Prognostic Impact of Tumor Budding on Moroccan Colon Cancer Patients

Fatima El Agy; Sanae el Bardai; Laila Bouguenouch; Nada Lahmidani; Mohammed El Abkari; El Bachir Benjelloun; Abdelmalek Ousadden; Khalid Mazaz; ImaneToughrai; Sidi Adil Ibrahimi; Zineb Benbrahim; Laila Chbani

doi:10.1155/2022/9334570

Prognostic Impact of Tumor Budding on Moroccan Colon Cancer Patients

El Agy, Fatima;Bardai, Sanae el;Bouguenouch, Laila;Lahmidani, Nada;El Abkari, Mohammed;Benjelloun, El Bachir;Ousadden, Abdelmalek;Mazaz, Khalid;ImaneToughrai;Ibrahimi, Sidi Adil;Benbrahim, Zineb;Chbani, Laila 2022-01-21 00:00:00 Hindawi International Journal of Surgical Oncology Volume 2022, Article ID 9334570, 10 pages https://doi.org/10.1155/2022/9334570 Research Article Prognostic Impact of Tumor Budding on Moroccan Colon Cancer Patients 1 2 3 4 Fatima El Agy , Sanae el Bardai, Laila Bouguenouch, Nada Lahmidani, 4 5 5 5 Mohammed El Abkari, El Bachir Benjelloun, Abdelmalek Ousadden, Khalid Mazaz, 5 4 6 7 ImaneToughrai, Sidi Adil Ibrahimi, Zineb Benbrahim, and Laila Chbani Laboratory of Biomedical and Translational Research, Faculty of Medicine and Pharmacy, Sidi Mohamed Ben Abdellah University, Fez, Morocco Laboratory of Anatomic and Molecular Pathology, University Hospital Hassan II, Fez, Morocco Laboratory of Medical Genetics and Oncogenetics, University Hospital Hassan II, Sidi Mohamed Ben Abdellah University, Fez, Morocco Department of Gastroenterology, University Hospital Hassan II, Sidi Mohamed Ben Abdellah University, Fez, Morocco Department of General Surgery, University Hospital Hassan II, Sidi Mohamed Ben Abdellah University, Fez, Morocco Department of Oncology, University Hospital Hassan II, Sidi Mohamed Ben Abdellah University, Fez, Morocco Laboratory of Anatomic and Molecular Pathology, University Hospital Hassan II, Sidi Mohamed Ben Abdellah University, Fez, Morocco Correspondence should be addressed to Fatima El Agy; fatima.elagy@usmba.ac.ma Received 10 November 2021; Accepted 5 January 2022; Published 21 January 2022 Academic Editor: Gaetano Gallo Copyright © 2022 Fatima El Agy et al. &is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background. Tumor budding is now emerging as one of the robust and promising histological factors that play an important role in colon cancer. In this study, we aimed to investigate the association between tumor budding and tumor clinicopathological factors, tumor molecular signature, and patient survival for the ﬁrst time in a Moroccan population. Methods. We collected data of 100 patients operated from colon adenocarcinoma. Tumor budding was assessed on HES slides, according to the International Tumor Budding Consensus Conference 2016 recommendations. &e expression of MMR proteins was performed by immu- nohistochemistry. KRAS and NRAS mutations testing was performed by Sanger sequencing and pyrosequencing. Results. High tumor budding grade (BUD 3) was found to be signiﬁcantly associated with adverse clinicopathological features including older age (P � 0.03), presence of perineural invasion (P � 0.02), presence of vascular invasion (P � 0.05), distant metastases (P< 0.001), advanced TNM stage (P � 0.001), the occurrence of relapse (P � 0.04), and the high number of deceased cases (P � 0.02). Interestingly, we found that tumors with high-grade tumor budding were more likely to be microsatellite stable (MSS) (P � 0.005) and harbor more KRAS mutations (P � 0.02). Tumors with high-grade tumor budding were strongly associated with KRAS G12D mutation (P � 0.007). In all stages, high tumor budding was correlated with poorer overall survival (P � 0.04) and decreased relapse-free survival with a diﬀerence close to signiﬁcance ((P � 0.09). We concluded that high tumor budding was strongly associated with unfavorable clinicopathological features and special molecular biomarkers and eﬀectively aﬀects the overall survival of CC patients. Conclusions. Based on these ﬁndings and the ITBCC group recommendations, tumor budding should be taken into account along with other clinicopathologic factors in the risk assessment of colorectal cancer. occurs consistently among males than females in the general 1. Introduction population with a median age at diagnosis of 55.56 years [2]. Colon cancer is the third most commonly diagnosed cancer &e tumor node metastasis (pTNM) stage is the primary and the second leading cause of cancer-related death in the factor used for prognostication purposes and to guide pa- Moroccan population [1]. A higher incidence of this disease tient management [3]. Indeed, the standard of care for colon 2 International Journal of Surgical Oncology factors, tumor molecular signature, and patient’s survival for cancer is surgical resection (stages I and II), and surgery is followed by adjuvant chemotherapy (ﬂuorouracil and folinic the ﬁrst time in a Moroccan population. acid) for stage II with high-risk factors tumors. Neoadjuvant chemotherapy and targeted therapies (anti-EGFR) are used 2. Materials and Methods for colon cancer with distant metastasis. Although and 2.1. Patients. We enrolled in the present study a hundred because of the survival heterogeneity seen in colon cancer patients with primary colon cancer resected between 2015 patients within the same pathological stage, the introduction and 2020, at Hassan II University Hospital, Fez, Morocco. of other molecular, immunological, and histological markers &e medical charts were prospectively and retrospectively to identify risk stratiﬁcation and disease outcome is now reviewed and patients were included according to the fol- mandatory for better management of colon cancer patients. lowing inclusion criterion: patients with histologically Tumor budding is now emerging as one of the robust and conﬁrmed primary adenocarcinoma, all cases with I-IV promising histological factors that play an important role in stage colon cancer, and patients with prognostic data. Pa- colon cancer. It is a histological manifestation of initiating tients were excluded from this study due to the following invasion and metastasis cascade in the invasive front of the exclusion criteria: All patients with incomplete clinical tumor. According to the International Tumor Budding records, patients without histological conﬁrmation of colon Consensus Conference 2016, ITBCC, it is deﬁned by the adenocarcinoma, and patients with rectal cancer (Figure 1). presence of individual cells and small clusters (<5) of tumor Demographic and clinicopathological data (e.g., age, gender, cells at the invasive front of carcinomas [4]. Several studies tumor grade, tumor localization histological subtype, tumor have demonstrated that tumor budding might be associated grade, disease stage, and number of examined regional with a high risk of relapse and poorer outcomes [5]. Its lymph nodes) and follow-up data were collected from the relationship with unfavorable clinicopathological features patient’s medical records and pathology reports. like nodal and distant metastases was also demonstrated [4, 5]. Indeed, higher grade of tumor budding was reported to be signiﬁcantly correlated with microsatellite stable tu- 2.2. Pathology Analysis. After ﬁrst-line therapy, fresh mors (MSS tumors) [6, 7]. Furthermore, Sammarco et al. specimens were transported to the department of pathology. have recently demonstrated that microsatellite “stable” &e tissue was ﬁxed in formalin (10%) and embedded in BRAF-mutated tumors show more aggressive morpholog- paraﬃn (FFPE). Histological slides based on hematoxylin ical behavior like tumor budding [8]. and eosin staining were prepared and examined by a pa- Recently, a study conducted by Anne et al. on 1320 colon thologist to deﬁne the histopathological characteristics of the cancer patients has shown that high tumor budding was tumor. associated with the presence of KRAS mutations and met- astatic tumors harboring a BRAF gene mutation [9]. 