Clinical and Translational Oncology (2020) 22:1976–1991 https://doi.org/10.1007/s12094-020-02357-z SPECIAL ARTICLE Update of the recommendations for the determination of biomarkers in colorectal carcinoma: National Consensus of the Spanish Society of Medical Oncology and the Spanish Society of Pathology P. García‑Alfonso1 · R. García‑Carbonero2 · J. García‑Foncillas3 · P. Pérez‑Segura4 · R. Salazar5 · R. Vera6 · S. Ramón y Cajal7 · J. Hernández‑Losa7 · S. Landolfi7 · E. Musulén8,9 · M. Cuatrecasas10 · S. Navarro11 Received: 18 December 2019 / Accepted: 28 April 2020 / Published online: 16 May 2020 © The Author(s) 2020 Abstract In this update of the consensus of the Spanish Society of Medical Oncology (Sociedad Española de Oncología Médica— SEOM) and the Spanish Society of Pathology (Sociedad Española de Anatomía Patológica—SEAP), advances in the analy- sis of biomarkers in advanced colorectal cancer (CRC) as well as susceptibility markers of hereditary CRC and molecular biomarkers of localized CRC are reviewed. Recently published information on the essential determination of KRAS, NRAS and BRAF mutations and the convenience of determining the amplification of human epidermal growth factor receptor 2 (HER2), the expression of proteins in the DNA repair pathway and the study of NTRK fusions are also evaluated. From the pathological point of view, the importance of analysing the tumour budding and poorly differentiated clusters, and its prog- nostic value in CRC is reviewed, as well as the impact of molecular lymph node analysis on lymph node staging in CRC. The incorporation of pan-genomic technologies, such as next-generation sequencing (NGS) and liquid biopsy in the clinical management of patients with CRC is also outlined. All these aspects are developed in this guide, which, like the previous one, will remain open to any necessary revision in the future. Keywords Diagnosis · Neoplastic disease · Gastrointestinal · Genetic prognosis · Tumour Introduction essential determination of KRAS, NRAS and BRAF muta- tions is also evaluated [2], as well as the convenience of During recent years, there have been advances related to determining other biomarkers, such as the amplification of the analysis of biomarkers with diagnostic, prognostic and the epidermal growth factor receptor (HER2), the expres- therapeutic value in colorectal cancer (CRC), especially sion of proteins in the DNA repair pathway and the study of in advanced disease. Five years have passed since the last NTRK fusions [2]. review of the consensus guide of the Spanish Society of From a pathology point of view, new data have been pub- Medical Oncology (Sociedad Española de Oncología lished with a high degree of evidence regarding the diag- Médica—SEOM) and the Spanish Society of Pathology nostic and prognostic value of some biomarkers that will be (Sociedad Española de Anatomía Patológica—SEAP) [1], detailed in this review. One example is the incorporation into and it is important to update it to incorporate the advances pathology diagnostic protocols the analysis of the tumour that facilitate the administration of precision therapies to budding (TB), defined as the presence of isolated tumour these patients. cells or small groups of less than five tumour cells at the In addition, this review examines the novelty of heredi- invasive tumour front, or the poorly differentiated clusters tary CRC susceptibility markers and the molecular biomark- (PDC), defined as groups of five or more tumour cells at ers of localized CRC. Recently published information on the the invasive front of the stroma of the CRC which does not form glandular structures. Both, TB and PDC are prognostic factors in CRC [3, 4]. * P. García‑Alfonso Another important pathological aspect is the introduc- pgarcaalfonso@gmail.com tion of new molecular assays for the analysis of lymph Extended author information available on the last page of the article nodes extracted from surgical specimens of CRC. The 13 Vol:.(1234567890) Clinical and Translational Oncology (2020) 22:1976–1991 1977 pooling-OSNA® (one-step nucleic acid amplification) The identification of these clinical pictures has a high method, based on the RT-LAMP (reverse transcription impact on the prevention of CRC, given that the identifica- loop-mediated isothermal amplification) of cytokeratin 19 tion of incipient neoplastic lesions leads to cure rates of up (CK19) mRNA for the detection of lymph node metastases, to 90% and affects comorbidities, quality of life and health surpassing the current performance of conventional morpho- efficiency. logical analysis with H&E. Molecular lymph node staging is relevant, especially in the early stages of the disease [5]. Lynch syndrome Finally, this review evaluates the future incorporation of new pan-genomic technologies, such as next-generation The identification of this type of syndrome is based on the sequencing (NGS) and liquid biopsy for the clinical manage- Amsterdam I and II criteria [6, 7], which are exclusively ment of patients with CRC. All these aspects are developed clinical. When a family complies, genetic testing should be in this guide, which, like the previous one, will remain open offered to identify the presence of germline mutations in to any necessary revision in the future. DNA repair genes (mismatch repair [MMR] genes), such as MLH1, MSH2, MSH6 and PMS2. This genetic study should always be accompanied by adequate genetic counselling. Clinical aspects The algorithm for the identification of these mutations is well described and accepted by different scientific socie- Molecular markers of hereditary CRC susceptibility ties (Fig. 1). Initial screening is performed by identifying the lack of expression of the corresponding proteins of the Of all CRC cases diagnosed each year, 25% present some genes involved in CRC and/or endometrium or by analysing characteristic indicating hereditary cancer susceptibility. Of microsatellite instability [MSI]. Any of these techniques is these, 5% correspond to Lynch syndrome, 1% to polyposis fully accepted today, and each has its specificities. In the and the rest to what could be called ‘family aggregations’. case that any of these alterations exist, and once it has been Fig. 1  Performance algorithm to detect Lynch syndrome. aAssess ject, as long as the impact on family management is important. dIn at the individual level. bWhen there is no available tumour, but the the event that the lack of expression is in MLH1, promoter methyla- Amsterdam criteria are met and the implications for the family are tion analysis and B6A6 V600E mutation should be performed. IHC important, the option of conducting a study of germline mutations in immunohistochemistry, MMR mismatch repair genes, MSI microsat- MMR genes should be evaluated. cWhen there are no living or avail- ellite instability able affects, the germinal study should be evaluated in a healthy sub- 13 1978 Clinical and Translational Oncology (2020) 22:1976–1991 ruled out that it is due to an exclusively somatic alteration, who may benefit more from chemotherapy. Only two tests, the germinal study of the MMR genes should be performed. Oncotype ­DX® and ­GeneFx® Colon [12], have been vali- Notably, at present, this type of study is being performed dated in cohorts of prospective and randomized patients by in any patient with CRC. This measure is justified not only multivariate analysis in formalin-fixed paraffin-embedded by the preventive implications for the patient and their fami- (FFPE) tumour samples. Oncotype ­DX® is the genetic lies but also because it helps to decide whether to administer test with the most evidence and has been developed as a adjuvant therapy in patients in whom the indication of such real-time polymerase chain reaction (RT-PCR) multigenic treatment is doubtful [8]. quantitative test. Its prognostic value has been observed in four independent prospective clinical trials that randomized Polyopsis syndromes patients with stage II CRC, observing hazard ratios (HR) between 1.43 and 1.68 [13–16]. It is important to note that The main characteristic of polyposis syndromes, which usu- Oncotype ­DX® maintains its prognostic value when other ally affect the younger population, is the development of a clinically relevant prognostic factors, such as T4 or MSI, are large number of polyps along the digestive tract, with differ- included in the multivariate analysis. However, its clinical ent histologies and associated with other systemic lesions, utility is not guaranteed due to the lack of predictive value with no histological markers to identify them. of the benefit of chemotherapy, as well as the small, but If a person and/or their family have a polyposis syndrome, significant, prognostic differentiation between low, interme- genetic counselling should be offered, and a germinal study diate and high risk. of MMR genes should be carried out according to the clini- In this context, Immunoscore® has recently emerged cal picture. as an alternative prognostic biomarker. It has been devel- Taking into account this information, this panel of experts oped as a quantitative measure of the presence of CD3+ / recommends the following: CD8+ lymphocytes that have invaded a tumour. Both its internal validity and its external clinical validation have been • Perform MSI and/or immunohistochemistry (IHC) analy- demonstrated in long series of patients with stages II and III sis for all patients diagnosed with CRC; and CRC through multivariate analysis, including when other • Continue with the germinal study of MMR genes in factors, such as clinical stage and MSI status, were included patients in which MSI is observed and/or loss of expres- in the multivariate analysis.