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. Author manuscript; available in PMC: 2020 Apr 1.
Published in final edited form as: Hum Pathol. 2018 Nov 24;86:12–20. doi: 10.1016/j.humpath.2018.11.014

Clinical validation of coexisting driver mutations in colorectal cancers

Gang Zheng a,*, Li-Hui Tseng a,b, Lisa Haley a, Junaid Ibrahim a, Jennifer Bynem a, Rena Xian a,c, Christopher D Gocke a,c, James R Eshleman a,c, Ming-Tseh Lin a
PMCID: PMC6467705  NIHMSID: NIHMS1522572  PMID: 30481508

Summary

Mutational profiling is recommended for selecting targeted therapy and predicting prognosis of metastatic colorectal cancer (CRC). Detection of coexisting mutations within the same pathway, which are usually mutually exclusive, raises the concern for potential laboratory errors. In this retrospective study for quality assessment of a next generation sequencing assay, we examined BRAF, KRAS and NRAS genes within the mitogen-activated protein kinase (MAPK) pathway and the PIK3CA gene within the phosphatidylinositol 3-kinase (mTOR) pathway in 744 CRC specimens submitted to our clinical diagnostics laboratory. While coexistence of mutations between the MAPK and mTOR pathways was observed, it rarely occurred within the MAPK pathway. Retrospective quality assessments identified false detection of coexisting activating KRAS and NRAS mutations in one specimen, and confirmed two activating KRAS mutations in 2 specimens and coexisting activating KRAS and NRAS mutations in 2 specimens, but no coexisting activating RAS and BRAF mutations. There were 15 CRCs with a kinase impaired BRAF mutation, including 3 with a coexisting activating KRAS mutation, which may have therapeutic implications. Multiregional analysis based on different histological features demonstrated that coexisting KRAS and NRAS mutations may be present in the same or different tumor populations, and showed that invasion of adenomas by synchronous adenocarcinomas of different clonal origin may result in detection of coexisting mutations within the MAPK pathway. In this study, we proposed an operating procedure for clinical validation of unexpected coexisting mutations. Further studies are warranted to elucidate the biological significance and clinical implications of coexisting mutations within the MAPK pathway.

Keywords: coexisting mutation, BRAF, KRAS, NRAS, PIK3CA, colorectal cancer

1. Introduction

Anti-EGFR monoclonal antibodies have been approved by the Food and Drug Administration (FDA) in the United States for targeted therapy of metastatic colorectal cancer (CRC) with wild-type KRAS and NRAS genes [1, 2]. Mutational profiling of these genes is part of the FDA label for these drugs and is considered standard-of-care for patients with metastatic CRC, along with BRAF mutational testing for prognostic stratification of CRC and evaluation of Lynch syndrome risk in mismatch repair-deficient CRC [2, 3].

KRAS mutations within the mitogen-activated protein kinase (MAPK) pathway promote the progression of small adenomas to large adenomas, while PIK3CA mutations within the phosphatidylinositol 3-kinase (mTOR) pathway promote the progression of large adenomas to adenocarcinomas [4, 5]. Therefore, coexistence of mutations within the MAPK pathway and PIK3CA mutations within the mTOR pathway are common in CRC, whereas activating KRAS, NRAS and BRAF mutations are thought to be mutually exclusive [6, 7]. This is also true for genes within the EGFR and MAPK pathway in non-small cell lung cancer [8].

We have previously shown an extremely low incidence (0.1%) of coexisting activating EGFR and KRAS mutations in non-small cell lung cancer in a clinical diagnostic setting [9]. However, a higher incidence (0.6–1.5%) has been reported in retrospective research studies [10, 11]. Detection of unusual coexisting mutations should raise the concern for potential laboratory errors, which may occur at any point during the pre-analytical, analytical and/or post-analytical phases of testing [12, 13]. In this retrospective study for quality assessment of coexisting mutations detected within the MAPK pathway (KRAS, NRAS and BRAF genes) and/or mTOR pathways (PIK3CA gene) in CRC, we propose a standard operating procedure to confirm the presence of coexisting mutations and to evaluate whether these coexisting mutations occur in the same tumor population or in different but physically adjacent tumor populations within the same tissue block.

2. Materials and methods

2.1. Materials

NGS results from 744 CRC specimens submitted to the Johns Hopkins Molecular Diagnostics Laboratory between April 2013 and December 2016 were analyzed for mutations within the MAPK and mTOR pathways. DNA was isolated from formalin-fixed paraffin-embedded (FFPE) tissues, purified, and measured as described previously [14]. The Johns Hopkins Medicine Institutional Review Board granted approval for this study.