2.3. Assessment of Tumor Budding. Tumor budding was Several studies have revealed the clinical implication of assessed on hematoxylin-eosin and Safran (HES) stained tumor budding in colon cancer management. First, in colon slides. For each CC case, one representative HES slide was cancer stage I, tumor budding is associated with lymph node metastasis. For this reason, patients with high tumor bud- used for scoring by the pathologist according to the ITBCC recommendations. Tumor buds were evaluated in a single ding may beneﬁt from oncologic resection [4]. Second, in stage II colon cancer, the presence of tumor budding is hotspot measuring 0.785 mm at the invasive front using microscopy at 20× objectives. associated with poorer survival. &erefore, adjuvant therapy should be discussed for stage II colon cancer patients with We then used a three-tier system which is recommended by the ITBCC group to provide tumor budding count and high-grade tumor budding [10]. &ird, the assessment of tumor budding in preoperative biopsies could be useful for tumor budding category. &e system of scoring is catego- rized as follows: selecting patients who may qualify for neoadjuvant therapy. However, in advanced colon cancer, the role of tumor (i) 0–4 buds: low budding (Bd 1) budding in clinical practice remains unclear and requires (ii) 5–9 buds: intermediate budding (Bd 2) more investigation [11]. &e lack of a standard quantiﬁcation method for tumor (iii) 10 or more buds: high budding (Bd 3) budding has limited its reporting in the clinical routine We grouped the patients to be low-intermediate (grade practice in CC as well as other histological factors. However, 1 + grade 2) and high tumor budding (grade 3). after the International Tumor Budding Consensus Con- ference (ITBCC), held in Bern in April 2016, a scoring system for assessing tumor budding has been reached 2.4. Determination of Mismatch Repair Protein Expression. according to conference recommendations [4]. &e ITBCC &e immunohistochemistry (IHC) method was used to group recommended that tumor budding should be in- establish the mismatch repair tumor status (MSS or MSI) cluded in guidelines/protocols and staging systems for the and to detect the intact or the loss expression of the MMR pathology reporting of colorectal cancer [4]. proteins (MLH1, PMS2, MSH2, and MSH6). &e IHC study &is study aimed to assess tumor budding according to was performed on unstained formalin-ﬁxed paraﬃn-em- the ITBCC recommendations and to investigate the associ- bedded (FFPE) tumor tissue sections of 5 μm thickness, on ation between tumor budding and tumor clinicopathological the automated immunostainer Ventana Benchmark International Journal of Surgical Oncology 3 PCR Master Mix 2x, 2.5 µl of Coral Load Concentrate 10x, 239 patients with colorectal cancer (I-IV) 4 µl of nuclease-free water, and 1 µl of the corresponding set of PCR primers (Qiagen). 10 µl of PCR products was immobilized to Streptavidin Sepharose High-Performance 104 patients excluded: Patients with rectum cancer beads (Qiagen) to prepare the single-stranded DNA. &e corresponding sequencing primers were allowed to anneal to the DNA using a PyroMark Q24 plate and a vacuum 135 colon cancer workstation (Qiagen). PyroMark Q24 reagents (enzyme mixture, substrate mixture, and nucleotide all from 18 patients excluded: No primary tumor resection Qiagen) were prepared and loaded into a cartridge to be dispensed during the sequencing process. Finally, the 17 patients excluded: sequences were analyzed using PyroMark Q24 software in Patients with incomplete histopathological records the AQ analysis mode. In each run, two controls were included: negative control (without template DNA) and 100 patients included in analysis an unmethylated control DNA, provided by the kit as a positive control for PCR, and sequencing reactions were Figure 1: Flow diagram for the study. included. ULTRA. We have employed monoclonal antibodies speciﬁc for each MMR protein, MLH1 (G168-728/CELL MAR- 2.6. Statistical Analysis. Clinical, pathological, and molec- QUE), MSH2 (G219-1129/CELL MARQUE), MSH6 (44/ ular variables collected at baseline were described as means CELL MARQUE), and PMS2 (MRQ-28/CELL MARQUE). and standard deviation (sd’s) for quantitative variables and Adjacent normal tissue (lymphocytes or normal glandular percentages for qualitative variables. Associations between cells) was used as an internal control for positive staining. tumor budding (assessing as a categorical variable) and categorical factors of tumor were assessed using the χ2-test or Fisher’s exact test variables. &e unpaired t-test was used 2.5. Detection of KRAS and NRAS Mutation for continuous variables. Tests were statistically signiﬁcant when P< 0.05. 2.5.1. DNA Extraction. Genomic DNA was extracted from 5 Overall survival was deﬁned as the time from the start of to 8 sections of 5 μm thickness of macrodissected formalin- diagnosis until death or until the last follow-up. Relapse-free ﬁxed paraﬃn-embedded (FFPE) tumor blocks, containing at survival was measured from the date of initial diagnosis until least 50% of tumor cells, as determined by an experienced the date of local relapse or regional relapse or last follow-up/ pathologist on H&E-stained paraﬃn slides. &e extraction death (all causes) whichever occurs ﬁrst. was eﬀected using the QIAamp DNA FFPE Tissue Kit RFS and OS rates according to tumor budding, clini- (Invitrogen) and according to the manufacturer’s instruc- copathological factors, and molecular features were deter- tions. DNA concentration (ng/ul) was assessed by Qubit mined using the Kaplan-Meier method, and survival ﬂuorometer. diﬀerences between groups were evaluated by log-rank test. Multivariate analysis was performed using a Cox pro- 2.5.2. PCR and Direct Sequencing. For each sample, mu- portional hazard model to identify independent risk factors tations of KRAS exons 2, 3, and 4 and NRAS exons 2 and 3 for survival. Factors that were signiﬁcant and nearly sig- were ampliﬁed by polymerase chain reaction (PCR). Brieﬂy, niﬁcant in univariate analysis (P< 0.1) were included in 10 ng of template DNA was ampliﬁed using 12× PCR mix multivariate analysis. platinum, 12.5 pmol primers, 50 μmol Mgcl , and 2.5 μl of Data from univariate and multivariate analyses were the corresponding set of PCR primers listed in Table 1. After reported as hazard ratios (HRs) with 95% conﬁdence in- the puriﬁcation of PCR products, the presence of mutations tervals (CIs). All statistics were assessed using 2-sided tests, was detected by direct sequencing using the BigDye Ter- with P values <0.05 considered statistically signiﬁcant. minator V3.1 Cycle Sequencing Kit (ABI Prism) and the Statistical analysis was performed using the IBM SPSS Applied Biosystems 3500Dx Genetic Analyzer (Applied Statistics 21. Biosystem). 