Mutant Allele-specific Imbalance Research Articles

P-290 Mutant allele specific imbalance in RAS as prognostic marker in colorectal cancer

All colorectal cancer patients who progress to metastatic stage have their tumor molecularly analyzed for mutations in RAS. The NGS results reveal the ratio between the mutant allele and the wild-type allele. RAS mutant allele specific imbalance (MASI) is defined as when the number of copies of the mutant RAS allele is higher than that of the wild-type RAS allele strain. The objectives of the present study are to estimate the rate of the RAS MASI phenomenon among patients with metastatic colorectal cancer and to examine the relationship between this phenomenon and survival rate of the patients.

Bi-allelic amplification of ATM gene in blastoid variant of mantle cell lymphoma: a novel mechanism of inactivation due to chromoanagenesis?

BackgroundMantle cell lymphoma (MCL) is derived from naïve CD5+ B-cells with the cytogenetic hallmark translocation 11;14. The presence of additional abnormalities is associated with blastoid variants in MCL (BMCL) and confers a poor prognosis. Many of these tumors also show deletion or loss of heterozygosity (LOH) of the ATM gene and biallelic ATM inactivation show significantly higher chromosomal imbalances.Case presentationHere we report a 52 year-old male who presented to the clinic with worsening dyspnea, fever, chills, diffuse lymphadenopathy, splenomegaly and leukocytosis with blastoid cells circulating in blood. The bone marrow aspirate showed about 40% abnormal blast-looking cells and biopsy revealed a remarkable lymphoid infiltrate. The patient was diagnosed with blastoid variant mantle cell lymphoma (BMCL). Chromosome analysis on bone marrow showed a complex karyotype. FISH analysis from B-cell lymphoma panel showed bi-allelic amplification of ATM gene. Other abnormalities were present including CCND1/IGH fusion, confirming the MCL diagnosis, in addition to RB1 and p53 deletion. High resolution SNP-microarray studies showed complex copy number changes, especially on chromosomes 7 and 11, consistent with chromoanagenesis. Microarray studies also showed LOH at the ATM locus indicating the amplification seen on FISH is not biallelic.ConclusionTo the best of our knowledge, ATM gene amplification is not previously reported in BMCL and our case suggests a novel mechanism of ATM inactivation caused by chromoanagenesis resulting in mutant allele specific imbalance with copy number gain.

BRCA2, ATM, and CDK12 Defects Differentially Shape Prostate Tumor Driver Genomics and Clinical Aggression.

DNA damage repair (DDR) defects are common across cancer types and can indicate therapeutic vulnerability. Optimal exploitation of DDR defects in prostate cancer requires new diagnostic strategies and a better understanding of associated clinical genomic features. We performed targeted sequencing of 1,615 plasma cell-free DNA samples from 879 patients with metastatic prostate cancer. Depth-based copy-number calls and heterozygous SNP imbalance were leveraged to expose DDR-mutant allelic configuration and categorize mechanisms of biallelic loss. We used split-read structural variation analysis to characterize tumor suppressor rearrangements. Patient-matched archival primary tissue was analyzed identically. BRCA2, ATM, and CDK12 were the most frequently disrupted DDR genes in circulating tumor DNA (ctDNA), collectively mutated in 15% of evaluable cases. Biallelic gene disruption via second somatic alteration or mutant allele-specific imbalance was identified in 79% of patients. A further 2% exhibited homozygous BRCA2 deletions. Tumor suppressors TP53, RB1, and PTEN were controlled via disruptive chromosomal rearrangements in BRCA2-defective samples, but via oncogene amplification in context of CDK12 defects. TP53 mutations were rare in cases with ATM defects. DDR mutations were re-detected across 94% of serial ctDNA samples and in all available archival primary tissues, indicating they arose prior to metastatic progression. Loss of BRCA2 and CDK12, but not ATM, was associated with poor clinical outcomes. BRCA2, ATM, and CDK12 defects are each linked to distinct prostate cancer driver genomics and aggression. The consistency of DDR status in longitudinal samples and resolution of allelic status underscores the potential for ctDNA as a diagnostic tool.

Clonal lineage from normal endometrium to ovarian clear cell carcinoma through ovarian endometriosis.