[17]. However, the predictive sion of repair proteins, provided that an exclusively value of the benefit of chemotherapy is uncertain and lacks somatic origin has been ruled out. independent evidence that demonstrates its prognostic value in stage II CRC, which is only observed when combined Molecular markers of localized CRC​ with known clinicopathological markers and MSI status. More promising is the analysis of circulating tumour The use of adjuvant chemotherapy in patients with stage II DNA (ctDNA), through which minimal residual disease can CRC is controversial and is only recommended if at least one be detected with high prognostic value. If this is validated in validated risk factor is observed. Among the clinical, patho- the studies of clinical utility that are being carried out, these logical and molecular markers that have been correlated with biomarkers may be the most robust in the near future [18]. progression-free survival (PFS) and overall survival (OS), Lastly, lack of CDX2 expression confers poor prognosis in only the evaluation of MSI versus stable microsatellites stage 2 and may also predict benefit from adjuvant chemo- (MSs), T3 versus T4 and the number of lymph nodes ana- therapy, but this must be confirmed in larger prospective lysed (≥ 12–14 versus < 12–14) have been validated repeat- randomized series [19]. edly in prospective randomized clinical trials [9]. However, in patients with stage III CRC, the prognostic Essential molecular markers of advanced CRC​ value of MSI is attenuated and is not observed in several large prospective cohort studies. The determination of altera- CRC is a disease characterized by its high heterogeneity, tions in RAS and BRAF has been shown to have prognostic a product of genomic instability and its interaction with value in recent randomized cohort studies but currently has multiple exogenous factors that influence carcinogenesis no clinical utility [10]. (diet, lifestyle, microbiome, etc.) and that constitutes the Several multigenic tests have been developed with a com- exposome. This genomic heterogeneity translates into dif- mercial vision, but their overlap is low, and their clinical ferent models of carcinogenesis and, ultimately, different application is not guaranteed [11]. These tests have focused phenotypes. Some of these genomic alterations can directly on patients with stage II CRC, in whom the evidence for influence the selection of treatments; therefore, it is standard administering adjuvant chemotherapy is conflictive, with practice to determine the mutational state of multiple genes, the aim of selecting patients with higher risk and, therefore, including the determination of the extended RAS mutation, 13 Clinical and Translational Oncology (2020) 22:1976–1991 1979 the BRAF V600E mutation and the MSI status, which is analysis of four phase III clinical trials of first-line treat- usually performed by IHC staining for MLH1, MSH2, MSH6 ment in patients with metastatic CRC evaluated the possible and PMS2. prognostic value of BRAF [27]. In a total of 3063 patients, of whom 8.2% had a BRAF mutation, the OS was 11.4 months, RAS mutations and the PFS was 6.2 months, compared to 17.2 months and 7.7 months, respectively, that were observed in patients The main biomarker of clinical utility in metastatic without BRAF mutations (p < 0.001). This same study CRC is the mutational state of genes in the RAS family analysed the possible prognostic value of MSI, and it was (KRAS/NRAS/HRAS). Mutations are present in approxi- observed that the OS for patients with MSI of high instabil- mately 50% of cases, the most frequent being those that ity (MSI-H) was 13.6 months, while in patients with MSs, occur in codons 12 and 13 of exon 2 and that condition a it was 16.8 months (p = 0.001). When the BRAF and MSI state of cellular expansion. statuses were analysed, there were no differences in terms RAS mutations are a predictor of the absence of response of PFS or OS in the MSI-H population regardless of BRAF to treatment with epidermal growth factor receptor (EGFR) status, but in the MSs population with mutated BRAF, a sig- inhibitors, such as cetuximab and panitumumab. In the nificant decrease in OS and PFS was observed. These data CRYSTAL study, the addition of cetuximab to the regimen support the negative prognostic value of BRAF mutations. with folinic acid, 5-fluorouracil and irinotecan (FOLFIRI) Recent data support the predictive value of BRAF muta- showed an increase in the response rate (57% versus 40%; tions [28]. Phase III BEACON Colorectal Cancer Study p < 0.001) and median OS (23.5 versus 20.0 months; HR enrolled 665 patients with BRAF V600E across three 0.796; p = 0.0093) in patients with tumours without KRAS cohorts, including the triplet regimen of encorafenib, bini- mutations [20]. However, in patients with tumours with metinib and cetuximab; the doublet regimen of encorafenib KRAS mutations, a 4% decrease in the response rate was and cetuximab; and a control cohort where patients could evidenced, and there were no significant differences in terms receive either cetuximab and irinotecan or cetuximab with of OS [20]. Similar findings were found when analysing the FOLFIRI. At the time of the interim analysis, the median OPUS trial, in which the chemotherapy regimen was folinic duration of follow-up was 7.8 months. The median OS acid, 5-fluorouracil and oxaliplatin (FOLFOX) with cetuxi- was 5.4 months, 8.4 months and 9.0 months for the con- mab, and in the PRIME trial, in which FOLFOX was com- trol group, the doublet regimen arm, and the triplet regi- bined with panitumumab [21, 22]. Taking into account these men arm, respectively. Comparing the results obtained in data [23], guidelines from the American Society of Clini- the triplet regimen arm with the control group, HR was cal Oncology (ASCO), the European Society for Medical 0.52 (p < 0.001). An independent central review committee Oncology (ESMO) and the National Comprehensive Cancer assessed the response rate in this analysis for the first 331 Network (NCCN) recommend the extended determination of randomized patients. The response rate in the control arm RAS mutations before the administration of EGFR inhibitors was 2% compared with 20% in the doublet regimen arm and [8, 24, 25]. The RAS analysis should include HRAS, KRAS 26% in the triplet regimen arm (control arm versus triplet and NRAS, exons 2 (codons 12 and 13), 3 (codons 59 and regimen arm; p < 0.001). 61) and 4 (codons 117 and 146) [8, 24, 25]. BRAF mutations MSI The most frequent BRAF mutation is the substitution of Mutations in the MLH1, MSH2, MSH6 and PMS2 genes are glutamic acid for valine in codon V600E, which produces related to poor MMR as well as MSI. They appear in 15% of constitutive activation of the MAPK pathway. This muta- stage II and III tumours but in only 4% of stage IV tumours. tion occurs in 8–10% of patients with metastatic CRC and In stages II–III, MSI is related to a better prognosis, and excludes the RAS mutation [26]. It is more common in specifically in stage II, the better prognosis occurs in the women, usually appears in more advanced stages and has absence of benefits from adjuvant treatment with fluoropy- been associated with tumours of the right colon, poorly dif- rimidines. However, MSI in stage IV is associated with an ferentiated, with mucinous histology and with MSI. Addi- unfavourable prognosis, most likely due to its association tionally, it seems that BRAF mutations lead to the develop- with BRAF mutations. ment of more peritoneal metastases and less disease limited Two recent studies show that the alteration of MMR to the liver or lung. may have a predictive role in response to immunotherapy; Regarding the prognostic role of BRAF, it seems that therefore, the NCCN recommends its determination in all mutation is a negative prognostic factor, although its asso- patients with metastatic CRC [29, 30]. The Food and Drug ciation with MSI has to be taken into account. A joint Administration (FDA) has approved the use of nivolumab 13 1980 Clinical and Translational Oncology (2020) 22:1976–1991 and pembrolizumab in patients with MMR-deficient and a recent series, in which 18,407 tumour samples and 513 MSI-H metastatic CRC. ctDNA samples from CRC patients were characterized by massive sequencing, genetic rearrangements of potentially Recommended molecular markers of advanced CRC​ treatable kinases were identified in 126 tumours (0.68%) and seven ctDNA samples (1.36%) [38]. The most frequently HER2 is overexpressed in a small proportion of CRC. The identified fusions in this series involved the RET (0.15%), Cancer Genome Atlas (TCGA) project identified somatic BRAF (0.12%), NTRK1 (0.14%) and ALK (0.09%) genes. amplifications or mutations of HER2 in 7% of CRC cases Ninety percent of the tumours with these gene rearrange- and in 5% of the non-hypermutated CRC subgroup (4% ments were native KRAS, with a high proportion of MSI- amplification, 3% mutation and 1% both), being more fre- H: 86.4%, 45.5% and 14.3% of the tumours with NTRK1, quent in native RAS tumours (8%) [31]. An analysis of 3256 RET and ALK fusions, respectively. The presence of MSI or patients with CRC from different clinical trials (QUASAR, the absence of RAS mutations may, therefore, help to better FOCUS and PICCOLO) confirmed a higher incidence select the subgroup of patients in which it is more profit- of HER2-positive CRC in native KRAS/BRAF tumours able to proceed to the molecular screening of these gene and advanced stages [32]. Some studies indicate that the alterations. amplification of HER2 is more frequent in distal tumours These fusions can be identified with different meth- (PETACC-3 and HERACLES-A), but others have not con- ods. Classically, the detection of ALK or ROS1 has been firmed that observation (PETACC-8) [33]. Its association performed using FISH or RT-PCR, but there is a growing with survival is also controversial [32, 33]. trend in the use of mass DNA sequencing techniques, which Protein overexpression and gene amplification are eas- has the advantage of allowing the simultaneous evaluation ily detectable by IHC or fluorescent in situ hybridization of multiple genes with potential utility as therapeutic tar- (FISH), respectively. The criteria developed and validated gets. Other useful techniques include complete or directed in the HERACLES programme defined HER2-positive sequencing of tumour RNA, sequencing of circulating DNA tumours as tumours with an IHC staining intensity of 3+ in or IHC (generally used as screening). more than 50% of cells or a staining intensity of 2+ with a The presence of gene rearrangements in ALK, ROS1 or HER2:CEP ratio (chromosome enumeration probe) greater NTRK has been associated with worse survival (15.6 versus than 2 per FISH in more than 50% of the cells [34]. Amplifi- 33.7 months for patients with [n = 27] or without [n = 319] cation of HER2 is involved in innate and acquired resistance tumour