Mismatch repair protein deficiency in triple-negative breast carcinomas

Abstract

Background

Breast cancer, particularly triple-negative breast cancer (TNBC), poses a significant global health burden. Chemotherapy was the mainstay treatment for TNBC patients until immunotherapy was introduced. Studies indicate a noteworthy prevalence (0.2% to 18.6%) of mismatch repair protein (MMRP) deficiency in TNBC, with recent research highlighting the potential of immunotherapy for MMRP-deficient metastatic breast cancer. This study aims to identify MMRP deficiency in TNBC patients using immunohistochemistry.

Methods

A retrospective cohort study design was used and included TNBC patients treated between 2015 and 2021 at King Hussein Cancer Center. Immunohistochemistry was conducted to assess MMRP expression

Results

Among 152 patients, 14 (9.2%) exhibited deficient MMR (dMMR). Loss of PMS2 expression was observed in 13 patients, 5 of whom showed loss of MLH1 expression. Loss of MSH6 and MSH2 expression was observed in one patient. The median follow-up duration was 44 (3–102) months. Despite the higher survival rate (80.8%, 5 years) of dMMR patients than of proficient MMR patients (62.3%), overall survival did not significantly differ between the two groups.

Conclusion

Approximately 9% of TNBC patients exhibit dMMR. dMMR could be used to predict outcomes and identify patients with TNBC who may benefit from immunotherapy.

Keywords

Triple negative breast cancer mismatch repair deficiency immunohistochemistry microsatellite instability immunotherapy

Introduction

Female breast cancer has overtaken lung cancer as the most commonly diagnosed cancer worldwide. Because of the estimated 2.3 million new cases, one in every eight cancers diagnosed in 2020 was breast cancer, accounting for 11.7% of all new cancer cases.¹ With 685,000 deaths from the disease in 2020, it is the seventh most common cause of cancer mortality globally.¹ In Jordan, breast cancer is the third leading cause of death, behind lung and colorectal cancers.² Approximately 10% to 15% of breast tumors are triple-negative breast cancer (TNBC).³ According to the King Hussein Cancer Registry and a study from a Jordanian tertiary facility, approximately 9% of breast cancer cases are triple negative.⁴

TNBCs are cancers that do not express the estrogen receptor, progesterone receptor, or human epidermal growth factor receptor 2. The current treatment is based on a combination of chemotherapy and immunotherapy.⁵ TNBC is associated with a worse prognosis than that of other breast cancer subtypes because it is often a high-grade, aggressive disease with a high proportion of distant metastases.⁶ The molecular complexity and immunological profile of this breast cancer subtype have recently been uncovered by genomics and immunology developments, which have led to the development of novel treatment approaches, such as immunotherapy. Immune-based treatment has increased the pathological complete response rate in the early setting and progression-free survival and overall survival (OS) in metastatic programmed cell death ligand 1 (PD-L1)-positive TNBC.^7,8 Currently, one of the most promising immune checkpoint inhibitor targets involves blocking the actions of programmed cell death receptor 1 (PD-1) and PD-L1, which leads to an enhanced attack on tumor cells through an immune-mediated mechanism.⁹ PD-L1 is frequently used to predict immune checkpoint inhibitor responses; nonetheless, many patients do not respond to immunotherapy. To enhance the treatment efficacy and prognosis of TNBCs, new sensitive biomarkers, other than PD-L1 expression, must be identified. Microsatellite instability/deficient mismatch repair (MSI/dMMR) status is an emerging possible indicator.