2.2. Next generation sequencing (NGS)

NGS was conducted using AmpliSeq Cancer Hotspot Panel (v2) (Life Technologies, Carlsbad, California) for targeted multi-gene amplification as described previously [7, 14]. Sequencing data of the targeted genes were analyzed using Torrent Suite (Life Technologies). Mutations were identified and annotated through both Torrent Variant Caller and direct visual inspection of the binary sequence alignment/map (BAM) file using the Broad Institute’s Integrative Genomics Viewer (IGV) [15]. CRC specimens were analyzed for BRAF (NM_004333), KRAS (NM_033360), NRAS (NM_002524) and PIK3CA (NM_006218) genes for clinical reporting. BRAF mutations were categorized into 3 classes according to the BRAF kinase activity (class 1: codon 600 mutants with high or intermediate kinase activity; class 2: non-codon 600 mutants with high or intermediate kinase activity; and class 3: low or no kinase activity) [16–18]. The analytic performance characteristics and the reportable ranges have been reported previously in the first 304 CRC specimens [7]. The limit of detection was 2% mutant alleles. An operating procedure was followed to confirm unusual coexisting mutations (Figure 1). In short, a tissue identification assay was performed to identify potential tissue or DNA mix-up. In the absence of mix-up, it was followed by multiregional analysis by pyrosequencing and NGS to confirm coexisting mutations, and to determine if mutations were present in the same or different subpopulations.

Figure 1.

Figure 1.

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A proposed operating procedure to evaluate unexpected coexisting mutations in a clinical diagnostics setting.

2.3. Prediction of clonality in specimens with coexisting mutations

The expected variant allele frequency (VAF) is half of the estimated tumor percentage for a heterozygous mutation. We have previously shown that a lower than expected VAF indicates the presence of the mutation in a subpopulation [7]. In this study, coexisting mutations detected in a specimen were predicted to be present in different neoplastic populations when the sum of the two observed VAFs was lower than or equivalent to the estimated VAF. In contrast, combined VAFs of coexisting mutations higher than the estimated VAF suggested that both mutations were present in the same population, particularly when the two VAFs were concordant with each other and the estimated VAF. Of note, subjective estimation of neoplastic cellularity and presence of mutant allele-specific imbalance (gain of the mutant allele or loss of the wild-type allele) may lead to a false prediction [19, 20].

2.4. Multiregional analysis to determine clonality

DNA was retrospectively re-isolated from multiple subareas within the originally designated area(s) of specimens with coexisting mutations. Subareas were defined based on physical separation and/or differences in histomorphology of the neoplastic cells, such as adenoma vs. adenocarcinoma or low-grade vs. high-grade, if present (Figure 1). DNA was isolated from 3 or more randomly-selected subareas if clearly distinctive morphologic features were not identifiable. Detection of coexisting mutations in each subarea with similar VAF ratios was suggestive of a neoplastic population containing both mutations.

2.5. Pyrosequencing

Pyrosequencing was performed as previously described [21]. The limit of detection of pyrosequencing is approximately 5% mutant alleles. The PCR primers were aggcctgctgaaaatgactgaatataa-3’ and 5’-caaagaatggtcctgcaccagtaatat-3’for KRAS codons 12/13 mutations, 5’-gactctgaagatgtacctatggtccta-3’ and 5’-cagatctgtatttatttcagtggtacttacct-3’ for KRAS codon 146 mutations, 5’-gttcttgctggtgtgaaatgactg-3’ and 5’-cctcacctctatggtgggatcatat-3’ for NRAS codons 12/13 mutations, and 5’-cacccccaggattcttacagaaaa-3’ and 5’-ttcgcctgtcctcatgtattgg-3’ for NRAS codon 61 mutations. The sequencing primer was 5’-cttgtggtagttggagct-3’ for KRAS codons 12/13 mutations, 5’-ggaattccttttattgaaacatca-3’ for KRAS codon 146 mutations, 5’-ctggtggtggttggagca-3’ for NRAS codons 12/13 mutations, and 5’-atactggatacagctgga-3’ for NRAS codon 61 mutations.

2.6. Tissue identity

Microsatellite analysis for tissue identification was performed using the AmpFlSTR Identifiler kit (Applied Biosystems, Foster City, CA), as described previously [22]. The assay has been clinically validated for detection of post-transplant chimerism. The limit of detection for the minor component is 1–5% alleles.