gene fusions, respectively) [39]. RET fusions to anti-EGFR drugs [33]. The dual inhibition of HER2 (lapa- have also been associated with worse survival in a series tinib and trastuzumab or pertuzumab), but not the simple of 24 positive RET patients [40]. In November 2018, the block, is capable of inducing durable responses in patient- FDA approved the specific TRK inhibitor larotrectinib for derived xenograft (PDX) models of native RAS/BRAF/PI3K the treatment of tumours with NTRK1/2/3 fusions, with cetuximab-resistant CRC [33]. Consistent with this, the an overall response rate of 75% (including two of three HERACLES-A trial documented a response rate of 30% patients with CRC) and a median duration of response and in 27 patients with native KRAS CRC, positive HER2, and PFS not attained after a median follow-up of 9.4 months anti-EGFR resistance treated with lapatinib and trastuzumab (PFS at 1 year: 55%) [39]. This is the second tumour-agnos- [35]. Similarly, in the MyPathway Phase II trial, a response tic approval in the history of antineoplastic therapy after rate of 38% was observed in 37 patients with HER2-positive approval of pembrolizumab for MSI tumours. Entrectinib metastatic CRC treated with trastuzumab and pertuzumab was approved in August 2019 for the treatment of tumours [36]. Other strategies being explored are anti-HER2 mono- with NTRK fusions. clonal antibody–drug conjugates, such as trastuzumab- emtansine (HERACLES-B and RESCUE assays), and tras- tuzumab-deruxtecan, at a 25% response rate documented in Pathological aspects a phase I study in 12 patients with HER2-positive CRC [37]. The determination of HER2 may, therefore, be of clinical What is relevant in the pathology diagnosis of CRC? utility and could be justified at least in native RAS tumours resistant to anti-EGFR treatment. Tumour budding (TB) and poorly differentiated clusters The constitutive activation of different kinases as a con- (PDC) sequence of gene translocations plays an essential role in the tumourigenesis of multiple neoplasias, including a The most important prognostic factors in CRC according subgroup of neoplasias of the colon and rectum, and their to the American Joint Committee on Cancer (AJCC) and pharmacological inhibition constitutes one of the greatest the 8th edition of the International Union Against Can- therapeutic successes in the field of precision oncology. In cer (UICC) are (i) TNM stage, (ii) venous, lymphatic or 13 Clinical and Translational Oncology (2020) 22:1976–1991 1981 perineural invasion and (iii) discontinuous tumour deposits in 11–50% of HE-determined pN0 patients. Likewise, the [41]. The determination of TB has been incorporated into total tumour load (TTL) or number of CK19 copies in the the pathology diagnosis protocol of the College of Ameri- lymph nodes is correlated with classical high-risk factors can Pathologists (CRC) v4.0.0.1 and the NCCN guidelines in CRC, such as pT and pN stage, histologic grade, muci- v2.2019. Other factors, such as type, histological grade, the nous or signet ring histology, tumour size, male sex, lym- configuration of the tumour border and intratumoural inflam- phatic invasion and number of lymph nodes assessed [55, matory infiltrate, influence the prognosis and treatment of 56]. Recently, TTL has been correlated with prognosis. The patients. Tumour budding or TB, defined by the presence of incorporation of molecular lymph node staging in CRC will isolated tumour cells and/or groups of less than five cells in allow to select stage II patients for adjuvant therapy and the stroma of an invasive front of the tumour, is the morpho- assist patient management. logical evidence of the epithelial-mesenchymal transition process and is an independent factor of poor prognosis in What are the requirements of an optimal sample? CRC. TB is relevant in different clinical contexts: (i) in pT1 CRC as an independent predictor of lymph node metastasis, For the study of biomarkers in CRC, a sample with enough (ii) in stage II CRC predicts recurrence risk and contributes amount of well-preserved tumour will guarantee the perfor- to therapeutic management [42, 43], and (iii) intratumoural mance of the analysis and quality of the results. TB in endoscopic biopsies allows the individualization of neoadjuvant treatment in rectal carcinomas [44, 45]. The International Tumour Budding Consensus Conference Preservation of tumour material (ITBCC) agreed to count TB with HE at the area of the tumour front with the most density using a 20× objective Currently, the primary source for biomarkers studies and a field of 0.785 mm2. TB is classified as Bd1-low (0–4 are FFPE tumours and normal tissues, stored at room buds), Bd2-intermediate (5–9 buds) and Bd3-high (10 or temperature. more buds). Providing the numerical value of buds [e.g., Although the use of formaldehyde solution is the uni- Bd3 (count 17)] is recommended [46]. TB should be evalu- versal method of tissue fixation, formaldehyde produces ated with caution in medullary carcinomas and can be very covalent bridges in aqueous solutions and interacts with difficult to count in tumours with glandular fragmentation the -NH2 groups of proteins, forming cross-linked meth- with marked acute inflammation. TB must be distinguished ylene bridges; the tertiary and quaternary conformations from PDCs, defined as groups of five or more tumour cells of these bridges are reversible (epitope retrieval), and they in the invasive tumour front that do not form glandular struc- interact with DNA by forming hydroxy methylene bridges tures [4]. TB should not be reported in rectal tumours treated between two amino groups. In addition, treatment with for- with neoadjuvancy, or in mucinous carcinomas with signet maldehyde can produce aberrant mutations by the forma- ring cells within mucus lakes. If TB cannot be assessed, it tion of apurinic and apyrimidinic sites, DNA degradation is indicated as no non-assessable with an explanatory note and cross-linking of cytosines and has recently been con- [47, 48]. sidered carcinogenic. Therefore, alternative fixatives have been developed to improve the preservation of nucleic acids. Lymph node staging Among these, P ­ AXgene® Tissue Fix (PreAnalytix GmbH Hombrechtikon, Switzerland) and glyoxal acid-free (GAF) Lymph node staging (pN) is an important prognostic factor stand out [57, 58]. The prolonged storage of paraffin blocks in CRC, a predictor of relapse and survival and determinant at room temperature causes the constant degradation of the of the therapeutic management of patients. The pN stage is nucleic acids regardless of the type of fixative used. To slow obtained from the analysis of lymph nodes stained with HE, this degradation process, the blocks should be stored at 4 ºC which has low sensitivity for detecting lymph node micro- or frozen [59]. metastases in stages I-II CRC. HE staining analyses less than 1% of lymph node tissue [49–53], resulting in 11–24% false Minimum sufficient quantity negatives. A meta-analysis concluded that the presence of micrometastases in lymph nodes undetected by HE is asso- It is crucial to select tumour material through macro- or ciated with worse survival [54]. To improve the sensitivity microdissection to avoid contamination with normal tissue. of lymph node analysis, molecular lymph node staging has To guarantee results, a tumour cell content 10% higher than been performed, which has shown an improvement in pN normal cell content is considered optimal, thus avoiding the staging, with respect to HE. possibility of false results. Currently, with the latest genera- The OSNA technique detects copies of CK19 messen- tion of sequencing technologies, a threshold of 5% could be ger RNA (mRNA) in lymph nodes, achieving overstaging established. 13 1982 Clinical and Translational Oncology (2020) 22:1976–1991 The ideal study sample contains the greatest possible Diagnostic (CE-IVD) at the expense of the tests developed number of tumour cells. Thus, it is preferable to compare by each laboratory (laboratory developed test [LDTs]), a resected piece of CRC with the content of a diagnostic whose use is limited in Spain in comparison with other endoscopic biopsy or cytological material [60]. European countries, such as Holland and Germany [64]. Notably, many of the kits used to determine Which techniques are most appropriate KRAS/NRAS mutations do not determine BRAF mutations for the determination of each biomarker? simultaneously; therefore, many laboratories have had to modify their workflows as well as combine molecular tech- Table 1 summarizes the most important biomarkers in CRC niques to analyse these mutations sequentially (e.g., first, along with the recommended techniques to evaluate them KRAS gene mutations are analysed, then NRAS gene muta- and whether its performance is optional or mandatory. tions and finally BRAF gene mutations), which increases response times, sample manipulation and complexity for RAS mutations obtaining these results. In the case of the BRAF gene, the majority of the muta- Currently available technologies for mutational analysis tions focus on the valine amino acid at position 600, which of the KRAS and NRAS genes have not been significantly can be affected in multiple ways (V600E/E2/K/D/R or M); modified compared to the last guide [61, 62], except for the however, given that the vast majority correspond to the p. incorporation of the mutational status of the BRAF gene V600E change, this mutation can be detected by a specific [25], as reflected in Table 2. antibody (Clone VE1) via IHC. However, although this A large number of laboratories continue to use molec- technique has shown high sensitivity and analytical speci- ular techniques based on sequencing as confirmation ficity in different studies [65, 66], confirmation is currently techniques of mutations (Sanger sequencing and pyrose- required by a molecular technique. quencing) or as screening techniques based on NGS, In recent years, there has been an increase in the use of where different platforms allow these determinations to commercial point of care (POC) techniques that minimize be performed simultaneously in both clinical trials and in and automate the process of determining gene mutations, research studies as well as in the assistance activities of significantly reducing response times, reducing