Microsatellites are genomic areas where mutations may develop during DNA replication. These mutations are often corrected by mismatch repair proteins (MMRPs). MSI-H cancers occur because of mutations in one DNA MMR gene.¹⁰ Immunohistochemical staining of paraffin-embedded tumor tissue sections can be used to reveal mutations in one or more of these MMRPs. MMRP deficiency is distinguished by the absence of nuclear immunostaining for at least one of four MMRPs, including MSH2, MSH6, MLH1, and PMS2. These proteins form two heterodimeric complexes, including MSH2 with MSH6 and MLH1 with PMS2. MSH2 and MLH1 are stable proteins, while MSH6 and PMS2 are unstable. The absence of immunostaining for MSH2/MSH6 or MLH1/PMS2 indicates the loss of MSH2 or MLH1, followed by the degradation of MSH6 or PMS2, respectively. The loss of MSH6 and PMS2 only leads to the loss of protein expression with no degradation of the respective partner protein (MSH2 or MLH1).¹¹

Immunohistochemistry has equivalent sensitivity to MSI analysis, with the added benefit of direct genetic testing for the appropriate MMR gene when a loss of MMRP expression is detected. Proficient MMR (pMMR) denotes an intact MMR system, whereas dMMR denotes a defective MMR system.¹² dMMR and MSI increase tumor mutational burden and immunogenicity, promote activation of the immune checkpoint system via the PD-1/PD-L1 pathway, and function as prognostic and predictive biomarkers.¹¹ Studies of MMRP deficiency in TNBC reported variable prevalence rates, ranging from 0.2% to 18.6%^13,14 Recent studies described an excellent response in patients with dMMR metastatic breast cancer treated with anti-PD1 monoclonal antibodies, illustrating the urgent need to test for dMMR as a predictive biomarker to select patients who could benefit from immunotherapy.^15–17 This study aims to examine the prevalence of dMMR in a cohort of TNBC patients using immunohistochemical staining for MMRPs.

Materials and methods

Study patients

This is a retrospective cohort study of TNBC occurrences recorded and managed at King Hussein Cancer Center (KHCC) between 2015 and 2021. The reporting of this study conforms to the STROBE guidelines.¹⁸ The study protocol was approved by the ethics committee and the institutional review board. Upon receiving approval from the KHCC Institutional Review Board (Ref# 21KHCC55), patient records were examined. All patients with TNBC treated at KHCC between 2015 and 2021 were included in the study, unless the corresponding paraffin block was unavailable or technically difficult. KHCC is the largest cancer research center in Jordan, and all participants were informed that their anonymized data could be used for research purposes. Patient consent was waived by the institutional review board because this was a retrospective study that did not use patient identifiers. Data were collected from approximately 152 individuals, encompassing variables such as age at diagnosis, familial history of breast or other cancer types, tumor characteristics (grade, stage), event-free survival (EFS), and OS. The patient records were anonymous when received by the research team.

Immunohistochemical analysis

A histopathological examination of breast mastectomy specimens was conducted. Whenever possible, slides containing tumors along with their corresponding paraffin blocks were retrieved; otherwise, diagnostic biopsy blocks were used.

Monoclonal antibodies targeting MLH1 (clone M1, Roche Tucson, AZ, USA), MSH2 (clone G219-1129, Roche), MSH6 (clone 44, Roche), and PMS2 (clone EPR3947, Roche) were employed. Staining was carried out using the BenchMark ULTRA system (Roche) with established protocols. Initial testing focused on PMS2 and MSH6, with only cases exhibiting abnormal expression proceeding to MLH1 and/or MSH2 evaluation. Aberrant expression was defined as loss of nuclear staining for one or more MMRPs while maintaining an internal positive control (lymphocytes, endothelial cells, or stromal cells). Two pathologists independently assessed all cases, with any discrepancies resolved through joint re-evaluation.

Statistical analysis

Structured spreadsheets and an Access database were used for systematic data collection, encompassing epidemiological and clinical factors, follow-up, and survival data. Descriptive statistics using frequencies and percentages were applied. Univariate analysis employed Student’s t-test for continuous variables and the χ² square or Fisher’s exact test for differences in proportions.

EFS and OS curves were estimated via the Kaplan–Meier method, with survival time comparisons conducted using the log-rank test. OS spanned from primary diagnosis to death from any cause, with censored data for patients alive at the last follow-up. EFS was calculated from the primary diagnosis to the first event (relapse or death), expressed as the median and 95% confidence interval (CI). Significance was determined at P ≤ 0.05. SAS software (SAS Institute Inc, Cary, NC, USA) was used for all analyses.

