<Emphasis Type="Italic">BRAF</Emphasis> V600E mutation and resistance to anti-EGFR monoclonal antibodies in patients with metastatic colorectal cancer: a meta-analysis

Epidemiologic studies have evaluated the association between BRAF mutations and resistance to the treatment of anti-EGFR monoclonal antibodies (MoAb) in patients with metastatic colorectal cancer (mCRC). However, the results are still inconclusive. To derive a more precise estimation of the relationship, we performed this meta-analysis. A total of 11 studies were included in the final meta-analysis. There were seven studies for unselected mCRC patients and four studies for patients with wild type KRAS mCRC. Among unselected mCRC patients, BRAF V600E mutation was detected in 48 of 546 primary tumors (8.8%). The objective response rate (ORR) of patients with mutant BRAF was 29.2% (14/48), whereas the ORR of patients with wild-type BRAF was 33.5% (158/472).The overall RR for ORR of mutant BRAF patients over wild-type BRAF patients was 0.86 (95% CI = 0.57–1.30; P = 0.48). For patients with KRAS wild-type mCRC, BRAF V600E mutation was detected in 40 of 376 primary tumors (10.6%). The ORR of patients with mutant BRAF was 0.0% (0/40), whereas the ORR of patients with wild-type BRAF was 36.3% (122/336). The pooled RR of mutant BRAF patients over wild-type BRAF patients was 0.14 (95% CI = 0.04–0.53; P = 0.004). In conclusion, this meta-analysis provides evidence that BRAF V600E mutation is associated with lack of response in wild-type KRAS mCRC treated with anti-EGFR MoAbs. BRAF mutation may be used as an additional biomarker for the selection of mCRC patients who might benefit from anti-EGFR MoAbs therapy.     

Significance of Liquid Biopsy for Monitoring and Therapy Decision of Colorectal Cancer

PURPOSE: Despite therapeutic improvements, all patients with nonresectable metastatic colorectal cancer (mCRC) acquire resistance to treatment probably due to the growth of mutated clones. In contrast to tissue-based studies, liquid biopsies have enabled the opportunity to reveal emerging resistance to treatment by detecting mutated clones and noninvasively monitoring clonal dynamics during therapy. METHODS: The courses of three patients with mCRC who were initially RAS wild-type were monitored longitudinally using liquid biopsy with long-term follow-up of up to 20 sequential samples. Detection of fragmented RAS mutated circulating cell-free DNA (cf)DNA in plasma was performed by BEAMing. In addition, plasma digital droplet PCR was used to detect and quantify BRAF and PIK3CA mutated cfDNA. Changes of mutational load were correlated with imaging data. RESULTS: A combination of liquid biopsy and radiological imaging enabled visualization of the occurrence of clonal redistribution after discontinuation of anti-EGFR mAb therapy, as well as emerging RAS mutations during therapy with anti-EGFR mAb indicating resistance. Furthermore, we found that growth of RAS mutated clones is independent of direct selective pressure by anti-EGFR therapy, which is a significant and new finding of this study. CONCLUSIONS: Our findings demonstrated the whole spectrum of clonal selection and redistribution of mutated cell clones leading to acquired resistance. Given our observation that the growth of RAS mutated clones can evolve even in the absence of anti-EGFR mAb therapy, there is a clear imperative to monitor RAS mutations in serial blood draws in all RAS wild-type patients in general and independent of the therapy.     

Single-Tubed Wild-Type Blocking Quantitative PCR Detection Assay for the Sensitive Detection of Codon 12 and 13 KRAS Mutations

The high degree of intra-tumor heterogeneity has meant that it is important to develop sensitive and selective assays to detect low-abundance KRAS mutations in metastatic colorectal carcinoma (mCRC) patients. As a major potential source of tumor DNA in the aforementioned genotyping assays, it was necessary to conduct an analysis on both the quality and quantity of DNA extracted from formalin-fixed paraffin-embedded (FFPE). Therefore, four commercial FFPE DNA extraction kits were initially compared with respect to their ability to facilitate extraction of amplifiable DNA. The results showed that TrimGen kits showed the greatest performance in relation to the quality and quantity of extracted FFPE DNA solutions. Using DNA extracted by TrimGen kits as a template for tumor genotyping, a real-time wild-type blocking PCR (WTB-PCR) assay was subsequently developed to detect the aforementioned KRAS mutations in mCRC patients. The results showed that WTB-PCR facilitated the detection of mutated alleles at a ratio of 1:10,000 (i.e. 0.01%) wild-type alleles. When the assay was subsequently used to test 49 mCRC patients, the results showed that the mutation detection levels of the WTB-PCR assay (61.8%; 30/49) were significantly higher than that of traditional PCR (38.8%; 19/49). Following the use of the real-time WTB-PCR assay, the ΔC _q method was used to quantitatively analyze the mutation levels associated with KRAS in each FFPE sample. The results showed that the mutant levels ranged from 53.74 to 0.12% in the patients analyzed. In conclusion, the current real-time WTB-PCR is a rapid, simple, and low-cost method that permits the detection of trace amounts of the mutated KRAS gene.     