the risk several molecular pathology laboratories in Europe [63]. of obtaining false positives/negatives and avoiding DNA However, the vast majority of laboratories have focused extraction because analyses can be performed directly with their activity on the use of specific assays to detect spe- an FFPE sample, with minimum manipulation by the tech- cific alterations in these genes (KRAS, NRAS and BRAF) nical staff and with validated results against conventional using commercial kits by Conformité Européenne-In Vitro Table 1  Main biomarkers used in CRC​ Biomarker Technique Indication CRC—universal MSI IHC and/or MSI analysis by qPCR, bPCR, NGS Required Immunoscore or immunodensity IHC and/or digital score Optional CRC—localized MSI IHC and/or PCR and MSI analysis Required Immunoscore or immunodensity IHC and/or digital score Optional CRC—advanced MSI IHC and/or PCR and MSI analysis Required Extended RAS KRAS, NRAS Required B-RAF V600E Required V600E2/K/D/R or M Optional HER2 IHC / FISH / SISH Optional Rearrangements of: NTRK1; NTRK2; IHC and FISH, NGS, RT-PCR, NanoString® Optional in MSI, MLH1 hyper- NTRK3 methylation and RAS WT Liquid biopsy qPCR, dPCR, NGS, cfDNA Idylla Optional for patient monitorization cfDNA cell fee DNA, FISH fluorescent in situ hybridization, IHC immunohistochemistry, MSI microsatellite instability, NGS next-generation sequencing, PCR polymerase chain reaction; bPCR bridging PCR, dPCR digital PCR, qPCR quantitative PCR, RT-PCR real-time PCR, SISH silver in situ hybridization, WT wild type 13 Clinical and Translational Oncology (2020) 22:1976–1991 1983 Table 2  Molecular techniques employed in CRC​ Techniques available Sensitivity (% Characteristics mutated DNA) Mutational study of isolated genes (KRAS, NRAS and BRAF) Methods of direct sequencing Sanger method 25 Detects any mutation Requires a greater amount of mutated DNA Inexpensive Pyrosequencing 5–10 Commercial trial available Requires pyrosequencer NGS 1–5 Commercial trial available Requires specific equipment Requires experience in molecular biology PCR-fragment analysis 1–5 Commercial trial available Requires specific equipment Requires experience in molecular biology Quantitative RT-PCR TaqMan ® PCR 10 Only detects specific mutations No commercial trial Requires real-time thermocycler Scorpions-ARMS 1 Only detects specific mutations Commercial trial available Requires real-time thermocycler Mutated allele enrichment techniques PNA-LNA PCR clamp 0,1–1 Only detects specific mutations Requires non-commercial LNA probes Requires experience in molecular biology COLD-PCR 0,1–1 Requires experience in molecular biology Can be associated with sequencing and pyrosequencing techniques PCR–RFLP 5 Only detects mutations that generate a restriction site Commercial trial available DHPLC 1 Detects any mutation Special equipment required Requires experience in HPLC HRM 1 Detects any mutation Requires specific equipment Requires experience in molecular biology PCR + hybridization 1–5 Only detects specific mutations Commercial trial available Requires specific equipment (in the case of hybridization in arrays or strips if there is a large volume) Requires experience in molecular biology Direct techniques for determining RAS Point of care 1–5 Only detects specific mutations Requires specific equipment Does not require experience in molecular biology IHQ BRAF (VE1) Only detects the mutation V600E of BRAF Requires specific automation equipment Does not require experience in molecular biology MSI study IHQ Detects mutations in the MLH1, MHS2, MSH3 and PMS2 genes Requires specific automation equipment Has internal controls for each staining PCR + MSI analysis 1–5 Detects MSI alterations Commercial trial available Requires sequencing and experience in molecular biology 13 1984 Clinical and Translational Oncology (2020) 22:1976–1991 Table 2  (continued) Techniques available Sensitivity (% Characteristics mutated DNA) NGS 1–5 Available commercial trials pending validation Requires specific equipment Requires experience in molecular biology HER2 amplification study IHQ High concordance with gene amplification in those overexpressed Requires specific automation equipment Has interpretation guides In situ hybridization 1–5 Detects amplifications in a specific way Commercial trials available Requires experience and fluorescence microscope (FISH) NGS 1–5 Available commercial trials pending validation Requires specific equipment Requires experience in molecular biology NTRK rearrangements study IHQ Overexpression associated with the presence of rearrangements of any of the three genes (NTRK1, NTRK2 and NRTK3) Requires specific automation equipment Confirmation with additional molecular techniques In situ hybridization 1–5 Detects amplifications in a specific way Commercial trials available Requires experience and fluorescence microscope (FISH) and the use of three probes NGS 1–5 Available commercial trials pending validation Requires specific equipment Requires experience in molecular biology ARMS mutation system refractory to amplification, COLD-PCR coamplification at lower denaturation temperatures, DHPLC denaturing high- performance liquid chromatography, FISH fluorescent in situ hybridization, HPLC high-performance liquid chromatography, HRM high-resolu- tion fusion, IHC immunohistochemistry, MSI microsatellite instability, NGS next-generation sequencing, PCR polymerase chain reaction, PNA- LNA peptide nucleic acid-blocked nucleic acid, RFLP restriction fragment length polymorphisms, RT-PCR real-time PCR techniques and NGS [67], even using DNA extracted from of markers that detect single base alterations (BAT25 and paraffin-embedded samples [68]. BAT26) as well as three dinucleotide markers (D2S123, In the coming years, it is believed that the use of specific D5S346 and D17S250), but recently, new mononucleotide algorithms based on the use of artificial intelligence will satellites have been incorporated, which have shown better increase and be used to analyse both radiological and HE prediction capabilities than those of previous dinucleotides images, which will produce RAS analyses with greater pre- [72]. cision [69, 70]. Finally, both recently implemented NGS techniques and new POC tools allow the determination of MSI with new MSI satellites included in their panels and are being validated in different studies [73]. Other indispensable markers in patients with CRC are MMR deficiency and the presence of MSI for identifying patients Alterations in HER2 with Lynch syndrome with prognostic purpose and to predict the response to different immunotherapeutic treatments. The determination of the amplification of HER2 in CRC For this purpose, in the majority of pathology laborato- has generated great interest because of its prognostic and ries, IHC analysis of the four repair genes (MLH1, MSH2, predictive value of response to specific treatments because MSH6 and PMS2) is performed; however, because the approximately 3% of patients have such alterations [74]. encoded proteins act in tandem, different groups have pro- The methods of analysis are the same as those used in posed that only IHQ determination of MLH1 and MSH2 can breast and gastric cancers, where IHC (­HercepTest® or be used as a screening technique [71]. Clone 4B5) stands out, showing great concordance with In addition, PCR techniques have been used for the detec- the gene amplification detected by FISH or in silver in situ tion of MSI. When developing the methodology, we began hybridization (SISH) [34]. Currently, both the determina- using a panel of microsatellites (Bethesda panel) composed tion of the copy number alterations (CNAs) in a tumour by 13 Clinical and Translational Oncology (2020) 22:1976–1991 1985 NGS techniques and the quantification of the levels of HER2 or tumour-derived platelets. The quantification of CTCs in mRNA are being validated in different studies before their peripheral blood has been shown to have prognostic value implementation in routine healthcare [39]. with a threshold level above or below three cells. The pos- sibility of extracting DNA from these cells for NGS has been NTRK rearrangements recently evaluated [81]. The most developed option in liquid biopsy is the study The rearrangements of the NTRK1, NTRK2 and NTRK3 of ctDNA in peripheral blood. The biomarker with the great- genes are of special relevance in CRC because, although est impact on CRC is the mutation in the RAS oncogene, their prevalence is very low (1.5%) [39], these alterations including KRAS and NRAS, in exons 2, 3 and 4, which indi- are associated with the presence of MSI, hypermethylated cates patients who may be candidates for treatment with MLH1 and native RAS [75]. In addition, it is important to anti-EGFR antibodies in case of the native or non-mutated mention that they have a great predictive value of response sequence. This technique has been studied using tumour tis- to TRK inhibitors. The classical techniques for the detection sue as a comparison. Different publications have confirmed of NTRK alterations are generally performed by IHC with that using high sensitivity molecular techniques [82], the pan-TRK clones as screening, with subsequent confirmation concordance between the analyses performed on tumour tis- by different tests (FISH, NGS, RT-PCR or NanoString®); sue and peripheral blood exceeds 90% [83]. Correctly select- however, each one has its limitations [76]. ing the analysis technique is critical. Liquid biopsy has two very significant advantages compared to tissue biopsy: (i) it indicates the heterogeneity of the tumour and (ii) it is a What is the current and future role of NGS real-time reflection of the molecular profile, which allows in CRC? determining the dynamic evolution of the tumour. Several studies have observed the convenience of using The implementation of NGS in the study of CRC will allow, liquid biopsy in the early determination of response in in the future, the diagnosis of Lynch syndrome through the patients with RAS mutations, in whom during the course mutational study of the MMR and EPCAM genes [77] and of treatment the basal mutation was not detected, and in will guide treatment by detecting the mutations of the main patients with a RAS native sequence, in whom during treat- genes involved in the pathways of CRC carcinogenesis, such ment a