Clear cell carcinoma of the ovary is thought to arise from endometriosis. In addition, retrograde menstruation of shed endometrium is considered the origin of endometriosis. However, little evidence supports cellular continuity from uterine endometrium to clear cell carcinoma through endometriosis at the genomic level. Here, we performed multiregional whole‐exome sequencing to clarify clonal relationships among uterine endometrium, ovarian endometriosis and ovarian clear cell carcinoma in a 56‐year‐old patient. Many somatic mutations including cancer‐associated gene mutations in ARID1A, ATM, CDH4, NRAS and PIK3CA were shared among epithelium samples from uterine endometrium, endometriotic lesions distant from and adjacent to the carcinoma, and the carcinoma. The mutant allele frequencies of shared mutations increased from uterine endometrium to distant endometriosis, adjacent endometriosis, and carcinoma. Although a splice site mutation of ARID1A was shared among the four epithelium samples, a frameshift insertion in ARID1A was shared by adjacent endometriosis and carcinoma samples, suggesting that the biallelic mutations triggered malignant transformation. Somatic copy number alterations, including loss of heterozygosity events at PIK3CA and ATM, were identified only in adjacent endometriosis and carcinoma, suggesting that mutant allele‐specific imbalance is another key factor driving malignant transformation. By reconstructing a clonal evolution tree based on the somatic mutations, we showed that the epithelium samples were derived from a single ancestral clone. Although the study was limited to a single patient, the results from this illustrative case could suggest the possibility that epithelial cells of ovarian endometriosis and clear cell carcinoma were descendants of uterine endometrial epithelium.

Activating AKT1 and PIK3CA Mutations in Metastatic Castration-Resistant Prostate Cancer

BackgroundActivating mutations in AKT1 and PIK3CA are undercharacterised in metastatic castration-resistant prostate cancer (mCRPC), but are linked to activation of phosphatidylinositol 3-kinase (PI3K) signalling and sensitivity to pathway inhibitors in other cancers. ObjectiveTo determine the prevalence, genomic context, and clinical associations of AKT1/PIK3CA activating mutations in mCRPC. Design, setting, and participantsWe analysed targeted cell-free DNA (cfDNA) sequencing data from 599 metastatic prostate cancer patients with circulating tumour DNA (ctDNA) content above 2%. Outcome measurements and statistical analysisIn patients with AKT1/PIK3CA mutations, cfDNA was subjected to PTEN intron sequencing and matched diagnostic tumour tissue was analysed when possible. Results and limitationsOf the patients, 6.0% (36/599) harboured somatic clonal activating mutation(s) in AKT1 or PIK3CA. Mutant allele-specific imbalance was common. Clonal mutations in mCRPC ctDNA were typically detected in pretreatment primary tissue and were consistent across serial ctDNA collections. AKT1/PIK3CA-mutant mCRPC had fewer androgen receptor (AR) gene copies than AKT1/PIK3CA wild-type mCRPC (median 4.7 vs 10.3, p = 0.003). AKT1 mutations were mutually exclusive with PTEN alterations. Patients with and without AKT1/PIK3CA mutations showed similar clinical outcomes with standard of care treatments. A heavily pretreated mCRPC patient with an AKT1 mutation experienced a 50% decline in prostate-specific antigen with Akt inhibitor (ipatasertib) monotherapy. Ipatasertib also had a marked antitumour effect in a patient-derived xenograft harbouring an AKT1 mutation. Limitations include the inability to assess AKT1/PIK3CA correlatives in ctDNA-negative patients. ConclusionsAKT1/PIK3CA activating mutations are relatively common and delineate a distinct mCRPC molecular subtype with low-level AR copy gain. Clonal prevalence and evidence of mutant allele selection propose PI3K pathway dependency in selected patients. The use of cfDNA screening enables prospective clinical trials to test PI3K pathway inhibitors in this population. Patient summaryOf advanced prostate cancer cases, 6% have activating mutations in the genes AKT1 or PIK3CA. These mutations can be identified using a blood test and may help select patients suitable for clinical trials of phosphatidylinositol 3-kinase inhibitors.

