*Article* **Clinical Score to Predict Recurrence in Patients with Stage II and Stage III Colon Cancer**

**David Viñal <sup>1</sup> , Sergio Martinez-Recio <sup>1</sup> , Daniel Martinez-Perez <sup>1</sup> , Iciar Ruiz-Gutierrez <sup>1</sup> , Diego Jimenez-Bou <sup>1</sup> , Jesús Peña-Lopez <sup>1</sup> , Maria Alameda-Guijarro <sup>1</sup> , Gema Martin-Montalvo <sup>1</sup> , Antonio Rueda-Lara <sup>1</sup> , Laura Gutierrez-Sainz <sup>1</sup> , Maria Elena Palacios <sup>2</sup> , Ana Belén Custodio <sup>1</sup> , Ismael Ghanem <sup>1</sup> , Jaime Feliu 3,\* and Nuria Rodríguez-Salas <sup>4</sup>**


**Simple Summary:** The prognosis of patients with stage II and stage III colon cancer is heterogeneous. Clinical and pathological characteristics may help to further refine the recurrence risk. We built a prognostic score and categorized patients into two risk groups in a training and validation cohort. We assigned two points to T4 and one point to N2 and high tumor budding based on the multivariate cox regression analysis for time to recurrence (TTR) in the training cohort. Forty-five percent of the patients were assigned to the low-risk group and compared to the high-risk group, had a significantly longer TTR. These results were confirmed in the validation cohort.

**Abstract:** Background: The prognosis of patients with stage II and stage III colon cancer is heterogeneous. Clinical and pathological characteristics, such as tumor budding, may help to further refine the recurrence risk. Methods: We included all the patients with localized colon cancer at Hospital Universitario La Paz from October 2016 to October 2021. We built a prognostic score for recurrence in the training cohort based on multivariate cox regression analysis and categorized the patients into two risk groups. Results: A total of 440 patients were included in the training cohort. After a median follow-up of 45 months, 81 (18%) patients had a first tumor recurrence. T4, N2, and high tumor budding remained with a *p* value <0.05 at the last step of the multivariate cox regression model for time to recurrence (TTR). We assigned 2 points to T4 and 1 point to N2 and high tumor budding. Forty-five percent of the patients were assigned to the low-risk group (score = 0). Compared to the high-risk group (score 1–4), patients in the low-risk group had a significantly longer TTR (hazard ratio for disease recurrence of 0.14 (95%CI: 0.00 to 0.90; *p* < 0.045)). The results were confirmed in the validation cohort. Conclusions: In our study, we built a simple score to predict tumor recurrence based on T4, N2, and high tumor budding. Patients in the low-risk group, that comprised 44% of the cohort, had an excellent prognosis.

**Keywords:** colonic neoplasms; chemotherapy; adjuvant; tumor budding

#### **1. Introduction**

Colorectal cancer is the third most common tumor and the second cause of cancerrelated cause of death globally [1]. In patients with stage II and stage III colon cancer, the prognosis is heterogeneous, and survival varies depending on numerous factors. Classically, for the pathologic stage at diagnosis, according to the American Joint Committee on Cancer (AJCC)/Union for International Cancer Control (UICC), the tumor, node, and metastasis (TNM) staging classification was considered the most important indicator of outcome [2]. However, patients with stage IIIa disease may have a more favorable prognosis

**Citation:** Viñal, D.; Martinez-Recio, S.; Martinez-Perez, D.; Ruiz-Gutierrez, I.; Jimenez-Bou, D.; Peña-Lopez, J.; Alameda-Guijarro, M.; Martin-Montalvo, G.; Rueda-Lara, A.; Gutierrez-Sainz, L.; et al. Clinical Score to Predict Recurrence in Patients with Stage II and Stage III

Colon Cancer. *Cancers* **2022**, *14*, 5891.

Academic Editor: Stephane Dedieu

https://doi.org/10.3390/ cancers14235891

Received: 10 June 2022 Accepted: 21 November 2022 Published: 29 November 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

than patients with IIb stage, which may indicate that other factors contribute significantly to the prognosis of the patient. Globally, it is estimated that 35% of the patients will eventually recur [3]. To further improve the outcome, chemotherapy has been established as a standard of care for stage III colon cancer with a 10 to 20% of survival benefit (depending on the regimen of chemotherapy) and is an option for patients with intermediate- or high-risk stage II [4,5]. The latest European Society for Medical Oncology (ESMO) guidelines include lymph node sampling <12 and T4 stage including perforation as major prognostic factors and high-grade tumor, vascular invasion, lymphatic invasion, perineural invasion, tumor presentation with obstruction, and high preoperative carcinoembryonic antigen (CEA) levels as minor prognostic factors. The National Comprehensive Cancer Network (NCCN) also includes high tumor budding and close, indeterminate, or positive margins as risk factors for recurrence. To better define the prognosis and recurrence risk of patients with resected colon cancer, several nomograms have been published [6–8]. One of the most widely used is the Memorial Sloan Kettering Cancer Center (MSKCC) colon cancer recurrence nomogram, which predicts freedom from recurrence based on nine clinicopathological features including age, tumor size, preoperative carcinoembryonic antigen (CEA), use of adjuvant chemotherapy, and other indicators of tumor invasiveness [6]. A recently published update simplified the score to five items; however, tumor-infiltrating lymphocytes were included in the nomogram, a feature not available in many centers [8].

The aim of this study is to create a simple clinical score to predict recurrence using clinical and pathological variables available in routine clinical practice and to select a subgroup of patients with excellent prognosis according to this score.

#### **2. Materials and Methods**

This is a single-institution retrospective observational study. We included all patients who underwent curative surgery for stage II and stage III colon cancer between October 2016 and October 2021 at Hospital Universitario La Paz (HULP), Madrid (Spain). The study protocol specified the inclusion criteria as follows: age above 18 years and completely resected colon adenocarcinoma located at >15 cm of the anal verge as determined by endoscopy or above the peritoneal reflection in the surgical resection without any evidence of metastatic disease. Main exclusion criteria were as follows: macroscopic evidence of residual tumor in the surgical specimen; no chemotherapy or radiotherapy were allowed before surgery; severe renal or hepatic disorder; bone marrow suppression; or disabling peripheral neuropathy. This study was approved by the Ethics Committee of HULP and was conducted in accordance with ethical standards of the Helsinki Declaration of the World Medical Association. Baseline disease, demographics, clinical data, treatment characteristics, and outcomes were analyzed from the medical record of each patient. Adjuvant chemotherapy was administered according to ESMO guidelines [4,9]. Patients were followed every 3 months with CT scan and CEA for the first 2 years from the surgery and every 6 months with CT scan and CEA from years 3 to 5. Colonoscopy was performed every 3 years starting 1 year after surgery.