Multi-Determinants Analysis of Molecular Alterations for Predicting Clinical Benefit to EGFR-Targeted Monoclonal Antibodies in Colorectal Cancer

Background

KRAS mutations occur in 35–45% of metastatic colorectal cancers (mCRC) and preclude responsiveness to EGFR-targeted therapy with cetuximab or panitumumab. However, less than 20% patients displaying wild-type KRAS tumors achieve objective response. Alterations in other effectors downstream of the EGFR, such as BRAF, and deregulation of the PIK3CA/PTEN pathway have independently been found to give rise to resistance. We present a comprehensive analysis of KRAS, BRAF, PIK3CA mutations, and PTEN expression in mCRC patients treated with cetuximab or panitumumab, with the aim of clarifying the relative contribution of these molecular alterations to resistance.

Methodology/Principal Findings

We retrospectively analyzed objective tumor response, progression-free (PFS) and overall survival (OS) together with the mutational status of KRAS, BRAF, PIK3CA and expression of PTEN in 132 tumors from cetuximab or panitumumab treated mCRC patients. Among the 106 non-responsive patients, 74 (70%) had tumors with at least one molecular alteration in the four markers. The probability of response was 51% (22/43) among patients with no alterations, 4% (2/47) among patients with 1 alteration, and 0% (0/24) for patients with ≥2 alterations (p<0.0001). Accordingly, PFS and OS were increasingly worse for patients with tumors harboring none, 1, or ≥2 molecular alteration(s) (p<0.001).

Conclusions/Significance

When expression of PTEN and mutations of KRAS, BRAF and PIK3CA are concomitantly ascertained, up to 70% of mCRC patients unlikely to respond to anti-EGFR therapies can be identified. We propose to define as ‘quadruple negative’, the CRCs lacking alterations in KRAS, BRAF, PTEN and PIK3CA. Comprehensive molecular dissection of the EGFR signaling pathways should be considered to select mCRC patients for cetuximab- or panitumumab-based therapies.     

KRAS Genotypic Changes of Circulating Tumor Cells during Treatment of Patients with Metastatic Colorectal Cancer

Introduction

Circulating tumor cells (CTCs) could represent a non-invasive source of cancer cells used for longitudinal monitoring of the tumoral mutation status throughout the course of the disease. The aims of the present study were to investigate the detection of KRAS mutations in CTCs from patients with metastatic colorectal cancer (mCRC) and to compare their mutation status during treatment or disease progression with that of the corresponding primary tumors.

Materials and Methods

Identification of the seven most common KRAS mutations on codons 12 and 13 was performed by Peptide Nucleic Acid (PNA)-based qPCR method. The sensitivity of the assay was determined after isolation of KRAS mutant cancer cells spiked into healthy donors'' blood, using the CellSearch Epithelial Cell kit. Consistent detection of KRAS mutations was achieved in samples containing at least 10 tumor cells/7.5 ml of blood.

Results

The clinical utility of the assay was assessed in 48 blood samples drawn from 31 patients with mCRC. All patients had PIK3CA and BRAF wild type primary tumors and 14 KRAS mutant tumors. CTCs were detected in 65% of specimens obtained from 74% of patients. KRAS mutation analysis in CTC-enriched specimens showed that 45% and 16.7% of patients with mutant and wild type primary tumors, respectively, had detectable mutations in their CTCs. Assessing KRAS mutations in serial blood samples revealed that individual patient''s CTCs exhibited different mutational status of KRAS during treatment.

