Professor Changlin Zhao: How to diagnose Lynch syndrome/Lynch syndrome-related tumors?

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Microsatellite instability (MSI) is a molecular marker of Lynch syndrome (LS), and it usually has a germline mutation in a mismatch repair (MMR) gene
.

LS is an autosomal dominant tumor syndrome caused by mutations in MMR
.

MMR genes mainly include MLH1 (3p21.
3), MSH2 (2p21-22), MSH6 (2p16), MSH3 (5q11-q12), PMS1 (2q31-q33), PMS2 (7p22), which are jointly responsible for the process of cell replication Correct the errors generated in the process
.

When the MMR gene is mutated, it leads to the loss of the corresponding repair function, causing DNA replication errors to be unable to be effectively corrected, resulting in the accumulation of DNA replication errors, and ultimately leading to the occurrence of cancer
.

The overall MMR gene mutation is MSI
.

1.
The risk of LS/LS-related tumors.
According to reports, in patients with LS caused by germline mutations in the MMR gene, up to 90% of the tumors exhibit high microsatellite instability (MSI-H)
.

Germline mutations in the MMR gene can also cause LS-related tumors (including colorectal, endometrium, ovary, stomach, small intestine, liver and gallbladder, urinary tract, brain and skin, etc.
), and its risk is higher than that of normal people
.

For males and females with LS, the average age at diagnosis of colorectal cancer is 42 and 47 years, respectively, and the cumulative risks are 66.
08% and 42.
7%, respectively.
Male individuals with MLH1 mutations have the highest risk of colorectal cancer
.

The average age of diagnosis of endometrial cancer in female individuals with LS is 47.
5 years, and the cumulative risk is 73.
42%
.

Among female individuals with LS, about 50% of patients with colorectal cancer and endometrial cancer have endometrial cancer first
.

The average age at diagnosis of gastric cancer in LS individuals is 56 years old.
Among the pathological types, small intestinal adenocarcinoma is the most common
.

The average age of diagnosis of ovarian cancer in female individuals with LS is 42.
5 years old, and about 30% of them have the onset of disease before the age of 40
.

National Comprehensive Cancer Network (NCCN) Colorectal Cancer Practice Guide: Colorectal Cancer Screening (2020.
V2) provides LS-related cancer risks with MLH1, MSH2, MSH6, and PMS2 deletions
.

(Table 1 can be enlarged)
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Table 1 MLH1, MSH2, MSH6, PMS2 deletions risk of LS-related cancer Tumor types MLH1/MSH2 defects have cancer risk MSH6 defects have cancer risk PMS2 defects have cancer risk Colon cancer 40%-80% 10%-22% 15%-20 % Endometrial cancer 25%-60% 16%-26% 15% Gastric cancer 1%-13% ≤ 3% Renal pelvis cancer, stomach cancer, ovarian cancer, small bowel cancer, ureter cancer, brain tumors, the combined risk is 6% ovarian cancer 4 %-24%1%-11%Biliary system cancer (gallbladder cancer, cholangiocarcinoma) 1.
4%-4%No report of urinary system cancer 1%-4%<1%Small bowel cancer 3%-6%No report of central nervous system Systemic malignant tumors 1%-3% No reports of sebaceous gland tumors 1%-9% No reports of pancreatic cancer 1%-6% No reports of pancreatic cancer : (1) The age of onset is earlier (median age is about 44 years); (2) Tumors are mostly located in the proximal colon; (3) Multiple primary colorectal cancers are significantly increased; (4) Extra-intestinal malignancies such as the endometrium Cancer, gastric cancer and pancreatic cancer have a high incidence; (5) Poorly differentiated adenocarcinoma and mucinous adenocarcinoma are common, often accompanied by lymphocyte infiltration or lymphoid cell aggregation; (6) Tumors tend to grow swelling rather than invasive Growth; (7) The prognosis is better than sporadic colorectal cancer
.

Pathologically, about two-thirds of LS tumors are located in the right colon.
The tumors are often large and well-defined, showing polyps, plaques or ulcers; the histology is often mucinous adenocarcinoma or poorly differentiated adenocarcinoma
.