The primary objective of the study was the identification of factors associated with time to recurrence (TTR). We chose TTR as the primary endpoint based on previous reports by other groups [8]. The sample was divided into a training cohort (patients diagnosed between October 2016 and September 2020, *n* = 440) and a validation cohort (patients diagnosed from October 2020 to September 2021, *n* = 100). TTR was calculated from the date of the surgery until the date of tumor recurrence or last follow-up. OS was defined as the time between the date of diagnosis and the date of death or last follow-up. The analysis was performed with a data cut-off of 15 September 2022. The relation between TTR and OS with each of the variables was analyzed using the log-rank test. Survival analysis was performed using the Kaplan–Meier method. Univariate cox regression analyses and multivariate proportional hazards regression model were carried out in the training cohort to identify independent prognostic factors for disease recurrence. We performed a correlation assessment using the Spearman's rho test. Multicollinearity among variables

was defined as a rho test value ≥ 0.50. In fact, we excluded adjuvant chemotherapy treatment as it positively correlates with the presence of high-risk features (Spearman's rho test = 0.533; *p* < 0.001). In the multivariate analysis, we included the variables significantly associated with TTR in the univariate analysis. Multivariate analysis was performed with backward elimination. Prognostic factors that yielded a *p* value < 0.05 at the last step of multivariate cox regression analysis were included in the score. For the development of the score, each factor was assigned a particular score based on its β coefficient. The β coefficient for each risk factor was divided by the lowest β coefficient and rounded to the nearest whole number. Model calibration and discrimination were assessed in the training cohort by the area under the receiver operating characteristic (ROC) curve [10,11]. The final score of each patient was the sum of the points. The prognostic score was then applied to each patient. Survival by prognostic group was represented by Kaplan–Meier curves, and *p* values were calculated using the log-rank test. The training sample was divided into two risk strata (low-risk group and high-risk group) based on the approximate median of risk score. Hazard ratios (HRs) were calculated using cox proportional hazard regression, with *p* values calculated using the Wald statistics. The performance of the two-risk group strategy was tested for TTR in the validation cohort. All statistical analyses were carried out using SPSS v.25.

#### **3. Results**

A total of 440 patients with stage II and stage III colon cancer underwent curative surgery between October 2016 and October 2020 and were included in the training cohort. The baseline characteristics are depicted in Table 1. The median age at diagnosis was 74 years (range 35–95), and 44% of the patients were female. The primary tumor was distributed equally in the right and left colon. Stage II and stage III were observed in 50% percent of the patients each. Of note, preoperative CEA was available in 219 patients and was high in 19% of them. Twenty-five percent of the patients had high tumor budding. A total of 225 (51%) patients received adjuvant chemotherapy: 61 patients with stage II (27%) and 164 patients with stage III (75%).

After a median follow-up of 45 months (range, 0,1 to 66 months), 81 (18%) patients had a first tumor recurrence: 27 (12%) patients with stage II and 54 (24%) patients with stage III. Ninety-six (17%) patients died: 39 (17%) patients with stage II and 57 (26%) patients with stage III. The median TTR and OS were not reached for the whole cohort. Univariate cox regression analysis showed that T4 (tumor invades the visceral peritoneum or invades or adheres to the adjacent organ or structure), N2 (four or more regional nodes are positive) [12], R1 (incomplete tumor resection with microscopic surgical resection margin involvement) [13], bowel obstruction and perforation at diagnosis, lymphovascular and perineural invasion, high tumor budding (defined as ≥10 buds) [14], grade 3, and deficient mismatch repair were significantly associated with TTR. Only T4 (hazard ratio (HR), 3.46 [95% confidence interval (CI): 1.68 to 7.13], *p* < 0.01), N2 (HR, 2.29 (95%CI, 1.19 to 4.38), *p* = 0.01), and high tumor budding (HR, 1.91 (95%CI, 1.02 to 3.54), *p* = 0.04) remained with a *p* value <0.05 at the last step of the multivariate cox regression model, and were selected to create the clinical score (see Table 2).

Based on the β coefficient of each feature (see Table 2), we assigned 2 points to T4, and 1 point to N2 and high tumor budding. Therefore, patients were assigned from 0 to 4 points (score 0 = 138, score 1 = 44, score 2 = 57, score 3 = 52, and score 4 = 13 patients). The area under the ROC curve for tumor recurrence at 36 months was 0.77 (95%CI, 0.70 to 0.84), *p* < 0.01 (Figure 1).


**Table 1.** Baseline characteristics of the patients.

CEA, carcinoembryonic antigen; dMMR, deficient mismatch repair; R0, complete tumor resection with all margins histologically uninvolved.

**Table 2.** Multivariate Cox Regression Analysis.


At 36 months, 95%, 83%, 73%, 60%, and 19% of the patients with scores 0, 1, 2, 3, and 4 were recurrence-free, respectively. The median TTR was not reached in patients with scores 0−3. Patients with score 4 had a median TTR of 29 months (95% confidence interval (CI): 0.1 to 60.23). Significant differences were observed between the groups (*p* < 0.001), see Figure 2. Patients were divided into a low-risk group (score = 0; *n* = 138; 45% of the patients) and a high-risk group (score = 1−4; *n* = 166; 55% of the patients). At 36 months, 95% and 67% of the patients in the low-risk and high-risk groups were recurrence-free, respectively. Patients assigned to the low-risk group had a significantly longer TTR than patients assigned to the high-risk group. The median TTR was not reached in either group, with a HR for disease recurrence of 0.13 (95%CI: 0.05 to 0.31; *p* <0.001), see Figure 3.

were selected to create the clinical score (see Table 2).

**Table 2.** Multivariate Cox Regression Analysis.

0.84), *p* < 0.01 (Figure 1).

