Comparison between Next-Generation Sequencing and Immunohistochemistry in the Diagnosis of Lynch Syndrome in Patients with Amsterdam II Clinical Criteria*

Abstract

Introduction

Colorectal cancer (CRC) ranks third as the most incidental in the world. One of its hereditary forms is Lynch syndrome (LS), characterized by mutations in one of the DNA mismatches repairs (MMR) genes (MLH1, MSH2, MSH6, PMS2, or EPCAM). Current protocols suggest screening with clinical criteria followed by immunohistochemistry (IHC) before next-generation sequencing (NGS) for an early diagnosis. However, using IHC as an isolated screening test may interfere with the diagnosis of LS.

Objective

To assess the correlation between IHC and NGS to diagnose LS in patients who meet the Amsterdam II clinical criteria.

Materials and Methods

Observational study comprising 32 patients of both genders, aged between 20 and 64 years at the time of CRC diagnosis. They must meet the Amsterdam II clinical criteria and be registered in the Hereditary Cancer Program of Hospital de Câncer de Pernambuco (HCP). All patients underwent IHC assessment of malignant tumor and blood sample NGS.

Results

The NGS yielded 11 (34%) positive results for LS. There was a correlation between IHC and NGS in 23 cases. In 8 (35%), there was diagnostic confirmation of IHC by NGS, and in 15 cases (65%) there was diagnostic exclusion of LS by IHC and NGS. In three cases, there was confirmation of a pathogenic variant in MLH1 by NGS. The cases presented the same large deletions, contradicting IHC with proficient MMR.

Conclusion

A moderate correlation between IHC and NGS has been established, as indicated by the Kappa coefficient of 0.410 utilizing the Amsterdam II clinical criteria.

Keywords

colorectal cancer - Lynch syndrome - microsatellite instability - complete genome sequencing - immunohistochemistry

Introduction

Lynch syndrome (LS) is a highly penetrant, autosomal, dominant, inherited disorder caused by deleterious germline mutations in one of the DNA mismatch repairs (MMR) genes: mutL homolog 1 (MLH1), mutS homolog 2 (MSH2), mutS homolog 6 (MSH6), postmeiotic segregation increased 2 (PMS2), or a deletion in the epithelial cell adhesion molecule (EPCAM). This syndrome results from a failure in the proofreading process that corrects base pair incompatibilities that may arise in the normal course of DNA replication.[1] [2] [3] [4] [5] [6] Such errors lead to a lifelong risk of cancer. There is a population estimate of one MMR protein mutation per 279 people.[7] [8]

This disorder has the following characteristics: high microsatellite instability, which causes rapid adenoma-carcinoma progression (between 1 and 3 years), triggering an increased risk of colorectal cancer (CRC); tumors with a preferential appearance in the right colon (60–70%) at an early age (approximately 45 years) with an increased rate of metachronous and synchronous tumors (20%); and presence of extracolonic tumors, such as in the endometrium, ovary, stomach, small intestine, and urinary tract, in patients carrying the germline mutation.[9] [10] [11] [12]

The lack of clinical indicators to forecast vulnerability to LS highlights family history as the primary means of identifying at-risk people.[12] [13] [14] In underdeveloped nations, limited financial resources require the utilization of familial history to identify cancer patients with hereditary forms, as it represents an affordable strategy and an effective risk-assessment instrument.[10] [14] [15]

The literature delineates some indicators for the clinical identification of LS, one of which is the Amsterdam II criteria.[12] [14] Patients fulfilling the Amsterdam II criteria should undergo testing to detect deficiencies in MMR proteins for the characterization of LS. The most effective instrument for LS screening is IHC, owing to its cost efficiency. It directly evaluates the expression of the four most pertinent MMR proteins (MLH1, MSH2, MSH6, and PMS2). The lack of protein expression in tumor tissue indicates which gene or genes may be impaired.[12] [14] [16] [17] [18] Certain studies indicate that the restrictive selection criterion (Amsterdam II) is more effective in detecting alterations in the germline than the Bethesda criterion. Amsterdam II exhibits low sensitivity (28–45%) and specificity (46–68%) for diagnosis via IHC in a general population.[17] [18] [19] [20]