Conclusions

The current findings support the rationale for using the CTCs as a dynamic source of tumor cells which, by re-evaluating their KRAS mutation status, could predict, perhaps more accurately, the response of mCRC patients to targeted therapy.     

Detection of KRAS Exon 2 Mutations in Circulating Tumor Cells Isolated by the ISET System from Patients with RAS Wild Type Metastatic Colorectal Cancer

INTRODUCTION: The presence of KRAS mutations in patients with metastatic colorectal cancer (mCRC) predicts poor response to agents targeting the EGFR. Even in patients with RAS wild type (WT) tumors, resistance eventually develops due to multiple mechanisms, including the expansion of previously undetected KRAS mutated clones. In this feasibility study, we aimed to detect KRAS exon 2 mutations in serial samples of circulating tumor cells (CTCs) of RAS WT patients with mCRC captured by the Isolation by Size of Epithelial Tumor cells (ISET) system. METHODS: CTC isolation using the ISET system was performed from prospectively collected blood samples obtained from patients with RAS and BRAF WT mCRC prior to first-line therapy initiation, at first imaging assessment and on disease progression. CTCs were enumerated using hematoxylin & eosin and CD45 double stain on a single membrane spot. DNA was extracted from 5 spots and KRAS exon 2 mutations were detected using a custom quantitative Polymerase Chain Reaction (qPCR) assay. RESULTS: Fifteen patients were enrolled and 28 blood samples were analyzed. In 9 (60%) patients, at least one sample was positive for the presence of a KRAS exon 2 mutation. In 11 out of 28 samples (39.2%) with detectable CTCs a KRAS mutation was detected; the corresponding percentages for baseline and on progression samples were 27% and 37.5%, respectively. The most commonly detected mutations were G13D and G12C (n = 3). The presence of KRAS mutated CTCs at baseline was not prognostic for either PFS (P = .950) or OS (P = .383). CTC kinetics did not follow tumor response patterns. CONCLUSION: The results demonstrate that using a qPCR-based assay, KRAS exon 2 mutations could be detected in CTCs captured by the ISET system from patients with RAS WT primary tumors. However, the clinical relevance of these CTCs remains to be determined in future studies.     

Validation of a Multiplex Allele-Specific Polymerase Chain Reaction Assay for Detection of KRAS Gene Mutations in Formalin-Fixed,Paraffin-Embedded Tissues from Colorectal Cancer Patients

Background

Patients with KRAS mutations do not respond to epidermal growth factor receptor (EGFR) inhibitors and fail to benefit from adjuvant chemotherapy. Mutation analysis of KRAS is needed before starting treatment with monoclonal anti-EGFR antibodies in patients with metastatic colorectal cancer (mCRC). The objective of this study is to develop a multiplex allele-specific PCR (MAS-PCR) assay to detect KRAS mutations.

Methods

We developed a single-tube MAS-PCR assay for the detection of seven KRAS mutations (G12D, G12A, G12R, G12C, G12S, G12V, and G13D). We performed MAS-PCR assay analysis for KRAS on DNA isolated from 270 formalin-fixed paraffin-embedded (FFPE) colorectal cancer tissues. Sequences of all 270 samples were determined by pyrosequencing. Seven known point-mutation DNA samples diluted with wild-type DNA were assayed to determine the limitation of detection and reproducibility of the MAS-PCR assay.

Results

Overall, the results of MAS-PCR assay were in good concordance with pyrosequencing, and only seven discordant samples were found. The MAS-PCR assay reproducibly detected 1 to 2% mutant alleles. The most common mutations were G13D in codon 13 (49.17%), G12D (25.83%) and G12V (12.50%) in codon 12.

Conclusion

The MAS-PCR assay provides a rapid, cost-effective, and reliable diagnostic tool for accurate detection of KRAS mutations in routine FFPE colorectal cancer tissues.     