2.7. Statistical analysis

χ2 test was performed to calculate P values.

3. Results

3.1. Coexisting mutations in CRC

Mutational profiling data of the first 304 CRCs have been reported previously [7]. Current expanded study populations showed a similar mutation rate in each gene (Table 1). Activating mutation of the BRAF, KRAS and NRAS genes were observed in 68 (9.1%), 351 (47%) and 34 (4.6%) of CRCs, respectively. Coexisting mutations within BRAF, KRAS, NRAS, and PIK3CA genes were observed in 114 (15%) CRCs (Table 2). These included 21 (2.8%) CRCs with coexisting mutations within the same gene (one with 2 BRAF mutations, 6 with 2 KRAS mutations, 13 with 2 PIK3CA mutations and one with 3 PIK3CA mutations) and 103 (14%) CRCs with coexisting mutations in different genes. Among the 13 CRCs with 2 PIK3CA mutations, coexistence with a KRAS or BRAF mutation was seen in 10 CRCs. PIK3CA-mutated CRC showed a significantly higher incidence of coexisting mutations (Table 2). The incidence of coexisting PIK3CA mutations were not significantly different among BRAF-, KRAS- or NRAS-mutated CRCs (21%, 20% and 17%, respectively).

Table 1.

Mutational profiling of 744 colorectal cancers

Gene Incidence of mutationa Incidence of activating mutation
BRAF 86 (12%) 68 (9.1%)b
KRAS 353 (47%) 351 (47%)c
NRAS 35 (4.7%) 34 (4.6%)c
PIK3CA 135 (18%) 90 (12%)d
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a

Incidence of mutation in each gene among the first 304 colorectal cancers has been reported previously [7].

b

defined as mutations with high kinase activity [16–18].

c

defined as mutations involving codons 12, 13, 59, 61, 117 and 146.

d

defined as mutations involving codons 542, 545 and 1047.

Table 2.

Coexisting mutations in 774 colorectal cancers (CRC)a

Geneb BRAF KRAS NRAS PIK3CA %CMc P valued
BRAF (86) 1 (1.1%) 5 (5.8%) 1 (1.1%) 18 (21%) 28% <0.001
KRAS (353) 5 (1.4%) 6 (1.7%) 4 (1.1%) 69 (20%) 22% <0.001
NRAS (35) 1 (2.9%) 4 (11%) 0 (0%) 6 (17%) 31% <0.001
PIK3CA (135) 18 (13%) 69 (51%) 6 (4.4%) 14 (10%)e 69%
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a

Incidence of coexisting mutations among the first 304 CRC have been reported previously [7].

b

Numbers in parentheses indicate case number of CRC with a gene-specific mutation.

c

Percentage of BRAF-, KRAS-, NRAS- or PIK3CA-mutated CRC containing coexisting mutations (CM) within other genes.

d

Compared to %CM of PIK3CA-mutated CRC.

e

Nine CRCs with two PIK3CA and one KRAS mutations, one CRC with two PIK3CA and one BRAF mutations, 3 CRC with two PIK3CA mutations, and one CRC with three PIK3CA mutations.

3.2. Coexisting mutations within the MAPK pathway

While coexisting mutations among genes in the mTOR pathway and the MAPK pathway were common, coexisting activating mutations within the MAPK pathway were rare and, therefore, raised the concern for potential laboratory errors (Table 3). Microsatellite-based identity testing showed no minor peaks to indicate a mixture of DNA from 2 or more individuals in all specimens listed in Table 3. Multiregional analysis was performed using pyrosequencing and NGS to evaluate whether the coexisting mutations occurred in the same or different tumor populations.

Table 3.

Coexisting activating mutations within the MAPK pathway in colorectal cancers

Case Tumor % mut 1 (VAF%) mut 2 (VAF%) Clonality (VAFp/MRA)a
KRAS + KRAS
CRC0256 (Re) 61–80% KRAS p.G12A (17%) KRAS p.G13D (17%) Different/Different
CRC0340 (Bx) 61–80% KRAS p.G13C (32%) KRAS p.A146V (30%) Same/NE
CRC0449 (Re) 61–80% KRAS p.G13V (14%) KRAS p.G13D (21%) Different/ Different
 