H3F3A mutant allele specific imbalance in an aggressive subtype of diffuse midline glioma, H3 K27M-mutant

Diffuse midline glioma, H3 K27M-mutant is a lethal brain tumor located in the thalamus, brain stem, or spinal cord. H3 K27M encoded by the mutation of a histone H3 gene such as H3F3A plays a pivotal role in the tumorigenesis of this type of glioma. Although several studies have revealed comprehensive genetic and epigenetic profiling, the prognostic factors of these tumors have not been identified to date. In various cancers, oncogenic driver genes have been found to exhibit characteristic copy number alterations termed mutant allele specific imbalance (MASI). Here, we showed that several diffuse midline glioma, H3 K27M-mutant exhibited high variant allele frequency (VAF) of the mutated H3F3A gene using droplet digital polymerase chain reaction (ddPCR) assays. Whole-genome sequencing (WGS) revealed that these cases had various copy number alterations that affected the mutant and/or wild-type alleles of the H3F3A gene. We also found that these MASI cases showed a significantly higher Ki-67 index and poorer survival compared with those in the lower VAF cases (P < 0.05). Our results indicated that the MASI of the H3F3A K27M mutation was associated with the aggressive phenotype of the diffuse midline glioma, H3 K27M-mutant via upregulation of the H3 K27M mutant protein, resulting in downregulation of H3K27me3 modification.

Molecular heterogeneity in lung cancer: from mechanisms of origin to clinical implications

Molecular heterogeneity is a frequent event in cancer responsible of several critical issues in diagnosis and treatment of oncologic patients. Lung tumours are characterized by high degree of molecular heterogeneity associated to different mechanisms of origin including genetic, epigenetic and non-genetic source. In this review, we provide an overview of recognized mechanisms underlying molecular heterogeneity in lung cancer, including epigenetic mechanisms, mutant allele specific imbalance, genomic instability, chromosomal aberrations, tumor mutational burden, somatic mutations. We focus on the role of spatial and temporal molecular heterogeneity involved in therapeutic implications in lung cancer patients.

BRAF Gene Copy Number and Mutant Allele Frequency Correlate with Time to Progression in Metastatic Melanoma Patients Treated with MAPK Inhibitors.

Metastatic melanoma is characterized by complex genomic alterations, including a high rate of mutations in driver genes and widespread deletions and amplifications encompassing various chromosome regions. Among them, chromosome 7 is frequently gained in BRAF-mutant melanoma, inducing a mutant allele-specific imbalance. Although BRAF amplification is a known mechanism of acquired resistance to therapy with MAPK inhibitors, it is still unclear if BRAF copy-number variation and BRAF mutant allele imbalance at baseline can be associated with response to treatment. In this study, we used a multimodal approach to assess BRAF copy number and mutant allele frequency in pretreatment melanoma samples from 46 patients who received MAPK inhibitor-based therapy, and we analyzed the association with progression-free survival. We found that 65% patients displayed BRAF gains, often supported by chromosome 7 polysomy. In addition, we observed that 64% patients had a balanced BRAF-mutant/wild-type allele ratio, whereas 14% and 23% patients had low and high BRAF mutant allele frequency, respectively. Notably, a significantly higher risk of progression was observed in patients with a diploid BRAF status versus those with BRAF gains [HR, 2.86; 95% confidence interval (CI), 1.29-6.35; P = 0.01] and in patients with low percentage versus those with a balanced BRAF mutant allele percentage (HR, 4.54; 95% CI, 1.33-15.53; P = 0.016). Our data suggest that quantitative analysis of the BRAF gene could be useful to select the melanoma patients who are most likely to benefit from therapy with MAPK inhibitors. Mol Cancer Ther; 17(6); 1332-40. ©2018 AACR.

Mutant allele-specific imbalance in patients with metastatic colorectal cancer.