3. Results 2.5.3. Pyrosequencing. &e analysis of RAS mutations was performed using the &eraScreen KRAS Pyro Kit (for 3.1. Patient Demographics and Pathological Characteristics. KRAS codons 12 and 13) and the &eraScreen RAS Ex- Patients and tumor characteristics of 100 patients are tension Pyro Kit (for KRAS codons 59/61, 117, and 146 and summarized in Table 2. Among 100 cases, 43 (43.0%) were NRAS codons 12, 13, 59, 61, 117, and 146) (Qiagen, Ger- women and 57 (57.0%) were men with a mean age of 54.9 many), according to the manufacturer’s instructions. As years. Our cohort was characterized by a predominance of described previously [2], 5 µl of template DNA (2–10 ng of the left-sided colon cancers (n � 62, 62.0%), compared to genomic DNA) was ampliﬁed by polymerase chain reaction right-sided CC (n � 38, 38.0%). Histologically, the adeno- (PCR) in a 20 µl volume containing 12.5 µl of PyroMark carcinoma subtype was documented in 86 tumors (86.0%), ® 4 International Journal of Surgical Oncology Table 1: Primer sequences used for PCR. Primer name Primer sequence KRAS-ex 2- F 5′-GGTGGAGTATTTGATAGTGTA- 3′ KRAS-ex 2- R 5′-TGCATATTACTGGTGCAGACC- 3′ KRAS-ex 3- F 5′-AGTAAAAGGTGCACTGTAATAA-3′ KRAS-ex 3- R 5′-ATAATAAGCTGACATTAAGGAG-3′ KRAS-ex 4- F 5′-TGTTACTAATGACTGTGCTATAACTTTT-3′ KRAS-ex 4- R 5′-TATGCTATACTATACTAGGAAATAAAA-3′ NRAS-ex2-F 5′-ATGACTGAGTACAAACTGGTGGTGGTTGGAGCA-3′ NRAS-ex2-R 5′-CACTTTGTAGATGAATATGATCCCACCATAGAG-3′ NRAS-ex3-F 5′-GATTCTTACAGAAAACAAGTGGTTA-3′ NRAS-ex3-R 5′-CATTTGCGGATATTAACCTCTACAG-3′ NRAS-ex4-F 5′-GGAGCAGATTAAGCGAG-3′ NRAS-ex4-R 5′-TCAGCCAAGACCAGACAG-3′ while only 9 (9.0%) tumors were mucinous adenocarcinoma. tumors showed high-grade tumor budding (grade 3). &e 46 (46.0%) tumors were classiﬁed as grade 2 (moderately results are shown in Table 4. diﬀerentiated) and grade 1 (well-diﬀerentiated), and 8 (8.0%) In correlation analysis, we divided the patients into two tumors were classiﬁed as grade 3 (poorly diﬀerentiated). groups, a group with low-grade tumor budding (grade Perineural invasion was observed in 15 (15.3%) tumors. In this 1 + grade 2) that represented 60% of all cases and a group study, the mean number of removed lymph nodes was 20.8 with high-grade tumor budding (grade 3) that represented (range, 1–57). 85 (87.6%) patients had more than 12 dissected 40% of all cases (Table 4). LN. Positive LNs were identiﬁed in 32 (32.6%) patients (mean � 1.3; rang, 0.1–16). According to the TNM classiﬁca- 3.5. Relationship between Tumor Budding and Clinicopath- tion, 4 (4.2%) of the tumors were stage I, 51 (53.1%) stage II, 25 ological Features. Table 5 shows the results of the association (26.0%) stage III, and 16 (16.7%) IV. In our cohort, 50% of between tumor budding and clinicopathologic factors. Tumor patients have received surgical treatment, and 46% have re- budding grades were signiﬁcantly associated with age, peri- ceived adjuvant chemotherapy. Neoadjuvant treatment was neural invasion, vascular invasion, distant metastases, TNM indicated for only 4% of cases. stage, the occurrence of relapse, and the number of deceased &e mean follow-up time of the patient’s OS was 49.4 cases. Indeed, compared with patients with low-grade tumor months (range, 6–119 months). Among 100 patients, 18 budding (grade 1 + grade 2), patients with high-grade tumor (18.2%) cases of death were recorded. Recurrence was ob- budding (grade 3) had more vascular invasion (28.2% vs. served in 29 (29.3%) patients. &e most frequent site of 15.3%; P � 0.05), more venous invasion (25.6% vs. 8.5%; recurrence was local recurrence (31.0%) followed by peri- P � 0.02), and a higher number of distant metastases (33.3% toneums (24.1%), lung (20.7%), and liver (17.4%). vs. 5.0%; P< 0.001). Also, these patients had a majority of advanced pathologic stage IV tumors (P � 0.001). Interestingly, patients with high-grade tumor budding 3.2. Molecular Features. Concerning molecular character- had signiﬁcantly high relapse rates (P � 0.04). Moreover, istics, the MSI tumors were found in 21 patients (21.0%), peritoneal recurrence was the most frequent recurrence site tumors with KRAS mutations in 31 patients (40.3%), and in tumors with high-grade tumor budding, with a diﬀerence NRAS mutations in 2 patients (2.6%). close to signiﬁcance (P � 0.07). Tumors with high-grade tumor budding were correlated with a higher risk of death (P � 0.02). 3.3. KRAS and NRAS Mutations Classes. All the basic data &ere was no correlation between tumor budding grade are presented in Table 3. Activating KRAS mutations were and gender, tumor localization, histologic subtype, histo- found in 31/77 examined tumor cases (40.3%). Among logic grade, and lymph nodes count. KRAS variants, G12D was the most frequent (11/31, 35.5%), followed by G13D (8/31, 25.8%), G12C (4/31, 12.9%), A146T 3.6. Tumor Budding and Molecular Biomarkers. (3/31, 9.7%), G12V (2/31, 6.5%), G12R (1/31, 3.2%), G12A (1/31, 3.2%), and G13V (1/31, 3.2%). Interestingly, we investigated the relationship between the diﬀerent grades of tumor budding and the molecular Out of 77 cases, two showed NRAS mutations (2/77, 2.6%). One mutation was detected in codon 12 (G12R) (1/2, characteristics of the tumor. &e diﬀerent results are shown in Table 6. According to our results, tumors with high-grade 50%) and the other in codon 61 (Q61L) (1/2, 50%). tumor budding were more likely to be microsatellite stable (MSS) (P � 0.005). &e mutation rate in the KRAS gene was signiﬁcantly 3.4. Incidence of Tumor Budding in Our Population. Among 100 CC cases, 28 (28%) tumors showed low-grade higher in the high-grade TB tumors compared to that in the low-grade TB tumors (P � 0.02). Tumors with KRAS codon tumor budding (grade 1), 32 (32%) tumors showed inter- mediate-grade tumor budding (grade 2), and 40 (40%) 12 mutations tented to have high tumor budding with a International Journal of Surgical Oncology 5 Table 2: Patient demographics, pathological, and molecular Table 2: Continued. features. Characteristics