RAS mutation is detected, predicting, presumably, as RAS and BRAF, as well as the genes associated with tar- the onset of radiological progression of the disease. The geted treatment, such as HER2. In addition, it will be possi- determination of BRAF mutations can be another important ble to classify CRC into molecular subtypes (CMS1, CMS2, application of liquid biopsy because it allows monitoring CMS3 and CMS4) with different biological behaviours and patients with CRC with this alteration [84]. therapeutic responses, which will open the possibility of Recent research has opened the way to new uses of liq- designing personalized treatment [9]. Likewise, NGS will uid biopsy. Some studies have proposed its possible use allow the identification of the tumour hypermutation status, for the early diagnosis of CRC; however, this option is still which will guarantee the efficacy of immunotherapy [78]. very preliminary and, for the moment, lacks an adequate Furthermore, through the study of ctDNA, it will be pos- cost-effectiveness relationship as a population screening sible to monitor response to treatment, anticipate the appear- technique [85]. Finally, the identification of low-incidence ance of local recurrence and metastasis, and detect resistance molecular alterations in CRC, such as the presence of HER2 to ongoing treatment [79]. amplification or fusions that affect NTRK, could be evaluated Finally, the possibility of implementing the combination via ctDNA by mass sequencing platforms [86]. of different molecular tools in the diagnostic routine will also improve the quality of tumour samples [68], without forgetting that IHC continues to be an indispensable diag- Conclusions nostic technique [80]. In recent decades, significant progress has been made in understanding the molecular characteristics of CRC and in What are the current and future roles the identification of specific mutations that have allowed the of liquid biopsy in CRC? development of new prognostic and predictive biomarkers in the different stages of the disease. These advances, together A liquid biopsy is based on the analysis of a biological liq- with those achieved through pathological knowledge, have uid, usually blood, but also cerebrospinal fluid or urine, to justified a new edition of this guide. reveal characteristics of a cancer, such as circulating tumour With respect to the susceptibility markers of hereditary cells (CTCs), free nucleic acids (DNA or RNA), exosomes CRC, this panel recommends the analysis of MSI and/ 13 1986 Clinical and Translational Oncology (2020) 22:1976–1991 or IHC in all patients diagnosed with CRC. In addition, Ethical approval (research involving human participants and/or ani- the germinal study of MMR genes should continue in mals) The study has been performed in accordance with the ethical standards of the Declaration of Helsinki and its later amendments. This patients with MSI and/or loss of repair protein expres- article does not contain any studies with human participants or animals sion. In localized stages, the determination of MSI as a performed by any of the authors. prognostic marker is required, especially in stage II CRC. The standardized implementation of genomic platforms Informed consent For this type of study formal consent is not required. is not recommended due to the lack of predictive value of response to chemotherapy. Open Access This article is licensed under a Creative Commons Attri- In patients with advanced CRC, it is essential to deter- bution 4.0 International License, which permits use, sharing, adapta- tion, distribution and reproduction in any medium or format, as long mine extended RAS mutations, the BRAF V600E mutation as you give appropriate credit to the original author(s) and the source, and the MSI status, which is usually performed via IHC provide a link to the Creative Commons licence, and indicate if changes for MLH1, MSH2, MSH6 and PMS2. The determination were made. The images or other third party material in this article are of extended RAS mutations is mandatory before mak- included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in ing therapeutic decisions (I-A). The RAS analysis should the article’s Creative Commons licence and your intended use is not include HRAS, KRAS and NRAS, exons 2 (codons 12 and permitted by statutory regulation or exceeds the permitted use, you will 13), 3 (codons 59 and 61) and 4, as negative predictive fac- need to obtain permission directly from the copyright holder. To view a tors of the anti-EGFR response. The BRAF V600E muta- copy of this licence, visit http://creat​iveco​mmons​.org/licen​ses/by/4.0/. tion is required due to its recognized negative prognostic value (I-A) and, recently, as a predictive marker of specific biological treatments (cetuximab, encorafenib and bini- metinib). The determination of MSI acquires predictive References value in advanced CRC for immunotherapy treatment with 1. Garcia-Alfonso P, Garcia-Foncillas J, Salazar R, Perez-Segura P, pembrolizumab or nivolumab (II-B). Garcia-Carbonero R, Musulen-Palet E, Cuatrecasas M, Landolfi Another recommended marker in advanced CRC is the S, Ramon YCS, Navarro S, Spanish Society of P, Spanish Society amplification of HER2 as a negative predictive marker of of Medical O. Updated guidelines for biomarker testing in colo- anti-EGFR response and as a response marker for dual anti- rectal carcinoma: a national consensus of the Spanish Society of Pathology and the Spanish Society of Medical Oncology. Clin HER2 therapy. Its determination is recommended, at least in Transl Oncol. 2015;17(4):264–73. https​://doi.org/10.1007/s1209​ native RAS tumours resistant to anti-EGFR. The ALK, ROS1 4-014-1252-0. and NTRK fusions, despite their low incidence, have clinical 2. DeStefanis RA, Kratz JD, Emmerich PB, Deming DA. Tar- interest due to recent approval for specific treatments. geted therapy in metastatic colorectal cancer: current stand- ards and novel agents in review. Curr Colorectal Cancer Rep. The greatest contribution to the genetic study of CRC 2019;15(2):61–9. https​://doi.org/10.1007/s1188​8-019-00430​-6. will come from liquid biopsies, with which minimal residual 3. Martinez-Ciarpaglini C, Fernandez-Sellers C, Tarazona N, disease can be detected after surgery. This technique reflects Rosello S, Huerta M, Roda D, Mongort C, Ferrandez A, Navarro tumour heterogeneity and molecular profiles in real time, S, Cervantes A. Improving tumour budding evaluation in colon cancer by extending the assessment area in colectomy specimens. reporting the dynamic evolution of the tumour. The imple- Histopathology. 2019. https​://doi.org/10.1111/his.13900​. mentation of NGS will improve genotypic knowledge, the 4. Konishi T, Shimada Y, Lee LH, Cavalcanti MS, Hsu M, Smith tumour hypermutation status and molecular subtypes. The JJ, Nash GM, Temple LK, Guillem JG, Paty PB, Garcia-Aguilar combination of different molecular tools in the diagnostic J, Vakiani E, Gonen M, Shia J, Weiser MR. Poorly differentiated clusters predict colon cancer recurrence: an in-depth comparative routine will improve the diagnosis using and quality of analysis of invasive-front prognostic markers. Am J Surg Pathol. tumour samples. 2018;42(6):705–14. https​://doi.org/10.1097/PAS.00000​00000​ 00105​9. Acknowledgements The authors are grateful for the editorial assis- 5. Rakislova N, Montironi C, Aldecoa I, Fernandez E, Bombi JA, tance of Beatriz Gil-Alberdi of HealthCo (Madrid, Spain) in the pro- Jimeno M, Balaguer F, Pellise M, Castells A, Cuatrecasas M. duction of this manuscript. Lymph node pooling: a feasible and efficient method of lymph node molecular staging in colorectal carcinoma. J Transl Med. Funding SEAP and SEOM have received financial support for this 2017;15(1):14. https​://doi.org/10.1186/s1296​7-016-1114-3. project in the form of unrestricted grants from Bayer, Merck, Pierre 6. American Gastroenterological A. American Gastroenterological Fabre and Roche. Association medical position statement: hereditary colorectal cancer and genetic testing. Gastroenterology. 2001;121(1):195–7. https​://doi.org/10.1053/gast.2001.25580​. Compliance with ethical standards 7. Vasen HF, Watson P, Mecklin JP, Lynch HT. New clinical criteria for hereditary nonpolyposis colorectal cancer (HNPCC, Lynch Conflict of interest The authors declare that, when writing and revis- syndrome) proposed by the International Collaborative group ing the text, they did not know the names of the pharmaceutical com- on HNPCC. Gastroenterology. 1999;116(6):1453–6. https​://doi. panies that provided financial support for this project, so this support org/10.1016/s0016​-5085(99)70510​-x. has not influenced the content of this article. 13 Clinical and Translational Oncology (2020) 22:1976–1991 1987 8. Sepulveda AR, Hamilton SR, Allegra CJ, Grody W, Cushman- 17. Pages F, Mlecnik B, Marliot F, Bindea G, Ou FS, Bifulco C, Lugli Vokoun AM, Funkhouser WK, Kopetz SE, Lieu C, Lindor NM, A, Zlobec I, Rau TT, Berger MD, Nagtegaal ID, Vink-Borger E, Minsky BD, Monzon FA, Sargent DJ, Singh VM, Willis J, Clark Hartmann A, Geppert C, Kolwelter J, Merkel S, Grutzmann R, J, Colasacco C, Rumble RB, Temple-Smolkin R, Ventura CB, Van den Eynde M, Jouret-Mourin A, Kartheuser A, Leonard D, Nowak JA. Molecular biomarkers for the evaluation of colorectal Remue C, Wang JY, Bavi P, Roehrl MHA, Ohashi PS, Nguyen cancer: guideline from the American Society for Clinical Pathol- LT, Han S, MacGregor HL, Hafezi-Bakhtiari S, Wouters BG, ogy, College of American Pathologists, Association for Molecular Masucci GV, Andersson EK, Zavadova E, Vocka M, Spacek J, Pathology, and American Society of Clinical Oncology. J Mol Petruzelka L, Konopasek B, Dundr P, Skalova H, Nemejcova K, Diagn. 2017;19(2):187–225. https​: //doi.org/10.1016/j.jmold​ Botti G, Tatangelo F, Delrio P, Ciliberto G, Maio M, Laghi L, x.2016.11.001. Grizzi F, Fredriksen T, Buttard B, Angelova M, Vasaturo A, Maby 9. Dienstmann R, Salazar R, Tabernero J. Personalizing colon P, Church SE, Angell HK, Lafontaine L, Bruni D, El Sissy C, Hai- cancer adjuvant therapy: selecting optimal treatments for indi- cheur N, Kirilovsky A, Berger A, Lagorce C, Meyers JP, Paustian vidual patients. J Clin Oncol. 