KRAS + NRAS
CRC0068 (Re) 61–80% NRAS p.Q61K (14%) KRAS p.G12D (15%) Different/Different
CRC0125 (Re) 61–80% NRAS p.Q61R (15%) KRAS p.A146T (57%) Same/no CMb
CRC0202 (Re) 61–80% NRAS p.G13C (36%) KRAS p.G12V (5.2%) Different/Samec
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Abbreviations: Bx, biopsy; CM, coexisting mutation; MAPK, mitogen-activated protein kinase; NE, not evaluated; Re, resection; VAF, variant allele frequency.

a

VAFp: prediction of clonality by variant allele frequency. MRA: evaluation of clonality by multiregional analysis.

b

Only KRAS p.A146T was detected in the repeated NGS assay and multiregional analysis.

c

Both KRAS and NRAS mutations in adenoma, but only NRAS in invasive adenocarcinoma.

3.3. Coexisting BRAF and RAS mutations

NGS detected 67 CRCs with a class 1 BRAF p.V600E mutation, 2 with a class 2 BRAF mutation (p.G469A and p.G469R), 15 with a class 3 BRAF mutation (9 involving codon 594), one with p.G606R of unknown kinase activity, and one with coexistence of activating p.V600E mutation (class 1) and kinase-impaired p.D594G mutation (class 3). Among the 6 BRAF mutations detected in KRAS- or NRAS-mutated CRC, 4 were kinase-impaired (BRAF p.G466E plus KRAS p.Q22K of uncertain activating status, BRAF p.Y472C plus KRAS p.G12V, BRAF p.D594G plus KRAS p.A59E, and BRAF p.D594G plus KRAS p.G12D), and one was a class 2 BRAF mutation with intermediate kinase activity (BRAF p.G469R plus NRAS p.G12D) [16–18, 23]. The remaining one with p.V600E was seen in CRC0145 with KRAS p.G15S of uncertain activating status. Thus, there were no cases with coexisting activating KRAS mutation and BRAF mutation with high kinase activity.

3.4. Dual activating KRAS mutations

Two activating KRAS mutations were seen in 3 (0.4%) of 744 CRCs (Table 3). There were no doublet activating NRAS mutations. Review of H/E slides from CRC0256 and CRC0449 showed an invasive adenocarcinoma component and an adjacent adenoma component within the originally designated tumor areas isolated for testing (Figure 2). Multiregional analysis of CRC0256 and CRC0449 showed a different KRAS mutations within the adenocarcinoma in comparison to the adjacent adenoma, with the sum of the two observed VAFs equivalent to the estimated VAF based on tumor cellularity (Table 2). Three specimens showed coexistence of an activating KRAS mutation and a KRAS mutation of unknown activating status (p.G12D plus p.Q22K, p.G12V plus p.V14I, and p.G12D plus p.G60S).

Figure 2.

Figure 2.

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Invasive adenocarcinoma and adjacent adenoma harboring different KRAS mutations. In CRC0256, the invasive adenocarcinoma has expanded to abut the adenoma (A) and invade the normal epithelium (B). Multiregional analysis by pyrosequencing and NGS showed KRAS p.G13D in the adenoma (subareas 6 and 7) and KRAS p.G12A in the adenocarcinoma (subareas 1–5) (not shown). The original magnifications of the H&E stained slides are 100x.

3.5. Coexisting activating KRAS and NRAS mutations

One specimen showed coexistence of activating KRAS p.G12D mutation and NRAS p.G60E of unknown activating status. Coexisting activating KRAS and NRAS mutations were seen in 3 CRCs (Table 3). In CRC0068, which had coexisting KRAS p.G12D and NRAS p.Q61K mutations, the sum of the VAFs predicted that each mutation was present in a different population. This was confirmed by multiregional analysis of the invasive adenocarcinoma (Figure 3).

Figure 3.

Figure 3.

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Two adjacent invasive adenocarcinomas harboring different RAS mutations. In specimen CRC0068, pyrosequencing showed NRAS p.Q61K in subareas 1–5 with an infiltrative growth pattern and KRAS p.G12D in subareas 6–8 with a nodular growth pattern. These were also confirmed by NGS analysis of the subareas 1 and 7. Percentage in parentheses indicates variant allele frequency.

Coexisting KRAS p.A146T and NRAS p.Q61R mutations were detected in CRC0125. Pyrosequencing, however, showed only the KRAS mutation from 5 subareas. NGS repeated on the originally examined DNA sample and on DNA samples from two subareas also showed only KRAS p.A146T. The results indicate that the NRAS mutation was not present in this CRC. False detection of NRAS mutation in the original NGS assay was most likely the result of contamination with an NRAS mutant tumor during library preparation or the sequencing processes.