Measuring KRAS Mutations in Circulating Tumor DNA by Droplet Digital PCR and Next-Generation Sequencing

Measuring total cell-free DNA (cfDNA) or cancer-specific mutations herein has presented as new tools in aiding the treatment of cancer patients. Studies show that total cfDNA bears prognostic value in metastatic colorectal cancer (mCRC) and that measuring cancer-specific mutations could supplement biopsies. However, limited information is available on the performance of different methods. Blood samples from 28 patients with mCRC and known KRAS mutation status were included. cfDNA was extracted and quantified with droplet digital polymerase chain reaction (ddPCR) measuring Beta-2 Microglobulin. KRAS mutation detection was performed using ddPCR (Bio-Rad) and next-generation sequencing (NGS, Ion Torrent PGM). Comparing KRAS mutation status in plasma and tissue revealed concordance rates of 79% and 89% for NGS and ddPCR. Strong correlation between the methods was observed. Most KRAS mutations were also detectable in 10-fold diluted samples using the ddPCR. We find that for detection of KRAS mutations in ctDNA ddPCR was superior to NGS both in analysis success rate and concordance to tissue. We further present results indicating that lower amount of plasma may be used for detection of KRAS mutations in mCRC.     

Effectors of Epidermal Growth Factor Receptor Pathway: The Genetic Profiling of KRAS,BRAF, PIK3CA,NRAS Mutations in Colorectal Cancer Characteristics and Personalized Medicine

Mutations in KRAS oncogene are recognized biomarkers that predict lack of response to anti- epidermal growth factor receptor (EGFR) antibody therapies. However, some patients with KRAS wild-type tumors still do not respond, so other downstream mutations in BRAF, PIK3CA and NRAS should be investigated. Herein we used direct sequencing to analyze mutation status for 676 patients in KRAS (codons 12, 13 and 61), BRAF (exon 11 and exon 15), PIK3CA (exon 9 and exon 20) and NRAS (codons12, 13 and 61). Clinicopathological characteristics associations were analyzed together with overall survival (OS) of metastatic colorectal cancer patients (mCRC). We found 35.9% (242/674) tumors harbored a KRAS mutation, 6.96% (47/675) harbored a BRAF mutation, 9.9% (62/625) harbored a PIK3CA mutation and 4.19% (26/621) harbored a NRAS mutation. KRAS mutation coexisted with BRAF, PIK3CA and NRAS mutation, PIK3CA exon9 mutation appeared more frequently in KRAS mutant tumors (P = 0.027) while NRAS mutation almost existed in KRAS wild-types (P<0.001). Female patients and older group harbored a higher KRAS mutation (P = 0.018 and P = 0.031, respectively); BRAF (V600E) mutation showed a higher frequency in colon cancer and poor differentiation tumors (P = 0.020 and P = 0.030, respectively); proximal tumors appeared a higher PIK3CA mutation (P<0.001) and distant metastatic tumors shared a higher NRAS mutation (P = 0.010). However, in this study no significant result was found between OS and gene mutation in mCRC group. To our knowledge, the first large-scale retrospective study on comprehensive genetic profile which associated with anti-EGFR MoAbs treatment selection in East Asian CRC population, appeared a specific genotype distribution picture, and the results provided a better understanding between clinicopathological characteristics and gene mutations in CRC patients.     

Changes in Colorectal Carcinoma Genomes under Anti-EGFR Therapy Identified by Whole-Genome Plasma DNA Sequencing

Monoclonal antibodies targeting the Epidermal Growth Factor Receptor (EGFR), such as cetuximab and panitumumab, have evolved to important therapeutic options in metastatic colorectal cancer (CRC). However, almost all patients with clinical response to anti-EGFR therapies show disease progression within a few months and little is known about mechanism and timing of resistance evolution. Here we analyzed plasma DNA from ten patients treated with anti-EGFR therapy by whole genome sequencing (plasma-Seq) and ultra-sensitive deep sequencing of genes associated with resistance to anti-EGFR treatment such as KRAS, BRAF, PIK3CA, and EGFR. Surprisingly, we observed that the development of resistance to anti-EGFR therapies was associated with acquired gains of KRAS in four patients (40%), which occurred either as novel focal amplifications (n = 3) or as high level polysomy of 12p (n = 1). In addition, we observed focal amplifications of other genes recently shown to be involved in acquired resistance to anti-EGFR therapies, such as MET (n = 2) and ERBB2 (n = 1). Overrepresentation of the EGFR gene was associated with a good initial anti-EGFR efficacy. Overall, we identified predictive biomarkers associated with anti-EGFR efficacy in seven patients (70%), which correlated well with treatment response. In contrast, ultra-sensitive deep sequencing of KRAS, BRAF, PIK3CA, and EGFR did not reveal the occurrence of novel, acquired mutations. Thus, plasma-Seq enables the identification of novel mutant clones and may therefore facilitate early adjustments of therapies that may delay or prevent disease progression.     