2015;33(16):1787–96. https​://doi. C, Feng Z, Ballesteros-Merino C, Dijkstra J, van de Water C, van org/10.1200/JCO.2014.60.0213. Lent-van Vliet S, Knijn N, Musina AM, Scripcariu DV, Popiv- 10. Taieb J, Kourie HR, Emile JF, Le Malicot K, Balogoun R, anova B, Xu M, Fujita T, Hazama S, Suzuki N, Nagano H, Okuno Tabernero J, Mini E, Folprecht G, Van Laethem JL, Mulot C, K, Torigoe T, Sato N, Furuhata T, Takemasa I, Itoh K, Patel PS, Bouche O, Aparicio T, Michel P, Thaler J, Bridgewater J, Van Vora HH, Shah B, Patel JB, Rajvik KN, Pandya SJ, Shukla SN, Cutsem E, Perkins G, Lepage C, Salazar R, Laurent-Puig P, Wang Y, Zhang G, Kawakami Y, Marincola FM, Ascierto PA, Pan-European Trials in Alimentary Tract Cancer I. Association Sargent DJ, Fox BA, Galon J. International validation of the con- of prognostic value of primary tumor location in stage III colon sensus Immunoscore for the classification of colon cancer: a prog- cancer with RAS and BRAF mutational status. JAMA Oncol. nostic and accuracy study. Lancet. 2018;391(10135):2128–39. 2018;4(7):e173695. https:​ //doi.org/10.1001/jamaon​ col.2017.3695. https​://doi.org/10.1016/S0140​-6736(18)30789​-X. 11. Sanz-Pamplona R, Berenguer A, Cordero D, Riccadonna S, Sole 18. Wong R, Tie J, Lee M, Cohen J, Wang Y, Li L, Ma S, Christie M, X, Crous-Bou M, Guino E, Sanjuan X, Biondo S, Soriano A, Kosmider S, Tomasetti C, Papadopoulos N, Kinzler KW, Vogel- Jurman G, Capella G, Furlanello C, Moreno V. Clinical value stein B, Gibbs P. The potential role of circulating tumor DNA of prognosis gene expression signatures in colorectal cancer: a (ctDNA) in the further investigation of colorectal cancer patients systematic review. PLoS ONE. 2012;7(11):e48877. https​://doi. with nonspecific findings on standard investigations. Int J Cancer. org/10.1371/journ​al.pone.00488​77. 2019;145(2):540–7. https​://doi.org/10.1002/ijc.32117​. 12. Niedzwiecki D, Frankel WL, Venook AP, Ye X, Friedman PN, 19. Dalerba P, Sahoo D, Paik S, Guo X, Yothers G, Song N, Wilcox- Goldberg RM, Mayer RJ, Colacchio TA, Mulligan JM, Davison Fogel N, Forgo E, Rajendran PS, Miranda SP, Hisamori S, Hutch- TS, O’Brien E, Kerr P, Johnston PG, Kennedy RD, Harkin DP, ison J, Kalisky T, Qian D, Wolmark N, Fisher GA, van de Rijn M, Schilsky RL, Bertagnolli MM, Warren RS, Innocenti F. Asso- Clarke MF. CDX2 as a prognostic biomarker in stage II and stage ciation between results of a gene expression signature assay and III colon cancer. N Engl J Med. 2016;374(3):211–22. https​://doi. recurrence-free interval in patients with stage II colon cancer org/10.1056/NEJMo​a1506​597. in cancer and leukemia group B 9581 (alliance). J Clin Oncol. 20. Van Cutsem E, Kohne CH, Lang I, Folprecht G, Nowacki MP, 2016;34(25):3047–53. https:​ //doi.org/10.1200/JCO.2015.65.4699. Cascinu S, Shchepotin I, Maurel J, Cunningham D, Tejpar S, 13. Gray RG, Quirke P, Handley K, Lopatin M, Magill L, Baehner FL, Schlichting M, Zubel A, Celik I, Rougier P, Ciardiello F. Cetuxi- Beaumont C, Clark-Langone KM, Yoshizawa CN, Lee M, Wat- mab plus irinotecan, fluorouracil, and leucovorin as first-line son D, Shak S, Kerr DJ. Validation study of a quantitative mul- treatment for metastatic colorectal cancer: updated analysis of tigene reverse transcriptase-polymerase chain reaction assay for overall survival according to tumor KRAS and BRAF mutation assessment of recurrence risk in patients with stage II colon can- status. J Clin Oncol. 2011;29(15):2011–9. https:​ //doi.org/10.1200/ cer. J Clin Oncol. 2011;29(35):4611–9. https​://doi.org/10.1200/ JCO.2010.33.5091. JCO.2010.32.8732. 21. Bokemeyer C, Bondarenko I, Hartmann JT, de Braud F, Schuch G, 14. Venook AP, Niedzwiecki D, Lopatin M, Ye X, Lee M, Fried- Zubel A, Celik I, Schlichting M, Koralewski P. Efficacy accord- man PN, Frankel W, Clark-Langone K, Millward C, Shak ing to biomarker status of cetuximab plus FOLFOX-4 as first- S, Goldberg RM, Mahmoud NN, Warren RS, Schilsky RL, line treatment for metastatic colorectal cancer: the OPUS study. Bertagnolli MM. Biologic determinants of tumor recurrence Ann Oncol. 2011;22(7):1535–46. https​://doi.org/10.1093/annon​ in stage II colon cancer: validation study of the 12-gene recur- c/mdq63​2. rence score in cancer and leukemia group B (CALGB) 9581. J 22. Douillard JY, Siena S, Cassidy J, Tabernero J, Burkes R, Barugel Clin Oncol. 2013;31(14):1775–811. https​://doi.org/10.1200/ M, Humblet Y, Bodoky G, Cunningham D, Jassem J, Rivera F, JCO.2012.45.1096. Kocakova I, Ruff P, Blasilska-Morawiec M, Smakal M, Canon 15. Yamanaka T, Oki E, Yamazaki K, Yamaguchi K, Muro K, Uetake JL, Rother M, Oliner KS, Tian Y, Xu F, Sidhu R. Final results H, Sato T, Nishina T, Ikeda M, Kato T, Kanazawa A, Kusumoto T, from PRIME: randomized phase 3 study of panitumumab with Chao C, Lopatin M, Krishnakumar J, Bailey H, Akagi K, Ochiai FOLFOX4 for first-line treatment of metastatic colorectal cancer. A, Ohtsu A, Ohashi Y, Yoshino T. 12-gene recurrence score assay Ann Oncol. 2014. https​://doi.org/10.1093/annon​c/mdu14​1. stratifies the recurrence risk in stage II/III colon cancer with sur- 23. Arnold D, Lueza B, Douillard JY, Peeters M, Lenz HJ, Venook gery alone: the SUNRISE Study. J Clin Oncol. 2016;34(24):2906– A, Heinemann V, Van Cutsem E, Pignon JP, Tabernero J, Cer- 13. https​://doi.org/10.1200/JCO.2016.67.0414. vantes A, Ciardiello F. Prognostic and predictive value of primary 16. Yothers G, O’Connell MJ, Lee M, Lopatin M, Clark-Langone tumour side in patients with RAS wild-type metastatic colorectal KM, Millward C, Paik S, Sharif S, Shak S, Wolmark N. Validation cancer treated with chemotherapy and EGFR directed antibodies of the 12-gene colon cancer recurrence score in NSABP C-07 as in six randomized trials. Ann Oncol. 2017;28(8):1713–29. https​ a predictor of recurrence in patients with stage II and III colon ://doi.org/10.1093/annon​c/mdx17​5. cancer treated with fluorouracil and leucovorin (FU/LV) and FU/ 24. NCCN (2019) NCCN Clinical Practice Guidelines in Oncol- LV plus oxaliplatin. J Clin Oncol. 2013;31(36):4512–9. https​:// ogy (NCCN Guidelines®): Colon Cancer Version 4.2018. https​ doi.org/10.1200/JCO.2012.47.3116. ://www.nccn.org/profe ​ s sion ​ a ls/physi ​ c ian_gls/pdf/colon ​ . pdf. Accessed 2 Jan 2019 13 1988 Clinical and Translational Oncology (2020) 22:1976–1991 25. Van Cutsem E, Cervantes A, Adam R, Sobrero A, Van Krieken 34. Valtorta E, Martino C, Sartore-Bianchi A, Penaullt-Llorca F, Viale JH, Aderka D, Aranda Aguilar E, Bardelli A, Benson A, Bodoky G, Risio M, Rugge M, Grigioni W, Bencardino K, Lonardi S, G, Ciardiello F, D’Hoore A, Diaz-Rubio E, Douillard JY, Ducreux Zagonel V, Leone F, Noe J, Ciardiello F, Pinto C, Labianca R, M, Falcone A, Grothey A, Gruenberger T, Haustermans K, Heine- Mosconi S, Graiff C, Aprile G, Frau B, Garufi C, Loupakis F, mann V, Hoff P, Kohne CH, Labianca R, Laurent-Puig P, Ma Racca P, Tonini G, Lauricella C, Veronese S, Truini M, Siena B, Maughan T, Muro K, Normanno N, Osterlund P, Oyen WJ, S, Marsoni S, Gambacorta M. Assessment of a HER2 scoring Papamichael D, Pentheroudakis G, Pfeiffer P, Price TJ, Punt C, system for colorectal cancer: results from a validation study. Mod Ricke J, Roth A, Salazar R, Scheithauer W, Schmoll HJ, Tabernero Pathol. 2015;28(11):1481–91. https​://doi.org/10.1038/modpa​ J, Taieb J, Tejpar S, Wasan H, Yoshino T, Zaanan A, Arnold D. thol.2015.98. ESMO consensus guidelines for the management of patients with 35. Sartore-Bianchi A, Trusolino L, Martino C, Bencardino K, metastatic colorectal cancer. Ann Oncol. 2016;27(8):1386–422. Lonardi S, Bergamo F, Zagonel V, Leone F, Depetris I, Mar- https​://doi.org/10.1093/annon​c/mdw23​5. tinelli E, Troiani T, Ciardiello F, Racca P, Bertotti A, Siravegna 26. Tran B, Kopetz S, Tie J, Gibbs P, Jiang ZQ, Lieu CH, Agar- G, Torri V, Amatu A, Ghezzi S, Marrapese G, Palmeri L, Valtorta wal A, Maru DM, Sieber O, Desai J. Impact of BRAF muta- E, Cassingena A, Lauricella C, Vanzulli A, Regge D, Veronese tion and microsatellite instability on the pattern of metastatic S, Comoglio PM, Bardelli A, Marsoni S, Siena S. Dual-targeted spread and prognosis in metastatic colorectal cancer. Cancer. therapy with trastuzumab and lapatinib in treatment-refractory, 2011;117(20):4623–32. https​://doi.org/10.1002/cncr.26086​. KRAS codon 12/13 wild-type, HER2-positive metastatic colorec- 27. Venderbosch S, Nagtegaal ID, Maughan TS, Smith CG, Chea- tal cancer (HERACLES): a proof-of-concept, multicentre, open- dle JP, Fisher D, Kaplan R, Quirke P, Seymour MT, Richman label, phase 2 trial. Lancet Oncol. 2016;17(6):738–46. https:​ //doi. SD, Meijer GA, Ylstra B, Heideman DA, de Haan AF, Punt CJ, org/10.1016/S1470​-2045(16)00150​-9. Koopman M. Mismatch repair status and BRAF mutation sta- 36. Meric-Bernstam F, Hurwitz H, Raghav KPS, McWilliams RR, tus in metastatic colorectal cancer patients: a pooled analysis of Fakih M, VanderWalde A, Swanton C, Kurzrock R, Burris H, the CAIRO, CAIRO2, COIN, and FOCUS studies. Clin Cancer Sweeney C, Bose R, Spigel DR, Beattie MS, Blotner S, Stone A, Res. 2014;20(20):5322–30. https​://doi.org/10.1158/1078-0432. Schulze K, Cuchelkar V, Hainsworth J. Pertuzumab plus trastu- CCR-14-0332. zumab for HER2-amplified metastatic colorectal cancer (MyPath- 28. Kopetz S, Grothey A, Yaeger R, Van Cutsem E, Desai J, Yoshino way): an updated report from a multicentre, open-label, phase 2a, T, Wasan H, Ciardiello F, Loupakis F, Hong YS, Steeghs N, Guren multiple basket study. Lancet Oncol. 2019;20(4):518–30. https​:// TK, Arkenau HT, Garcia-Alfonso P, Pfeiffer P, Orlov S, Lonardi doi.org/10.1016/S1470​-2045(18)30904​-5. S, Elez E, Kim TW, Schellens JHM, Guo C, Krishnan A, Dekervel 37. Yoshino T, Iwata H, Tamura K, Takahashi S, Redfern C, Modi J, Morris V, Calvo Ferrandiz A, Tarpgaard LS, Braun M, Gollerk- S, Doi T, Kawakami H, Taniguchi H, Takashima A, Yamaguchi eri A, Keir C, Maharry K, Pickard M, Christy-Bittel J, Anderson K, Fisher J, Li B, Saito K, Fujisaki Y, Sugihara M, Tsurutani J. L, Sandor V, Tabernero J. Encorafenib, binimetinib, and cetuxi- 563PUpdated results of phase I study of trastuzumab deruxtecan mab in BRAF V600E-mutated colorectal cancer. N Engl J Med. (DS-8201a) in HER2-expressing advanced colorectal cancer. Ann 2019;381(17):1632–43. https:​ //doi.org/10.1056/NEJMoa​ 19080​ 75. Oncol. 2018. https​://doi.org/10.1093/annon​c/mdy28​1.109. 