Microsatellite instability, reflective of a defective mismatch repair system, has been implicated as one of the main pathways involved in the pathogenesis of colorectal carcinoma. Herein, we describe the role of the mismatch repair system in the development of colorectal carcinoma, the advantages and disadvantages of using immunohistochemistry as the primary method of determining mismatch repair status, and compare the suitability of colorectal endoscopic biopsy versus resection specimens as the testing material of choice.
by Dr Odharnaith O’Brien, Dr Éanna Ryan and Prof. Kieran Sheahan             

Introduction
Owing to recent remarkable advances in our understanding of the molecular and genetic basis of disease, it is now known that colorectal carcinoma (CRC) is a heterogenous clinical entity characterized by multiple molecular subtypes [1]. One such molecular pathway involved in CRC pathogenesis is the microsatellite instability (MSI) pathway, where a deficient mismatch repair (dMMR) system leads to unchecked errors in DNA replication [2]. These errors result in a propensity for abnormal insertion or deletion of short, repetitive sequences of DNA (microsatellites), resulting in mutations in cancer-related genes and ultimately neoplasia. Up to 15–20% of colorectal carcinomas are of MSI phenotype. An inherited predisposition to dMMR cancers, particularly CRC, is present in Lynch syndrome, the most common heritable cancer syndrome. It is due to autosomal dominant mutations in four mismatch repair (MMR) genes (MLH1, MSH2, MSH6, PMS2) or more rarely by mutations in EPCAM, a gene upstream of MSH2. Patients present at an earlier age and have an increased incidence of synchronous and metachronous CRCs. Histologically, tumours are poorly differentiated, frequently exhibiting a mucinous or signet ring cell morphology. Tumour infiltrating lymphocytes are often prominent and a Crohn’s-like inflammatory response may be present at the tumour periphery. However, the majority of dMMR CRCs arise sporadically and are a result of MLH1 promoter hypermethylation. Unlike in Lynch syndrome, these tumours affect the right side of the colon, are diagnosed at advanced age and have a female preponderance. They are, however, histologically similar to Lynch syndrome CRCs. Mutation of the BRAF V600E gene is present in 60–70% of sporadic dMMR tumours and is almost never seen in Lynch syndrome. As such, incorporating BRAF and/or MLH1 methylation status into MMR diagnostic algorithms offers potential exclusion criteria for genetic testing [3–5].

Why is it important to identify dMMR in colorectal carcinoma?
Diagnosing a patient with a dMMR cancer has a number of advantages:

1. Identification of patients with Lynch syndrome
Once diagnosed, these patients benefit from increased surveillance, prophylactic aspirin therapy and more radical surgery in order to facilitate the prevention and/or early detection of potential tumours (both colonic and extracolonic) [5].
2. It provides prognostic information
Several studies have shown that dMMR CRC has a better prognosis than MMR proficient (pMMR) CRC. dMMR tumours are less likely to develop lymph node and liver metastases. However, in advanced disease (stage IV) dMMR status can portend a poorer prognosis [6–8].
3. It provides predictive information
dMMR tumours likely have a reduced response to 5-flurouracil based chemotherapy. In addition, advanced dMMR tumours have been shown to have a better response rate and progression free survival to the anti PD-1 drug pembrolizumab when compared to pMMR tumours [7–9].
Reliance by clinicians on clinical criteria such as the revised Bethesda guidelines to determine which patients should undergo screening for Lynch syndrome results in inaccurate determination of eligibility for screening in up to 28% of cases [10]. Consequently, a number of organizations have recently published guidelines endorsing reflex MMR testing of all diagnosed CRCs, including the National Institute for Health and Care Excellence (NICE), the American Society for Clinical Pathology (ASCP) and the American Society for Clinical Oncology (ASCP), among others [11–12]. The cost effectiveness of such a screening approach has been proven by several studies [13].