Total (%) Characteristics Total (%) Mortality Age Death cases 18 (18.2%) ≤57 53 (53.0%) Censored cases 81 (81.8%) ≥57 47 (47.0%) MSI status Gender MSS 79 (79.0%) Female 43 (43.0%) MSI 21 (21.0%) Male 57 (57.0%) KRAS mutation Tumor site Presence 31 (40.3%) Right colon 38 (38.0%) Absence 46 (59.7%) Left colon 62 (62.0%) NRAS mutation Histological subtype Presence 2 (2.6%) Adenocarcinoma 86 (86.0%) Absence 75 (97.4%) Mucinous adenocarcinoma 9 (9.0%) Others 5 (5.0%) Histological grade Table 3: &e frequencies of genetic alteration classes. Well 46 (46.0%) Moderate 46 (46.0%) Mutations Number % Poor 8 (8.0%) KRAS 31 40.3 Venous invasion Codon 12 19 61.3 Presence 20 (20.4%) G12D 11 35.5 Absence 78 (79.6%) G12C 4 12.9 Perineural invasion G12V 2 6.5 Presence 15 (15.3%) G12A 1 3.2 Absence 83 (84.7%) G12R 1 3.2 Number of removed lymph nodes Codon 13 9 29.0 Mean (±SD) 20.8 (±10.8) G13D 8 25.8 <12 12 (12.4%) G13V 1 3.2 ≥12 85 (87.6%) Codon 146 3 9.7 Range 1–57 A146T 3 9.7 Positive lymph node NRAS 2 2.6 Mean (±SD) 1.3 (±2.9) Codon 12 1 50 Presence 32 (8.6%) G12R 1 50 Absence 66 (32.7%) Codon 61 1 50 Average 0.1–16 Q61L 1 50 Lymph node ratio Mean (±SD) 0.1 (±0.14) Range 0.01–0.81 Table 4: Incidence of tumor budding. Distant metastases (M) Tumor budding grades Frequency (%) M0 83 (83.8%) M1 16 (16.2%) Grade 1 (low) 28 (28%) Disease stages Grade 2 (intermediate) 32 (32%) I 4 (4.2%) Grade 3 (high) 40 (40%) II 51 (53.1%) Low-grade tumor budding 60 (60%) III 25 (26.0%) High-grade tumor budding 40 (40%) IV 16 (16.7%) Treatment diﬀerence close to signiﬁcance, as compared with tumors Surgery 50 (50.0%) harboring other KRAS codon mutations (P � 0.05). Surgery + adjuvant chemotherapy 46 (46.0%) Moreover, KRAS G12D mutation was found to be sig- Neoadjuvant therapy 4 (4.0%) niﬁcantly correlated with high-grade TB compared to the Follow-up time (months) other KRAS codon 12 variants (P � 0.007). However, Mean (SD) 49.4 (±29.3) Range 6–119 there was no correlation between KRAS codon 13 variants Recurrence and tumor budding grade. (+) 29 (29.3%) &ere was no signiﬁcant association between tumor (−) 70 (70.7%) budding and NRAS status. Recurrence patterns Liver 5 (17.4%) Lung 6 (20.7%) 3.7. Survival Outcomes according to Tumor Budding. Peritoneum 7 (24.1%) Table 7 shows associations of tumor budding with Local recurrence 9 (31.0%) overall survival and relapse-free survival of CC patients Others 2 (6.8%) when tumor budding is stratiﬁed into three groups 6 International Journal of Surgical Oncology Table 5: Association between tumor budding and clinicopathological features. Variables Low-grade tumor budding High-grade tumor budding P value Age <57 36 (60.0%) 17 (42.5%) 0.03 ≥57 24 (40.0%) 23 (57.5%) Genre Female 28 (46.7%) 15 (37.5%) 0.2 Male 32 (53.3%) 25 (62.5%) Tumor site Right colon 22 (36.7%) 16 (40.0%) 0.4 Left colon 38 (63.3%) 24 (60.0%) Histologic subtype Adenocarcinoma 54 (90.0%) 32 (80.0%) 0.2 Mucinous 3 (5.0%) 6 (15.0%) Others 3 (5.0%) 2 (5.0%) Histologic grade Well 28 (46.7%) 18 (45.0%) 0.5 Moderate 26 (43.3%) 20 (50.0%) Poor 6 (10.0%) 2 (5.0%) Venous invasion Presence 9 (15.3%) 11 (28.2%) 0.05 Absence 50 (84.7%) 28 (71.8%) Perineural invasion Presence 5 (8.5%) 10 (25.6%) 0.02 Absence 54 (91.5%) 29 (74.4%) Number of removed lymph nodes Mean (SD) 21.7 (±8.5) 19.2 (±6.9) 0.2 ˂12 5 (8.6%) 7 (17.9%) 0.1 ˃12 53 (91.4%) 32 (82.1%) Positive lymph node Mean (SD) 1.2 (±0.0) 1.4 (±2.9) 0.8 Presence 19 (32.2%) 13 (33.3%) 0.5 Absence 40 (67.8%) 26 (66.7%) RGL Mean (SD) 0.06 (±0.03) 0.063 (±0.2) 0.9 Synchronous metastasis (M) M0 57 (95.0%) 26 (66.7%) ˂0.001 M1 3 (5.0%) 13 (33.3%) Disease stages I 2 (3.3%) 2 ((5.0%) II 37 (61.7%) 15 (37.5%) 0.001 III 18 (30.0%) 9 (22.5%) IV 3 (5.0%) 14 (35.0%) Follow-up time (months) 27.0 (±19.3) 24.8 (±13.3) 0.7 Recurrence (+) 14 (23.3%) 15 (38.5%) 0.04 (−) 46 (76.7%) 24 (61.5%) Recurrence patterns Liver 2 (14.3%) 3 (20%) 0.07 Lung 3 (21.4%) 3 (20%) Peritoneum 2 (14.3%) 5 (33.3%) Local recurrence 6 (42.9%) 3 (20%) Others 1 (7.1%) 1 (6.7%) Mortality Death cases 7 (11.7%) 11 (28.2%) 0.02 Censored cases 53 (88.3%) 28 (71.8%) (BD1, BD2, BD3) or two groups (low (BD1 + 2), high tumors with high-grade tumor budding were signiﬁcantly (BD3)). correlated with shorter OS (P � 0.04; Figure 2(a)). In the three-tier analysis, tumor budding was not Moreover, these tumors tended to be associated with associated with OS and RFS (BD1 versus BD2 versus shorter RFS with a diﬀerence close to signiﬁcance BD3). In the 2-tier approach (BD1 + 2 versus BD3), (P � 0.09; Figure 2(b)). International Journal of Surgical Oncology 7 Table 6: Association between tumor budding and molecular biomarkers. Variables Low-grade tumor budding High-grade tumor budding P value MSI status MSS 42 (70.0%) 37 (92.5%) 0.005 MSI 18 (30.0%) 3 (7.5%) KRAS status Mutant 14 (23.3%) 17 (42.5%) 0.02 Wild-type 46 (76.7%) 23 (57.5%) KRAS codon types Codon 12 7 (50%) 12 (70.6%) 0.05 Codon 13 5 (35.7%) 4 (23.5%) Codon 146 2 (14.3%) 1 (5.9%) KRAS codon 12 variants G12D 1 (14.3%) 10 (83.3%) 0.007 G12C 2 (28.6%) 2 (16.7%) G12V 2 (28.6%) 0 (0.0%) G12A 1 (14.3%) 0 (0.0%) G12R 1 (14.3%) 0 (0.0%) KRAS codon 13 variants G13D 4 (80%) 4 (100%) 0.3 G13V 1 (20%) 0 (0.0%) NRAS gene status Mutant 1 (1.7%) 1 (2.5%) 0.4 Wild-type 59 (98.3%) 59 (97.5%) Table 7: Analysis of OS and RFS according to tumor budding. Tumor budding Mean OS month (95% CI) P value Mean RFS month (95% CI) P value BD1 85.7 (76.9–94.5) 0.1 88.7 (69.7–107.7) 0.1 BD2 100.5 (84.0–116.9) 108.4 (90.8–126.1) BD3 82.6 (67.2–97.9) 81.4 (62.7–100.1) High 82.5 (67.2–97.8) 0.04 81.4 (62.7–100.1) 0.09 Low 104.2 (94.0–114.4) 99.4 (80.9–103.6) associations have fueled the hypothesis that tumor buds can 4. Discussion pervade the extracellular matrix (ECM) and migrate and &e present study was designed to investigate the rela- disseminate into blood vessels [16]. It was suggested that tionship of tumor budding with the clinicopathological tumor budding harbors the properties of cells undergoing an characteristics and molecular biomarkers of CC. Also, we epithelial-mesenchymal transition (EMT) or a partial-EMT evaluate the prognostic impact of tumor budding on hun- state [16]. In this process, epithelial cells lose intracellular dred CC patients using the ITBCC scoring method of TB on and cell-matrix contacts mediated by E-cadherin, leading to HES slides for the ﬁrst time in the Moroccan population and invasion and metastatic cancer spread [17]. the Middle East and Nord Africa region. Additionally, we observed a signiﬁcant correlation be- In recent years, several reports showed that tumor tween high-grade tumor budding and advanced TNM stage, budding is characterized by diﬀerent clinicopathological and which further supports the results of previous studies as histological features. In this study, we were able to dem- reported by Van et al. [18]. onstrate that high-grade tumor budding (BD3) grade un- Interestingly, our