29. Le DT, Uram JN, Wang H, Bartlett BR, Kemberling H, Eyring 38. Madison R, Pietrantonio F, Juckett L, Cremolini C, Chung J, AD, Skora AD, Luber BS, Azad NS, Laheru D, Biedrzycki B, Albacker LA, Miller VA, Klempner SJ, Resnick MB, Yakirevich Donehower RC, Zaheer A, Fisher GA, Crocenzi TS, Lee JJ, E, Ou S-HI, Fakih M, Schrock AB, Ross JS, Ali SM. 457PDKi- Duffy SM, Goldberg RM, de la Chapelle A, Koshiji M, Bhai- nase fusions in colorectal cancers: a unique biologic subset. Ann jee F, Huebner T, Hruban RH, Wood LD, Cuka N, Pardoll DM, Oncol. 2018. https​://doi.org/10.1093/annon​c/mdy28​1.005. Papadopoulos N, Kinzler KW, Zhou S, Cornish TC, Taube JM, 39. Pietrantonio F, Di Nicolantonio F, Schrock AB, Lee J, Tejpar Anders RA, Eshleman JR, Vogelstein B, Diaz LA Jr. PD-1 block- S, Sartore-Bianchi A, Hechtman JF, Christiansen J, Novara L, ade in tumors with mismatch-repair deficiency. N Engl J Med. Tebbutt N, Fuca G, Antoniotti C, Kim ST, Murphy D, Berenato 2015;372(26):2509–20. https:​ //doi.org/10.1056/NEJMoa​ 15005​ 96. R, Morano F, Sun J, Min B, Stephens PJ, Chen M, Lazzari L, 30. Overman MJ, Lonardi S, Wong KYM, Lenz HJ, Gelsomino F, Miller VA, Shoemaker R, Amatu A, Milione M, Ross JS, Siena Aglietta M, Morse MA, Van Cutsem E, McDermott R, Hill A, S, Bardelli A, Ali SM, Falcone A, de Braud F, Cremolini C. ALK, Sawyer MB, Hendlisz A, Neyns B, Svrcek M, Moss RA, Ledeine ROS1, and NTRK rearrangements in metastatic colorectal cancer. JM, Cao ZA, Kamble S, Kopetz S, Andre T. Durable clinical ben- J Natl Cancer Inst. 2017. https​://doi.org/10.1093/jnci/djx08​9. efit with nivolumab plus ipilimumab in DNA mismatch repair- 40. Pietrantonio F, Di Nicolantonio F, Schrock AB, Lee J, Morano F, deficient/microsatellite instability-high metastatic colorectal Fuca G, Nikolinakos P, Drilon A, Hechtman JF, Christiansen J, cancer. J Clin Oncol. 2018;36(8):773–9. https​://doi.org/10.1200/ Gowen K, Frampton GM, Gasparini P, Rossini D, Gigliotti C, Kim JCO.2017.76.9901. ST, Prisciandaro M, Hodgson J, Zaniboni A, Chiu VK, Milione 31. Cancer Genome Atlas N. Comprehensive molecular char- M, Patel R, Miller V, Bardelli A, Novara L, Wang L, Pupa SM, acterization of human colon and rectal cancer. Nature. Sozzi G, Ross J, Di Bartolomeo M, Bertotti A, Ali S, Trusolino 2012;487(7407):330–7. https​://doi.org/10.1038/natur​e1125​2. L, Falcone A, de Braud F, Cremolini C. RET fusions in a small 32. Richman SD, Southward K, Chambers P, Cross D, Barrett J, Hem- subset of advanced colorectal cancers at risk of being neglected. mings G, Taylor M, Wood H, Hutchins G, Foster JM, Oumie A, Ann Oncol. 2018;29(6):1394–401. https​://doi.org/10.1093/annon​ Spink KG, Brown SR, Jones M, Kerr D, Handley K, Gray R, c/mdy09​0. Seymour M, Quirke P. HER2 overexpression and amplification 41. Amin MB, Edge S, Greene F, Byrd DR, Brookland RK, Wash- as a potential therapeutic target in colorectal cancer: analysis of ington MK, Gershenwald JE, Compton CC, Hess KR, Sullivan 3256 patients enrolled in the QUASAR, FOCUS and PICCOLO DC, Jessup JM, Brierley JD, Gaspar LE, Schilsky RL, Balch CM. colorectal cancer trials. J Pathol. 2016;238(4):562–70. https:​ //doi. AJCC cancer staging manual. 8th ed. New York: American Joint org/10.1002/path.4679. Commission on Cancer Springer International Publishing; 2017. 33. La Salvia A, Lopez-Gomez V, Garcia-Carbonero R. HER2-tar- 42. Nakamura T, Mitomi H, Kanazawa H, Ohkura Y, Watanabe M. geted therapy: an emerging strategy in advanced colorectal can- Tumor budding as an index to identify high-risk patients with cer. Expert Opin Investig Drugs. 2019;28(1):29–38. https​://doi. stage II colon cancer. Dis Colon Rectum. 2008;51(5):568–72. org/10.1080/13543​784.2019.15555​83. https​://doi.org/10.1007/s1035​0-008-9192-9. 13 Clinical and Translational Oncology (2020) 22:1976–1991 1989 43. van Wyk HC, Park J, Roxburgh C, Horgan P, Foulis A, McMillan Sugihara K, Japanese Society for Cancer of the C, Rectum. Japa- DC. The role of tumour budding in predicting survival in patients nese Society for Cancer of the Colon and Rectum (JSCCR) guide- with primary operable colorectal cancer: a systematic review. lines 2010 for the treatment of colorectal cancer. Int J Clin Oncol. Cancer Treat Rev. 2015;41(2):151–9. https​://doi.org/10.1016/j. 2012;17(1):1–29. https​://doi.org/10.1007/s1014​7-011-0315-2. ctrv.2014.12.007. 54. Rahbari NN, Bork U, Motschall E, Thorlund K, Buchler MW, 44. De Smedt L, Palmans S, Sagaert X. Tumour budding in colo- Koch M, Weitz J. Molecular detection of tumor cells in regional rectal cancer: what do we know and what can we do? Virchows lymph nodes is associated with disease recurrence and poor sur- Arch. 2016;468(4):397–408. https ​ : //doi.org/10.1007/s0042​ vival in node-negative colorectal cancer: a systematic review 8-015-1886-5. and meta-analysis. J Clin Oncol. 2012;30(1):60–70. https​://doi. 45. Koelzer VH, Zlobec I, Lugli A. Tumor budding in colorectal can- org/10.1200/JCO.2011.36.9504. cer–ready for diagnostic practice? Hum Pathol. 2016;47(1):4–19. 55. Aldecoa I, Atares B, Tarragona J, Bernet L, Sardon JD, Pereda T, https​://doi.org/10.1016/j.humpa​th.2015.08.007. Villar C, Mendez MC, Gonzalez-Obeso E, Elorriaga K, Alonso 46. Lugli A, Kirsch R, Ajioka Y, Bosman F, Cathomas G, Daw- GL, Zamora J, Planell N, Palacios J, Castells A, Matias-Guiu son H, El Zimaity H, Flejou JF, Hansen TP, Hartmann A, X, Cuatrecasas M. Molecularly determined total tumour load in Kakar S, Langner C, Nagtegaal I, Puppa G, Riddell R, Risti- lymph nodes of stage I-II colon cancer patients correlates with maki A, Sheahan K, Smyrk T, Sugihara K, Terris B, Ueno H, high-risk factors. A multicentre prospective study. Virchows Arch. Vieth M, Zlobec I, Quirke P. Recommendations for reporting 2016;469(4):385–94. https:​ //doi.org/10.1007/s00428​ -016-1990-1. tumor budding in colorectal cancer based on the International 56. Yamamoto H, Tomita N, Inomata M, Furuhata T, Miyake Y, Tumor Budding Consensus Conference (ITBCC) 2016. Mod Noura S, Kato T, Murata K, Hayashi S, Igarashi S, Itabashi M, Pathol. 2017;30(9):1299–311. https​://doi.org/10.1038/modpa​ Kameoka S, Matsuura N. OSNA-assisted molecular staging in thol.2017.46. colorectal cancer: a prospective multicenter trial in Japan. Ann 47. Rogers AC, Gibbons D, Hanly AM, Hyland JM, O’Connell PR, Surg Oncol. 2016;23(2):391–6. https​://doi.org/10.1245/s1043​ Winter DC, Sheahan K. Prognostic significance of tumor budding 4-015-4880-x. in rectal cancer biopsies before neoadjuvant therapy. Mod Pathol. 57. Hognas G, Kivinummi K, Kallio HML, Hieta R, Ruusuvuori P, 2014;27(1):156–62. https:​ //doi.org/10.1038/modpat​ hol.2013.124. Koskenalho A, Kesseli J, Tammela TLJ, Riikonen J, Ilvesaro J, 48. Rogers AC, Winter DC, Heeney A, Gibbons D, Lugli A, Puppa Kares S, Hirvikoski PP, Laurila M, Mirtti T, Nykter M, Kujala G, Sheahan K. Systematic review and meta-analysis of the PM, Visakorpi T, Tolonen T, Bova GS. Feasibility of prostate impact of tumour budding in colorectal cancer. Br J Cancer. PAXgene fixation for molecular research and diagnostic surgi- 2016;115(7):831–40. https​://doi.org/10.1038/bjc.2016.274. cal pathology: comparison of matched fresh frozen, FFPE, and 49. Compton C.C., Tanabe K K, Savarese D M (2015) UptoDate: PFPE tissues. Am J Surg Pathol. 2018;42(1):103–15. https​://doi. pathology and prognostic determinants of colorectal cancer. https​ org/10.1097/PAS.00000​00000​00096​1. ://www.uptod​ate.com/conte​nts/patho​logy-and-progn​ostic​-deter​ 58. Bussolati G, Annaratone L, Berrino E, Miglio U, Panero M, Cupo minan​ts-of-color​ectal​-cance​r. Accessed 15 May 2019 M, Gugliotta P, Venesio T, Sapino A, Marchio C. Acid-free gly- 50. Schmoll HJ, Van Cutsem E, Stein A, Valentini V, Glimelius B, oxal as a substitute of formalin for structural and molecular pres- Haustermans K, Nordlinger B, van de Velde CJ, Balmana J, Reg- ervation in tissue samples. PLoS ONE. 2017;12(8):e0182965. ula J, Nagtegaal ID, Beets-Tan RG, Arnold D, Ciardiello F, Hoff P, https​://doi.org/10.1371/journ​al.pone.01829​65. Kerr D, Kohne CH, Labianca R, Price T, Scheithauer W, Sobrero 59. Groelz D, Viertler C, Pabst D, Dettmann N, Zatloukal K. Impact A, Tabernero J, Aderka D, Barroso S, Bodoky G, Douillard JY, of storage conditions on the quality of nucleic acids in paraffin El Ghazaly H, Gallardo J, Garin A, Glynne-Jones R, Jordan K, embedded tissues. PLoS ONE. 2018;13(9):e0203608. https​://doi. Meshcheryakov A, Papamichail D, Pfeiffer P, Souglakos I, Turhal org/10.1371/journ​al.pone.02036​08. S, Cervantes A. ESMO Consensus Guidelines for management 60. Bellevicine C, Malapelle U, Vigliar E, Pisapia P, Vita G, Troncone of patients with colon and rectal cancer: a personalized approach G. How to prepare cytological samples for molecular testing. J to clinical decision making. Ann Oncol. 2012;23(10):2479–516. Clin Pathol. 2017;70(10):819–26. https​://doi.org/10.1136/jclin​ https​://doi.org/10.1093/annon​c/mds23​6. path-2017-20456​1. 51. van de Velde CJ, Boelens PG, Borras JM, Coebergh JW, Cervantes 61. Douillard JY, Oliner KS, Siena S, Tabernero J, Burkes R, Barugel A, Blomqvist L, Beets-Tan RG, van den Broek CB, Brown G, M, Humblet Y, Bodoky G, Cunningham D, Jassem J, Rivera F, Van Cutsem E, Espin E, Haustermans K, Glimelius B, Iversen Kocakova I, Ruff P, Blasinska-Morawiec M, Smakal M, Canon JL, LH, van Krieken JH, Marijnen CA, Henning G, Gore-Booth J, Rother M, Williams R, Rong A, Wiezorek J, Sidhu R, Patterson Meldolesi E, Mroczkowski P, Nagtegaal I, Naredi P, Ortiz H, SD. Panitumumab-FOLFOX4 treatment and RAS mutations in Pahlman L, Quirke P, Rodel C, Roth A, Rutten H, Schmoll HJ, colorectal cancer. N Engl J Med. 2013;369(11):1023–34. https​:// Smith JJ, Tanis PJ, Taylor C, Wibe A, Wiggers T, Gambacorta doi.org/10.1056/NEJMo​a1305​275. MA, Aristei C, Valentini V. EURECCA colorectal: multidiscipli- 62. Karapetis CS, Khambata-Ford S, Jonker DJ, O’Callaghan CJ, Tu nary management: European consensus conference colon and rec- D, Tebbutt NC, Simes RJ, Chalchal H, Shapiro JD, Robitaille S, tum. Eur J Cancer. 2014;50(1):e1–e34. https​://doi.org/10.1016/j. Price TJ, Shepherd L, Au HJ, Langer C, Moore MJ, Zalcberg JR. ejca.2013.06.048. K-ras mutations and benefit from cetuximab in advanced colo- 52. Vieth M, Quirke P, Lambert R, von Karsa L, Risio M, Interna- rectal cancer. N Engl J Med. 2008;359(17):1757–65. https​://doi. tional Agency for Research on C. European guidelines for quality org/10.1056/NEJMo​a0804​385. assurance in colorectal cancer screening and diagnosis First Edi- 63. Richman SD, Fairley J, Butler R, Deans ZC. RAS screening in tion-Annotations of colorectal lesions. Endoscopy. 