Diagnosis
Diagnosis of dMMR tumours is either via PCR amplification of specific microsatellite repeats in formalin-fixed, paraffin-embedded tumour tissue or by immunohistochemistry (IHC) which confirms the absence or presence of MMR proteins. Both MSI testing and IHC have virtually equivalent informative value in predicting germline mutation [3, 14]. Given that IHC is more widely available in general pathology laboratories and is a rapid, efficient and cost-effective method of testing, it is the more frequently used test. It also has the added benefit of directing germline testing to the particular mutated gene.
A number of commercially available MMR IHC antibodies are available for laboratory use. A protocol using a panel of four immunohistochemical antibodies to the four mismatch repair gene proteins (MLH1, MSH2, MSH6, PMS2) is recommended (Fig. 1). Complete loss of expression of one or more MMR protein is suggestive of dMMR. Loss of MLH1 often occurs in conjunction with loss of PMS2. This is due to the fact that MLH1 protein forms a heterodimer complex with PMS2. Isolated loss of PMS2 Is indicative of a defect in the PMS2 gene. However, combined loss of PMS2 and MLH1 indicates the defect lies in MLH1, as MLH1 confers stability to PMS2. A similar situation is seen with MSH2 and MSH6; isolated loss of MSH6 indicating defective MSH6, whereas loss of expression of both proteins indicates the defect involves MSH2. Background positive IHC staining in intratumoural lymphocytes or of adjacent normal colonic epithelium, if present, serve as reliable internal positive controls [5].
Once loss of expression of any IHC MMRP is confirmed, further testing is required. In cases where there is loss of MLH1, testing for the presence of BRAF V600E mutation and MLH1 hypermethylation, as mentioned previously, can further stratify those patients who likely have sporadic dMMR tumours. Patients demonstrating loss of MSH2, MSH6 or PMS2, and patients demonstrating loss of MLH1 who are BRAF V600E negative and MLH hypermethylation negative, should undergo germline testing to confirm Lynch syndrome (Fig. 2).
MMR IHC testing is typically performed on CRC resection specimens. Data has recently begun to accumulate that the yield of IHC testing performed on endoscopic biopsy material may be as good as that performed on surgical resections. We recently published a study evaluating the reliability of MMR IHC in CRC from preoperative endoscopic biopsy tissue when compared to matched surgical resection specimens and demonstrated 100% concordance in 53 cases of dMMR (n=10) and pMMR (n=43) tumours [14]. Our results corroborate the results of other studies that indicate endoscopic biopsies are a suitable source of tissue for MMR IHC analysis [15–17].
Preferential testing of MMR status on endoscopic biopsy samples over resection specimens carries a number of advantages. Immunostaining is highly sensitive to the degree of tissue fixation; given the small size of biopsy samples, faster and more thorough fixation may result in superior quality staining. Additionally, neoadjuvant chemoradiotherapy used in the standard treatment of locally advanced rectal tumours may result in a complete pathologic response, with no residual tumour available for testing. Neoadjuvant treatment can also occasionally alter the MMRP status of the tumour. In these two scenarios, the pretreatment biopsy could provide reliable testing material.
Endoscopic biopsies could also be used to initiate earlier and indeed preoperative genetic testing, allowing informed clinical decisions regarding the extent of resection to be made before surgery in those patients confirmed as having Lynch syndrome. The option of total colectomy as an alternative to segmental colectomy could be discussed, particularly with younger patients, to reduce the risk of metachronous CRC and the need for intense postoperative surveillance. In addition, females identified as having Lynch syndrome, who have completed their families, could be considered for concurrent hysterectomy, with/without bilateral salpingo-oophorectomy, in order to prevent the development of a gynecological tract malignancy and spare them a potential additional future procedure.
Recent studies suggest that dMMR tumours may respond well to immunotherapy in patients with advanced disease [9]. In the instance that an advanced tumour is inoperable at diagnosis, metastatic or endoscopic biopsy tissue could be used to screen for dMMR and Lynch syndrome, and direct immunotherapy.
Despite these advantages, some limitations exist in the use of IHC to determine MMR status which are not just specific to biopsy tissue. Rare missense mutations have been reported in MLH1 and MSH6 genes that affect MMR protein function but not translation and antigenicity – in this scenario the tumour harbours a defective protein, but one which demonstrates retention of IHC staining, giving a false result [19].
Intratumoural heterogeneity, where there is heterogeneity of MMR protein expression within a single tumour, also represents a potential pitfall [20]. This may be of particular concern in biopsy samples as they represent only a small proportion of a tumour and could erroneously misclassify the MMR status by virtue of inadequate sampling. Another issue is the small size of endoscopic biopsies; adequate material may not be available for IHC. Encouraging generous tumour sampling at the time of biopsy could reduce the risk of such limitations. Heterogeneity in the MMR status of CRC is rare and is thought in many instances to be a result of suboptimal tissue fixation. Given biopsies are usually of small size, adequate fixation of tissue can be assured.

Conclusion
Up to 15–20% of CRCs are of MSI phenotype, secondary to either sporadic methylation-induced silencing or inherited mutations in MMR-related genes. IHC is an effective and reliable testing modality for determining MMR status in CRC. Colorectal endoscopic biopsy and resection specimens are both suitable sources of testing material, with resection specimens currently the preferred specimen type. Endoscopic biopsy samples may become increasingly important as a testing material as the potential of tailored approaches to surgery, chemotherapy and immunotherapy becomes a standard of care in this era of personalized medicine.