706 Background: Patients with colorectal cancer(CRC) harboring RAS mutations do not respond to antiepidermal growth factor receptor (anti-EGFR) therapy. Next generation sequencing (NGS) identifies and quantifies mutant and wild type alleles as seen on the sequencing electropherograms. RAS mutant allele-specific imbalance (MASI) occurs when the mutant allele peak is higher than the wild type one. The aims of this study were to verify the rate and tissue distribution of RAS MASI as well as its clinical relevance. Methods: Results of NGS study of patients with metastatic colorectal cancer were collected retrospectively. The clinical charts of patients with RAS mutations were reviewed by the investigators who were blinded to the MASI results. Results: Between August 2014 and Jan 2016, 26 out of 52 (50%)patients tested, were found to harbor RAS mutations. Among them, 11 patients were detected with KRAS MASI(42.3%) Table 1 shows the different characteristics. 6 out of 11 patients presented with synchronous metastatic disease. This correlated with a statistically significant difference in the mean disease free survival that was 9 months in the KRAS MASI group compared with 27.5 months in the non MASI group. A few patients had more than one mutation. ie KRAS Ex2- G12D, 62.3%, PKI3CA Ex9-E545K, 24.6%, Ex20-H1047Y, 0.6% in the MASI group and KRAS Ex2-G12C+G12V - 10.85%, KRAS Ex4-A146T - 10.3%, NRAS gene Ex2-G12D - 1.1%, in non MASI group. Patients in the MASI group had a different clinical profile. (See Table 1) The overall survival of patients was calculated from diagnosis of metastatic disease, till death, 24 months-this was not significantly nor statistically different amongst groups. Conclusions: With the caveat of a small study and a retrospective analysis we showed that patients with MASI have worse characteristics that were translated to a significant shorter disease free survival. OS was not different amongst the groups. [Table: see text]

Mutant allele specific imbalance in oncogenes with copy number alterations: Occurrence, mechanisms, and potential clinical implications

Mutant allele specific imbalance (MASI) was initially coined to describe copy number alterations associated with the mutant allele of an oncogene. The copy number gain (CNG) specific to the mutant allele can be readily observed in electropherograms. With the development of genome-wide analyses at base-pair resolution with copy number counts, we can now further differentiate MASI into those with CNG, with copy neutral alteration (also termed acquired uniparental disomy; UPD), or with loss of heterozygosity (LOH) due to the loss of the wild-type (WT) allele. Here we summarize the occurrence of MASI with CNG, aUPD, or MASI with LOH in some major oncogenes (such as EGFR, KRAS, PIK3CA, and BRAF). We also discuss how these various classifications of MASI have been demonstrated to impact tumorigenesis, progression, metastasis, prognosis, and potentially therapeutic responses in cancer, notably in lung, colorectal, and pancreatic cancers.

Pancreatic carcinosarcoma with the same KRAS gene mutation in both carcinomatous and sarcomatous components: molecular evidence for monoclonal origin of the tumour.

To better understand the histogenesis, prognosis and feasible treatment of pancreatic carcinosarcoma, a rare type of neoplasia. We investigated eight additional cases of pancreatic carcinosarcoma at a single institution, including the clinicopathological, immunohistochemical, and KRAS mutation characteristics. We have also reviewed the current literature on this rare type of neoplasia, and summarized the clinicopathological features and feasible treatments. As a result, concordant strong nuclear immunoreactivity for P53 protein and the same type of KRAS gene mutation, c.35G>A (p.G12D) or c.35G>T (p.G12V), were showed in both carcinomatous and sarcomatous components in five of eight cases. Furthermore, we found that the patients treated with surgery plus postoperative chemotherapy had longer survival than those treated with surgery only (P = 0.034 and P = 0.131 for overall survival and disease-free survival, respectively), although Cox regression multivariate analysis indicated that it was not an independent predictor. In addition, we found KRAS mutant allele-specific imbalance in four of our five cases of pancreatic carcinosarcoma, which was associated with advanced disease and a worse prognosis. This is the largest panel of cases of pancreatic carcinosarcoma studied so far, including clinicopathological, immunohistochemical and molecular cytogenetic features. Our findings indicate that the tumour could have been of monoclonal origin, and that the sarcomatous components might have arisen from metaplastic transformation of the carcinomatous components. Our results also suggest that surgery plus POC including gemcitabine may be a good choice for patients with pancreatic carcinosarcoma.

Performance characteristics of next-generation sequencing in clinical mutation detection of colorectal cancers

