Downloaded from http://jcp.bmj.com/ on May 29, 2015 - Published by group.bmj.com
JCP Online First, published on April 29, 2015 as 10.1136/jclinpath-2015-202969 Short report
Are all RAS mutations the same? Coexisting KRAS and NRAS mutations in a caecal adenocarcinoma and contiguous tubulovillous adenoma N N Vagaja,1,2 J Parry,1,2 D McCallum,1,2 M A Thomas,1,2 J M Bentel1,2 1
Anatomical Pathology, PathWest Laboratory Medicine, Royal Perth Hospital, Perth, Western Australia, Australia 2 Anatomical Pathology, PathWest Laboratory Medicine, Fiona Stanley Hospital Network, Murdoch, Western Australia, Australia Correspondence to Dr J M Bentel, Anatomical Pathology, PathWest Laboratory Medicine, Royal Perth Hospital, Wellington Street, Perth, WA 6000, Australia; Jacky.Bentel@ health.wa.gov.au Received 12 March 2015 Revised 7 April 2015 Accepted 9 April 2015
ABSTRACT Mutations of the human Kirsten rat sarcoma viral oncogene homologue (KRAS) and the highly homologous human neuroblastoma RAS viral oncogene homologue (NRAS) are associated with resistance to antiepidermal growth factor receptor therapies in patients with colorectal cancer. In this report, we describe a caecal adenocarcinoma that contains both KRAS c.35G>T (G12V) and NRAS c.34G>A (G12S) mutations. The adenocarcinoma arises from a contiguous high-grade tubulovillous adenoma, which also carries the identical KRAS and NRAS mutations, supporting their common origin. While KRAS mutations are common in colorectal cancers, NRAS mutations are relatively rare and the coexistence of multiple RAS mutations is not documented, presumably reflecting similar functions of wild-type and mutant forms of RAS. Recent experimental evidence has suggested that KRAS and NRAS may in fact mediate distinct biological processes in the colon, and this unusual case potentially illustrates the hypothesis clinically. Characterisation of the diverse and divergent functions of RAS family members and mutant forms of RAS in the colon form important considerations for the development of RAS-targeting therapeutics.
INTRODUCTION
To cite: Vagaja NN, Parry J, McCallum D, et al. J Clin Pathol Published Online First: [ please include Day Month Year] doi:10.1136/ jclinpath-2015-202969
Monoclonal antibodies to the epidermal growth factor receptor (EGFR), including Cetuximab and Panitumumab are widely used in the management of metastatic colorectal cancer, with a response rate of approximately 10% for metastatic, pretreated colorectal tumours.1 Binding of ligand (eg, EGF) to the EGFR results in the activation of downstream signalling cascades including the RAS/mitogenactivated protein kinase (MAPK) and phosphatidylinositol-3-kinase (PI3K) pathways.2 Evidence that mutations in KRAS, a member of the RAS/MAPK signalling pathway, were associated with resistance to anti-EGFR antibodies led to the routine screening of colorectal tumours for KRAS mutations prior to administration of EGFR-targeted therapies.2–4 Additional studies have reported that mutations in other components of EGFR-induced signalling pathways, v-raf murine sarcoma viral oncogene homologue B (BRAF), NRAS and phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA), were associated with reduced efficacy of Cetuximab, providing support for more comprehensive mutation testing of colorectal tumours to improve the response rates of this and other EGFR-targeted agents.5 KRAS mutations, principally affecting codons 12, 13 and to a lesser extent 61, are evident in 35%–
40% of colorectal cancers, while NRAS mutations are detected in only ∼5%, also predominantly affecting codons 12, 13 and 61.2 6 KRAS, NRAS and BRAF mutations are reported to be mutually exclusive in colorectal tumours, potentially reflecting a lack of selective advantage or redundancy of mutations in multiple members of the same signalling pathway; however, PIK3CA mutations may be present in KRAS, NRAS or BRAF mutant tumours, feasibly representing divergent but complementary roles of the RAS/MAPK and PI3K pathways in colorectal tumorigenesis.5 Recent characterisation of RAS family members, KRAS (KRAS 4A, KRAS 4B), NRAS and human Harvey RAS viral oncogene homologue (HRAS) has identified overlapping and distinct roles of each, with experimental models highlighting fundamentally divergent activities of mutant KRAS and NRAS in the colon.7 8 In this report, we present a molecularly unusual case of colon cancer arising from a contiguous high-grade tubulovillous adenoma, both of which contain KRAS c.35G>T (G12V) and NRAS c.34G>A (G12S) mutations. As the practice of personalised medicine is finetuned to include multiple molecular and immunohistochemical markers of prognostic and predictive significance, and with major academic and pharmaceutical company efforts underway to develop RAS or mutant RAS-targeting therapies, this rare case illustrates in a clinical setting, the emerging evidence from basic research that RAS family members have distinct biological activities in the colon.