3.
Diagnosis of LS (1) Clinical diagnosis of LS There are currently three main diagnostic criteria for LS, namely the Chinese LS family standard, Amsterdam standard I and Amsterdam standard II
.

According to the incidence of LS in China, this article only introduces the family standards of Chinese LS
.

The Chinese LS family standard is that there are more than 2 cases of colorectal cancer patients with a clear diagnosis of histopathology in the family, of which 2 cases are the relationship between parents and children or siblings (first-degree blood relatives), and meet any of the following conditions: ( 1) ≥1 case of multiple colorectal cancer patients (including adenoma); (2) ≥1 case of colorectal cancer onset age <50 years; (3) ≥1 case of family with LS-related extraintestinal malignancies (including Gastric cancer, endometrial cancer, small bowel cancer, ureter and renal pelvis cancer, ovarian cancer and hepatobiliary system cancer)
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Therefore, all patients who meet the above criteria or have a family with one of the three to be highly suspected of LS should be further screened for LS-related genes
.

(2) Molecular diagnosis of LS The molecular diagnosis of LS mainly includes two approaches: immunohistochemistry (Immunohistochemistr, IHC) detection of MMR, polymerase chain reaction (PCR) of MSI or next-generation sequencing (Next- generation" sequencing technology, NGS) detection
.

1.
IHC detection of MMR protein deletion.
According to the statistics of LS International Cooperation Organization, different MMR gene mutations account for different proportions of individuals with LS.
About 49% of them are MLH1 gene mutations and 38% are MSH2 gene mutations, 9% are MSH6 gene mutations, 2% are PMS2 gene mutations
.

1.
1 How to treat IHC showing negative expression of MLH1 and/or MLH2? IHC showing negative expression of MLH1 protein does not effectively predict the presence of mutations in MLH1, because Negative MLH1 protein expression can also be seen in the gene promoter region of the double nucleotide "cytosine-guanine (CG)" region, that is, CPG island methylation caused MLH1 expression silence, MLH1 gene promoter region CPG island methylation is usually It is the main cause of MSI in sporadic colorectal cancer.
Therefore, patients with negative MLH1 protein expression need to be tested for the presence of methylation in the MLH1 promoter region, or BRAF V600E mutations need to be excluded
.

It is generally believed that patients with detection of methylation in the MLH1 promoter region or BRAF V600E mutation can basically exclude LS
.

IHC showing negative expression of MLH2 protein does not effectively predict the presence of mutations in MLH1, because the EPCAM (epithelial cell adhesion molecule, EPCAM) gene located upstream of MSH2 encodes cancer-associated antigens and is a member of a family containing at least two type I membrane proteins.

.

This antigen is expressed on most normal epithelial cells and gastrointestinal cancer cells, and acts as a homotype calcium-dependent cell adhesion molecule
.

The deletion of this gene can cause hypermethylation of the MSH2 promoter and cause the silencing of the MSH2 gene.
The expression of MLH2 protein is negative, which is a sporadic tumor
.

If hypermethylation of the MLH2 promoter is detected, LS can be ruled out.
Therefore, patients with negative MSH2 protein expression also need to detect the EPCAM gene
.

1.
2 How to treat the problem of negative expression of MLH1 and PMS2 in IHC? Under normal circumstances, MLH1 and PMS2, MSH2 and MSH6 are coupled together.
The MSH2 protein first recognizes and binds the mismatched DNA sequence, and forms a dimer with MSH6, and then MLH1 and PMS2 form a heterodimer.
, Adjust base mismatches to complete DNA repair
.

How to treat the negative expression of MLH1 and PMS2 in IHC? There are two situations: (1) Germline mutations from MLH1 and PMS2 genes cause Lynch syndrome/Lynch syndrome-related tumors; (2) Methylation in the promoter region of MLH1 gene or BRAF V600E gene mutations, resulting in Because the MLH1 gene is silent, the PMS2 protein coupled with it is negatively expressed, which is a sporadic tumor
.