Results

The median age of the patients at diagnosis was 49 years. Fourteen of 152 (9.2%) patients exhibited dMMR. Specifically, loss of PMS2 expression was observed in 13 patients, of whom 5 also showed loss of MLH1 expression. Loss of MSH6 and MSH2 expression was observed in one patient (Figures 1 and 2). The median duration of follow-up was 44 (3–102) months. Six of the dMMR patients had a positive family history of cancer (three with isolated loss of PMS2, two with loss of PMS2 and MLH1, and one with loss of MSH6 and MSH2). The last patient had a family history and a personal history of a second malignancy (lung cancer). Only five patients had genetic testing for MMR genes, one of whom showed a variant of uncertain significance of MLH1, and the others were negative.

Figure 1.

Distribution of cases according to mismatch repair (MMR) status (a) and distribution of deficient mismatch repair (dMMR) cases (b).

Figure 2.

Immunohistochemical staining for mismatch repair proteins. (a) Case 1 hematoxylin and eosin 40×. (b) PMS2 immunostaining shows loss of nuclear staining in the tumor cells (arrowheads) in the presence of the internal control (arrows) (40×). (c) MLH1 immunostaining shows loss of nuclear staining in the tumor cells (arrowheads), while immunostaining is retained in infiltrating lymphocytes (arrows) (40×). (d) Case 2 hematoxylin and eosin 40×. (e) MSH6 immunostaining shows loss of nuclear staining in the tumor (40×) and (f) MSH2 immunostaining shows loss of nuclear staining in the tumor.

The overall 5-year survival rate was 63.7%, and 52.5% [95% CI (43.6–61.3)] of patients had no events during the 5-year follow-up period (Figure 3(a) and (b)).

Figure 3.

Overall survival (a) and event-free survival (b).

When comparing dMMR and pMMR in terms of survival and EFS, although 80.8% [95% CI (52.7–97.8)] of dMMR patients survived for 5 years and only 62.3% [95% CI (52.5–71.7)] of pMMR patients survived for the same period, the Kaplan–Meier plot revealed no significant difference in OS between the two groups. There was no significant difference between the dMMR and pMMR groups in terms of 5-year EFS (74.1%; 95% CI [46.2–94.1] and 50.8% 95% CI [41.6%–60.0%], respectively) (Figure 4(a) and (b)). In addition, no correlation was detected between the MMR results and clinicopathological parameters, including age at diagnosis, positive family history of cancer, T stage, and lymph node status (Table 1).

Figure 4.

Overall survival (a) and event-free survival (b) in patients with deficient mismatch repair (dMMR) vs patients with proficient mismatch repair (pMMR).

Table 1.

Relationship between deficient mismatch repair and clinicopathological features.

Clinicopathological features	Total cases (n)	pMMR (n)	dMMR (n)	P value
Age
Age <50 years	84	76 (55.1%)	8 (57.1%)	0.882
Age ≥50 years	68	62 (44.9%)	6 (42.9%)	0.882
Family history of cancer
Positive	66	60 (44.4%)	6 (50.0%)	0.711
Negative	81	75 (55.6%)	6 (50.0%)	0.711
T stage
T0	3	3 (2.2%)	0 (0%)	0.608
T1	52	49 (36.0%)	3 (23.1%)
T2	58	52 (38.2%)	6 (46.2%)
T3	27	23 (16.9%)	4 (30.8%)
T4	9	9 (6.6%)	0 (0%)
N stage
0	77	69 (54.3%)	8 (61.5%)	0.912
1	29	27 (21.3%)	2 (15.4%)
2	17	16 (12.6%)	1 (7.7%)
3	17	15 (11.8%)	2 (15.4%)
Patient status
Alive	111	101 (74.8%)	10 (83.3%)	0.731
Dead	36	34 (25.2%)	2 (16.7%)	0.731

pMMR, proficient mismatch repair; dMMR, deficient mismatch repair.