(HR), 3.46 [95% confidence interval (CI): 1.68 to 7.13], *p* < 0.01), N2 (HR, 2.29 (95%CI, 1.19 to 4.38), *p* = 0.01), and high tumor budding (HR, 1.91 (95%CI, 1.02 to 3.54), *p* = 0.04) remained with a *p* value <0.05 at the last step of the multivariate cox regression model, and

**Characteristic β Coefficient HR** (**95%CI**) *p* **Value** T4 1.243 3.46 (1.68–7.13) 0.001 N2 0.829 2.29 (1.19–4.38) 0.012

Based on the β coefficient of each feature (see Table 2), we assigned 2 points to T4, and 1 point to N2 and high tumor budding. Therefore, patients were assigned from 0 to 4 points (score 0 = 138, score 1 = 44, score 2 = 57, score 3 = 52, and score 4 = 13 patients). The area under the ROC curve for tumor recurrence at 36 months was 0.77 (95%CI, 0.70 to

High tumor budding 0.647 1.91 (1.02–3.54) 0.041

**Figure 1.** ROC curve of prognostic score (0 to 4 points) for recurrence at 24 months. **Figure 1.** ROC curve of prognostic score (0 to 4 points) for recurrence at 24 months.

**Figure 2.** Time to recurrence according to the score (0–4) in the training cohort. **Figure 2.** Time to recurrence according to the score (0–4) in the training cohort.

A total of 100 patients were included in the validation cohort. The baseline characteristics are depicted in Table 1. The median age at diagnosis was 75 years (range 45–97), and 50% of the patients were female. The primary tumor was distributed equally in the right and left colon. Stage II was observed in 54% percent of the patients each. Twenty-one percent of the patients had high tumor budding. A total of 43 patients received adjuvant chemotherapy. Patients were assigned to the low-risk (*n* = 46; 46%) and high-risk (*n* = 54;

**Figure 3.** Time to recurrence according to risk groups (low-risk vs high-risk) in the training cohort.

A total of 100 patients were included in the validation cohort. The baseline characteristics are depicted in Table 1. The median age at diagnosis was 75 years (range 45–97), and 50% of the patients were female. The primary tumor was distributed equally in the right and left colon. Stage II was observed in 54% percent of the patients each. Twenty-one percent of the patients had high tumor budding. A total of 43 patients received adjuvant chemotherapy. Patients were assigned to the low-risk (*n* = 46; 46%) and high-risk (*n* = 54; 54%) groups. After a median follow-up of 15 months (range, 2 to 25 months), 15 (15%) of the patients had a first tumor recurrence. Recurrences were observed in five (9%) patients

HR, hazard ratio; CI, confidence interval.

54%) groups. After a median follow-up of 15 months (range, 2 to 25 months), 15 (15%) of the patients had a first tumor recurrence. Recurrences were observed in five (9%) patients with stage II and 10 (22%) patients with stage III. According to our score, all the recurrences were observed in the high-risk group. At 12 months, 100% and 79% of the patients in the low-risk and high-risk groups were recurrence-free, respectively. Patients assigned to the low-risk group had significantly longer TTR than patients assigned to the high-risk group. The median TTR was not reached in either group. HR for disease recurrence of 0.14 (95%CI: 0.00 to 0.90; *p* <0.045), see Figure 4. **Figure 2.** Time to recurrence according to the score (0–4) in the training cohort.

*Cancers* **2022**, *14*, x FOR PEER REVIEW 6 of 11

**Figure 3.** Time to recurrence according to risk groups (low-risk vs high-risk) in the training cohort. HR, hazard ratio; CI, confidence interval. **Figure 3.** Time to recurrence according to risk groups (low-risk vs. high-risk) in the training cohort. HR, hazard ratio; CI, confidence interval. group. The median TTR was not reached in either group. HR for disease recurrence of 0.14 (95%CI: 0.00 to 0.90; *p* <0.045), see Figure 4.

In this study, we created a simple score using three clinicopathological parameters available in routine clinical practice to better estimate the recurrence risk in patients with

Multiple scores and nomograms have attempted to overcome the aforementioned limitations of the AJCC's TNM staging system for the prediction of outcomes. One of the most relevant is the MSKCC nomogram published in 2008 for the estimation of the recurrence risk of patients with stages I to III colon cancer after a complete resection (R0) of the tumor [6]. The nomogram was based on nine variables including patient age, tumor location, preoperative carcinoembryonic antigen, T stage, number of positive and negative lymph nodes, lymphovascular invasion, perineural invasion, and use of postoperative chemotherapy. The nomogram successfully predicted relapse with a concordance index of 0.77, improving the stratification provided by the AJCC staging scheme and was externally validated in multiple cohorts [15–17]. However, the high number of features and its complexity may prevent it from being used as a practical tool in clinical practice. The MSKCC clinical calculator was updated in 2019 [8]. The nomogram was simplified to six variables and incorporated recently validated molecular and histologic factors, including microsatellite genomic phenotype; AJCC T category; number of tumors involved; lymph nodes; presence of high-risk pathologic features, such as venous, lymphatic, or perineural

ranges from 5% in patients with a score = 0 to 81% in patients with a score = 4, with an AUC of 0.77. More importantly, the score can discriminate a subgroup of patients (lowrisk group, score = 0), so that even with locally advanced disease, they will have an excellent prognosis after completing the standard treatment recommendations according to their stage. This low-risk group comprises the 45% of the training cohort included in the

**Figure 4.** Time to recurrence according to risk groups in the validation cohort. **Figure 4.** Time to recurrence according to risk groups in the validation cohort.

multivariate analysis and 46% of the patients in the validation cohorts.

**4. Discussion**

#### **4. Discussion**

In this study, we created a simple score using three clinicopathological parameters available in routine clinical practice to better estimate the recurrence risk in patients with stage II and stage III colon cancer. This score shows that the probability of recurrence ranges from 5% in patients with a score = 0 to 81% in patients with a score = 4, with an AUC of 0.77. More importantly, the score can discriminate a subgroup of patients (low-risk group, score = 0), so that even with locally advanced disease, they will have an excellent prognosis after completing the standard treatment recommendations according to their stage. This low-risk group comprises the 45% of the training cohort included in the multivariate analysis and 46% of the patients in the validation cohorts.