results indicate that patients exhibiting derlines special clinicopathological parameters. In our high-grade tumor budding had a higher rate of recurrence. context, BD3 was signiﬁcantly greater in older age patients In a study conducted on 138 patients, Tanaka et al. [19] and we are the ﬁrst to report this result because none of the reported that tumor budding was signiﬁcantly associated previous studies have found a signiﬁcant association be- with disease recurrence. Another study evaluated 200 pa- tween age and tumor budding grades [5, 12, 13]. tients with CC and reported the same result [20]. Okuyama As reported in many studies [5, 10, 14], we documented a et al. showed a statistically signiﬁcant relationship between positive relationship between high-grade tumor budding, high-grade tumor budding and local recurrence [21]. the presence of vascular invasion, and the presence of &e majority of studies investigating the relationship perineural invasion. Furthermore, we found that tumors between tumor budding and clinicopathological features with high-grade tumor budding were signiﬁcantly charac- have documented its positive correlation with lymph node terized by an increased frequency of distant metastases. &e involvement [22]. Conversely, in our study, we did not ﬁnd same result was reported by Jayasinghe et al. [15]. &ese any association. 8 International Journal of Surgical Oncology 1.0 1.0 0.8 0.8 0.6 0.6 0.4 0.4 0.2 0.2 0.0 0.0 0.0 20.0 40.0 60.0 80.0 100.0 120.0 0.0 25.0 50.0 75.0 100.0 125.0 Time (month) Time (month) Budding Budding Low Low High High (a) (b) Figure 2: Survival curves of CC patients stratiﬁed by budding grades. (a) Overall survival in all patients stratiﬁed by BD1 + 2 (low) versus BD3 (high). (b) Relapse-free survival in all patients stratiﬁed by BD1 + 2 (low) versus BD3 (high). As a point of interest, we also investigated the association According to our results, it can be reported that high between tumor budding and molecular biomarkers, such as grade of tumor budding is generally associated with poor MSI status, KRAS, and NRAS mutations. We observed that prognostic factors (vascular and perineural invasion, distant high-grade tumor budding was more common in MSS tu- metastases, MSS status, and KRAS mutations). mors, which is consistent with ﬁndings reported by previous Secondly, we aimed to evaluate the relationship be- studies [12, 23, 24]. In addition, Lugli et al. have recently tween tumor budding and clinical outcome in CC pa- tients, for the ﬁrst time in the Middle East and North showed that tumor buds are infrequently found in colorectal cancers with microsatellite instability (MSI) [7]. It was Africa region. We validated the prognostic eﬀect of tumor budding on overall survival in our cohort using the newly showed that MSS tumors were signiﬁcantly correlated with shorter overall survival of CC patients [25]. established ITBCC criteria for the scoring of tumor We found that tumors with high-grade tumor budding budding on H&E slides. We found that OS was better in had signiﬁcantly more KRAS mutations. &e relationship patients with low-grade tumor budding (BD1/2 versus between KRAS mutation and tumor budding has previously BD3) at all stages; we also observed that high-grade tumor been reported [26, 27]. Jang and colleagues found that 61.8% budding was linked to an increased risk of death. &is of colorectal cancers with high-grade tumor budding harbor method is already included in the Japanese Guidelines for more KRAS mutations [28]. Recently, Trinh et al. [9] and the reporting of CRC. However, many reports suggest that Lugli et al. have reported the same results [7]. Similar to Jang whichever scoring method is utilized, the presence of high-grade tumor budding is correlated with worse et al. [28], we found that the G12D substitution in the KRAS gene was strongly associated with high-grade tumor budding. clinical outcomes [10, 29, 30]. In the literature, several studies are investigating the According to this result, the KRAS G12D mutation could be proposed as a high-grade tumor budding biomarker. impact of tumor budding on CC patient’s survival. Similar to Of note, our group has previously reported KRAS our results, Oh and colleagues pooled results from more mutations as a predictor factor of worse OS [2]. than 4000 Japanese patients from all stages and conﬁrmed We did not ﬁnd any association between NRAS status the positive association of high-grade budding with worse and tumor budding grade in our context. Considering we OS [31]. Trinh et al. also validated the prognostic impact of found only 2 patients harboring NRAS mutations, this tumor budding independent of age, stage, and sex in a cohort ﬁnding does not yet allow us to draw ﬁrm conclusions. including 1320 colorectal cancers [9]. Barresi et al. were also limited by a small number of NRAS Regarding the association between tumor budding and RFS, we observed that patients with high tumor budding had mutated cases (N � 4) and they did not ﬁnd any correlation between NRAS status and tumor budding grade [27]. a shorter RFS but with a diﬀerence close to signiﬁcant Overall survival Relapse free survival International Journal of Surgical Oncology 9 although several reports have conﬁrmed this correlation Authors’ Contributions with signiﬁcant diﬀerences [31, 32]. All authors read and approved the ﬁnal manuscript. A preprint has previously been published [33]. &ere were some limitations to the present study. First is the size of the study cohort. Indeed, the number of cases References included in our study is relatively small, in comparison with some previous reports. Second, our study represents a single [1] H. Sung, J. Ferlay, R. L. Siegel, M. Laversanne, I. Soerjomataram, and A. Jemal, “Global cancer statistics 2020: institution and thus carries the possibility of selection bias GLOBOCAN estimates of incidence and mortality worldwide and does not allow us to generalize our results in the overall for 36 cancers in 185 countries,” CA: A Cancer Journal for population of our country. &ird, it has been demonstrated Clinicians, vol. 71, no. 3, pp. 209–249, 2021. that high-grade tumor budding is signiﬁcantly associated [2] F. El Agy, S. El Bardai, I. El Otmani, Z. Benbrahim, with lymph node metastasis in colon cancer. However, we I. M. H. Karim, and K. Mazaz, “Mutation status and prog- were unable to produce similar results. &is could be likely nostic value of KRAS and NRAS mutations in Moroccan attributable to the size and the characteristics of our sample. colon cancer patients: a ﬁrst report,” PLoS One, vol. 16, no. 3, Above all, our study is the ﬁrst report investigating the prognostic impact on colon cancer patients in the Middle [3] C. Zhang, Z. Mei, J. Pei, M. Abe, X. Zeng, and Q. Huang, “A East and Nord Africa region. modiﬁed tumor-node-metastasis classiﬁcation for primary operable colorectal cancer,” JNCI Cancer