Therefore, patients with negative MLH1 and PMS2 protein expressions shown by IHC alone cannot be diagnosed with LS.
Further testing of BRAF V600E gene status or MLH1 gene promoter methylation status, or MMR gene germline mutation testing is needed to diagnose LS.
Or LS-related tumors
.

The steps of IHC to detect MMR in the diagnosis of LS/LS-related tumors
.

(Table 2 can be enlarged) Table 2 IHC detection of MMR to diagnose LS/LS-related tumors The interpretation of MMR IHC test results and recommendations for the diagnosis of LS/LS-related tumors are shown in Table 3 (can be enlarged)
.

Table 3 Interpretation of MMR IHC test results and recommendations for the diagnosis of LS/LS-related tumors 2.
PCR capillary electrophoresis or NGS detection of MSI status Currently recommended PCR capillary electrophoresis for the detection of MSI 5 common sites are BAT- 25.
BAT-26, D2S123, D5S346 and D17S250, of which 2 sites are unstable are called MSI-H; 1 site is unstable is called microsatellite low instability (MSI-L); 0 sites are not stable Stability is called microsatellite stabilization (MSS)
.

In recent years, the NGS platform has also been used to detect MSI status
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Current studies have shown that the coincidence rate of NGS-based MSI detection results compared with traditional PCR detection MSI is higher than 95%
.

PCR capillary electrophoresis or NGS detection MSI diagnosis of LS/LS-related tumor steps
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(Table 4 can be enlarged) Table 4 Steps of PCR capillary electrophoresis or NGS detection of MSI to diagnose LS/LS-related tumors 3.
How to treat inconsistent results of MMR/MSI? The relationship between MSI and MMR is close, but there are problems of inconsistency
.

Due to factors such as detection accuracy and tumor heterogeneity, the coincidence rate of MMR and MSI detection results is about 90%
.

IHC detection itself is also subjective, and is affected by factors such as the quality of the detection antibody and the detection process (fixation, staining), etc.
, which are prone to misjudgment, resulting in different detection accuracy rates of each center, and inconsistent MMR/MSI detection results
.

How to deal with inconsistent MMR/MSI test results? What strategy should be adopted? A meta-study analysis showed that the detection rate of MMR evaluated by IHC alone was 88%~89%, and the detection rate of MSI evaluated by PCR alone was 95%~99.
7%
.

Studies have also shown that the coincidence rate between NGS detection MSI results and PCR detection MSI results is higher than 95%
.

The research results suggest that the use of IHC/PCR or NGS combined detection of MSI/MMR can maximize the accuracy of the detection results
.

In addition, you can also use the multi-disicipline team (MDT) to make a comprehensive assessment based on the clinical characteristics and clinical diagnostic criteria of LS, combined with the MSI/MMR test results, and decide whether to proceed with BRAF V600E gene status or MLH1 gene activation.
Detection of daughter methylation status, or detection of MMR gene germline mutations
.

As scientists and researchers have realized that the essence of LS lies in the genetic defects of the four genes of MLH1, MSH2, MSH6, and PMS2, genetic diagnosis has become the "gold standard" of LS
.

Therefore, NCCN and The European Society for Medical Oncology (ESMO) developed guidelines for the diagnosis of LS genetic genes
.

The guidelines emphasize that all newly diagnosed colorectal cancer patients under the age of 70 must be included in the LS genetic screening, first through IHC testing for MMR protein, NGS testing for MSI, and then undergoing methylation testing.
Those with genetic defects cannot be ruled out.
, Then enter MLH1, MSH2, MSH6, PMS2 genetic gene germline mutation screening
.

If it is found that there is a genetic defect, then LS family screening will be carried out, that is, all relatives of a genetic defect person must be inspected for the corresponding genetic defect
.

LS/LS-related tumor diagnosis and genetic gene germline mutation inspection process
.