Multiple scores and nomograms have attempted to overcome the aforementioned limitations of the AJCC's TNM staging system for the prediction of outcomes. One of the most relevant is the MSKCC nomogram published in 2008 for the estimation of the recurrence risk of patients with stages I to III colon cancer after a complete resection (R0) of the tumor [6]. The nomogram was based on nine variables including patient age, tumor location, preoperative carcinoembryonic antigen, T stage, number of positive and negative lymph nodes, lymphovascular invasion, perineural invasion, and use of postoperative chemotherapy. The nomogram successfully predicted relapse with a concordance index of 0.77, improving the stratification provided by the AJCC staging scheme and was externally validated in multiple cohorts [15–17]. However, the high number of features and its complexity may prevent it from being used as a practical tool in clinical practice. The MSKCC clinical calculator was updated in 2019 [8]. The nomogram was simplified to six variables and incorporated recently validated molecular and histologic factors, including microsatellite genomic phenotype; AJCC T category; number of tumors involved; lymph nodes; presence of high-risk pathologic features, such as venous, lymphatic, or perineural invasion; presence of tumor-infiltrating lymphocytes; and use of adjuvant chemotherapy. The concordance index was 0.792, and external validation confirmed the utility for the prediction of recurrence. Unfortunately, the generalization of this nomogram was hampered because tumor-infiltrating lymphocytes are not reflexively measured in many centers, including ours.

Our score was built with variables that showed a *p* value < 0.05 in the multivariate cox regression model and included T4, N2, and budding. Primary colon cancer is classified as T4 per the AJCC TNM staging 8th edition when it invades the visceral peritoneum or invades or adheres to an adjacent organ or structure [12]. T4 has classically been considered a negative prognostic factor. In fact, patients with T4 stage II disease have worse outcomes than patients with stage IIIa disease. A recent subanalysis of patients with stage II colon cancer included in the IDEA collaboration showed that high-risk stage II patients with T4 disease have a worse outcome than those with T3 disease [18]. The IDEA collaboration also highlighted that those patients with stage III with T4 and/or N2 are a different population with a worse prognosis than the other patients with stage III (T1−3 and N1) and suggested the use of these risk groups as stratification categories in randomized trials [19]. Tumor budding refers to isolated or clusters of up to four cancer cells located at the invasive tumor front [20]. A growing amount of evidence has confirmed its prognostic value in localized colon cancer, independent of the tumor grade [21,22]. A recently published subanalysis from the IDEA-France phase III trial [23] showed that tumor budding is an independent prognostic factor in stage III colon cancer patients. The DFS at 3 years was 79% vs 67% (*p* = 0.001) in patients with budding grade 1 vs 2−3 with a HR for recurrence or death of 1.41 (95% CI, 1.12 to 1.77), *p* = 0.003, after adjustment for relevant clinicopathological features. Interestingly, high tumor budding was associated with perineural (*p* < 0.01) and vascular (*p* = 0.002) invasions, which may explain that these well-known adverse prognostic features are not present in the last step of our multivariate analysis. The role of tumor budding in predicting benefit from adjuvant chemotherapy is still controversial. In a subanalysis of the SACURA trial [24], a nonsignificant improvement of 5% in the 5-year recurrence rate was observed in patients with stage II and stage III colon cancer treated with adjuvant

chemotherapy vs surgery alone. In patients with pT1, tumor budding currently influences decision making. More recently, the ASCO guidelines were updated and added high tumor budding (≥10 buds, high grade) to the list of adverse prognostic factors to classify patients in the high-risk subpopulation that may derive more benefit from chemotherapy [25]. However, the ESMO guidelines for the management of localized stage II colon cancer still do not consider tumor budding in the decision making. In light of the results of our group and those of previous groups, high tumor budding might be considered as a risk factor.

Prognostic characterization and subgroup categorization in patients with localized colon cancer have more implications than providing the patient a tailored risk of recurrence. Some authors suggest that stratification categories based on T and N should be included in randomized trials for localized colon cancer. Assessing the risk of recurrence may also have implications for the follow-up. The ESMO guidelines recommend a CT scan of the chest and abdomen every 6 to 12 months for the first 3 years in patients who are at higher risk of recurrence according to the TNM classification. Other authors suggest that the preferred approach should be performing two CT scans at 12 and 36 months independent of the stage and risk groups due to the lack of survival benefit of a more intensive approach [26]. We suggest that due to the significantly different risk of recurrence according to subgroups, and the possible benefit of early treatment of oligometastatic disease, the follow-up should be tailored accordingly, or at least taken into account in future follow-up trials.

The limitations to our study are mostly due to its retrospective and unicentric nature. A significant amount of data are missing including tumor budding and preoperative CEA, mainly in the training cohort. Preoperative CEA should be performed before surgery; however, data are missing in half of the patients due to multiple reasons including emergency surgery or even human error. The advantage of our score is that it is based on three features that should be available in every patient with a colorectal cancer diagnosis. Adjuvant treatment was given to the patients following indications by the ESMO guidelines [4,9]. This feature was not considered in the analysis because the benefit of therapy may be masked by the administration in the high-risk subgroup. In fact, we found a correlation between the presence of high-risk features as defined by the ESMO guidelines and the administration of adjuvant chemotherapy (Spearman's rho test = 0.533; *p* < 0.001). Therefore, this score should not be interpreted as a predictive marker of benefit for adjuvant chemotherapy but rather as a predictive marker for recurrence in patients that have followed the standard treatment strategy for localized colon cancer. Nevertheless, we consider that tumor budding is such a strong predictive marker for recurrence that should also be considered as a risk factor and should be included in the guidelines for adjuvant chemotherapy. Only patients with complete (R0) resection were included in the initial MSKCC nomogram [6]; however, approximately 10% of patients have involved resection margins at the pathological report of the surgery. We therefore consider that this feature should be included in a real-world analysis. Finally, although the results of the internal validation cohort seem to confirm the performance of our score, the sample size was small, and the cohort was still immature. We did not perform an external validation and thus an accurate determination of the AUC and calibration of the model was not possible.

#### **5. Conclusions**

In conclusion, defining subgroups of patients with localized colon cancer at a high risk of recurrence has implications in the treatment strategy, trial designs, and follow-up. Although the traditional AJCC TNM staging provides adequate prognostic estimation, a more personalized approach using high-risk clinicopathological features may be more precise and practical. In our study, we built a simple score to accurately predict tumor recurrence based on T4, N2, and high tumor budding. Patients with a score = 0, that comprises 44% of the cohort, had an excellent prognosis. A longer follow-up is needed, and an external validation is recommended to confirm our results.