HER2+ mCRC patients with exon 20 R784G substitution mutation do not respond to the cetuximab therapy

The human epidermal growth factor 2 (HER2) gene undergoes various mutations that could alter its activity or respond to the antibody therapies. Cetuximab, a known anti-EGFR monoclonal antibody (mAB), is widely administered in metastatic colorectal cancer (mCRC) cases. Here we identified mCRC patients who did not respond to cetuximab (500 mg/m², q2w) after fluoropyrimidine/oxaliplatin regimen failure. Tumor samples were examined with immunohistochemistry for protein distribution, polymerase chain reaction (PCR) sequencing for mutation detection and real-time PCR for mRNA expression pattern analysis between cetuximab sensitive and resistance patients. The conformational differences of normal and mutated protein structures were predicted by bioinformatics analysis. The 5-year survival rates of target groups were estimated using the Kaplan–Meier method. Immunohistochemistry showed that all cases had high level of HER2 protein. No K-Ras or B-Raf mutation was observed among the study population; however, cetuximab resistance patients harbored a somatic mutation R784G at the exon 20 region of HER2 coding sequence. According to bioinformatics analysis, this mutation caused a notable misfold in protein conformation. Meanwhile, survival analysis showed R784G mutated mCRC patients had shortened survival rate compared with the mCRC cases with wild-type HER2. Collectively, these data report a new mechanism of resistance to cetuximab and might be applicable in modifying new therapeutic strategies for HER2 involved cancers.     

BRAF,PIK3CA,and HER2 Oncogenic Alterations According to KRAS Mutation Status in Advanced Colorectal Cancers with Distant Metastasis

Background

Anti-EGFR antibody–based treatment is an important therapeutic strategy for advanced colorectal cancer (CRC); despite this, several mutations—including KRAS, BRAF, and PIK3CA mutations, and HER2 amplification—are associated with the mechanisms underlying the development of resistance to anti-EGFR therapy. The aim of our study was to investigate the frequencies and clinical implications of these genetic alterations in advanced CRC.

Methods

KRAS, BRAF, and PIK3CA mutations were determined by Cobas real-time polymerase chain reaction (PCR) in 191 advanced CRC patients with distant metastasis. Microsatellite instability (MSI) status was determined by a fragmentation assay and HER2 amplification was assessed by silver in situ hybridization. In addition, KRAS mutations were investigated by the Sanger sequencing method in 97 of 191 CRC cases.

Results

Mutations in KRAS, BRAF, and PIK3CA were found in 104 (54.5%), 6 (3.1%), and 25 (13.1%) cases of advanced CRC, respectively. MSI-high status and HER2 amplification were observed in 3 (1.6%) and 16 (8.4%) cases, respectively. PIK3CA mutations were more frequently found in KRAS mutant type (18.3%) than KRAS wild type (6.9%) (P = 0.020). In contrast, HER2 amplifications and BRAF mutations were associated with KRAS wild type with borderline significance (P = 0.052 and 0.094, respectively). In combined analyses with KRAS, BRAF and HER2 status, BRAF mutations or HER2 amplifications were associated with the worst prognosis in the wild type KRAS group (P = 0.004). When comparing the efficacy of detection methods, the results of real time PCR analysis revealed 56 of 97 (57.7%) CRC cases with KRAS mutations, whereas Sanger sequencing revealed 49 cases (50.5%).

Conclusions

KRAS mutations were found in 54.5% of advanced CRC patients. Our results support that subgrouping using PIK3CA and BRAF mutation or HER2 amplification status, in addition to KRAS mutation status, is helpful for managing advanced CRC patients.     

Resistance to EGFR blockade in colorectal cancer: liquid biopsies and latent subclones