2012;44(Suppl colorectal cancer: a comprehensive analysis of the results from the 3):SE131–139. https​://doi.org/10.1055/s-0032-13097​98. UK NEQAS colorectal cancer external quality assurance schemes 53. Watanabe T, Itabashi M, Shimada Y, Tanaka S, Ito Y, Ajioka (2009–2016). Virchows Arch. 2017;471(6):721–9. https​://doi. Y, Hamaguchi T, Hyodo I, Igarashi M, Ishida H, Ishiguro M, org/10.1007/s0042​8-017-2162-7. Kanemitsu Y, Kokudo N, Muro K, Ochiai A, Oguchi M, Ohkura 64. Tack V, Ligtenberg MJL, Siebers AG, Rombout PDM, Dabir PD, Y, Saito Y, Sakai Y, Ueno H, Yoshino T, Fujimori T, Koinuma Weren RDA, van Krieken J, Dequeker EMC. RAS testing for colo- N, Morita T, Nishimura G, Sakata Y, Takahashi K, Takiuchi H, rectal cancer patients is reliable in European laboratories that pass Tsuruta O, Yamaguchi T, Yoshida M, Yamaguchi N, Kotake K, 13 1990 Clinical and Translational Oncology (2020) 22:1976–1991 external quality assessment. Virchows Arch. 2018;472(5):717–25. BRAF/KRAS are enriched for targetable kinase fusions. Cancer https​://doi.org/10.1007/s0042​8-017-2291-z. Res. 2019;79(6):1047–53. https​://doi.org/10.1158/0008-5472. 65. Dvorak K, Higgins A, Palting J, Cohen M, Brunhoeber P. Immu- CAN-18-3126. nohistochemistry with anti-BRAF V600E (VE1) mouse mono- 76. Marchio C, Scaltriti M, Ladanyi M, Iafrate AJ, Bibeau F, Dietel clonal antibody is a sensitive method for detection of the BRAF M, Hechtman JF, Troiani T, Lopez-Rios F, Douillard JY, Andre F, V600E mutation in colon cancer: evaluation of 120 cases with and Reis-Filho JS. ESMO recommendations on the standard methods without KRAS mutation and literature review. Pathol Oncol Res. to detect NTRK fusions in daily practice and clinical research. 2019;25(1):349–59. https​://doi.org/10.1007/s1225​3-017-0344-x. Ann Oncol. 2019. https​://doi.org/10.1093/annon​c/mdz20​4. 66. Gonzalez-Colunga KJ, Lino-Silva LS, Salcedo-Hernandez RA, 77. Yurgelun MB, Hampel H. Recent advances in lynch syndrome: Ruiz-Garcia EB, Zepeda-Najar C. BRAF V600E expression by diagnosis, treatment, and cancer prevention. Am Soc Clin immunohistochemistry in colon cancer and clinico-pathologic fea- Oncol Educ Book. 2018;38:101–9. https​: //doi.org/10.1200/ tures associated with BRAF-mutated colonic cancers in Mexican EDBK_20834​1. patients. J Gastrointest Cancer. 2019. https​://doi.org/10.1007/ 78. Schrock AB, Ouyang C, Sandhu J, Sokol E, Jin D, Ross JS, Miller s1202​9-018-00191​-9. VA, Lim D, Amanam I, Chao J, Catenacci D, Cho M, Braiteh 67. Prieto-Potin I, Montagut C, Bellosillo B, Evans M, Smith M, Mel- F, Klempner SJ, Ali SM, Fakih M. Tumor mutational burden is chior L, Reiltin W, Bennett M, Pennati V, Castiglione F, Burrig predictive of response to immune checkpoint inhibitors in MSI- KF, Cooper U, Dockhorn-Dworniczak B, Rossenbach C, Luna- high metastatic colorectal cancer. Ann Oncol. 2019. https​://doi. Aguirre CM, Barrera-Saldana HA, Machado JC, Costa JL, Yacobi org/10.1093/annon​c/mdz13​4. R, Tabibian-Keissar H, Buglioni S, Ronchetti L, Douglas-Berger 79. Ou SI, Nagasaka M, Zhu VW. Liquid biopsy to identify action- L, Dubbink HJ, Alorini M, Sabourin JC, Rojo F. Multicenter able genomic alterations. Am Soc Clin Oncol Educ Book. evaluation of the idylla NRAS-BRAF mutation test in metastatic 2018;38:978–97. https​://doi.org/10.1200/EDBK_19976​5. colorectal cancer. J Mol Diagn. 2018;20(5):664–76. https​://doi. 80. Cenaj O, Ligon AH, Hornick JL, Sholl LM. Detection of org/10.1016/j.jmold​x.2018.05.008. ERBB2 amplification by next-generation sequencing predicts 68. Franczak C, Dubouis L, Gilson P, Husson M, Rouyer M, Demange HER2 expression in colorectal carcinoma. Am J Clin Pathol. J, Leroux A, Merlin JL, Harle A. Integrated routine workflow 2019;152(1):97–108. https​://doi.org/10.1093/ajcp/aqz03​1. using next-generation sequencing and a fully-automated plat- 81. Tan K, Leong SM, Kee Z, Caramat PV, Teo J, Blanco MVM, Koay form for the detection of KRAS, NRAS and BRAF mutations ESC, Cheong WK, Soh TI, Yong WP, Pang A. Longitudinal moni- in formalin-fixed paraffin embedded samples with poor DNA toring reveals dynamic changes in circulating tumor cells (CTCs) quality in patients with colorectal carcinoma. PLoS ONE. and CTC-associated miRNAs in response to chemotherapy in 2019;14(2):e0212801. https:​ //doi.org/10.1371/journa​ l.pone.02128​ metastatic colorectal cancer patients. Cancer Lett. 2018;423:1–8. 01. https​://doi.org/10.1016/j.canle​t.2018.02.039. 69. Cui Y, Cui X, Yang X, Zhuo Z, Du X, Xin L, Yang Z, Cheng X. 82. Garcia-Foncillas J, Alba E, Aranda E, Diaz-Rubio E, Lopez-Lopez Diffusion kurtosis imaging-derived histogram metrics for predic- R, Tabernero J, Vivancos A. Incorporating BEAMing technology tion of KRAS mutation in rectal adenocarcinoma: preliminary as a liquid biopsy into clinical practice for the management of findings. J Magn Reson Imaging. 2019;50(3):930–9. https​://doi. colorectal cancer patients: an expert taskforce review. Ann Oncol. org/10.1002/jmri.26653​. 2017;28(12):2943–9. https​://doi.org/10.1093/annon​c/mdx50​1. 70. Oh JE, Kim MJ, Lee J, Hur BY, Kim B, Kim DY, Baek JY, Chang 83. Garcia-Foncillas J, Tabernero J, Elez E, Aranda E, Benavides HJ, Park SC, Oh JH, Cho SA, Sohn DK. Magnetic resonance- M, Camps C, Jantus-Lewintre E, Lopez R, Muinelo-Romay L, based texture analysis differentiating KRAS mutation status in Montagut C, Anton A, Lopez G, Diaz-Rubio E, Rojo F, Vivancos rectal cancer. Cancer Res Treat. 2019. https​://doi.org/10.4143/ A. Prospective multicenter real-world RAS mutation comparison crt.2019.050. between OncoBEAM-based liquid biopsy and tissue analysis in 71. Jover R, Paya A, Alenda C, Poveda MJ, Peiro G, Aranda FI, Perez- metastatic colorectal cancer. Br J Cancer. 2018;119(12):1464–70. Mateo M. Defective mismatch-repair colorectal cancer: clinico- https​://doi.org/10.1038/s4141​6-018-0293-5. pathologic characteristics and usefulness of immunohistochemical 84. Sartore-Bianchi A, Loupakis F, Argiles G, Prager GW. Chal- analysis for diagnosis. Am J Clin Pathol. 2004;122(3):389–94. lenging chemoresistant metastatic colorectal cancer: therapeutic https​://doi.org/10.1309/V9PG-K2Y2-60VF-VULR. strategies from the clinic and from the laboratory. Ann Oncol. 72. Murphy KM, Zhang S, Geiger T, Hafez MJ, Bacher J, Berg 2016;27(8):1456–66. https​://doi.org/10.1093/annon​c/mdw19​1. KD, Eshleman JR. Comparison of the microsatellite instability 85. Marcuello M, Vymetalkova V, Neves RPL, Duran-Sanchon S, analysis system and the Bethesda panel for the determination Vedeld HM, Tham E, van Dalum G, Flugen G, Garcia-Barberan of microsatellite instability in colorectal cancers. J Mol Diagn. V, Fijneman RJ, Castells A, Vodicka P, Lind GE, Stoecklein NH, 2006;8(3):305–11. https​://doi.org/10.2353/jmold​x.2006.05009​2. Heitzer E, Gironella M. Circulating biomarkers for early detection 73. Samaison L, Grall M, Staroz F, Uguen A. Microsatellite instability and clinical management of colorectal cancer. Mol Aspects Med. diagnosis using the fully automated Idylla platform: feasibility 2019. https​://doi.org/10.1016/j.mam.2019.06.002. study of an in-house rapid molecular testing ancillary to immu- 86. Olmedillas-Lopez S, Garcia-Olmo DC, Garcia-Arranz M, Peiro- nohistochemistry in pathology laboratories. J Clin Pathol. 2019. Pastor R, Aguado B, Garcia-Olmo D. Liquid biopsy by NGS: https​://doi.org/10.1136/jclin​path-2019-20593​5. differential presence of exons (DPE) in cell-free DNA reveals 74. Fusco N, Bosari S. HER2 aberrations and heterogeneity in cancers different patterns in metastatic and nonmetastatic colorectal can- of the digestive system: implications for pathologists and gastro- cer. Cancer Med. 2018;7(5):1706–16. https​://doi.org/10.1002/ enterologists. World J Gastroenterol. 2016;22(35):7926–37. https​ cam4.1399. ://doi.org/10.3748/wjg.v22.i35.7926. 75. Cocco E, Benhamida J, Middha S, Zehir A, Mullaney K, Shia Publisher’s Note Springer Nature remains neutral with regard to J, Yaeger R, Zhang L, Wong D, Villafania L, Nafa K, Scaltriti jurisdictional claims in published maps and institutional affiliations. M, Drilon A, Saltz L, Schram AM, Stadler ZK, Hyman DM, Benayed R, Ladanyi M, Hechtman JF. Colorectal carcinomas containing hypermethylated MLH1 promoter and wild-type 13 Clinical and Translational Oncology (2020) 22:1976–1991 1991 Affiliations P. García‑Alfonso1 · R. García‑Carbonero2 · J. García‑Foncillas3 · P. Pérez‑Segura4 · R. Salazar5 · R. Vera6 · S. Ramón y Cajal7 · J. Hernández‑Losa7 · S. Landolfi7 · E. Musulén8,9 · M. Cuatrecasas10 · S. Navarro11 2 R. García‑Carbonero Departament of Medical Oncology, Hospital Universitario rgcarbonero@gmail.com 12 de Octubre, Instituto de Investigación Sanitaria Hospital 12 de Octubre (imas12), UCM, CNIO, CIBERONC, Madrid, J. García‑Foncillas Spain jgfoncillas@gmail.com 3 Departament of Medical Oncology, Hospital Universitario P. Pérez‑Segura Fundación Jiménez Díaz, Universidad Autónoma de Madrid, perezsegura09@gmail.com Madrid, Spain R. Salazar 4 Departament of Medical Oncology, Hospital Clínico ramonsalazar@iconcologia.net Universitario San Carlos, CIBERONC, Madrid, Spain R. Vera 5 Departament of Medical Oncology, ICO L’Hospitalet, ruth.vera.garcia@cfnavarra.es Oncobell Program (IDIBELL), CIBERONC, S. Ramón y Cajal Hospitalet de Llobregat, Spain sramon@vhebron.net 6 Departament of Medical Oncology, Complejo Hospitalario J. Hernández‑Losa de Navarra; Navarrabiomed, IDISNA, Pamplona, Spain javierhernandezlosa@gmail.com 7 Department of Pathology, Hospital Universitario Vall S. Landolfi D’Hebron, CIBERONC, Barcelona, Spain slandolfi@vhebron.net 8 Department of Pathology, Hospital Universitari General de E. Musulén Catalunya, Grupo Quirónsalud, Sant Cugat del Vallès, Spain eva.musulen@quironsalud.es; 9 Cancer Epigenetics Group, Institut de Recerca Contra La emusulen@carrerasresearch.org Leucèmia Josep Carreras, Badalona, Spain M. Cuatrecasas 10 Department of Pathology, Hospital Clinic, CIBERehd, mcuatrec@clinic.cat Barcelona, Spain S. Navarro 11 Department of Pathology, University of Valencia, Hospital samuel.navarro@uv.es Clínico Universitario de Valencia, CIBERONC, Valencia, 1 Spain Departament of Medical Oncology, Hospital General Universitario Gregorio Marañón, Madrid, Spain 13