**Author Contributions:** Conceptualization, D.V., N.R.-S. and J.F.; methodology, D.V., L.G.-S. and J.F.; formal analysis, D.V.; investigation, D.V., S.M.-R., D.M.-P., I.R.-G., D.J.-B., J.P.-L., M.A.-G., G.M.-M. and A.R.-L.; data curation, D.V., S.M.-R. and D.M.-P.; writing—original draft preparation, D.V.; writing review and editing, L.G.-S., M.E.P., A.B.C., I.G. and J.F.; visualization, D.V. and J.F.; supervision, J.F. and N.R.-S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** The study was conducted in accordance with the Declaration of Helsinki and approved by the Institutional Review Board (or Ethics Committee) of Hospital Universitario La Paz (PI-3607).

**Informed Consent Statement:** The local ethical committee approved the use of anonymized historic samples and data for the study and waived informed consent from the patients.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


## *Article* **Routine Immunohistochemical Analysis of Mismatch Repair Proteins in Colorectal Cancer—A Prospective Analysis**

**Joana Lemos Garcia 1,\*, Isadora Rosa 1,2, Sofia Saraiva <sup>1</sup> , Inês Marques <sup>1</sup> , Ricardo Fonseca <sup>3</sup> , Pedro Lage 1,2 , Inês Francisco 2,4, Patrícia Silva 2,4, Bruno Filipe 2,4, Cristina Albuquerque 2,4 and Isabel Claro 1,2**


**Simple Summary:** Recognition of a hereditary colorectal cancer (CRC) syndrome is crucial. Our aim was to assess the value of routine immunohistochemistry screening for mismatch repair proteins deficiency in CRC patients under 70 years-old. In our cohort, this inclusive strategy allowed the identification of Lynch Syndrome patients that could otherwise be missed using a restrictive approach that relies only on Amsterdam and Bethesda criteria. This study strengthens current recommendations and highlights the role of universal CRC screening for MMR protein status.

**Abstract:** Recognition of a hereditary colorectal cancer (CRC) syndrome is crucial and Lynch Syndrome (LS) is the most frequent immunohistochemistry (IHC)—screening for mismatch repair proteins (MMR) deficiency in CRC is therefore advocated. An unicentric cohort study was conducted in a central Oncological Hospital to assess its results. All patients under 70 years-old admitted between July 2017–June 2019 and submitted to surgery for CRC were included. Of 275 patients, 56.0% were male, median age 61.0 (IQR:54.5–65.0), with synchronous tumors in six. Histology revealed high grade adenocarcinoma in 8.4%; mucinous and/or signet ring differentiation in 11.3%; and lymphocytic infiltration in 29.8%. Amsterdam (AC) and Bethesda (BC) Criteria were fulfilled in 11 and 74 patients, respectively. IHC revealed loss of expression of MMR proteins in 24 (8.7%), mostly MLH1 and PMS2 (*n* = 15) and PMS2 (*n* = 4). Among these, no patients fulfilled AC and 13 fulfilled BC. BRAF mutation or MLH1 promoter hypermethylation was found in four patients with MLH1 loss of expression. Genetic diagnosis was performed in 51 patients, 11 of them with altered IHC. LS was diagnosed in four, and BC was present in three. One patient would not have been diagnosed without routine IHC screening. These results strengthen the important role of IHC screening for MMR proteins loss of expression in CRC.

**Keywords:** colorectal cancer; Lynch Syndrome; mismatch repair proteins

## **1. Introduction**

Colorectal cancer (CRC) is the third most common cancer type [1–3] and its incidence in some developed countries is increasing among the young (less than 50 years-old) [4–8]. Hereditary syndromes may be responsible for 15–22% of CRC cases [7,9,10].

Recognition of a hereditary CRC syndrome is of paramount importance, since it impacts on patients' surgical management and surveillance as well as on their families

**Citation:** Lemos Garcia, J.; Rosa, I.; Saraiva, S.; Marques, I.; Fonseca, R.; Lage, P.; Francisco, I.; Silva, P.; Filipe, B.; Albuquerque, C.; et al. Routine Immunohistochemical Analysis of Mismatch Repair Proteins in Colorectal Cancer—A Prospective Analysis. *Cancers* **2022**, *14*, 3730. https://doi.org/10.3390/ cancers14153730

Academic Editor: Stephane Dedieu

Received: 10 June 2022 Accepted: 29 July 2022 Published: 31 July 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

screening and surveillance programs [11]. Lynch Syndrome (LS) is the most frequent hereditary CRC syndrome, accounting for 1–3% of all CRC. It occurs due to autosomal dominant mutations in the mismatch repair (MMR) genes *MLH1*, *MSH2*, *MSH6* and *PMS2* or deletions on the cell adhesion molecule (*EPCAM*) gene, which is located upstream of *MSH2*. The MMR defect (which may also be somatic, mostly due to MLH1 promoter hypermethylation) will lead to failure to correct DNA replication errors with accumulation of mutations, resulting in a microsatellite instability (MSI) phenotype. Diagnosis of MSI is via polymerase chain reaction (PCR) amplification of specific microsatellite repeats. Alternatively, immunohistochemistry (IHC) can show absence of expression of MMR proteins in the tumor [12].

Lynch Syndrome can be suspected through family history and clinical data collection, considering the Amsterdam criteria and the revised Bethesda guidelines (Table 1), or using computer-based calculators [12]. However, this strategy lacks sensitivity and specificity. Clinical criteria limitations are overcome by routine IHC staining for MMR proteins in all CRC samples [13–15] in a cost-effective manner [16–18]. International guidelines recommend tumor screening for MMR deficiency for all colorectal cancers regardless of age at diagnosis [19,20] or in patients bellow 70 years-old [21]. In case of MSH2, MSH6 or PMS2 loss of expression, germline testing should ensue. If there is loss of MLH1 or MLH1/PMS2 expression, somatic tumor mutations should be ruled-out first, by searching for *BRAF V600E* mutation and/or *MLH1* promoter hypermethylation [12].

**Table 1.** Clinical Criteria for Lynch Syndrome Screening (adapted from [22,23]).

#### **Amsterdam II**


#### **Revised Bethesda Guidelines**

Colorectal tumors from individuals should be tested for MSI in the following situations

1. CRC diagnosed in a patient who is <50 years of age


4. CRC diagnosed in one or more first-degree relatives with an HNPCC-related tumor, with one of the cancers being diagnosed under age 50 years.

5. CRC diagnosed in two or more first- or second-degree relatives with HNPCC-related tumors, regardless of age.

HNPCC—Hereditary Non-polyposis Colorectal Cancer, CRC—Colorectal cancer, FAP—Familial Adenomatous Polyposis, MSI-H—Microsatellite Instability-High.