Two recent papers identify KRAS activation as a mechanism of acquired resistance to EGFR blockade in colorectal cancer. In doing so, they suggest that resistance to single-agent EGFR blockade will be unavoidable because these alterations exist as latent subclones within the tumor even prior to the initiation of therapy.Among the important molecular alterations in colon cancer is constitutive activation of the epidermal growth factor (EGFR) receptor tyrosine kinase, which promotes constitutive cellular proliferation and tumor progression. Recently, cetuximab and panitumumab, two monoclonal antibodies against EGFR, have shown promising results in the treatment of metastatic colorectal cancer (mCRC)^1,2. However, only a subset of patients respond to these anti-EGFR therapies. Strikingly, mutations in KRAS— a signaling effector downstream of EGFR — predict a lack of response to EGFR blockade^3,4. In other words, in the presence of a downstream KRAS activating mutation, upstream signaling by EGFR is dispensable. This phenomenon — in which a patient never responds to a therapy — is termed intrinsic (or de novo) resistance.However, even among patients with KRAS wild-type tumors, the efficacy of EGFR blockade is limited. Not all KRAS “wild type” patients respond, and those that do inevitably develop resistance to anti-EGFR therapy within a matter of months. This latter phenomenon of acquired — as opposed to intrinsic — drug resistance raises several important questions. What are the mechanisms of acquired resistance? Are they related to the observation of KRAS as a biomarker of intrinsic resistance? More generally, how does acquired resistance arise? Are new resistance-causing alterations generated during the course of treatment? Alternatively, do they pre-exist at low frequency in the initial tumor, expanding during the course of treatment due to the selective pressure of the therapy?Two recent studies in Nature begin to address these and other questions^5,6. In a paper by Misale et al.⁵, colorectal cancer cell lines initially sensitive to cetuximab were grown under continuous cetuximab treatment. The cetuximab-resistant daughter lines that ultimately emerged had gained either KRAS amplification or KRAS activating mutations—alterations sufficient to confer cetuximab resistance. Intriguingly, the authors showed that these alterations actually existed prior to treatment at low frequency in the parental cell lines, which suggests that the selective pressure of cetuximab treatment led to expansion of this previously latent sub-population and the emergence of resistance. To establish the clinical relevance of these findings, the authors queried whether KRAS alterations could be detected in previously KRAS wild-type mCRC patients following failure of cetuximab therapy. In 8/11 patients, cetuximab-resistant tumors had gained either KRAS mutation or amplification; moreover, by analyzing plasma samples for circulating tumor DNA (ctDNA), the authors were able to detect emergence of KRAS-mutant alleles up to 10 months before radiographic evidence of progression.In contrast to Misale et al.''s translational approach, a study by Diaz et al.⁶ began with a cohort of KRASwild-type mCRC patients who received panitumumab. Starting prior to pantiumumab therapy, serum samples were collected from each patient monthly until disease progression. These so-called “liquid biopsies” enabled the investigators to establish that, among patients with initially KRAS wild-type tumors, 38% developed detectable KRAS mutations during the course of therapy.However, whereas a conventional post-relapse tumor biopsy allows only an after-the-fact analysis of resistance, this longitudinal collection of serum samples allowed the investigators to monitor the evolution of resistance over time. By relating computed tomography measurements of tumor volume to ctDNA read counts, and then monitoring the increase in KRAS-mutant ctDNA reads over time, the authors estimated the growth rate of these panitimumab-resistant subclones. This information, together with the length of time between the initiation of panitumumab therapy and first detection of KRAS-mutant ctDNA reads, allowed the authors to calculate that KRAS mutations preceded the initiation of therapy.Cumulatively, these studies offer several insights. First, while it was previously known that KRAS mutations predicted intrinsic resistance to anti-EGFR therapy, the studies described here identify a causal role for KRAS in acquired resistance. Second, the use of ctDNA rather than conventional biopsy circumvents two critical problems: spatial bias of sample collection, and limited availability of matched pre-treatment and post-treatment tissue. This advantage was critical to the retrospective time course approach taken by Diaz et al.⁶; it also suggests tantalizing prospective applications for monitoring patients during therapy and anticipating clinical relapse. Third, these studies take markedly different approaches to the same question yet arrive at strikingly similar answers. Misale et al.⁵ began with in vitro selection, followed by validation of their findings in patients; in contrast, Diaz et al.⁶ began directly with patient samples and proceed to develop a mathematical argument for the pre-existence of KRAS-mutant resistant sub-populations. The convergence of these distinct approaches lends significant weight to both groups'' results. Finally, both papers support the premise (now well-established) that many resistance mutations exist prior to the initiation of therapy. In this sense, they describe a mechanistic convergence of intrinsic and acquired resistance: resistance can simultaneously be acquired (from the perspective of the behavior of the overall tumor burden) and intrinsic (in that it was present early on, within a latent subclone of the tumor). Unfortunately, this also means that relapse following single-agent targeted therapy seems virtually assured, since treatment-resistant cells may be present in the tumor even prior to the initiation of therapy.While these studies lay a solid foundation for understanding acquired resistance to EGFR blockade in mCRC, much future work remains. KRAS activation is unlikely to be the only mechanism of acquired resistance in this context. Indeed, both papers provide evidence that only a minority of the tumor cells within a relapsing lesion actually harbored mutant KRAS. Conceptually, this could imply that the relapsing subclone somehow “drives” rescue of the remaining “passenger” tumor tissue in a paracrine fashion, or that other mechanisms of resistance are also at play in the tumor as a whole. Under either model, future work must define the dynamics of interaction between these multiple subclones within a tumor as well as between the tumor and the circulation — particularly as ctDNA gains importance as a diagnostic tool. It will also be necessary to catalog the full spectrum of alterations that are sufficient to cause resistance. In particular, while the current studies focused on DNA-level changes—mutation and amplification—other mechanisms such as gene expression changes and epigenetic alterations must also be explored. The potential for stromal contribution to resistance adds yet another layer of complexity to this picture. Ultimately, as a more comprehensive view of mechanisms of resistance to EGFR blockade becomes available, it should become possible to group them by mechanism, identify therapeutic strategies to overcome these various classes of resistance, and achieve more durable responses for patients with mCRC.     