CASE PRESENTATION The primary tumour was diagnosed following a 12-month history of gastrointestinal symptoms and presented on CT as a caecal mass at the base of the appendix. Right hemicolectomy was carried out and histopathological examination, performed by two pathologists, showed a well-differentiated 43 mm adenocarcinoma at the appendiceal orifice. The tumour arose from a sessile high-grade tubulovillous adenoma (figure 1) and extended into the muscularis propria ( pT2N0MX). Acute inflammation, focal ulceration and abscess formation were also seen in the bowel wall extending into the muscularis propria. Epithelial cells from both the adenoma and the adenocarcinoma were positive for CDX2 and CK20 and negative for CK7, with positive immunostaining for MLH1, PMS2, MSH2 and MSH6 indicating no evidence of microsatellite instability (figures 2 and 3). Mutation analysis of the adenocarcinoma identified a KRAS c.35G>T G12V (Gly12Val) and an NRAS c.34G>A G12S
Vagaja NN, et al. J Clin Pathol 2015;0:1–4. doi:10.1136/jclinpath-2015-202969
Copyright Article author (or their employer) 2015. Produced by BMJ Publishing Group Ltd under licence.
1
Downloaded from http://jcp.bmj.com/ on May 29, 2015 - Published by group.bmj.com
Short report
Figure 1 H&E images of the originally diagnosed caecal adenoma, adenocarcinoma and adjacent non-malignant (normal) colon, and the subsequently diagnosed rectal polyp indicating KRAS exon 2 (left) and NRAS exon 2 (right) sequencing chromatograms for each. Forward sequences are presented in the upper panel and reverse sequences in the lower panel for each KRAS and NRAS sequence. Mutant bases encoding the KRAS c.35G>T and NRAS c.34G>A base substitutions for the adenoma and adenocarcinoma (*), and the corresponding wild-type only bases in the normal colon and rectal polyp (°) are indicated. Bar=200 μm (rectal polyp=600 μm). (Gly12Ser) mutation (figure 1). No mutations in BRAF exon 15 were detected. The contiguous adenoma contained both the KRAS c.35G>T G12V (Gly12Val) and NRAS c.34G>A G12S (Gly12Ser) mutations, while adjacent non-malignant colon contained no detectable KRAS, NRAS or BRAF mutations (figure 1). A rectal polyp, biopsied 9 months following the original surgery was identified as a tubular adenoma with low-grade dysplasia and contained no mutations in KRAS, NRAS or BRAF (figure 1).
DISCUSSION KRAS mutation analysis of colorectal cancers, and the recent testing to include NRAS and in some centres BRAF, has been pivotal in the tailoring of anti-EGFR monoclonal antibody therapies for individual patients. In addition to indicating cancers that are highly unlikely to respond to these costly treatments, the accumulation of molecular data is informing both basic scientific research into the function of RAS and oncogenic RAS in 2
the colon, as well as defining potential pharmacotherapeutic targets for future development. The current case, in which KRAS and NRAS mutations coexist in both the adenoma and the invasive cancer components of disease, presents an unusual molecular signature with currently unknown clinical or biological significance, except to indicate unsuitability for EGFR-targeted treatments. A lack of evidence of KRAS or NRAS mutations in adjacent non-malignant tissues provides evidence that both RAS mutations are somatic, with the presence of KRAS and NRAS mutations in contiguous adenoma and carcinoma tissues supporting their common origin. Although it is feasible that the adenoma and invasive tumour contain (at least) two prominent subpopulations of cells, each with a distinct RAS mutation, the presence and preservation of both populations during emergence of the invasive cancer from the adenoma are highly unlikely. This scenario cannot be discounted, however, as the heterogeneity of KRAS mutation status in primary colorectal cancers and in their metastases has been well-documented.9 Vagaja NN, et al. J Clin Pathol 2015;0:1–4. doi:10.1136/jclinpath-2015-202969
Downloaded from http://jcp.bmj.com/ on May 29, 2015 - Published by group.bmj.com
Short report Figure 2 Immunophenotype of the KRAS/NRAS mutant caecal adenoma and adenocarcinoma. Strong diffuse cytoplasmic expression of CK20 was detected in both the caecal adenoma (A) and the caecal adenocarcinoma (B). No expression of CK7 was evident in either the caecal adenoma (C) or the caecal adenocarcinoma (D). Both lesions showed strong nuclear expression of CDX2 (E: caecal adenoma and F: caecal adenocarcinoma). Bar=500 μm.