Activating mutations in downstream genes of the epidermal growth factor receptor (EGFR) pathway may cause anti-EGFR resistance in patients with colorectal cancers. We present performance characteristics of a next-generation sequencing assay designed to detect such mutations. In this retrospective quality assessment study, we analyzed mutation detected in the KRAS, NRAS, BRAF, and PIK3CA genes by a clinically validated next-generation sequencing assay in 310 colorectal cancer specimens. Tumor cellularity and mutant allele frequency were analyzed to identify tumor heterogeneity and mutant allele-specific imbalance. Next-generation sequencing showed precise measurement of mutant allele frequencies and detected 23% of mutations with 2–20% mutant allele frequencies. Of the KRAS mutations detected, 17% were outside of codons 12 and 13. Among PIK3CA mutations, 48% were outside of codons 542, 545, and 1047. The percentage of tumors with predicted resistance to anti-EGFR therapy increased from 40% when testing for only mutations in KRAS exon 2 to 47% when testing for KRAS exons 2–4, 48% when testing for KRAS and NRAS exons 2–4, 58% when including BRAF codon 600 mutations, and 59% when adding PIK3CA exon 20 mutations. Right-sided colorectal cancers carried a higher risk of predicted anti-EGFR resistance. A concomitant KRAS mutation was detected in 51% of PIK3CA, 23% of NRAS, and 33% of kinase-impaired BRAF-mutated tumors. Lower than expected mutant allele frequency indicated tumor heterogeneity, while higher than expected mutant allele frequency indicated mutant allele-specific imbalance. Two paired neuroendocrine carcinomas and adjacent adenomas showed identical KRAS mutations, but only PIK3CA mutations in neuroendocrine carcinomas. Next-generation sequencing is a robust tool for mutation detection in clinical laboratories. It demonstrates high analytic sensitivity and broad reportable range, and it provides simultaneous detection of concomitant mutations and a quantitative measurement of mutant allele frequencies to predict tumor heterogeneity and mutant allele-specific imbalance.

EGFR and KRAS Mutations in ALK-Positive Lung Adenocarcinomas: Biological and Clinical Effect

IntroductionIn lung adenocarcinoma (ADC), anaplastic lymphoma receptor tyrosine kinase (ALK) rearrangements are mutually exclusive with epidermal growth factor receptor (EGFR) and Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations. However, the existence of double-positive (DP) patients have been sporadically described. We identified DP cases in therapy-naive ALK-rearranged ADC and characterized the biology of these tumors to better understand the clinical response to tyrosine kinase inhibitors (TKIs). Materials and MethodsWe selected 42 ALK-positive ADCs from a multicentric series of 301 cases of ADCs. A mutational analysis was performed using Sanger and/or pyrosequencing to address exons 18-21 of EGFR and codons 12-13 of the KRAS gene. In addition, the KRAS and EGFR copy number was investigated using fluorescent in situ hybridization. DP patients were treated with TKIs, and their response was evaluated according to the Response Evaluation Criteria in Solid Tumors criteria. ResultsEight of 42 ALK-positive ADCs (19%) demonstrated a concomitant mutation in the EGFR (3 cases) or KRAS (5 cases) genes and were classified as DP. All DP cases displayed copy number gains in the EGFR or KRAS gene because of polysomy or gene amplification. In the latter cases, a mutant allele-specific imbalance was observed. Four patients were treated with TKIs. The 2 EGFR-mutant DP patients demonstrated a better response to crizotinib compared with erlotinib. The 2 KRAS-mutant DP patients experienced opposite responses to crizotinib. ConclusionThe incidence of DP ADC is not negligible. Patients with ALK/EGFR might benefit more from crizotinib compared with erlotinib administration, although the efficacy of TKIs in patients with ALK/KRAS remains unclear. An integrated targeted therapy should be considered for patients with DP ADC.

KRAS Mutant Allele-Specific Imbalance (MASI) assessment in routine samples of patients with metastatic colorectal cancer

AimsPatients with colorectal cancer harbouring KRAS mutations do not respond to antiepidermal growth factor receptor (anti-EGFR) therapy. Community screening for KRAS mutation selects patients for treatment. When a KRAS mutation...

Lymph node metastases of melanoma: challenges for BRAF mutation detection.