Germline genetic testing may be proposed for patients who complete the Amsterdam II criteria, regardless of the MMR protein status in immunohistochemistry (IHC). The test may be applicable despite the limitations in sensitivity and specificity of clinical criteria, potentially resulting in the endorsement of universal molecular screening for CRC with IHC. When all Amsterdam II clinical criteria are met alongside the loss of MMR protein expression, the likelihood of identifying a germline mutation rises dramatically, to approximately 70 to 80%.[17] [19] [20] [21]

In the context described above, this study aims to establish an accessible screening and diagnostic method for LS. As well as to evaluate the correlation between IHC and next-generation sequencing (NGS) for the diagnosis of LS in patients who meet the Amsterdam II criteria for this syndrome.

Materials and Methods

The present is a cross-sectional study conducted at Hospital Barão de Lucena (HBL) and Universidade Federal de Pernambuco (UFPE) between 2021 and 2023, and it was registered in the Hereditary Cancer Program of Hospital de Câncer de Pernambuco (HCP). We aimed to evaluate the correlation between IHC and NGS for the diagnosis of LS among patients who met the Amsterdam II clinical criteria.

The sample comprised patients of both genders, aged between 20 and 64 years, registered in the project “Rare Genomes: Application of genomics for the diagnosis of rare diseases and the hereditary risk of cancer in Brazil in public health services,” who met Amsterdam II clinical criteria for LS. Patients with familial adenomatous polyposis (FAP) and those without record of genetic sequencing were excluded from the analysis.

The specific diagnosis assessment involved obtaining the following materials from all patients diagnosed with LS: (a) paraffin block containing colorectal tumor from a surgical specimen and, if possible, adjacent normal colonic tissue for IHC analysis; (b) genomic DNA extracted from peripheral blood; (c) clinical data; and (d) genogram showing at least three generations.

The Pathological Anatomy laboratories of the HCP and HBL conducted the IHC examination. The protein products from the MSH2, MSH6, MLH1, and PMS2 genes were detected, according to the protocol described by Kim et al., which employs monoclonal antibodies against the MLH1 (Anti-MLH1), MSH2 (Anti-MSH2), MSH6 (Anti-MSH6), and PMS2 proteins (Anti-PMS2).

The positive control consisted of normal tissue adjacent to the tumor. The assessment considered positive nuclear staining as follows: (a) Pattern 1: < 5%, (b) Pattern 2: 5 to 10%, and (c) Pattern 3: > 10% nuclear staining. Negative protein expression was defined by the complete absence of nuclear staining in the tumor tissue (Pattern 4, referred to as “lost”). One pathologist analyzed the IHC slides.

Genomic DNA from each patient was extracted from a 5 mL sample of peripheral blood collected in an EDTA tube. The extracted DNA underwent NGS on the Illumina (Illumina, Inc.) platform. Data was processed for SNV variant calling (small indels) and copy number analysis (CNV) for large deletions and exon duplications in the Genomika-Einstein pipeline. Variant numbering was performed using the reference transcript from base A of the ATG initiation codon aligned against the GRCh38/hg38 reference genome. Variant classification followed the criteria of the American College of Genomics and Clinical Genetics (ACMG) and updates proposed by the ClinGen workgroup.

The sample consisted of 217 families diagnosed with CRC registered in the Hereditary Cancer Program of the HCP. Among these patients, the analysis included 48 cases that allowed the identification of Amsterdam II criteria for LS. This facilitated the confirmation of the Amsterdam II clinical criteria for LS, followed by the signature of the informed consent form (ICF) and collection of peripheral blood for analysis at Hospital Israelita Albert Einstein, where genomic DNA was extracted. The NGS testing was funded by the same hospital (PROADI-SUS, no. 01/2017, process no. 25000.083098/2019-71).