EGF-Induced Acetylation of Heterogeneous Nuclear Ribonucleoproteins Is Dependent on KRAS Mutational Status in Colorectal Cancer Cells

KRAS mutational status is considered a negative predictive marker of the response to anti-EGFR therapies in colorectal cancer (CRC) patients. However, conflicting data exist regarding the variable response to EGFR-targeted therapy. The effects of oncogenic KRAS on downstream targets were studied in cell lines with different KRAS mutations. Cells harboring a single KRAS^G13D allele showed the most tumorigenic profile, with constitutive activation of the downstream pathway, rendering them EGF-unresponsive. Conversely, KRAS^A146T cells showed a full EGF-response in terms of signal transduction pathways, cell proliferation, migration or adhesion. Moreover, the global acetylome of CRC cells was also dependent on KRAS mutational status. Several hnRNP family members were identified within the 36 acetylated-proteins. Acetylation status is known to be involved in the modulation of EGF-response. In agreement with results presented herein, hnRNPA1 and L acetylation was induced in response to EGF in KRAS^A146T cells, whereas acetyl-hnRNPA1 and L levels remained unchanged after growth factor treatment in KRAS^G13D unresponsive cells. Our results showed that hnRNPs induced-acetylation is dependent on KRAS mutational status. Nevertheless hnRNPs acetylation might also be the point where different oncogenic pathways converge.     

No evidence for PML-RARa bcr1 fusion gene in colorectal cancer

Colorectal cancer is the third most prevalent cancer and a leading cause of cancer death. Metastatic colorectal cancer patients are treated with anti-EGFR monoclonal antibodies in combination with chemotherapy; however, the efficiency is only 10-20% of such patients. An increasing amount of data has demonstrated that response to anti-EGFR therapies is confined to patients with KRAS and BRAF wild type tumors but still some of these patients are non responders to this treatment. The presence of oncogenic deregulation of different members of EGFR downstream signaling or crosstalk molecules could predict the lack of response in these patients. In this study, 40 wild type KRAS and BRAF colorectal tumors were analyzed to elucidate whether PML-RARa bcr1 fusion gene may play a role in colorectal carcinogenesis. Specifically we want to determine if this fusion could be responsible for the inability to respond to anti-EGFR monoclonal antibodies. Here, for the first time it is reported, that PML-RARa bcr1 fusion is not responsible for colorectal tumor development and also, this translocation is not predicting the lack of efficacy of anti-EGFR therapies in wild type KRAS and BRAF colorectal cancer patients. These results also suggest that PML-RARa is unlikely to be a promising target for adjuvant therapy in colorectal cancer patients.     