(Figure 1 can be enlarged) Figure 1 LS/LS-related tumor diagnosis and genetic gene germline mutation inspection process 4.
Different MMR gene germline mutations cause different risks of LS LS is a type of disease related to genetics.
Susceptibility to a variety of tumors, including colorectal cancer, endometrial cancer, gastric cancer, etc.
, is characterized by its early onset time
.

About 50% to 80% of LS occurs in the colorectal, accounting for about 2% to 3% of all colorectal cancers
.

The lifetime risk of colorectal cancer caused by different MMR gene germline mutations is also different.
Among them, MLH1 and MSH2 are 30% to 74%, MSH6 is only 10% to 22%, and PMS2 is 15% to 20%
.

In addition, germline mutations in the MMR gene have significantly increased the risk of extraintestinal tumors in patients with colorectal cancer.
LS-related endometrial cancer is the most common, accounting for about 2% of all endometrial cancers
.

The lifetime risk of endometrial cancer caused by different MMR gene germline mutations is also different.
Among them, MLH1 and MSH2 are 25%-60%, MSH6 is 16%~26%, and PMS2 is 15%
.

Other susceptibility include ovarian cancer, stomach cancer, prostate cancer and so on
.

5.
Management after genetic genetic germline mutation examination.
Many guidelines recommend that carriers of MLH1 and MSH2 germline mutations should be followed up with colonoscopy from 20 to 25 years old or 2 to 5 years earlier than the youngest patient in the family; and It is recommended that carriers of germline mutations of MSH6 and PMS2 be followed up with colonoscopy from 25 to 30 years old or 2 to 5 years earlier than the youngest patient in the family
.

6.
Summary LS is a type of disease related to genetics.
It is characterized by susceptibility to multiple tumors including colorectal cancer, endometrial cancer, ovarian cancer, gastric cancer, and early onset
.

To fully understand and understand the clinical characteristics of LS, genetic diagnosis is the "gold standard" for LS on the basis of clinical diagnosis
.

The molecular diagnosis of LS mainly includes two approaches.
Whether using IHC to detect MMR, or using PCR or NGS to detect MSI, the detection steps and procedures must be followed.
High attention should be paid to the differential diagnosis of LS/LS-related tumors and sporadic tumors.
Never make any subjective judgments
.

The strategies for screening and diagnosis of LS/LS-related tumors and detection of genetic mutations in LS families are shown in Figure 2
.

(Enlarged) Figure 2.
LS Screening and Diagnosis and Strategies after LS Family Genetic Mutation Checking Or LS-related tumors and sporadic tumors have different treatment strategies
.

Only by fully understanding and understanding the clinical characteristics of LS, and accurately grasping the steps and procedures of LS/LS-related tumor diagnosis and genetic germline mutation examination can we accurately diagnose LS/LS-related tumors and develop LS/LS-related tumors.
The treatment strategy and the management after genetic germline mutation examination provide scientific basis
.

Prof.
Changlin Zhao National Class III Professor, Chief Physician, Doctor of Medicine, Master Supervisor Director of the Department of Gastrointestinal Oncology, Xinhua Hospital Affiliated to Dalian University Head of Dalian Colon and Rectal Cancer Diagnosis and Treatment Base, Chinese Anti-Cancer Association, Standing Committee of Liaoning Colorectal Cancer Professional Committee, Chinese Anti-Cancer Association Member of the Standing Committee of the Liaoning Provincial Gastric Cancer Professional Committee Member of the Standing Committee of the Liaoning Base of the Abdominal Tumor Committee of the Chinese Medical Education Association Member of the Standing Committee of the Liaoning Provincial Tumor Marker Professional Committee Member of the Eighth and Ninth Committees of the Science Society Chinese General Surgery Literature (Electronic Edition) Editorial Board Member of the Chinese Electronic Journal of Colorectal Diseases Special Expert Reviewer, National Natural Science Foundation of China Project Reviewer, National Health Commission, Science and Technology Project Reviewer, National Ministry of Education Science and Technology Project review expert, Dalian Community Health Service Research Association, Chairman of the First Dalian Community Tumor Prevention and Treatment Professional Committee