Although not a common occurrence, specific investigation of the molecular heterogeneity of primary and secondary colorectal tumours is limited and therefore its frequency with regards to RAS mutation status is unknown. Nevertheless, this has obvious relevance when determining factors that may diminish clinical responses to anti-EGFR therapies. Antibodies to mutant forms of KRAS and NRAS have been developed and are commercially available from several companies including NewEast Biosciences (King of Prussia, Pennsylvania, USA) and Spring Bioscience (Pleasanton, California, USA), respectively. Specific antibodies to KRAS G12V or NRAS G12S were not
commercially available at the time of writing this manuscript; however, their development and use in cases such as the present specimen will provide additional evidence of the co-expression of mutant KRAS and mutant NRAS in colorectal cancer cells or the coexistence of subpopulations of malignant cells carrying either mutant KRAS or mutant NRAS. Apart from this case, coexisting mutations in members of the RAS/MAPK pathway have not been identified in our laboratory, which is in line with other reports examining patients with colorectal cancer in Western countries.5 However, in a study investigating KRAS, BRAF, PIK3CA and NRAS mutations in 676
Figure 3 Mismatch repair enzyme expression in the KRAS/NRAS mutant caecal adenoma and adenocarcinoma. Immunohistochemical evaluation of the expression of mismatch repair enzymes (MLH1, MSH2, MSH6 and PMS2) showed no abnormal loss of expression in either the caecal adenoma or the caecal adenocarcinoma (A–D: caecal adenoma; E–H: caecal adenocarcinoma). Bar=100 μm. Vagaja NN, et al. J Clin Pathol 2015;0:1–4. doi:10.1136/jclinpath-2015-202969
3
Downloaded from http://jcp.bmj.com/ on May 29, 2015 - Published by group.bmj.com
Short report colorectal cancer cases from China, coexistent KRAS and BRAF mutations were detected in 11 of the cases, with one case harbouring BRAF (V600E) and NRAS (G15W) mutations, and a single tumour (with no clinical or histopathological data included) carrying unspecified KRAS and NRAS mutations.10 These findings potentially indicate distinct genetic profiles of colorectal cancers in Western and Asian patients; however, additional larger scale studies and meta analyses will be required to confirm this. While the functional consequences of coexisting KRAS G12V and NRAS G12S mutations are unknown at this time, maintenance of both mutations in the adenoma and invasive cancer imply that together, these mutant forms of RAS provide a selective advantage for the tumour cells compared with either mutation alone. The KRAS G12V mutation is frequently identified in colorectal cancers and has been associated with poorer prognosis compared with KRAS G12D mutations.9 11 12 In contrast, the NRAS G12S mutation has been documented to occur in colorectal cancers,6 but the effects of this amino acid change on NRAS function have not been characterised. A recent study reporting differential expression of KRAS and NRAS and distinct effects of mutant forms of KRAS and NRAS in the colon indicate that rather than mutant forms of KRAS and NRAS resulting in similar oncogenic effects, each regulates different cellular processes that together can contribute to carcinogenesis and/or tumour progression.8 If this process underlies the occurrence and maintenance of both RAS mutations during progression of the adenoma to adenocarcinoma in the present case, then it may be speculated that the rarity of dual KRAS and NRAS mutations in colorectal cancers is due to mutations affecting one of the cellular functions more commonly occurring in other members of that pathway. For example, if KRAS predominantly regulates proliferation and NRAS cell survival in the colon, as indicated in the study of Haigis et al,8 then mutations in the PI3K pathway, a regulator of cell survival, may more frequently co-occur in KRAS mutant tumours.