Regarding the 48 initially selected patients, contact could not be established in 8 cases. Another 6 patients did not fill out the informed consent form or did not want to participate in the research. In another 2 cases, NGS results are pending due to an unforeseen delay in the delivery of genetic sequencing results.

Statistical Analysis

Descriptive and inferential statistical methods were utilized for data analysis. The descriptive ones included absolute distributions, percentages, and measurements such as mean, standard deviation (SD), and coefficient of variation. Numerical variables were represented by measures of central tendency and dispersion.

Inferential statistical methods consisted of Fisher's exact test for categorical variables and Student's t-test for paired data. The Shapiro-Wilk test assessed the normality of quantitative variables. Notably, the selection of Fisher's exact test for categorical variables and Student's t-test for normally distributed data was based on the number of observations and the assumption of normality of the variables of interest (MLH1, MSH2, MSH6, and PMS2), all at a 95% confidence level.

The agreement between IHC and NGS in patients with LS who met the Amsterdam II criteria was evaluated using the Kappa index, with correlation categorizations as follows: < 0 (none), 0 to 0.19 (poor), 0.20 to 0.39 (fair), 0.40 to 0.59 (moderate), 0.60 to 0.79 (substantial), and 0.80 to 1.00 (almost perfect).

Results

The assessment comprised 32 patients who underwent both IHC and NGS. The mean age at CRC diagnosis was of 44 ± 10.6 (range: 20–64) years. Regarding gender distribution, there was a higher frequency of CRC among female subjects (60%) [Table 1].

Table 1 Demographic data of the study sample

Variable
Female gender (%)	60
Male gender (%)	40
Mean age at diagnosis (years)	44 ± 10.6
Age range (years)	20–64

In the IHC evaluation of tumor tissue, 18 patients exhibited MMR protein expression (56%), while it was absent in 14 (44%), as presented in [Table 2]. Among those without this expression, one case involved MLH1 (7%) and one involved PMS2 (7%). Additionally, there was no associated expression between MLH1 and PMS2 in 6 cases (43%) and between MSH2 and MSH6 in 6 cases (43%).

Table 2 Immunohistochemistry distribution according to frequency

Immunohistochemistry	Frequency	%
Presence of expression	18	56
Absence of expression	14	44
Total	32	100.0

Genetic sequencing (NGS) resulted in 21 negative findings for LS (66%) among patients meeting the Amsterdam II clinical criteria. Among the 11 positive results for LS, 4 revealed a pathogenic variant in MLH1 and 4 in MSH2, as detailed in [Table 3].

Table 3 Distribution of next-generation sequencing according to frequency

Next-generation sequencing	Frequency	%
Negative	21	66
MLH1	7	22
MSH2	4	12
Total	32	100.0

Abbreviations: MLH1, mutL homolog 1; MSH2, mutS homolog 2.

[Table 4] demonstrates the presence of a statistically significant Kappa value. The coefficient obtained was 0.410, indicating a moderate agreement between IHC and NGS. A correlation was found in 23 cases; 8 cases confirmed the diagnosis by both methods, while 15 did not confirm the diagnosis of LS either.

Table 4 Correlation between immunohistochemistry and next-generation sequencing

Variable	Immunohistochemistry		Kappa	Kappa p-value
	Deficient: n (%)	Proficient: n (%)
Next-generation sequencing
Positive	8 (25.0)	3 (9.0)	0.415	0.017
Negative	6 (19.0)	15 (47.0)

The positive predictive value of IHC was 57%, and the negative predictive value was 83% in patients meeting Amsterdam II clinical criteria. The specificity was 71% and the sensitivity was 73% in this study.

The analysis also suggested that 9 cases (28%) exhibited discordance between IHC and NGS, primarily due to the absence of MMR repair proteins (n = 6), although NGS did not identify pathogenic variants in these genes.