Spectrum, vol. 5, 5. Conclusions [4] A. Lugli, R. Kirsch, Y. Ajioka et al., “Recommendations for reporting tumor budding in colorectal cancer based on the In this study, we concluded that high-grade tumor budding International Tumor Budding Consensus Conference was strongly associated with unfavorable clinicopathological (ITBCC) 2016,” Modern Pathology, vol. 30, no. 9, features, like a perineural invasion, venous invasion, and pp. 1299–1311, 2017. distant metastases, and special molecular biomarkers which [5] H. C. van Wyk, J. H. Park, J. Edwards, P. G. Horgan, are MSS status and KRAS mutations. We deﬁned KRAS D. C. McMillan, and J. J. Going, “&e relationship between G12D mutation as a biomarker of high-grade tumor tumour budding, the tumour microenvironment and survival budding. in patients with primary operable colorectal cancer,” British Our results also indicate that high-grade tumor budding Journal of Cancer, vol. 115, no. 2, pp. 156–163, 2016. eﬀectively aﬀects the overall survival of CC patients. Based [6] I. Centeno, A. Paasinen Sohns, M. Flury et al., “DNA proﬁling of tumor buds in colorectal cancer indicates that they have the on these ﬁndings and the ITBCC group recommendations, same mutation proﬁle as the tumor from which they derive,” tumor budding should be taken into account along with Virchows Archiv, vol. 470, no. 3, pp. 341–346, 2017. other clinicopathologic factors in the risk assessment of [7] A. Lugli, I. Zlobec, M. D. Berger, R. Kirsch, and colorectal cancer. I. D. Nagtegaal, “Tumour budding in solid cancers,” Nature Reviews Clinical Oncology, vol. 18, no. 2, pp. 101–115, 2021. Data Availability [8] G. Sammarco, G. Gallo, G. Vescio et al., “Mast cells, microRNAs and others: the role of translational research on &e datasets used/or analyzed during the current study are colorectal cancer in the forthcoming era of precision medi- available from the corresponding author on reasonable cine,” Journal of Clinical Medicine, vol. 3, no. 9, p. 2852, 2020. request. [9] A. Trinh, C. Ladrach, ¨ H. E. Dawson et al., “Tumour budding is associated with the mesenchymal colon cancer subtype and Ethical Approval RAS/RAF mutations: a study of 1320 colorectal cancers with Consensus Molecular Subgroup (CMS) data,” British Journal &is study protocol was reviewed and approved by Hassan II of Cancer, vol. 119, no. 10, pp. 1244–1251, 2018. [10] A. C. Rogers, D. C. Winter, A. Heeney et al., “Systematic University Hospital Ethics Committee of Fez, Morocco, review and meta-analysis of the impact of tumour budding in under reference no. 13/18 according to the World Medical colorectal cancer,” British Journal of Cancer, vol. 115, no. 7, Association Declaration of Helsinki. pp. 831–840, 2016. [11] I. Zlobec and A. Lugli, “Tumour budding in colorectal cancer: Consent molecular rationale for clinical translation,” Nature Reviews Cancer, vol. 18, no. 4, pp. 203-204, 2018. All patients gave verbal informed consent before the start of [12] R. P. Graham, R. A. Vierkant, L. S. Tillmans et al., “Tumor the study. budding in colorectal carcinoma: conﬁrmation of prognostic signiﬁcance and histologic cutoﬀ in a population-based co- Disclosure hort,” =e American Journal of Surgical Pathology, vol. 39, no. 10, pp. 1340–1346, 2015. &e authors alone are responsible for the content and [13] M. Karlberg, K. Stenstedt, M. Hallstrom, ¨ P. Ragnhammar, writing of the paper. C. Lenander, and D. Edler, “Tumor budding versus mismatch repair status in colorectal cancer - an exploratory analysis,” Anticancer Research, vol. 38, no. 8, pp. 4713–4721, 2018. Conflicts of Interest [14] H. Dawson, F. Galuppini, P. Trager ¨ et al., “Validation of the &e authors declare no conﬂicts of interest. International Tumor Budding Consensus Conference 2016 10 International Journal of Surgical Oncology recommendations on tumor budding in stage I-IV colorectal [30] R. Cappellesso, C. Luchini, N. Veronese et al., “Tumor cancer,” Human Pathology, vol. 85, pp. 145–151, 2019. budding as a risk factor for nodal metastasis in pT1 colorectal [15] C. Jayasinghe, N. Simiantonaki, and C. J. Kirkpatrick, “His- cancers: a meta-analysis,” Human Pathology, vol. 65, pp. 62–70, 2017. topathological features predict metastatic potential in locally advanced colon carcinomas,” BMC Cancer, vol. 15, no. 1, p. 14, [31] B. Y. Oh, Y. A. Park, J. W. Huh et al., “Prognostic impact of 2015. tumor-budding grade in stages 1-3 colon cancer: a retro- [16] A. D. Grigore, M. K. Jolly, D. Jia, M. C. Farach-Carson, and spective cohort study,” Annals of Surgical Oncology, vol. 25, H. Levine, “Tumor budding: the name is EMT. Partial EMT,” no. 1, pp. 204–211, 2018. Journal of Clinical Medicine, vol. 5, no. 5, 2021. [32] F. Petrelli, E. Pezzica, M. Cabiddu et al., “Tumour budding [17] A. Lugli, E. Karamitopoulou, and I. Zlobec, “Tumour bud- and survival in stage II colorectal cancer: a systematic review ding: a promising parameter in colorectal cancer,” British and pooled analysis,” Journal of Gastrointestinal Cancer, Journal of Cancer, vol. 106, no. 11, pp. 1713–1717, 2012. vol. 46, no. 3, pp. 212–218, 2015. [18] H. C. van Wyk, J. Park, C. Roxburgh, P. Horgan, A. Foulis, [33] F. E. agy, S. E. bardai, L. Bouguenouch, N. Lahmidani, and D. C. McMillan, “&e role of tumour budding in pre- M. E. abkari, and B. E. bachir, “Prognostic impact of tumor dicting survival in patients with primary operable colorectal budding on moroccan colon cancer patients,” 2021, https:// cancer: a systematic review,” Cancer Treatment Reviews, www.researchsquare.com/article/rs-546470/v1. vol. 41, no. 2, pp. 151–159, 2015. [19] M. Tanaka, Y. Hashiguchi, H. Ueno, K. Hase, and H. Mochizuki, “Tumor budding at the invasive margin can predict patients at high risk of recurrence after curative surgery for stage II, T3 colon cancer,” Diseases of the Colon & Rectum, vol. 46, no. 8, pp. 1054–1059, 2003. [20] T. Nakamura, H. Mitomi, H. Kanazawa, Y. Ohkura, and M. Watanabe, “Tumor budding as an index to identify high- risk patients with stage II colon cancer,” Diseases of the Colon & Rectum, vol. 51, no. 5, pp. 568–572, 2008. [21] T. Okuyama, M. Oya, and H. Ishikawa, “Budding as a useful prognostic marker in pT3 well- or moderately-diﬀerentiated rectal adenocarcinoma,” Journal of Surgical Oncology, vol. 83, no. 1, pp. 42–47, 2003. [22] A. Mehta, M. Goswami, R. Sinha, and A. Dogra, “Histo- pathological signiﬁcance and prognostic impact of tumor budding in colorectal cancer,” Asian Paciﬁc Journal of Cancer Prevention: Asian Paciﬁc Journal of Cancer Prevention, vol. 19, no. 9, pp. 2447–2453, 2018. [23] J. R. Jass, M. Barker, L. Fraser, M. D. Walsh, V. L. J. Whitehall, and B. Gabrielli, “APC mutation and tumour budding in colorectal cancer,” Journal of Clinical Pathology, vol. 56, no. 1, pp. 69–73, 2003. [24] I. Zlobec, M. P. Bihl, A. Foerster, A. Ruﬂe, and A. Lugli, “&e impact of CpG island methylator phenotype and micro- satellite