emerging experimental evidence of distinct functions of wildtype and mutant KRAS and NRAS in the colon and in colorectal cancers. Handling editor Runjan Chetty Acknowledgements The authors acknowledge the technical assistance of Nathan Acott and Ian Sinclair for immunohistochemical staining and imaging of slides. Contributors NV, JP and DM performed the histopathological analysis of the case, NV and JP wrote the case report, JMB and MAT performed the molecular analysis of the case and wrote the remainder of the manuscript. NV, JMB and MAT prepared the figures and all authors approved the final manuscript. Competing interests None declared. Provenance and peer review Not commissioned; externally peer reviewed. Data sharing statement Comprehensive methods for immunohistochemical staining, KRAS and NRAS mutation detection, including primer sequences and cycling conditions are available to all researchers by emailing the corresponding author.
REFERENCES 1
2 3 4
5
6
7 8
CONCLUSIONS Coexistent mutations in multiple RAS family members are rarely documented in colorectal cancers. Their occurrence supports
9
10
Take home message 11
Histological and molecular pathological characterisation of clinical tumours remains important tools that inform basic scientific research and drug development.
4
12
Cunningham D, Humblet Y, Siena S, et al. Cetuximab monotherapy and cetuximab plus irinotecan in irinotecan-refractory metastatic colorectal cancer. N Engl J Med 2004;351:337–45. Pylayeva-Gupta Y, Grabocka E, Bar-Sagi D. RAS oncogenes: weaving a tumorigenic web. Nat Rev Cancer 2011;11:761–74. Karapetis CS, Khambata-Ford S, Jonker DJ, et al. K-ras mutations and benefit from cetuximab in advanced colorectal cancer. N Engl J Med 2008;35:757–65. Amado RG, Wolf M, Peeters M, et al. Wild-type KRAS is required for panitumumab efficacy in patients with metastatic colorectal cancer. J Clin Oncol 2008;26:1626–34. De Roock W, Claes B, Bermasconi D, et al. Effects of KRAS, BRAF, NRAS, and PIK3CA mutations on the efficacy of cetuximab plus chemotherapy in chemotherapy-refractory metastatic colorectal cancer: a retrospective consortium analysis. Lancet Oncol 2010;11:753–62. Forbes SA, Beare D, Gunasekaran P, et al. COSMIC: exploring the world’s knowledge of somatic mutations in human cancer. Nucleic Acids Res 2015;43 (Database issue):D805–11. (COSMIC: ). Ahearn IM, Haigis K, Bar-Sagi D, et al. Regulating the regulator: post-translational modification of RAS. Nature Rev Mol Cell Biol 2012;13:39–51. Haigis KM, Kendall KR, Wang Y, et al. Differential effects of oncogenic K-Ras and N-Ras on proliferation, differentiation and tumor progression in the colon. Nat Genet 2008;40:600–8. Al-Mulla F, Going JJ, Sowden E, et al. Heterogeneity of mutant versus wild-type Ki-ras in primary and metastatic colorectal carcinomas, and association of codon-12 valine with early mortality. J Pathol 1998;185:130–8. Shen Y, Wang J, Han X, et al. Effectors of epidermal growth factor receptor pathway: the genetic profiling of KRAS, BRAF, PIK3CA, NRAS mutations in colorectal cancer characteristics and personalized medicine. PLoS ONE 2013;8: e81628. Keohavong P, DeMichele MA, Melacrinos AC, et al. Detection of K-ras mutations in lung carcinomas: relationship to prognosis. Clin Cancer Res 1996;2:411–18. Andreyev HJ, Norman AR, Cunningham D, et al. Kirsten ras mutations in patients with colorectal cancer: the multicentre “RASCAL” study. J Natl Cancer Inst 1998;90:675–84.