Currently, MMR defects' identification in CRC and has a role beyond LS identification selection of stage II patients for chemotherapy (CT), choice of the type of adjuvant CT and selection of stage IV patients for immunotherapy all depend on MSI status.

The goal of this study was to assess the importance of routine IHC screening for MMR defects in CRC patients in the identification of Lynch Syndrome patients, in a real-world setting.

#### **2. Materials and Methods**

A unicentric cohort study was conducted at the Portuguese Oncological Institute of Lisbon, Portugal, which integrates a Familial Risk Clinic. In this hospital, around 290 new colorectal cancer patients are admitted per year by the Multidisciplinary Colorectal Cancer Group. In their first appointment, relevant personal and clinical data are collected, including family history of neoplasia. All CRC cases are reviewed in a weekly multidisciplinary

meeting. All tumors are classified according to the World Health Organization (WHO) Classification of Tumors (2019) [24] and staged using the American Joint Committee on Cancer (AJCC) (8th edition) [25] TNM system. plinary meeting. All tumors are classified according to the World Health Organization (WHO) Classification of Tumors (2019) [24] and staged using the American Joint Committee on Cancer (AJCC) (8th edition) [25] TNM system.

A unicentric cohort study was conducted at the Portuguese Oncological Institute of Lisbon, Portugal, which integrates a Familial Risk Clinic. In this hospital, around 290 new colorectal cancer patients are admitted per year by the Multidisciplinary Colorectal Cancer Group. In their first appointment, relevant personal and clinical data are collected, including family history of neoplasia. All CRC cases are reviewed in a weekly multidisci-

*Cancers* **2022**, *14*, x 3 of 11

#### *2.1. Patient Selection 2.1. Patient Selection*

All patients reviewed in the multidisciplinary CRC meeting from 01-07-2016 to 30-06- 2019 who were 70 years-old or younger and underwent primary tumor resection surgery were included, in a total of 275 patients. All patients reviewed in the multidisciplinary CRC meeting from 01-07-2016 to 30- 06-2019 who were 70 years-old or younger and underwent primary tumor resection surgery were included, in a total of 275 patients.

#### *2.2. Data Collection 2.2. Data Collection*

Data collected included demographic information, tumor location, radiological and pathological staging, therapeutic modalities performed, family history of CRC and other LS-spectrum cancers, MMR protein status, *BRAF V600E* mutation status, MMR gene promotor methylation and germline mutation analysis. For stage at diagnosis classification, pathological staging was the gold standard, except in patients who underwent neoadjuvant treatment, for whom radiological staging at diagnosis was preferred. Data collected included demographic information, tumor location, radiological and pathological staging, therapeutic modalities performed, family history of CRC and other LS-spectrum cancers, MMR protein status, *BRAF V600E* mutation status, MMR gene promotor methylation and germline mutation analysis. For stage at diagnosis classification, pathological staging was the gold standard, except in patients who underwent neoadjuvant treatment, for whom radiological staging at diagnosis was preferred.

#### *2.3. Hospital Standard Procedures*

#### 2.3.1. CRC Sample Processing *2.3. Hospital Standard Procedures*

In our institution, until 2021, according to the 2009 Jerusalem Workshop recommendations [21], in all patients 70 years old or younger who underwent surgery for CRC, the tumor was screened for loss of expression of MMR proteins by immunohistochemistry. To assess the expression of MLH1, PMS2, MSH2 and MSH6 proteins, IHC analysis is performed using Ventana CC1 equipment (sample in 10% formalin buffer, using thermal recuperation method) and monoclonal antibodies anti-MLH1 (clone ES05), anti-PMS2 (clone EP51), anti-MSH2 (clone G219-1129) and anti-MSH6 (clone EP49) (Figure 1). 2.3.1. CRC Sample Processing In our institution, until 2021, according to the 2009 Jerusalem Workshop recommendations [21], in all patients 70 years old or younger who underwent surgery for CRC, the tumor was screened for loss of expression of MMR proteins by immunohistochemistry. To assess the expression of MLH1, PMS2, MSH2 and MSH6 proteins, IHC analysis is performed using Ventana CC1 equipment (sample in 10% formalin buffer, using thermal recuperation method) and monoclonal antibodies anti-MLH1 (clone ES05), anti-PMS2

(clone EP51), anti-MSH2 (clone G219-1129) and anti-MSH6 (clone EP49) (Figure 1).

**Figure 1.** Immunohistochemistry showing loss of MLH1 (left) and maintained MSH2 (right) staining **Figure 1.** Immunohistochemistry showing loss of MLH1 (**a**) and maintained MSH2 (**b**) staining (10×).

To exclude somatic mutations that lead to MLH1-defective cases, since 2019, tumors with MLH1 loss of expression are further investigated for *BRAF V600E* mutation: DNA from samples of tumor tissue is amplified by PCR using primers for BRAF exon 15 and the product is sequenced using Sanger sequencing on Big Dye terminator v1.1 sequencing kit (Applied Biosystems) on an automatic ABI PrismTM 3130 Genetic Analyzer (Applied To exclude somatic mutations that lead to MLH1-defective cases, since 2019, tumors with MLH1 loss of expression are further investigated for *BRAF V600E* mutation: DNA from samples of tumor tissue is amplified by PCR using primers for BRAF exon 15 and the product is sequenced using Sanger sequencing on Big Dye terminator v1.1 sequencing kit (Applied Biosystems) on an automatic ABI PrismTM 3130 Genetic Analyzer (Applied Biosystems).

Biosystems). BRAF V600E mutation analysis results were also available in some stage IV (at diagnosis or during follow-up) patients, in whom the test was performed for chemotherapy selection, regardless of IHC results.

### 2.3.2. Family Risk Clinic Referral

(10x).

In case Amsterdam or revised Bethesda criteria are fulfilled or when germline MMR genes' mutations are suspected after IHC analysis, the patients are referred to the Familial Risk Clinic. All patients with 10 or more adenomas or those who fulfil the World Health Organization criteria for Serrated Polyposis Syndrome are also referred.

In cases referred for evaluation in the Familial Risk Clinic, additional tumor testing before genetic diagnosis may be done, at physician's discretion, according to available evidence and international recommendations.