instability on tumour budding in colorectal cancer,” Histopathology, vol. 61, no. 5, pp. 777–787, 2012. [25] F. El Agy, I. E. Otmani, A. Mazti, N. Lahmidani, A. Oussaden, and M. El Abkari, “Implication of microsatellite instability pathway in outcome of colon cancer in Moroccan pop- ulation,” Disease Markers, vol. 2019, Article ID 3210710, 10 pages, 2019. [26] F. Prall and C. Ostwald, “High-degree tumor budding and podia-formation in sporadic colorectal carcinomas with K-ras gene mutations,” Human Pathology, vol. 38, no. 11, pp. 1696–1702, 2007. [27] V. Barresi, L. Reggiani Bonetti, and S. Bettelli, “KRAS, NRAS, BRAF mutations and high counts of poorly diﬀerentiated clusters of neoplastic cells in colorectal cancer: observational analysis of 175 cases,” Pathology, vol. 47, no. 6, pp. 551–556, [28] S. Jang, M. Hong, M. K. Shin et al., “KRAS and PIK3CA mutations in colorectal adenocarcinomas correlate with ag- gressive histological features and behavior,” Human Pathol- ogy, vol. 65, pp. 21–30, 2017. [29] V. H. Koelzer, I. Zlobec, and A. Lugli, “Tumor budding in colorectal cancer-ready for diagnostic practice?” Human Pathology, vol. 47, no. 1, pp. 4–19, 2016. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png International Journal of Surgical Oncology Hindawi Publishing Corporation http://www.deepdyve.com/lp/hindawi-publishing-corporation/prognostic-impact-of-tumor-budding-on-moroccan-colon-cancer-patients-CAKDoJXawn

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Publisher: Hindawi Publishing Corporation
Copyright: Copyright © 2022 Fatima El Agy et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ISSN: 2090-1402
eISSN: 2090-1410
DOI: 10.1155/2022/9334570
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Abstract

Hindawi International Journal of Surgical Oncology Volume 2022, Article ID 9334570, 10 pages https://doi.org/10.1155/2022/9334570 Research Article Prognostic Impact of Tumor Budding on Moroccan Colon Cancer Patients 1 2 3 4 Fatima El Agy , Sanae el Bardai, Laila Bouguenouch, Nada Lahmidani, 4 5 5 5 Mohammed El Abkari, El Bachir Benjelloun, Abdelmalek Ousadden, Khalid Mazaz, 5 4 6 7 ImaneToughrai, Sidi Adil Ibrahimi, Zineb Benbrahim, and Laila Chbani Laboratory of Biomedical and Translational Research, Faculty of Medicine and Pharmacy, Sidi Mohamed Ben Abdellah University, Fez, Morocco Laboratory of Anatomic and Molecular Pathology, University Hospital Hassan II, Fez, Morocco Laboratory of Medical Genetics and Oncogenetics, University Hospital Hassan II, Sidi Mohamed Ben Abdellah University, Fez, Morocco Department of Gastroenterology, University Hospital Hassan II, Sidi Mohamed Ben Abdellah University, Fez, Morocco Department of General Surgery, University Hospital Hassan II, Sidi Mohamed Ben Abdellah University, Fez, Morocco Department of Oncology, University Hospital Hassan II, Sidi Mohamed Ben Abdellah University, Fez, Morocco Laboratory of Anatomic and Molecular Pathology, University Hospital Hassan II, Sidi Mohamed Ben Abdellah University, Fez, Morocco Correspondence should be addressed to Fatima El Agy; fatima.elagy@usmba.ac.ma Received 10 November 2021; Accepted 5 January 2022; Published 21 January 2022 Academic Editor: Gaetano Gallo Copyright © 2022 Fatima El Agy et al. &is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background. Tumor budding is now emerging as one of the robust and promising histological factors that play an important role in colon cancer. In this study, we aimed to investigate the association between tumor budding and tumor clinicopathological factors, tumor molecular signature, and patient survival for the ﬁrst time in a Moroccan population. Methods. We collected data of 100 patients operated from colon adenocarcinoma. Tumor budding was assessed on HES slides, according to the International Tumor Budding Consensus Conference 2016 recommendations. &e expression of MMR proteins was performed by immu- nohistochemistry. KRAS and NRAS mutations testing was performed by Sanger sequencing and pyrosequencing. Results. High tumor budding grade (BUD 3) was found to be signiﬁcantly associated with adverse clinicopathological features including older age (P � 0.03), presence of perineural invasion (P � 0.02), presence of vascular invasion (P � 0.05), distant metastases (P< 0.001), advanced TNM stage (P � 0.001), the occurrence of relapse (P � 0.04), and the high number of deceased cases (P � 0.02). Interestingly, we found that tumors with high-grade tumor budding were more likely to be microsatellite stable (MSS) (P � 0.005) and harbor more KRAS mutations (P � 0.02). Tumors with high-grade tumor budding were strongly associated with KRAS G12D mutation (P � 0.007). In all stages, high tumor budding was correlated with poorer overall survival (P � 0.04) and decreased relapse-free survival with a diﬀerence close to signiﬁcance ((P � 0.09). We concluded that high tumor budding was strongly associated with unfavorable clinicopathological features and special molecular biomarkers and eﬀectively aﬀects the overall survival of CC patients. Conclusions. Based on these ﬁndings and the ITBCC group recommendations, tumor budding should be taken into account along with other clinicopathologic factors in the risk assessment of colorectal cancer. occurs consistently among males than females in the general 1. Introduction population with a median age at diagnosis of 55.56 years [2]. Colon cancer is the third most commonly diagnosed cancer &e tumor node metastasis (pTNM) stage is the primary and the second leading cause of cancer-related death in the factor used for prognostication purposes and to guide pa- Moroccan population [1]. A higher incidence of this disease tient management [3]. Indeed, the standard of care for colon 2 International Journal of Surgical Oncology factors, tumor molecular signature, and patient’s survival for cancer is surgical resection (stages I and II), and surgery is followed by adjuvant chemotherapy (ﬂuorouracil and folinic the ﬁrst time in a Moroccan population. acid) for stage II with high-risk factors tumors. Neoadjuvant chemotherapy and targeted therapies (anti-EGFR) are used 2. Materials and Methods for colon cancer with distant metastasis. Although and 2.1. Patients. We enrolled in the present study a hundred because of the survival heterogeneity seen in colon cancer patients with primary colon cancer resected between 2015 patients within the same pathological stage, the introduction and 2020, at Hassan II University Hospital, Fez, Morocco. of other molecular, immunological, and histological markers &e medical charts were prospectively and retrospectively to identify risk stratiﬁcation and disease outcome is now reviewed and patients were included according to the fol- mandatory for better management of colon cancer patients. lowing inclusion criterion: patients with histologically Tumor budding is now emerging as one of the robust and