Vagaja NN, et al. J Clin Pathol 2015;0:1–4. doi:10.1136/jclinpath-2015-202969
Downloaded from http://jcp.bmj.com/ on May 29, 2015 - Published by group.bmj.com
Are all RAS mutations the same? Coexisting KRAS and NRAS mutations in a caecal adenocarcinoma and contiguous tubulovillous adenoma N N Vagaja, J Parry, D McCallum, M A Thomas and J M Bentel J Clin Pathol published online April 29, 2015
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JCP Online First, published on April 29, 2015 as 10.1136/jclinpath-2015-202969 Short report
Are all RAS mutations the same? Coexisting KRAS and NRAS mutations in a caecal adenocarcinoma and contiguous tubulovillous adenoma N N Vagaja,1,2 J Parry,1,2 D McCallum,1,2 M A Thomas,1,2 J M Bentel1,2 1
Anatomical Pathology, PathWest Laboratory Medicine, Royal Perth Hospital, Perth, Western Australia, Australia 2 Anatomical Pathology, PathWest Laboratory Medicine, Fiona Stanley Hospital Network, Murdoch, Western Australia, Australia Correspondence to Dr J M Bentel, Anatomical Pathology, PathWest Laboratory Medicine, Royal Perth Hospital, Wellington Street, Perth, WA 6000, Australia; Jacky.Bentel@ health.wa.gov.au Received 12 March 2015 Revised 7 April 2015 Accepted 9 April 2015
ABSTRACT Mutations of the human Kirsten rat sarcoma viral oncogene homologue (KRAS) and the highly homologous human neuroblastoma RAS viral oncogene homologue (NRAS) are associated with resistance to antiepidermal growth factor receptor therapies in patients with colorectal cancer. In this report, we describe a caecal adenocarcinoma that contains both KRAS c.35G>T (G12V) and NRAS c.34G>A (G12S) mutations. The adenocarcinoma arises from a contiguous high-grade tubulovillous adenoma, which also carries the identical KRAS and NRAS mutations, supporting their common origin. While KRAS mutations are common in colorectal cancers, NRAS mutations are relatively rare and the coexistence of multiple RAS mutations is not documented, presumably reflecting similar functions of wild-type and mutant forms of RAS. Recent experimental evidence has suggested that KRAS and NRAS may in fact mediate distinct biological processes in the colon, and this unusual case potentially illustrates the hypothesis clinically. Characterisation of the diverse and divergent functions of RAS family members and mutant forms of RAS in the colon form important considerations for the development of RAS-targeting therapeutics.
INTRODUCTION
To cite: Vagaja NN, Parry J, McCallum D, et al. J Clin Pathol Published Online First: [ please include Day Month Year] doi:10.1136/ jclinpath-2015-202969
Monoclonal antibodies to the epidermal growth factor receptor (EGFR), including Cetuximab and Panitumumab are widely used in the management of metastatic colorectal cancer, with a response rate of approximately 10% for metastatic, pretreated colorectal tumours.1 Binding of ligand (eg, EGF) to the EGFR results in the activation of downstream signalling cascades including the RAS/mitogenactivated protein kinase (MAPK) and phosphatidylinositol-3-kinase (PI3K) pathways.2 Evidence that mutations in KRAS, a member of the RAS/MAPK signalling pathway, were associated with resistance to anti-EGFR antibodies led to the routine screening of colorectal tumours for KRAS mutations prior to administration of EGFR-targeted therapies.2–4 Additional studies have reported that mutations in other components of EGFR-induced signalling pathways, v-raf murine sarcoma viral oncogene homologue B (BRAF), NRAS and phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA), were associated with reduced efficacy of Cetuximab, providing support for more comprehensive mutation testing of colorectal tumours to improve the response rates of this and other EGFR-targeted agents.5 KRAS mutations, principally affecting codons 12, 13 and to a lesser extent 61, are evident in 35%–
40% of colorectal cancers, while NRAS mutations are detected in only ∼5%, also predominantly affecting codons 12, 13 and 61.2 6 KRAS, NRAS and BRAF mutations are reported to be mutually exclusive in colorectal tumours, potentially reflecting a lack of selective advantage or redundancy of mutations in multiple members of the same signalling pathway; however, PIK3CA mutations may be present in KRAS, NRAS or BRAF mutant tumours, feasibly representing divergent but complementary roles of the RAS/MAPK and PI3K pathways in colorectal tumorigenesis.5 Recent characterisation of RAS family members, KRAS (KRAS 4A, KRAS 4B), NRAS and human Harvey RAS viral oncogene homologue (HRAS) has identified overlapping and distinct roles of each, with experimental models highlighting fundamentally divergent activities of mutant KRAS and NRAS in the colon.7 8 In this report, we present a molecularly unusual case of colon cancer arising from a contiguous high-grade tubulovillous adenoma, both of which contain KRAS c.35G>T (G12V) and NRAS c.34G>A (G12S) mutations. As the practice of personalised medicine is finetuned to include multiple molecular and immunohistochemical markers of prognostic and predictive significance, and with major academic and pharmaceutical company efforts underway to develop RAS or mutant RAS-targeting therapies, this rare case illustrates in a clinical setting, the emerging evidence from basic research that RAS family members have distinct biological activities in the colon.