Detection of B-Raf proto-oncogene, serine/threonine kinase (BRAF) mutations is required to predict response to BRAF or mitogen-activated protein kinase kinase 1 and 2 inhibitors in metastatic melanoma. Lymph node (LN) specimens carrying melanoma cells intermingled with abundant lymphocytes often contain low tumor cellularity. This study is aimed to examine challenges in the clinical detection of BRAF mutations in LN specimens with metastatic melanoma and to illustrate characteristic features of p.V600E and non-p.V600E mutations. In this retrospective study for quality assessment of the pyrosequencing assay, we compared characteristics of 53 LN and 135 non-LN formalin-fixed, paraffin-embedded specimens with metastatic melanoma submitted for BRAF mutation detection over a 40-month period. LN specimens showed a significantly higher incidence of p.V600E mutations than non-LN specimens (49% versus 22%, P < .01) but a significantly lower tumor cellularity, particularly in the case of subcapsular or infiltrative metastases. Mutant allele-specific imbalance of the p.V600E mutation was predominantly present in specimens with distant organ metastases (79% versus 27% in LN metastases versus 13% in primary cutaneous tumors or adjacent soft tissue, P < .001). p.V600K was detected in 23% of men older than 60 years old, compared with 6% in women older than 60 years old and 2% in both men and women younger than 60 years old (P < .001). LN specimens with low tumor cellularity due to numerous adjacent lymphocytes may pose a challenge to clinical detection of BRAF mutations of melanoma. The higher incidence of p.V600E mutations in LNs may prompt further studies to elucidate if the p.V600E mutation in primary tumors is associated with a higher risk of LN metastasis.

KRAS mutant allele-specific imbalance is associated with worse prognosis in pancreatic cancer and progression to undifferentiated carcinoma of the pancreas

KRAS codon 12 mutations are present in about 90% of ductal adenocarcinomas and in undifferentiated carcinomas of the pancreas. The role of KRAS copy number changes and resulting KRAS mutant allele-specific imbalance (MASI) in ductal adenocarcinoma (n=94), and its progression into undifferentiated carcinoma of the pancreas (n=25) was studied by direct sequencing and KRAS fluorescence in situ hybridization (FISH). Semi-quantitative evaluation of sequencing electropherograms showed KRAS MASI (ie, mutant allele peak higher than or equal to the wild-type allele peak) in 22 (18.4%) cases. KRAS FISH (performed on 45 cases) revealed a trend for more frequent KRAS amplification among cases with KRAS MASI (7/20, 35% vs 3/25, 12%, P=0.08). KRAS amplification by FISH was seen only in undifferentiated carcinomas (10/24, 42% vs 0/21 pancreatic ductal adenocarcinoma, 0%, P=0.0007). In 6 of 11 cases with both undifferentiated and well-differentiated components, transition to undifferentiated carcinoma was associated with an increase in KRAS copy number, due to amplification and/or chromosome 12 hyperploidy. Pancreatic carcinomas with KRAS MASI (compared to those without MASI) were predominantly undifferentiated (16/22, 73% vs 9/97, 9%, P<0.001), more likely to present at clinical stage IV (5/22, 23% vs 7/97, 7%, P=0.009), and were associated with shorter overall survival (9 months, 95% confidence interval, 5–13, vs 22 months, 95% confidence interval, 17–27; P=0.015) and shorter disease-free survival (5 months, 95% confidence interval, 2–8 vs 13 months, 95% confidence interval, 10–16; P=0.02). Our findings suggest that in a subset of ductal adenocarcinomas, KRAS MASI correlates with the progression to undifferentiated carcinoma of the pancreas.

Colorectal carcinomas, KRAS p.G13D mutant allele–specific imbalance, and anti–epidermal growth factor receptor therapy

Woo et al have comprehensively reviewed controversies surrounding anti–epidermal growth factor receptor (anti-EGFR) treatment in metastatic colon cancer, specifically the unresolved issue of anti-EGFR therapy in patients with colorectal carcinomas (CRC) harboring KRAS Gly13Asp (G13D) mutation.1 Even before the report by De Roock et al on the potential benefit of cetuximab in CRC with KRAS p.G13D,2 there were contradicting data on the prognostic significance of this mutation (reviewed in Hartman et al3). The apparently discrepant clinical and cell line data may be reconciled by taking into account the phenomenon of mutant allele–specific imbalance (MASI). Introduced by Soh et al, the term “MASI” summarizes a variety of mechanisms that may lead to the predominance of the mutated allele over the wild-type allele.4 Such an allelic imbalance favoring the mutant allele may result from the deletion of the wild-type allele, and/or copy number gain of the mutant allele. It was shown that KRAS MASI is associated with increased ras guanine triphosphatase activity in cell lines and worse outcome in KRAS-mutated CRC. In a study of 394 patients with KRAS-mutated CRC, KRAS MASI developed by chromosome 12 hyperploidy or KRAS amplification, most frequently in cases with KRAS codon 13 mutations. In a multivariate analysis, KRAS codon 13 MASI represented an adverse prognostic factor independent of clinical stage and tumor site.3 We would like to suggest for future reports on anti-EGFR therapy in KRAS p.G13D-mutated CRC to explicitly control for KRAS MASI. Two scenarios are likely. First, the presence or absence of KRAS MASI may correlate with response to anti-EGFR treatment. Alternatively, anti-EGFR treatment may have no therapeutic effect and the outcome differences attributed to anti-EGFR therapy in some studies may merely reflect the unaccounted effect of KRAS MASI. Practically, KRAS MASI can be estimated from a semiquantitative review of routine sequencing electropherograms or by more sophisticated methods aimed at detecting KRAS allelic imbalances, chromosome 12 hyperploidy, or KRAS amplification. Douglas J. Hartman, MD Simion I. Chiosea, MD Department of Pathology University of Pittsburgh Medical Center Pittsburgh, Pennsylvania