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The authors
Dr Odharnaith O’Brien* MB BCh BAO, Dr Éanna Ryan MB BCh BAO, and Prof. Kieran Sheahan MB BCh BAO
Department of Pathology, St. Vincent’s
University Hospital, Dublin, Ireland
*Corresponding author
E-mail: odharnaithobrien@ gmail.com

The relevance of BRCA analysis
The identification of BRCA pathogenic variants (PVs) is the major concern for the genetic counselling in families with a high risk of breast (BC) and ovarian cancer (OC). BRCA1 (breast cancer early onset 1) and BRCA2 (breast cancer early onset 2) are the two major susceptibility genes in BC/OC, conferring a lifetime risk up to 87% for BC and up to 44% for OC. BRCA mutations have been found in 4–14% of all OC, with a higher occurrence of about 22% in the high-grade serous OC [1]. The clinical relevance of the identification of BRCA PV carriers concerns many aspects of a patient’s evaluation. The first relevant implication is the assessment of the lifetime cancer risk. Additionally, BRCA testing has a relevant impact on the therapeutic approach and on the treatment outcomes owing to the possibility of selecting patients for biomarker-directed therapy based on the mutational status [2]. BRCA-positive patients with OC, particularly, respond well to platinum-based chemotherapy, especially in the high-grade serous OC subtype, and tend to retain platinum-sensitivity for longer than those without BRCA PVs. Additionally, the treatment with poly (ADP-ribose) polymerase (PARP) inhibitor (e.g. olaparib) was approved as a target therapy in patients with both germline and somatic BRCA PVs. PARP inhibitor therapy is able to improve progression-free survival in response to a recent platinum-based chemotherapy [3]. To date, licensed PARP inhibitor is part of the standard care and, consequently, BRCA evaluation is considered a routine investigation tool, useful before treatment management. With respect to these benefits, BRCA testing should be offered to all patients with OC on the basis of histological subtype, regardless of age, or family and personal history of malignancy. This issue causes an increase of the demand for BRCA testing with a strong challenge into the diagnostic laboratories committed in fulfilling the need of an efficient and rapid molecular evaluation [4].

The challenge of BRCA testing
To date 1700 PVs in BRCA1 and 1900 PVs in BRCA2 have been reported. Most of them are single nucleotide polymorphisms (SNPs) or small insertion-deletion mutations (indels), with a significant impact on the structure and function of the protein. Also large genomic rearrangements (LGRs), consisting mainly in large deletions or duplications, represent an important part of BRCA molecular lesions. To date, a total of 98 different BRCA LGRs have been reported, 81 in BRCA1 and 17 in BRCA2 [5] with a prevalence that varies considerably. Interestingly, deletion of BRCA1 exon 1a-2 is reported in several populations worldwide and is considered a recurrent BRCA LGRs in BC/OC patients [6]. Owing to the broad complexity in the mutational landscape of BRCA genes, comprehensive screening including the efficient assessment of both qualitative (SNPs, indels) and quantitative (LGRs) alterations is mandatory (Fig. 1). Diagnostic laboratories are adopting next-generation sequencing (NGS) technology for BRCA testing, which offers the potential of fast, cost-efficient and comprehensive sequencing. By choosing NGS technology, many considerations should be made, such as the selection of an NGS platform, including the enrichment methods, the sequencing chemistries, the analytical procedures and the variant calling for both germline and somatic PVs [2]. NGS is highly recommended as the reference sequencing method for BRCA testing because of the size of coding region and the method’s sensitivity in tumour sample evaluation. In fact, Sanger sequencing is not suitable for the analysis of somatic mutations, especially in samples where the percentage of tumour cells is under 50%, and it requires also a large amount of starting DNA [4]. Several methods are commonly used for LGR analysis, including multiplex ligation-dependent probe amplification (MLPA) and multiplex amplicon quantification (MAQ). However, these approaches are expensive and time-consuming, and consequently these are not always suitable for all laboratories. In this case, LGR evaluation of BRCA genes represents a bottleneck in terms of time and costs. In this context, the great benefit of the NGS approach is the opportunity to obtain both qualitative and quantitative information from the same sequencing data by using tailored bioinformatics algorithms [7]. Only a positive bioinformatics result needs to be confirmed using an alternative conventional method. In order to optimize our routine diagnostic procedures for BRCA testing, we recently developed a new molecular approach called competitive PCR-high resolution melting analysis (cPCR-HRMA) [8], as an alternative method for LGR identification in BRCA genes. HRMA is a simple and robust closed-tube method commonly used for diagnostics, forensic and research purposes. This method consists of a PCR amplification performed in the presence of saturating binding dyes followed by a melting reaction. Specifically, the incremental increase of the reaction temperature causes the denaturation of double-stranded DNA with the concomitant release of intercalated dye and a decrease of fluorescence signal. The specific sequence of the analysed amplicon, primarily relating to the GC content and the length, determines the melting behaviour observed in a fluorescence signal versus temperature plot. Additionally, the melting temperature (Tm) may be calculated as the derivative of the melting curve. The shape of the curves and the specific Tm value obtained in the output plots is used for the genotyping. The advantages of this technique include rapid turn-around times and a closed-system environment that decrease the risk of laboratory contamination [9].

