patients younger than 50 years ( p < 0.001). KRAS G12C mutations were largely mutually exclusive with other driver mutations; only 11 NSCLC (3.2%) and 1 CRC (0.6%) cases had relevant co-mutations. Conclusions KRAS G12C presents in frequencies higher than several other driver mutations, and may represent a large volume of patients in absolute numbers. KRAS testing should be considered in all CRC and NSCLC patients, independently of clinical or demographic characteristics.
Surgical oncology
Pyrosequencing
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4065 Background: KRAS mutation is rare (< 5%) in gastroesophageal cancer (GEC). However, the incidence of KRAS gene amplification (amp+), consequent protein levels, and prognostic and/or therapeutic implications are unknown. Methods: 410 GEC samples and 30 cell lines were assessed for KRAS gene copy number (GCN) by fluorescence in situ hybridization (FISH) (n = 90), Kras expression by selected reaction monitoring mass spectrometry (Kras-SRM-MS) (n = 393), and Kras-SRM level evaluated for correlation with KRAS amp+ status (n = 73). Survival analysis was performed comparing KRAS amp+ versus non-amp+ patients. When possible, concurrent 315 gene next-generation sequencing was also performed. Four KRAS-amplified xenograft lines (CAT-2,12,14,15) were established from malignant effusions. Tumorigenic activity of KRAS amp+ lines (CAT lines, MKN-1) were assessed using MTT and soft agar assays in vitro and subcutaneous xenograft models, compared to non-amp+ lines. Inhibitory assays were performed using KRAS siRNA and CRIPSR, and commercial inhibitors targeting downstream effectors MEK and/or PIK3CA. Results: KRAS FISH revealed clustered gene amp+ in 28.9% (26/90); these patients had worse prognosis than non-amp+ patients. GCN significantly correlated with Kras expression. All KRAS amp+ cell lines significantly overexpressed Kras protein and were tumorigenic in xenograft subcutaneous models. KRAS siRNA and KRAS CRISPR of KRAS amp+ cell lines demonstrated inhibition in MTT viability and soft agar assays, compared to appropriate controls, and demonstrated significant and durable xenograft growth reduction. Conversely, inhibition using MEK and/or PI3K inhibitors demonstrated only transient growth reduction in vivo. Conclusions: KRAS gene amp+ was observed in a large subset (26%) of GEC patients, which correlated with extreme expression by mass spectrometry. Established xenograft lines serve as models to investigate therapeutic strategies for KRAS amp+ patients. Inhibition using MEK/PIK3CA inhibitors provided transient benefit for KRAS amp+ tumors while durable inhibition was observed with Kras protein knockdown, suggesting potential benefit from novel siRNA therapeutics currently in development.
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