An Unusual Immunohistochemical “Null” Pattern of Four MMRs Proteins of Gastric Cancer and Literature Review

Meng Yue Hebei Medical University Fourth A liated Hospital and Hebei Provincial Tumor Hospital https://orcid.org/0000-0002-0326-2140 Junying Liu Hebei Medical University Fourth A liated Hospital and Hebei Provincial Tumor Hospital Qingwei Liu Hebei Medical University Fourth A liated Hospital and Hebei Provincial Tumor Hospital Yueping Liu (  annama@163.com ) Hebei Medical University Fourth A liated Hospital and Hebei Provincial Tumor Hospital https://orcid.org/0000-0002-4582-114X


Introduction
Gastric cancer is one of the most common cancers and a main cause of cancer deaths worldwide. Its molecular and clinical characteristics are complicated by histological and etiological heterogeneity. Gastric adenocarcinomas are divided into four subtypes according to their molecular features: tumors exhibiting chromosomal instability, microsatellite instability high (MSI-H), Epstein-Barr virus (EBV) positivity, and genomic stability [1,2]. Microsatellite instability (MSI) and molecular typing are important in the treatment and prognosis of gastric cancer [3,4].
The maintenance of mismatch repair (MMR) pathway plays an important role in maintaining accurate DNA replication and genome stability. Base pair insertion or loss occurs in the microsatellite region due to mismatch repair system defects, the replication errors cause MSI [5,6]. Four representative MMR-related proteins, including MLH1, PMS2, MSH2, and MSH6 tested by Immunohistochemistry(IHC), can direct testing to that speci c gene and assist in the identi cation of patients with Lynch syndrome (LS). LS is an autosomal dominant disease caused by germline mutations of MMR related genes [7]. IHC is a convenient and affordable method for MSI analysis, and it has high sensitivity and speci city. Different IHC staining patterns re ect the complex biological phenomena underlying MMR de ciency. Therefore, it is greatly important to improve the understanding of abnormal IHC ndings and their biological signi cance. We found an abnormal and rare expression of IHC in four MMR-related proteins by an unusual "null" pattern, and these MMR-related genes exhibited changes tested by gene testing.

Case Presentation
We present the case of a 67-year-old man with upper abdominal discomfort and vomiting for 4 months. He had no personal or family history of other tumor. Gastroscopy showed a large mass on gastric body. Total gastrectomy was performed. The result showed a 5 cm T3N3aM0 adenocarcinoma.
Para n-embedded block of gastric cancer was performed for pathologic diagnosis, IHC, and molecular analysis. The tumor showed an in ltrative growth pattern in gross (Fig. 1A). Histologically, the tumor was poorly differentiated. It has a solid pattern with necrosis. It was heterogeneous with glandular differentiation and prominent tumor-in ltrating lymphocytes ( Fig. 1B and C). Subsequently, the methylation-speci c polymerase chain reaction assay of the MLH1 promoter region was performed. There was promoter hypermethylation of MLH1. Many studies had shown that MSI-H is associated with MLHl promoter methylation. However, the presence of MSI-H is not certain in gastric cancer patients with MLHl promoter methylation, and not all patients with MSI-H gastric cancer have MLHl promoter methylation [8,9].
Simultaneously, next generation sequencing (NGS), which included 41 genes, was performed and demonstrated that MLH1, MSH2, MSH6, and PMS2 genes exhibited changes. There were the mutation of the clipping region in exon 12 of MLH1 (c.1039-13_1039-8del), missense mutation in exon 11 of PMS2 (c.1799T > C, p.Met600Thr), missing copy number in exon 14 of MSH2 (Fig. 3), and missense mutation in exon 4 of MSH6 (c.2693C > A, p.Pro898His). This mutation of MSH2 may lead to the shift of the subsequent coding frame of MSH2 gene, and then lead to the loss of protein expression. Mutation analysis using peripheral blood showed no germline mutation in the these four genes. MLHl promoter methylation and the loss-of-function mutation of MSH2 support to MSI-H. The patient had no personal or family tumor history indicated that this MMR de ciency was highly likely sporadic in nature.
The tumor showed a high number of other mutations, including the copy number ampli ed by PIK3CA gene (Fig. 3). It may lead to up regulation of PIK3CA gene expression. The PTEN gene had a frameshift mutation in exon 7 (c.800del, p.Lys267fs) and nonsense mutation in exon 5 (c.388C > T, p.Arg130). The exon began to shift from the 267 amino acid lysine in exon 7. It is likely that terminators will be introduced into the new reading frame. The 130 amino acid of exon 5 was mutated from arginine to terminator. These two premature terminator may lead to meaningless mRNA degradation, resulting in protein loss. It had a missense mutation of p.g245s of the TP53 gene in exon 7, and mutations in the shear region of the ATM gene in exon 6 (c.497-5_497-4del) and MET gene in exon 12(c. 2584-13_2584-9del). The genes of PTEN, TP53, and ATM were identi ed as signi cantly mutated in all types of adenocarcinomas, but the mutation of the MET gene is relatively rare in gastric cancer [1]. Its EBER was negative. An increased frequency of mutant-pattern TP53 expression was found in EBV-negative carcinomas, consistent with this case. This case belongs to MSI-H of four subtypes of gastric adenocarcinomas [1]. PD-L1 is highly expressed due to the presence of more lymphoid stroma.
The patient returned to normal after the operation. 9 months after operation, patients were admitted to hospital and no signs of tumor metastasis and recurrence were found. After 6 cycles of adjuvant chemotherapy, the patients and was discharged home in a stable condition.