## 2.3.3. Molecular and Genetic Testing

## Microsatellite Instability Analysis

Between 2016 and 2017, this was carried out using the Bethesda microsatellite markers: BAT26, BAT25, D17S250, D2S123 and D5S346 [26–28]. In tumor samples exhibiting microsatellite instability (MSI) in only one marker, or without a conclusive result in at least one marker, two additional markers were analyzed (BAT40 and MYCL1). From 2017 onwards, the MSI analysis was performed with 10 microsatellite markers (the above mentioned and 3 additional mononucleotide repeat marker—NR21, NR24 and NR27).

Between 2016 and 2017, DNA was isolated from CRC-PDEs samples using the KAPA Express Extract Kit (KAPABIOSYSTEMS, Potters Bar, United Kingdom) and from paraffinembedded tissue (FFPE) colorectal cancer and normal colonic mucosa using proteinase K digestion, which was followed by phenol/chloroform extraction and ethanol precipitation [29]. From 2017, the Maxwell® RSC DNA FFPE Kit (Promega, Madison, WI, USA) was used to isolate DNA from FFPE samples in the Maxwell® RSC Instrument (Promega). Each tumor and paired normal DNA were amplified by PCR for each of the microsatellite markers, using fluorescent labelled primers (Applied Biosystems, Foster City, CA USA), specific for each locus [30,31]. PCR products were analyzed in the ABI PrismTM 3130 Genetic Analyzer using the GeneMapper software (Applied Biosystems). Tumors presenting MSI in >40% of the markers analyzed were classified as MSI-High (MSI-H); otherwise they were classified as MSI-Low (MSI-L) [32]. Tumors without MSI in any of the markers were considered to be microsatellite stable (MSS).

#### MMR Gene Promoter Methylation Analysis

The analysis of MMR gene promoters methylation was performed by methylationspecific multiplex ligation-dependent probe amplification (MS\_MLPA) [33], using the MS-MLPA kits ME011 MMR (MRC-Holland, Amsterdam, the Netherlands). MS-MLPA reactions were performed as described by the manufacturer. MS-MLPA fragments were analyzed on the ABI Prism 3130TM Genetic Analyzer (Applied Biosystems) and normalized using the Coffalyser. NET software (MRC-Holland, Amsterdam, the Netherlands). A baseline for positive methylation was calculated for each gene as described previously [34]. A ratio of 0.15 or higher, corresponding to 15% of methylated DNA, was indicative of *MLH1* promoter methylation.

#### Germline Mutation Analysis

In case of MMR proteins' deficiency in IHC analysis, mutations in *MLH1*, *MSH2*, *MSH6*, *PMS2* and *EPCAM* were investigated. In other cases, Next Generation Sequencing (NGS) multigene panels were used, according to clinical data and family history.

Germline mutation analysis was performed after signed informed consent, by NGS using multigene panels (TruSight Cancer kit (Illumina, San Diego, CA, USA)) and MLPA (multiplex ligation-dependent probe amplification) analysis (MRC-Holland, Amsterdam, the Netherlands). All pathogenic, probably pathogenic or of uncertain pathogenicity mutations (frequency less than 1% in the population) are confirmed by Sanger sequencing, from an independent DNA sample. The interpretation of the variants is performed according to the rules established by LOVD-InSIGHT (International Society for Gastrointestinal Hereditary Tumors—http://www.insight-group.org/criteria last accessed on the 1 June 2022).

#### *2.4. Statistical Analysis*

For statistical analysis, SPSS Statistics 26 (IBM) was used. Demographic and clinical characteristics were presented as frequencies. Continuous variables were expressed as median and standard deviation or as median and interquartile range, according to data distribution, and were compared using t-Student or Wilcoxon tests, respectively. Qualitative variables were compared using chi-square or Fisher Exact tests. Multiple variables were analyzed using logistic regression models. A *p* value lower than 0.05 was considered statistically significant.

#### **3. Results**

#### *3.1. Clinical Characterization*

A total of 275 patients were included, 56.0% males, with a median age at diagnosis of 61.0 (IQR 54.5–65.0) years old. Tumors were mostly (53.1%) stage III at diagnosis and histological report revealed high grade (G3) tumors in 8.4%, mucinous and/or signed ring morphology in 11.3% and lymphocytic infiltrate in 29.8%. Population and tumor characteristics are depicted in Tables 2 and 3. Mean follow-up time was 40.6 ± 15.6 months. After personal and family history investigation, 11 (4.0%) patients fulfilled Amsterdam criteria (AC) and 74 (26.9%) revised Bethesda criteria (BC).

**Table 2.** Clinical characteristics.


CRC—colorectal cancer. In case of synchronous CRC, location and staging of the more advanced neoplasia was selected to present in the table.



N/A—not available. NOS—no other specification.

#### *3.2. Immunohistochemical Analysis*

IHC evaluation revealed loss of MMR proteins' expression in 24 cases (8.7%)– MLH1 and PMS2 (*n* = 15) (Figure 1); PMS2 (*n* = 4); MSH2 and MSH6 (*n* = 1); MSH2 (*n* = 1); MSH6 (*n* = 2); MLH1, PMS2 and MSH6 (*n* = 1). AC and BC were fulfilled in 0 and 13 of such cases, respectively (Table 4).

Altered IHC analysis showed a significant association with tumor location in the right colon (*p <* 0.001), poor differentiation (*p =* 0.015) and mucinous histology (*p =* 0.016), but not with gender (*p =* 0.157), age (*p =* 0.709), stage (*p =* 0.44), lympho-vascular (*p* = 0.279) or perineural invasion (*p =* 0.567), lymphocytic infiltrate (*p =* 0.052) or tumor budding (*p =* 0.499).

#### *3.3. Analysis of MMR Deficient Cases—BRAFV600E Mutation Status, MMR Gene Methylation and Germline Mutation Analysis*

From the 16 patients with MLH1 loss of expression (15 with MLH1/PMS2 loss of expression, one with MLH1/PMS2/MSH6 loss of expression), somatic BRAF V600E mutation testing was carried out in seven, and found in one patient—the IHC alteration was considered somatic and the patient was not referred for genetic testing. From the remaining six patients, three had MLH1 promoter hypermethylation and three did not show either of the somatic alterations. Genetic testing was performed in these last three patients, of whom one had confirmed LS; in the other two, no germline mutation was detected (Table 4).

BRAF V600E mutation testing results were also available in three other patients in whom the analysis was requested by oncologists, for chemotherapy selection (Table 4).

Five patients with altered IHC died before the Family Risk Clinic appointment/germline mutation analysis and one refused genetic testing. Family Risk Clinic appointment is pending or genetic testing is still ongoing in six patients.