CASE PRESENTATION The primary tumour was diagnosed following a 12-month history of gastrointestinal symptoms and presented on CT as a caecal mass at the base of the appendix. Right hemicolectomy was carried out and histopathological examination, performed by two pathologists, showed a well-differentiated 43 mm adenocarcinoma at the appendiceal orifice. The tumour arose from a sessile high-grade tubulovillous adenoma (figure 1) and extended into the muscularis propria ( pT2N0MX). Acute inflammation, focal ulceration and abscess formation were also seen in the bowel wall extending into the muscularis propria. Epithelial cells from both the adenoma and the adenocarcinoma were positive for CDX2 and CK20 and negative for CK7, with positive immunostaining for MLH1, PMS2, MSH2 and MSH6 indicating no evidence of microsatellite instability (figures 2 and 3). Mutation analysis of the adenocarcinoma identified a KRAS c.35G>T G12V (Gly12Val) and an NRAS c.34G>A G12S
Vagaja NN, et al. J Clin Pathol 2015;0:1–4. doi:10.1136/jclinpath-2015-202969
Copyright Article author (or their employer) 2015. Produced by BMJ Publishing Group Ltd under licence.
1
Downloaded from http://jcp.bmj.com/ on May 29, 2015 - Published by group.bmj.com
Short report
Figure 1 H&E images of the originally diagnosed caecal adenoma, adenocarcinoma and adjacent non-malignant (normal) colon, and the subsequently diagnosed rectal polyp indicating KRAS exon 2 (left) and NRAS exon 2 (right) sequencing chromatograms for each. Forward sequences are presented in the upper panel and reverse sequences in the lower panel for each KRAS and NRAS sequence. Mutant bases encoding the KRAS c.35G>T and NRAS c.34G>A base substitutions for the adenoma and adenocarcinoma (*), and the corresponding wild-type only bases in the normal colon and rectal polyp (°) are indicated. Bar=200 μm (rectal polyp=600 μm). (Gly12Ser) mutation (figure 1). No mutations in BRAF exon 15 were detected. The contiguous adenoma contained both the KRAS c.35G>T G12V (Gly12Val) and NRAS c.34G>A G12S (Gly12Ser) mutations, while adjacent non-malignant colon contained no detectable KRAS, NRAS or BRAF mutations (figure 1). A rectal polyp, biopsied 9 months following the original surgery was identified as a tubular adenoma with low-grade dysplasia and contained no mutations in KRAS, NRAS or BRAF (figure 1).
DISCUSSION KRAS mutation analysis of colorectal cancers, and the recent testing to include NRAS and in some centres BRAF, has been pivotal in the tailoring of anti-EGFR monoclonal antibody therapies for individual patients. In addition to indicating cancers that are highly unlikely to respond to these costly treatments, the accumulation of molecular data is informing both basic scientific research into the function of RAS and oncogenic RAS in 2
the colon, as well as defining potential pharmacotherapeutic targets for future development. The current case, in which KRAS and NRAS mutations coexist in both the adenoma and the invasive cancer components of disease, presents an unusual molecular signature with currently unknown clinical or biological significance, except to indicate unsuitability for EGFR-targeted treatments. A lack of evidence of KRAS or NRAS mutations in adjacent non-malignant tissues provides evidence that both RAS mutations are somatic, with the presence of KRAS and NRAS mutations in contiguous adenoma and carcinoma tissues supporting their common origin. Although it is feasible that the adenoma and invasive tumour contain (at least) two prominent subpopulations of cells, each with a distinct RAS mutation, the presence and preservation of both populations during emergence of the invasive cancer from the adenoma are highly unlikely. This scenario cannot be discounted, however, as the heterogeneity of KRAS mutation status in primary colorectal cancers and in their metastases has been well-documented.9 Vagaja NN, et al. J Clin Pathol 2015;0:1–4. doi:10.1136/jclinpath-2015-202969
Downloaded from http://jcp.bmj.com/ on May 29, 2015 - Published by group.bmj.com
Short report Figure 2 Immunophenotype of the KRAS/NRAS mutant caecal adenoma and adenocarcinoma. Strong diffuse cytoplasmic expression of CK20 was detected in both the caecal adenoma (A) and the caecal adenocarcinoma (B). No expression of CK7 was evident in either the caecal adenoma (C) or the caecal adenocarcinoma (D). Both lesions showed strong nuclear expression of CDX2 (E: caecal adenoma and F: caecal adenocarcinoma). Bar=500 μm.