cPCR-HRMA for LGR evaluation
HRMA technology is typically applied to detect a single substitution, as well as small indels variants [6, 10]. The new cPCR-HRMA represents an optimized HRMA method that allows an efficient evaluation of BRCA1 copy number variation (CNV) by relying on the melting behaviour of target BRCA amplicon compared to a reference amplicon in the same HRMA reaction. In particular, specific albumin sequences were chosen as unchanged CNV references and analysed by coupling them with specific BRCA1 exons in a duplex PCR assay preceding the melting analyses. The landmarks of this new HRMA rely on the primers and the amplification protocol design. First of all, primer pairs for the simultaneous amplification of target and reference sequences are selected in order to produce paired amplicons with comparable lengths (similar amplification efficiencies) and different melting temperatures (no overlap between amplicons melting peaks). Furthermore, the primer concentration used for both target and reference amplification was set in order to produce comparable PCR performance between the two amplicon types and to obtain melting profiles more suggestive of CNV. In addition, the PCR thermal cycling was carried on until the exponential phase, in which the amplification performance reflects the CNV status of the target region. These optimized features lead to melting profiles specifically tailored for CNV investigations allowing a rapid detection of samples affected by a change in copy number. Genotype association was assessed by direct interpretation of melting profiles, as shown in Figure 2: samples with similar profiles were clustered into the same genotype group and CNV positive samples showed a typical melting profile with a detectable shape comparing to the wild type. In addition to the qualitative evaluation, we provide also a semi-quantitative analysis of melting behaviour with the calculation of the fluorescence peak height ratio (R) of target the amplicon (BRCA1) to the reference amplicon (albumin), according to the formula:
The mean and the standard deviation of the R values calculated in a consistent number of control CNV samples allowed the identification of the reference range for the R parameter: WT sample (mean±2SD; 2 copies), deletion (≤mean−2SD; n copies) and duplication (≥mean+2SD; 3n copies). The R value calculated in each analysed sample is normalized with the average of the ratios calculated in the WT sample group, obtaining the normalized fluorescence peak height ratio (Rn). The latter is compared to the reference range in order to obtain the copy number interpretation. Taken together, the qualitative and the semi-quantitative evaluations of the cPCR-HRMA assay allow the correct identification of copy number status in BRCA gene, resulting as a rapid and alternative method for the analysis of LGRs. Advantages of cPCR-HRMA are the ease and fast handling of samples. Furthermore, this application needs the same reagents and equipment for standard HRMA protocols commonly used in many laboratories routines. By introducing this efficient alternative method, our first aim was the optimization of the BRCA workflow, promoting a more rational use of confirmatory testing, such as MLPA and MAQ. Finally, we are confident that a general implementation of BRCA testing is now necessary as an emerging challenge. After a complete genetic counselling and a multidisciplinary activity that involves geneticists, oncologist and all other professionals, the patient should be directed to specialized laboratories. The complexity of the potential BRCA mutations, coupled with their clinical relevance, leads to the mandatory adoption of a comprehensive molecular workflow for BRCA analysis that must be characterized by a low wait-time and efficient clinical reporting in order to guarantee a useful medical application.

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8. Minucci A, De Paolis E, Concolino P, et al. Competitive PCR-high resolution melting analysis (C-PCR-HRMA) for large genomic rearrangements (LGRs) detection: a new approach to assess quantitative status of BRCA1 gene in a reference laboratory. Clin Chim Acta 2017; 470: 83–92.
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10. De Paolis E, Minucci A, De Bonis M,  et al. A rapid screening of a recurrent CYP24A1 pathogenic variant opens the way to molecular testing for idiopathic infantile hypercalcemia (IIH). Clin Chim Acta. (2018) Mar 21; 482: 8–13.

The authors
Elisa De Paolis MSc, Angelo Minucci PhD, Giovanni Luca Scaglione PhD, Maria De Bonis MSc, Ettore Capoluongo* PhD
Catholic University of The Sacred Heart, Rome, Italy
*Corresponding author
E-mail: ettoredomenico.capoluongo@policlinicogemelli.it