Tumor microenvironment disparity in multiple primary lung cancers: Impact of non-intrinsic factors,histological subtypes,and genetic aberrations

IntroductionMultiple primary lung cancers (MPLCs) occur in common carcinogenetic risks such as lifestyle, biological aging, immune responses, hormones, and metabolism. Although MPLCs harbor various genetic profiles within the same individuals, differences in the tumor microenvironment (TME) are unclear. We investigated the impact of genetic aberrations, non-intrinsic factors, and pathological subtypes on tumor immunity.Materials and MethodsIn total, 73 surgically resected specimens from 32 patients with MPLC were analyzed. PD-L1 expression in tumor cells (TCs) and immune cells (ICs), CD3-positive tumor-infiltrating lymphocytes (TILs), CD8/CD3 ratios, and FOXP3-positive TILs that compose TMEs were evaluated by immunohistochemistry and classified on a score of 0–2. 38 tumors were sequenced for somatic mutations in 409 cancer-associated genes.ResultsFemales and never or light smokers had a higher incidence of PD-L1-negative tumors and a higher concordance rate. PD-L1 positivity in TCs and ICs was significantly less frequent in EGFR-mutated than in wild-type tumors. Differences in the score of TMEs were observed between the KRAS-mutated-only tumor and the KRAS and TP53-co-mutated tumors, and between the KRAS-mutated-only tumor and the KRAS and STK11-co-mutated tumors. Significantly more FOXP3-high TILs were observed in invasive pathological subtypes than in non-invasive ones.ConclusionComparing TMEs among MPLCs revealed that non-smokers or light smokers and females were unlikely to express PD-L1 regardless of tumor site and confirmed that the EGFR mutations and co-occurring KRAS and STK11 or TP53 mutations were associated with TME. Pathological subtypes may impact the efficacy of immune therapy due to their potential correlations with regulatory T cells.     

Extended RAS and BRAF Mutation Analysis Using Next-Generation Sequencing

Somatic mutations in KRAS, NRAS, and BRAF genes are related to resistance to anti-EGFR antibodies in colorectal cancer. We have established an extended RAS and BRAF mutation assay using a next-generation sequencer to analyze these mutations. Multiplexed deep sequencing was performed to detect somatic mutations within KRAS, NRAS, and BRAF, including minor mutated components. We first validated the technical performance of the multiplexed deep sequencing using 10 normal DNA and 20 formalin-fixed, paraffin-embedded (FFPE) tumor samples. To demonstrate the potential clinical utility of our assay, we profiled 100 FFPE tumor samples and 15 plasma samples obtained from colorectal cancer patients. We used a variant calling approach based on a Poisson distribution. The distribution of the mutation-positive population was hypothesized to follow a Poisson distribution, and a mutation-positive status was defined as a value greater than the significance level of the error rate (α = 2 x 10^-5). The cut-off value was determined to be the average error rate plus 7 standard deviations. Mutation analysis of 100 clinical FFPE tumor specimens was performed without any invalid cases. Mutations were detected at a frequency of 59% (59/100). KRAS mutation concordance between this assay and Scorpion-ARMS was 92% (92/100). DNA obtained from 15 plasma samples was also analyzed. KRAS and BRAF mutations were identified in both the plasma and tissue samples of 6 patients. The genetic screening assay using next-generation sequencer was validated for the detection of clinically relevant RAS and BRAF mutations using FFPE and liquid samples.     

Colorectal cancer spheroid biobanks: multi-level approaches to drug sensitivity studies

Biobanking of molecularly characterized colorectal cancer stem cells (CSCs) generated from individual patients and growing as spheroids in defined serum-free media offer a fast, feasible, and multi-level approach for the screening of targeted therapies and drug resistance molecular studies. By combining in vitro and in vivo analyses of cetuximab efficacy with genetic data on an ongoing collection of stem cell-enriched spheroids, we describe the identification and preliminary characterization of microsatellite stable (MSS) CSCs that, despite the presence of the KRAS (G12D) mutation, display epidermal growth factor (EGF)-dependent growth and are strongly inhibited by anti-EGF-receptor (EGFR) treatment. In parallel, we detected an increased resistance to anti-EGFR therapy of microsatellite instable (MSI) CSC lines irrespective of KRAS mutational status. MSI CSC lines carried mutations in genes coding for proteins with a role in RAS and calcium signaling, highlighting the role of a genomically unstable context in determining anti-EGFR resistance. Altogether, these results argue for a multifactorial origin of anti-EGFR resistance that emerges as the effect of multiple events targeting direct and indirect regulators of the EGFR pathway. An improved understanding of key molecular determinants of sensitivity/resistance to EGFR inhibition will be instrumental to optimize the clinical efficacy of anti-EGFR agents, representing a further step towards personalized treatments.