Discussion

TNBC is a challenging breast cancer subtype that lacks targeted therapy options. Immunotherapy has emerged as a promising treatment option, but not all patients benefit. Immunohistochemical analysis of MMRPs can help identify patients who are likely to respond to immunotherapy.^7,19,20

The main aim of this study was to evaluate whether regular testing for these proteins is necessary in all TNBC cases to ensure that only those who are likely to benefit receive the treatment. This approach can also help avoid unnecessary complications and reduce therapy costs.

The methodology of this study consisted of retrospective selection of TNBC patients with MMRP deficiency, who were compared with the majority of TNBC patients regarding survival and outcome.

We found that 9.2% of TNBC patients had dMMR. This finding is consistent with previous studies that have found a range of 0.2% to 18% of dMMR in TNBC patients.^{13,14,21–23} It is worth noting that the prevalence of dMMR in TNBC can be influenced by several factors, including the study population, poor tissue fixation, artifacts, the criteria used to define dMMR, and the methods employed to assess MMRP expression. Variations in these factors among different studies may lead to the observed wide range of dMMR prevalence variations.

We also found no statistically significant difference in EFS or OS between patients with dMMR and pMMR. However, there was a trend towards improved survival in the dMMR group, even though none of our dMMR patients received immunotherapy. This is consistent with previous studies that have shown improved EFS and OS in patients with dMMR TNBC.¹³ However, it is essential to note that there are conflicting reports in the literature. For instance, studies by Ren et al. and Horimoto et al. did not observe any significant differences in survival outcomes between TNBC patients with pMMR and dMMR.^21,24 In addition, a prior study found that dMMR/MSI-H is associated with treatment resistance and poor prognosis.²⁵

The discrepancy between our findings and those of previous studies could be explained by several factors. The small number of dMMR patients in this study may make it difficult to draw firm conclusions about the clinical significance of dMMR. Another possibility is that the survival differences between dMMR and pMMR patients are caused by factors other than MMR status, such as tumor biology or treatment.

In addition, we did not find any correlation between MMR status and clinicopathological parameters, including age at diagnosis, family history of cancer, T stage, or lymph node status. This is consistent with other studies that have reported that MMR status is not associated with these factors in TNBC.²¹

The study suggests that dMMR is not a predictive biomarker for TNBC because of the need for a correlation between MMR status and clinicopathological parameters. However, MMR testing should be considered for all TNBC patients to identify those more likely to benefit from immunotherapy.²⁶ The Food and Drug Administration approved pembrolizumab for TNBC with MSI-H/dMMR, but its use as a prognostic or predictive biomarker is still under investigation.²⁷

This study had several limitations, including its retrospective design, small sample size, and lack of genetic testing for all patients. Our study is based on secondary data from a pathology laboratory and lacks comprehensiveness for treatment modalities and other variables that may confound our results. Furthermore, the majority of the included patients were diagnosed and treated before the introduction of immunotherapy-based treatment in our institution. Future studies are needed to confirm these findings in a larger, prospective cohort of patients. In addition, studies are needed to investigate the role of dMMR in predicting the response of TNBC to immunotherapy.

Conclusion

Our study found that 9.2% of TNBC patients at KHCC exhibited dMMR. Our findings suggest that dMMR could be used to predict outcomes and identify patients with TNBC who may benefit from immunotherapy. Further studies are needed to confirm these findings and to identify the optimal treatment strategies for patients with dMMR TNBC.

Footnotes

Author contributions

Bayan Maraqa was responsible for the conceptualization, data acquisition, formal analysis, funding acquisition, and writing of the original draft. Hamza Zughaier, Maxim Al-Ashhab, Fareed Barakat, Majd Khader, Hussein Al Maaitah, and Ruba Alabweh were responsible for data collection and investigation. Maher Sughayer was responsible for the critical review of the manuscript. All authors read and approved the final manuscript.

Declaration of conflicting interests

The authors declare that there is no conflict of interest.

Funding

This research was supported by funds from the Intramural Research Grants Program of the King Hussein Cancer Center.

ORCID iDs

Bayan Maraqa

Maher Sughayer

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