Discussion
This "null" IHC staining pattern of all four MMR proteins was rst reported in colorectal cancer by Hagen [10] due to germline MSH2 mutation and somatic MLH1 hypermethylation in a 71-year-old woman with LS. The morphology under the microscope was similar to our case, but the gene expression was inconsistent. The patient had personal tumour history of colon cancer and ureteral cancer. Besides, she also had a strong family tumour history. All these demonstrated this was a patient with LS. But our case supported sporadic case.
Wang [11] et al deemed the four MMR proteins de ciency in an 80-year-old woman with colorectal cancer was highly likely sporadic, and no high-risk surveillance protocols were recommended to the patient or her family members. Its morphology and gene expression were similar to our case. They found promoter hypermethylation of MLH1 and double somatic truncating mutations in MSH2. They also found BRAFV600E mutation. We found promoter hypermethylation of MLH1 and missing copy number in exon 14 of MSH2. Westwood [12] found the the percentage of additional Partly or completely loss of MSH2 and MSH6 in MLH1-de cient CRC of patients was 0.48% (4/829). In addition, there only one case showed a complete null expression of MMR proteins by IHC in colorectal cancer, but it showed strong staining for all four proteins in rectal cancer. Unfortunately, there only biopsy was available for testing in colorectal cancer. Further genetic analysis showed MLH1 promoter hypermethylation and BRAFV600E mutation. Summary of "null" IHC staining pattern of all four MMR proteins reported in the literature can be seen in Table 1.
Research showed MSI tumors with MLH1 methylation were associated with BRAFV600E mutation only in the colon, not in the stomach [13]. Our case proved this point, the BRAFV600E mutation was not detected in our case. In addition, no abnormalities were found at the detection site of KRAS, NRAS, and HRAS. It's also different from colon cancer.
All of the above three literatures were found in colorectal cancer, none in gastric cancer. Junhun [14]. found 5 cases showed all four MMR proteins negative and none showed a single MMR protein loss of 580 cases by IHC, further genetic testing was not carried out except PCR tests of MMR genes. But We retrospectively collected 2808 cases of postoperative gastric cancers from the Fourth Hospital of Hebei Medical University, from May, 2017 to August, 2020. Only this case was completely negative. The current incidence is 0.0356%. And 15 cases showed only negative of PMS2, and 3 cases only negative of MSH6. What are the reasons for the differences? Junhun used the standards that completely loss or < 20% focal weak equivocal nuclear staining. We consider that the negative judgment standards used for our cases were more strict, so the total negative case is more rare. In addition, we tested more cases, so we found the cases with single negative of MMR protein. In another 464 cases of gastric cancer study, the conegetive percent of MLH1 and MSH2 was 4.4% [15]. They uesd tissue microarray for test. However, there was a potential heterogeneity in the use of tissue microarray. The in-depth genetic testing was not performed. Therefore, our case rst revealed this rare IHC staining pattern of four MMR proteins in gastric cancer and showed the difference of gene expression with colorectal cancer. We do not think it is a common phenomenon in gasteic cancer, even has not been reported in detail in the stomach before.
The patients with MSI-H gastric cancer frequently are older, majority of them are women, the cancer is mostly in the distal part of the stomach, most of them are intestinal type in Lauren's classi cation, and less lymph node metastasis. They have lower recurrence rate, and the best prognosis compared with other subgroups [1]. But our patient is old man, the cancer located near cardia. Although the tumor are heterogeneous, but the intestinal type part of Lauren's classi cation only occupied a small part in the tumour. Kawazoe [16] showed that PD-L1 expression (22C3) in immune cells (ICs) was associated with EBV positivity and lymph-node metastasis. Our case showed the expression of PD-L1 only on ICs and high CPS with EBV negative and lymph-node metastasis. MSI status is considered to be a practical alternative to immunotherapy response. The immunotherapy is recommended for this MSI-H patienrs [17]. But the patient did not receive immunotherapy. However, the patient's condition is still stable after 9 months, and there was no sign of recurrence and metastasis. Besides, this patient had the large tumor size, many lymph node metastasis and the speci city of gene expression. These are unusual compared with others, so the patient was asked to follow up regularly just to be on the safe side.
In summary, we reported a sporadic case with unusual MMR IHC and genes pattern in gastric adenocarcinoma, needed no clinical management implications for his family. The intimate understanding of the abnormal expression of MMR is helpful for individualized follow-up treatment.