Although not a common occurrence, specific investigation of the molecular heterogeneity of primary and secondary colorectal tumours is limited and therefore its frequency with regards to RAS mutation status is unknown. Nevertheless, this has obvious relevance when determining factors that may diminish clinical responses to anti-EGFR therapies. Antibodies to mutant forms of KRAS and NRAS have been developed and are commercially available from several companies including NewEast Biosciences (King of Prussia, Pennsylvania, USA) and Spring Bioscience (Pleasanton, California, USA), respectively. Specific antibodies to KRAS G12V or NRAS G12S were not
commercially available at the time of writing this manuscript; however, their development and use in cases such as the present specimen will provide additional evidence of the co-expression of mutant KRAS and mutant NRAS in colorectal cancer cells or the coexistence of subpopulations of malignant cells carrying either mutant KRAS or mutant NRAS. Apart from this case, coexisting mutations in members of the RAS/MAPK pathway have not been identified in our laboratory, which is in line with other reports examining patients with colorectal cancer in Western countries.5 However, in a study investigating KRAS, BRAF, PIK3CA and NRAS mutations in 676
Figure 3 Mismatch repair enzyme expression in the KRAS/NRAS mutant caecal adenoma and adenocarcinoma. Immunohistochemical evaluation of the expression of mismatch repair enzymes (MLH1, MSH2, MSH6 and PMS2) showed no abnormal loss of expression in either the caecal adenoma or the caecal adenocarcinoma (A–D: caecal adenoma; E–H: caecal adenocarcinoma). Bar=100 μm. Vagaja NN, et al. J Clin Pathol 2015;0:1–4. doi:10.1136/jclinpath-2015-202969
3
Downloaded from http://jcp.bmj.com/ on May 29, 2015 - Published by group.bmj.com
Short report colorectal cancer cases from China, coexistent KRAS and BRAF mutations were detected in 11 of the cases, with one case harbouring BRAF (V600E) and NRAS (G15W) mutations, and a single tumour (with no clinical or histopathological data included) carrying unspecified KRAS and NRAS mutations.10 These findings potentially indicate distinct genetic profiles of colorectal cancers in Western and Asian patients; however, additional larger scale studies and meta analyses will be required to confirm this. While the functional consequences of coexisting KRAS G12V and NRAS G12S mutations are unknown at this time, maintenance of both mutations in the adenoma and invasive cancer imply that together, these mutant forms of RAS provide a selective advantage for the tumour cells compared with either mutation alone. The KRAS G12V mutation is frequently identified in colorectal cancers and has been associated with poorer prognosis compared with KRAS G12D mutations.9 11 12 In contrast, the NRAS G12S mutation has been documented to occur in colorectal cancers,6 but the effects of this amino acid change on NRAS function have not been characterised. A recent study reporting differential expression of KRAS and NRAS and distinct effects of mutant forms of KRAS and NRAS in the colon indicate that rather than mutant forms of KRAS and NRAS resulting in similar oncogenic effects, each regulates different cellular processes that together can contribute to carcinogenesis and/or tumour progression.8 If this process underlies the occurrence and maintenance of both RAS mutations during progression of the adenoma to adenocarcinoma in the present case, then it may be speculated that the rarity of dual KRAS and NRAS mutations in colorectal cancers is due to mutations affecting one of the cellular functions more commonly occurring in other members of that pathway. For example, if KRAS predominantly regulates proliferation and NRAS cell survival in the colon, as indicated in the study of Haigis et al,8 then mutations in the PI3K pathway, a regulator of cell survival, may more frequently co-occur in KRAS mutant tumours.