Among 14 patients with IHC showing no MMR protein expression, 6 cases exhibited no identification of pathogenic variants by NGS, and 3 cases displayed absence of MLH1/PMS2, 1 involving PMS2 and 2 involving MSH2/MSH6. Among these 6 cases, three had variants of uncertain significance (VUSs), warranting further investigation.

Notably, in the three cases with confirmed pathogenic variants of the MMR gene for LS in MLH1, with IHC indicating pMMR (proficient MMR), it is noteworthy that the pathogenic variants resulted from the same deletion, as detailed in [Table 5].

Table 5 Correlation between MMR proteins in immunohistochemistry and MMR genes by next-generation sequencing

		Immunohistochemistry						Total
Next-generation sequencing		Stable	MLH1	MSH2	PMS2	MLH1 + PMS2 a	MSH2 + MSH6 b
	Negative	15	0	0	1	3	2	21
	MLH1	3*	1	0	0	3	0	7
	MSH2	0	0	0	0	0	4	4
Total		18	1	0	1	6	6	32

Abbreviations: MLH1, mutL homolog 1; MMR, DNA mismatch repair; MSH2, mutS homolog 2; MSH6, mutS homolog 6; PMS2, postmeiotic segregation increased 2.

Notes: ^aOne patient: variant of uncertain significance (VUS) MLH1: c.4484G > T p. (Arg1495Met) heterozigose + c.1731 + 3A > C p.(?) heterozigose (VUS). ^bOne patient: VUS MSH6: c.3598A > G p. (Ile1200Val) heterozigose. ^* MLH1: Seq [GRCh38] del (3) (p22.1p22.2) NC_000003.12: g.(37047963_37059588) del.

Discussion

As the most common hereditary CRC, LS occurs in approximately one every 35 cases, with a slightly greater prevalence in females. This syndrome is characterized by heterozygous germline mutations in MMR genes (MLH1, MSH2, MSH6, PMS2, or EPCAM), impairing the ability to correct mismatched base pairs during cell division.[9] [17] In terms of gender distribution, the incidence of CRC is greater among female (n = 18; 56%) than male subjects (n = 14; 44%). This result is consistent with international and Brazilian literature about CRC and LS.[15] [22] [23]

This study examined 32 individuals with a mean age of 44 years at the time of CRC diagnosis, with ages ranging from 20 to 64 years. These findings validate the average ages documented in the literature, which range from 32 to 45 years for CRC diagnosis in LS patients. Early onset is noted in MLH1/MSH mutations.[14] [16] [24]

Patient selection for genetic testing remains a significant challenge. Current clinical criteria exhibit low sensitivity, potentially leaving between 39 and 50% of cases undiagnosed.[ 18] [25] [26] The absence of MLH1 and MSH2 expression accounts for approximately 70% of LS cases identified via IHC. In this study, among patients meeting the Amsterdam II criteria, 14 (44%) displayed deficient expression in DNA repair proteins, comprising 6 cases (43%) in MSH2 alone or in conjunction with MSH6, 7 (50%) in MLH1 alone or in conjunction with PMS2, and 1 in PMS2 alone (7%).

Loss of expression in MLH1 (MLH1/PMS2 heterodimer) and MSH2 (MSH2/MSH6 heterodimer) in IHC leads to the loss of entire heterodimer formation. However, mutation in MSH6 or PMS2 induces isolated lack of MSH6 or PMS2 expression.[7] [8] [12] [14]

Among the available results, the correlation between the two evaluated diagnostic methods (IHC and NGS) is moderately statistically significant (p = 0.017), as indicated in [Table 4]. There was a correlation between IHC and NGS in 23 cases, with eight presenting diagnostic confirmation by both methods.