In CRC202, which had coexisting KRAS p.G12V and NRAS p.G13C mutations, the originally examined DNA was isolated from a designated area containing a dominant invasive adenocarcinoma component and an adjacent minor adenoma component. NGS detected a 5.2% KRAS mutation and a 36% NRAS mutation in the context of 61–80% estimated neoplastic cellularity. Multiregional analysis by pyrosequencing showed only the NRAS mutation in 3 adenocarcinoma subareas but both KRAS and NRAS mutations in a small adenoma subarea. NGS analysis of the adenoma subarea showed a 34% KRAS p.G12V and a 37% NRAS p.G13C in a context of 61–80% estimated neoplastic cellularity, suggesting that KRAS and NRAS mutations were present in the same adenoma population (Figure 4). Pyrosequencing of 2 additional adenoma subareas, which were not included in the originally designated area for examination and physically separated from the invasive adenocarcinoma in a 2-dimentional section, also showed both RAS mutations.

Figure 4.

Figure 4.

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Coexistence of KRAS and NRAS mutations in the adenoma, but only NRAS mutation in the adenocarcinoma. In CRC202, pyrosequencing showed coexistence of KRAS and NRAS mutations in a small adenoma subarea (subarea 1), but only the NRAS mutation in 3 adenocarcinoma subareas (subareas 2–4). These were also confirmed by NGS analysis of subareas 1 and 4. Percentage in parentheses indicates variant allele frequency.

4. Discussion

Coexisting mutations within the same signal transduction pathway are often mutually exclusive [6–8, 24]. Observation of unexpected coexisting mutations raises the concern for potential laboratory errors, which may occur during any step of pre-analytical, analytical or post-analytical phases. This includes contamination of analytes (tissues, DNA or PCR products) [12, 13]. Swapping of analytes or data files can also lead to the false detection of coexisting mutations, especially when simplex assays, such as Sanger sequencing or pyrosequencing, are applied to retrospective large cohort studies.

We propose an operating procedure to validate coexisting mutations (Figure 1). When unusual coexisting mutations are observed, previously isolated DNA is examined by microsatellite analysis to confirm tissue identity. If coexisting mutations are detected by a simplex assay, this is followed by either repeating the original assay or using an alternative assay to confirm results. Simultaneous mutational profiling by multiplex assays, such as NGS, may reduce the false detection of coexisting mutations caused by swapping of analytes and/or data files. In this retrospective study, we showed a false detection of coexisting KRAS and NRAS mutations in one specimen. In the presence of coexisting mutations that may affect clinical decision-making, multiregional analysis based on histomorphology not only confirms coexistence of mutations but also determines whether these mutations are present in the same or different populations of tumor cells.

BRAF mutations can be categorized according to their kinase activity [16, 25]. Coexistence of activating RAS mutations was seen in 3 of 15 CRCs with a kinase-impaired BRAF mutation in this study, supporting the reported interaction of oncogenic RAS proteins and kinase-impaired BRAF leading to hyperactivation of the CRAF/MEK/ERK cascade [17, 18]. Preclinical studies suggest tumors with coexisting RAS and kinase-impaired BRAF mutations may be sensitive to MEK or ERK inhibitors, and tumors with a kinase-impaired BRAF mutation and a wild-type RAS are predicted to be sensitive to anti-EGFR therapy [17, 18]. Response to anti-EGFR therapy in CRC patients with kinase-impaired BRAF mutation and wild-type RAS has also been reported [6, 17].

Coexistence of activating RAS mutation and BRAF p.V600E was not observed in 744 CRC and in 1006 lung cancers in our clinical laboratory [9, 13]. These findings were consistent with several previous reports with 500–1500 Caucasian or Asian patients examined for both KRAS and BRAF mutation in each study. Only one patient with coexisting activating RAS and BRAF p.V600E mutations was detected in a total of more than 200 CRC with class 1 BRAF mutations (mainly p.V600E) [6, 25–30]. In another report with 4411 CRC patients, KRAS mutations within codons 12 and 13 were not seen in 480 BRAF p.V600E-muated CRC. However, coexistence of activating KRAS mutation and BRAF mutation involving codon 600 or 601 was reported in 9 (32%) of 28 LN metastases of CRC [31], and in 6 (0.5%) of 1,261 CRC or 6 (3.3%) of 181 BRAF-mutated CRC [32]. Rare cases with coexistence of an activating RAS mutation and BRAF p.V600E have also been reported [33–36].