emerging experimental evidence of distinct functions of wildtype and mutant KRAS and NRAS in the colon and in colorectal cancers. Handling editor Runjan Chetty Acknowledgements The authors acknowledge the technical assistance of Nathan Acott and Ian Sinclair for immunohistochemical staining and imaging of slides. Contributors NV, JP and DM performed the histopathological analysis of the case, NV and JP wrote the case report, JMB and MAT performed the molecular analysis of the case and wrote the remainder of the manuscript. NV, JMB and MAT prepared the figures and all authors approved the final manuscript. Competing interests None declared. Provenance and peer review Not commissioned; externally peer reviewed. Data sharing statement Comprehensive methods for immunohistochemical staining, KRAS and NRAS mutation detection, including primer sequences and cycling conditions are available to all researchers by emailing the corresponding author.
REFERENCES 1
2 3 4
5
6
7 8
CONCLUSIONS Coexistent mutations in multiple RAS family members are rarely documented in colorectal cancers. Their occurrence supports
9
10
Take home message 11
Histological and molecular pathological characterisation of clinical tumours remains important tools that inform basic scientific research and drug development.
4
12
Cunningham D, Humblet Y, Siena S, et al. Cetuximab monotherapy and cetuximab plus irinotecan in irinotecan-refractory metastatic colorectal cancer. N Engl J Med 2004;351:337–45. Pylayeva-Gupta Y, Grabocka E, Bar-Sagi D. RAS oncogenes: weaving a tumorigenic web. Nat Rev Cancer 2011;11:761–74. Karapetis CS, Khambata-Ford S, Jonker DJ, et al. K-ras mutations and benefit from cetuximab in advanced colorectal cancer. N Engl J Med 2008;35:757–65. Amado RG, Wolf M, Peeters M, et al. Wild-type KRAS is required for panitumumab efficacy in patients with metastatic colorectal cancer. J Clin Oncol 2008;26:1626–34. De Roock W, Claes B, Bermasconi D, et al. Effects of KRAS, BRAF, NRAS, and PIK3CA mutations on the efficacy of cetuximab plus chemotherapy in chemotherapy-refractory metastatic colorectal cancer: a retrospective consortium analysis. Lancet Oncol 2010;11:753–62. Forbes SA, Beare D, Gunasekaran P, et al. COSMIC: exploring the world’s knowledge of somatic mutations in human cancer. Nucleic Acids Res 2015;43 (Database issue):D805–11. (COSMIC: ). Ahearn IM, Haigis K, Bar-Sagi D, et al. Regulating the regulator: post-translational modification of RAS. Nature Rev Mol Cell Biol 2012;13:39–51. Haigis KM, Kendall KR, Wang Y, et al. Differential effects of oncogenic K-Ras and N-Ras on proliferation, differentiation and tumor progression in the colon. Nat Genet 2008;40:600–8. Al-Mulla F, Going JJ, Sowden E, et al. Heterogeneity of mutant versus wild-type Ki-ras in primary and metastatic colorectal carcinomas, and association of codon-12 valine with early mortality. J Pathol 1998;185:130–8. Shen Y, Wang J, Han X, et al. Effectors of epidermal growth factor receptor pathway: the genetic profiling of KRAS, BRAF, PIK3CA, NRAS mutations in colorectal cancer characteristics and personalized medicine. PLoS ONE 2013;8: e81628. Keohavong P, DeMichele MA, Melacrinos AC, et al. Detection of K-ras mutations in lung carcinomas: relationship to prognosis. Clin Cancer Res 1996;2:411–18. Andreyev HJ, Norman AR, Cunningham D, et al. Kirsten ras mutations in patients with colorectal cancer: the multicentre “RASCAL” study. J Natl Cancer Inst 1998;90:675–84.
Vagaja NN, et al. J Clin Pathol 2015;0:1–4. doi:10.1136/jclinpath-2015-202969
Downloaded from http://jcp.bmj.com/ on May 29, 2015 - Published by group.bmj.com
Are all RAS mutations the same? Coexisting KRAS and NRAS mutations in a caecal adenocarcinoma and contiguous tubulovillous adenoma N N Vagaja, J Parry, D McCallum, M A Thomas and J M Bentel J Clin Pathol published online April 29, 2015
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