Of the 11 LS-positive cases identified by NGS, seven displayed mutations in MLH1 (64%) and four in MSH2 (36%). There were 21 cases that yielded no LS diagnostic confirmation, as depicted in [Table 4]. In Brazil, 80 to 90% of cases entail mutations in MLH1 or MSH2, with around 50% of cases in MLH1, 40% in MSH2, and 10% related to MSH6 or PMS2.[15] [22] [25] [26] The advent of NGS has heightened the significance of LS detection tests for patients' screening, treatment guidance, and family member segregation for active surveillance, with a progressive reduction in costs.[20] [21] [27]

In 6 cases, IHC exhibited alterations in MMR proteins, while NGS detected no pathogenic variant. In this study, the positive predictive value of IHC was 57%, and the negative was 83% in patients meeting Amsterdam II clinical criteria. Assessing repair proteins via IHC necessitates testing all 4 MMR proteins with a sensitivity of 83% and specificity of 89%, surpassing the values found in the present study. While IHC-based screening for colorectal tumors is crucial in identifying LS carriers, its efficacy is inherently confined to individuals already afflicted by cancer who can provide a tumor sample.[10] [17] [18] [22]

Presently, two primary methodologies are employed to identify individuals and families at risk for LS. The first is universal criteria applicable to all CRC cases. The second is a targeted method based on age or familial history criteria for individuals at elevated risk, followed by confirmation by identifying mutations in gene sequencing, as abnormalities in IHC only suggest LS.[10] [12] [18]

When considering the 11 LS-positive cases identified by NGS, as indicated in [Table 5], there are 3 with a pathogenic variant of the MMR gene for LS in MLH1 that wasn't detected in IHC. The IHC result with the presence of MMR would lead to the absence of a diagnosis of LS in 27% of confirmed cases, based on the current consensus for evaluating LS.[10] [12] [14] [17] [18] [22]

Although the universal strategy is cost-effective, it may not detect instances where mutations in repair genes impair their function without resulting in microsatellite instability, as seen in MSH6 mutation cases, or when IHC results are normal despite the presence of a nonfunctional mismatch repair protein (functionally inactive but antigenically intact proteins).[18] [25] [28]

Recent extensive national and international research reveals a 6% rate of germline pathogenic mutations identified alongside somatic MLH1 hypermethylation in. This study indicates that diagnosing 10% of cases using NGS may result in the oversight of MLH1 germline mutation carriers when somatic hypermethylation of MLH1 is assessed via IHC to exclude LS.

The evaluation of the diagnostic modalities' efficacy for LS facilitates a comparison of the Amsterdam II criteria, IHC and NGS. This study has shown that using full genome sequencing in the analysis of this cohort augmented and broadened the existing framework, improving diagnostic capabilities and facilitating the assessment of the cost-effectiveness of this tool across several health system situations.[13] [16] [22] [29] [30]

The present study has several limitations. The main one is the small sample size. Due to the high processing costs associated with NGS, the sample size was limited, and the selection process was conducted by convenience in a nonprobabilistic manner. Despite these limitations, this study furnishes significant insights into the utilization of NGS for confirming LS diagnoses in conjunction with clinical screening using the Amsterdam II criteria and correlation with IHC findings. It suggests that while there's some concordance, discrepancies remain, reinforcing the need for NGS confirmation.

Conclusion

A moderate correlation between IHC and NGS has been established, as indicated by the Kappa coefficient of 0.410 utilizing the Amsterdam II clinical criteria.

Conflict of Interests

The authors report no conflict of interest.

Authors' Contributions

PMB: conceptualization, formal analysis, investigation, methodology, project administration, visualization, writing – original draft, review and editing; FSC: formal analysis, writing – review and editing; JSB: formal analysis, writing – review and editing; MJMS: writing – review and editing; VC: data curation, funding acquisition, investigation, methodology, project administration, supervision, writing – review and editing; AF: conceptualization, supervision, visualization, writing – review and editing.

^* Study conducted at Hospital Barão de Lucena and Universidade Federal de Pernambuco, Recife, PE, Brazil.

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Publication History

Received: 09 July 2025

Accepted: 04 August 2025

Article published online:
29 December 2025