KRAS and NRAS mutations are also expected to be mutually exclusive, although preclinical data has shown divergent oncogenic properties of the KRAS p.G12D and NRAS p.G12D mutations [37]. Coexisting KRAS and NRAS mutations were not seen in two studies with approximately 500–700 CRC patients [6, 38], but was reported in 11 (0.9%) of 1294 European CRC patients [26], and in 1 (0.2%) of 621 and 8 (0.7%) of 1110 Chinese CRC patients [28, 29]. In this study, the coexistence of activating KRAS and NRAS mutation was initially reported in 3 of 744 CRC. Retrospective quality assessment confirmed false detection of coexistence in one of these specimens. Multiregional analysis revealed presence of KRAS and NRAS in separate invasive adenocarcinoma components submitted within the same tissue blocks (CRC0068) and presence of both KRAS and NRAS mutations in an adenoma component (CRC0202).

Dual KRAS mutations have also been reported in 4 (0.5%) of 747 European CRC patients and 8 (0.7%) of 1110 Chinese CRC patients [6, 29]. Whether the presence of two KRAS mutations were seen in the same or different tumor populations is not reported. In this study, NGS detected 3 (0.4%) of 744 CRC with coexistence of two activating KRAS mutations. Multiregional analysis confirmed different KRAS mutations within the invasive adenocarcinoma component and the adjacent adenoma component in CRC0256 and CRC0449. Coexistence of 2 activating KRAS mutations within an individual adenocarcinoma component was not seen in 744 CRC of this study. Analysis of the entire NGS panel revealed an APC mutation (p.R1450* in CRC0265 and p.P1443fs in CRC0449, data not shown) in the adenoma but not the adenocarcinoma, indicating CRC0256 and CRC0449 each contained a collisional adenoma and adenocarcinoma originating from different ancestral clones. The adenocarcinoma may have expanded laterally and upwards to invade a synchronous adenoma. In the absence of molecular profiling, the histomorphology could have been simply interpreted as colonic adenocarcinoma arising from an adenoma.

Invasion or abutting of a colonic adenocarcinoma into an adenoma of different clonal origin may have clinical implications. In the current guidelines for standard-of-care of metastatic CRC published by the College of American Pathologists and the Association of Molecular Pathology, KRAS, NRAS and BRAF are the only 3 genes recommended for mutational profiling [3]. In the multistep model for colorectal tumorigeneisis [4, 5], KRAS, NRAS and BRAF mutations, which are all part of the MAPK pathway, represent trunk (initiating) drivers to promote progression from small adenoma to large adenoma, a step before the formation of the founder cell of adenocarcinomas. These trunk drivers should be present in the invasive adenocarcinomas and their adjacent precursor adenomas. Therefore, inclusion of the precursor adenoma for mutational profiling of trunk drivers, such as the KRAS mutational status, is expected to be concordant with those from the invasive adenocarcinoma component. However, two or more colonic polyps may develop in close proximity, which may lead an adenocarcinomas to invade upward and collide with a neighboring unrelated adenomas. Endoscopic biopsy of a superficial lesion may, therefore, contain a minor invasive adenocarcinoma component insufficient for molecular profiling, and a dominant adenoma component originating from a different ancestral clone carrying different trunk driver mutations.

We propose an operating procedure for clinical validation of coexisting activating mutations of the BRAF, KRAS and NRAS genes within the MAPK pathway, which are expected to be mutually exclusive in colorectal tumorigenesis. Multiregional analysis according to this operating procedure confirmed a rare incidence of coexisting activating RAS mutations, determined their presence in the same or different tumor populations, and identified invasion of a synchronous adenoma by a collisional adenocarcinoma of different clonal original. Further studies are warranted to elucidate the biological significance and clinical implications of coexisting activating mutations within the MAPK pathway.

Highlights.

  • Quality assessment of coexisting mutations within the same pathway in colon cancers

  • An operating procedure proposed to validate unexpected coexisting mutations

  • False detection of coexisting mutations within the MAPK pathway demonstrated

  • Rare incidences of coexisting activating mutations within the MAPK pathway identified

  • Coexisting activating RAS and kinase impaired BRAF mutations shown

Acknowledgments

Funding: This retrospective analysis for quality improvement was supported by 1UM1CA186691–01 from the NIH-National Cancer Institute of the United States.

Footnotes

Conflict of interest: The authors declare that they have no competing interests.

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