Reply to colorectal carcinomas, KRAS p.G13D mutant allele–specific imbalance, and anti–epidermal growth factor receptor therapy

Reply to colorectal carcinomas, KRAS p.G13D mutant allele–specific imbalance, and anti–epidermal growth factor receptor therapy

The prognostic and predictive value of KRAS oncogene substitutions in lung adenocarcinoma

The prognostic and therapeutic implications of the spectrum of v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) oncogene substitutions in lung cancer remain poorly understood. The objective of this study was to determine whether KRAS oncogene substitutions differed with regard to prognosis or predictive value in lung adenocarcinoma. KRAS oncogene substitutions and mutant allele-specific imbalance (MASI) were determined in patients with lung adenocarcinoma, and the associations with overall survival (OS), recurrence-free survival (RFS), and chemotherapy interactions were assessed. KRAS mutational analysis was performed on 988 lung adenocarcinomas, and 318 KRAS mutations were identified. In this predominantly early stage cohort (78.6% of patients had stage I-III disease), OS and RFS did not differ according to the type of KRAS substitution (OS, P = .612; RFS, P = .089). There was a trend toward better OS in the subset of patients with KRAS codon 13 mutations (P = .052), but that trend was not significant in multivariate analysis (P = .076). RFS did not differ according to codon type in univariate analysis (P = .322). There was a marked difference in RFS based on the presence of MASI in univariate analysis (P = .004) and multivariate analysis (P = .009). A test for interaction was performed to determine whether the effect of chemotherapy on OS and RFS differed based on KRAS substitution type, codon type, or the presence of MASI. That test indicated that there were no differences in the effects of chemotherapy for any of variables examined. KRAS codon 13 mutations and MASI were identified as candidate biomarkers for prognosis, and it may be useful to incorporate them into prospective studies evaluating novel therapies in KRAS-mutant lung adenocarcinoma.

Mutant allele‐specific imbalance modulates prognostic impact of KRAS mutations in colorectal adenocarcinoma and is associated with worse overall survival

The prognostic impact of distinct KRAS mutations in colorectal carcinomas is not fully characterized. We hypothesized that the prognostic impact of KRAS mutations is modulated by KRAS mutant allele-specific imbalance (MASI). KRAS MASI was assessed by sequencing electropherograms in KRAS-mutated colorectal carcinomas (N = 394, prospectively tested). The mechanism of KRAS MASI was studied by fluorescence in situ hybridization (FISH; N = 50). FISH showed that KRAS MASI developed by chromosome 12 hyperploidy (9/18, 50%) or KRAS amplification (1/18, 5.5%). KRAS MASI was more common in tumors with KRAS codon 13 than with codon 12 mutations [24/81, 30% vs. 54/313, 17%; odds ratio (OR), 2.0, 95% confidence interval (CI), 1.2-3.5; p = 0.01]. KRAS MASI was correlated with overall survival (N = 358, median follow-up = 21 months). In a multivariate analysis, KRAS codon 13 MASI was an independent adverse prognostic factor (compared to codon 13 mutants without MASI combined with all codon 12 mutants; adjusted hazard ratio, 2.2, 95% CI: 1.2-3.9; p = 0.01). KRAS MASI arises through chromosome 12 hyperploidy or KRAS amplification and, when affects KRAS codon 13, is associated with worse overall survival.