Therefore, in total, genetic test results were available in 11 of the 24 patients with altered IHC and in one with artifacts, and Lynch Syndrome was diagnosed in four of them.

Patients with Lynch Syndrome were men in three cases, and aged less than 50 yearsold in three (median age 37.0 (IQR 27.5–51.8)). AC were not fulfilled in any of the patients, and three met BC; IHC was altered in three and unavailable in one due to artifacts (Table 4).

Tumor was in the right colon in three and rectum in one, stage I in one and III in three cases. Histology report revealed low-grade (G1/G2) tumors with no other specification, no lymphocytic infiltrate and no unfavorable invasions in all cases.

All patients were alive without evidence of cancer relapse at last follow-up (median follow-up = 33.0 months (IQR: 26.8–54.3)).

The presence of Lynch Syndrome had a significant association with younger age at diagnosis (*p <* 0.001) and right-sided tumors (*p* = 0.037), but not with gender (*p =* 0.634), stage (*p =* 0.718), differentiation (*p =* 1.000), histological subtype (*p =* 1.000), lympho-vascular invasion (*p =* 0.575), perineural invasion (*p =* 1.000), lymphocytic infiltrate (*p =* 0.323) or tumor budding (*p =* 1.000).

*Cancers* **2022**, *14*, 3730


BC–Revised Bethesda criteria; N/A–non applicable/ non available; LS–Lynch Syndrome.

**Table 4.** Clinical and molecular characterization of cases with altered MMR status by immunohistochemical analysis.

#### *3.4. Germline Mutation Analysis in MMR Proficient Cases*

In 10 patients with altered IHC and in one with artifacts, germline MMR mutation analysis was performed and in 40 patients a multigene panel was used. From these, one Familial Adenomatous Polyposis and one *MUTYH*-associated Polyposis were diagnosed (both in patients with multiple adenomas). A *MUTYH* heterozygote mutation was found in a patient with CRC at the age of 47 with family history of colonic adenomas. Familial Colorectal Cancer Type X) was diagnosed in a patient in whom no mutation was found after multigene panel testing.

#### **4. Discussion**

This study presents the clinical picture of CRC in an adult population under 70 years old. As expected, most cases were sporadic cancers. Nevertheless, the use of IHC, combined with personal and familial data, allowed the attending physicians to diagnose Lynch Syndrome in four (1.5%) cases. It is important to notice that one of these patients did not fulfill Amsterdam II or Bethesda criteria and genetic diagnosis would have been missed if IHC analysis had not been performed.

Accurate and timely identification of Lynch Syndrome patients is extremely important, since surveillance for colonic and extra-colonic malignancies can increase survival and improve quality of live. This is relevant both for the patients and for at-risk relatives that may benefit from genetic study [13,35,36]. Even if LS is a rare entity, the cost of missing this diagnosis is significant.

Altered IHC was detected in 9.6% of the cases, a rate that is lower than expected, given that deficient-MMR protein status can be found in 15–30% of sporadic CRC [37]. The rates found may be due to the young population studied, where all CRC in patients aged more than 70 years old were excluded. Indeed, microsatellite instability in sporadic cases is frequently associated to MLH1 promoter methylation and these features are more frequently detected in older female patients, some of them often older than 70 years old [38].

In 16 patients, there was MLH1 ± PMS2 loss of expression in the tumor. A major limitation of our study was the fact that somatic *BRAF V600E* mutation/MLH1 promoter hypermethylation analysis' results were available in only a minority of these cases. Routine *BRAF* testing after a MLH1 loss of expression result has only been implemented in our hospital in the last year of the study. Nevertheless, from seven patients with available results, one had *BRAF V600E* mutation and three others had *MLH1* promoter hypermethylation. These findings highlight the benefits of a step-up approach [20,39], that prevents a significant proportion of patients from undergoing most likely inconclusive genetic testing. This strategy makes sense not only in an economic standpoint, but also considering the psychological burden associated with genetic testing [39].

Further advantages of MMR status investigation are the possibility of personalized therapies. MSI tumors may have a reduced response to 5-FU chemotherapy and a better overall prognosis in early stages. Therefore, most stage II MMR deficient CRC patients do not seem to benefit from adjuvant chemotherapy, namely, with 5-FU [40]. Another scenario is metastatic MSI CRC, where therapy with immune checkpoint inhibitors may be proposed, since these patients often show sustained responses to this class of drugs. This is explained by the increased expression of several immune checkpoints in MMR deficient tumors, resulting from the production of abnormal proteins which elicit antigen-driven immune responses [40,41].

Although IHC analysis, molecular and genetic studies' results were prospectively recorded, clinical data collection was retrospective, which is a limitation of the study, which may be relevant in details such as family history that may not have been carefully reported in all cases. However, the study was conducted in an oncological center which integrates a Family RiskClinic, in strict interaction with a Molecular Biology Laboratory and therefore has the means and expertise to pursue genetic investigation when indicated, limiting bias due to unrecognized hereditary cancer patients. Furthermore, this is a sequential series of patients with a relevant number of cases included, reflecting real-life practice.

#### **5. Conclusions**

This study strengthens current recommendations and highlights the role of universal CRC screening for MMR protein status. This inclusive strategy allows the identification of Lynch Syndrome patients that could otherwise be missed using a restrictive approach that relies only on Amsterdam and Bethesda criteria.

**Author Contributions:** Conceptualization, J.L.G. and I.R.; methodology, J.L.G. and I.R.; validation, J.L.G. and I.R.; formal analysis, J.L.G.; investigation, J.L.G. and S.S.; resources, C.A., I.F., P.S., B.F. and R.F.; writing—original draft preparation, J.L.G., I.R, C.A., I.F., P.S., B.F.; writing—review and editing, S.S., C.A., R.F., P.L., I.M. and I.C.; supervision, I.C.; project administration, I.R. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** The study was conducted in accordance with the Declaration of Helsinki. Approval was obtained from the Ethical Committee of Instituto Português de Oncologia de Lisboa Francisco Gentil (Document Number: UIC/1538; date of approval 26 July 2022).

**Informed Consent Statement:** Patients gave informed consent for all genetic studies.

**Data Availability Statement:** Data not shared due to confidentiality.

**Acknowledgments:** The authors would like to thank David Carvalho Fiel who was responsible for proofreading the article.

**Conflicts of Interest:** The authors have no conflicts of interest to declare.

#### **References**