conﬁrmed primary adenocarcinoma, all cases with I-IV promising histological factors that play an important role in stage colon cancer, and patients with prognostic data. Pa- colon cancer. It is a histological manifestation of initiating tients were excluded from this study due to the following invasion and metastasis cascade in the invasive front of the exclusion criteria: All patients with incomplete clinical tumor. According to the International Tumor Budding records, patients without histological conﬁrmation of colon Consensus Conference 2016, ITBCC, it is deﬁned by the adenocarcinoma, and patients with rectal cancer (Figure 1). presence of individual cells and small clusters (<5) of tumor Demographic and clinicopathological data (e.g., age, gender, cells at the invasive front of carcinomas [4]. Several studies tumor grade, tumor localization histological subtype, tumor have demonstrated that tumor budding might be associated grade, disease stage, and number of examined regional with a high risk of relapse and poorer outcomes [5]. Its lymph nodes) and follow-up data were collected from the relationship with unfavorable clinicopathological features patient’s medical records and pathology reports. like nodal and distant metastases was also demonstrated [4, 5]. Indeed, higher grade of tumor budding was reported to be signiﬁcantly correlated with microsatellite stable tu- 2.2. Pathology Analysis. After ﬁrst-line therapy, fresh mors (MSS tumors) [6, 7]. Furthermore, Sammarco et al. specimens were transported to the department of pathology. have recently demonstrated that microsatellite “stable” &e tissue was ﬁxed in formalin (10%) and embedded in BRAF-mutated tumors show more aggressive morpholog- paraﬃn (FFPE). Histological slides based on hematoxylin ical behavior like tumor budding [8]. and eosin staining were prepared and examined by a pa- Recently, a study conducted by Anne et al. on 1320 colon thologist to deﬁne the histopathological characteristics of the cancer patients has shown that high tumor budding was tumor. associated with the presence of KRAS mutations and met- astatic tumors harboring a BRAF gene mutation [9]. 2.3. Assessment of Tumor Budding. Tumor budding was Several studies have revealed the clinical implication of assessed on hematoxylin-eosin and Safran (HES) stained tumor budding in colon cancer management. First, in colon slides. For each CC case, one representative HES slide was cancer stage I, tumor budding is associated with lymph node metastasis. For this reason, patients with high tumor bud- used for scoring by the pathologist according to the ITBCC recommendations. Tumor buds were evaluated in a single ding may beneﬁt from oncologic resection [4]. Second, in stage II colon cancer, the presence of tumor budding is hotspot measuring 0.785 mm at the invasive front using microscopy at 20× objectives. associated with poorer survival. &erefore, adjuvant therapy should be discussed for stage II colon cancer patients with We then used a three-tier system which is recommended by the ITBCC group to provide tumor budding count and high-grade tumor budding [10]. &ird, the assessment of tumor budding in preoperative biopsies could be useful for tumor budding category. &e system of scoring is catego- rized as follows: selecting patients who may qualify for neoadjuvant therapy. However, in advanced colon cancer, the role of tumor (i) 0–4 buds: low budding (Bd 1) budding in clinical practice remains unclear and requires (ii) 5–9 buds: intermediate budding (Bd 2) more investigation [11]. &e lack of a standard quantiﬁcation method for tumor (iii) 10 or more buds: high budding (Bd 3) budding has limited its reporting in the clinical routine We grouped the patients to be low-intermediate (grade practice in CC as well as other histological factors. However, 1 + grade 2) and high tumor budding (grade 3). after the International Tumor Budding Consensus Con- ference (ITBCC), held in Bern in April 2016, a scoring system for assessing tumor budding has been reached 2.4. Determination of Mismatch Repair Protein Expression. according to conference recommendations [4]. &e ITBCC &e immunohistochemistry (IHC) method was used to group recommended that tumor budding should be in- establish the mismatch repair tumor status (MSS or MSI) cluded in guidelines/protocols and staging systems for the and to detect the intact or the loss expression of the MMR pathology reporting of colorectal cancer [4]. proteins (MLH1, PMS2, MSH2, and MSH6). &e IHC study &is study aimed to assess tumor budding according to was performed on unstained formalin-ﬁxed paraﬃn-em- the ITBCC recommendations and to investigate the associ- bedded (FFPE) tumor tissue sections of 5 μm thickness, on ation between tumor budding and tumor clinicopathological the automated immunostainer Ventana Benchmark International Journal of Surgical Oncology 3 PCR Master Mix 2x, 2.5 µl of Coral Load Concentrate 10x, 239 patients with colorectal cancer (I-IV) 4 µl of nuclease-free water, and 1 µl of the corresponding set of PCR primers (Qiagen). 10 µl of PCR products was immobilized to Streptavidin Sepharose High-Performance 104 patients excluded: Patients with rectum cancer beads (Qiagen) to prepare the single-stranded DNA. &e corresponding sequencing primers were allowed to anneal to the DNA using a PyroMark Q24 plate and a vacuum 135 colon cancer workstation (Qiagen). PyroMark Q24 reagents (enzyme mixture, substrate mixture, and nucleotide all from 18 patients excluded: No primary tumor resection Qiagen) were prepared and loaded into a cartridge to be dispensed during the sequencing process. Finally, the 17 patients excluded: sequences were analyzed using PyroMark Q24 software in Patients with incomplete histopathological records the AQ analysis mode. In each run, two controls were included: negative control (without template DNA) and 100 patients included in analysis an unmethylated control DNA, provided by the kit as a positive control for PCR, and sequencing reactions were Figure 1: Flow diagram for the study. included. ULTRA. We have employed monoclonal antibodies speciﬁc for each MMR protein, MLH1 (G168-728/CELL MAR- 2.6. Statistical Analysis. Clinical, pathological, and molec- QUE), MSH2 (G219-1129/CELL MARQUE), MSH6 (44/ ular variables collected at baseline were described as means CELL MARQUE), and PMS2 (MRQ-28/CELL MARQUE). and standard deviation (sd’s) for quantitative variables and Adjacent normal tissue (lymphocytes or normal glandular percentages for qualitative variables. Associations between cells) was used as an internal control for positive staining. tumor budding (assessing as a categorical variable) and categorical factors of tumor were assessed using the χ2-test or Fisher’s exact test variables. &e unpaired t-test was used 2.5. Detection of KRAS and NRAS Mutation for continuous variables. Tests were statistically signiﬁcant when P< 0.05. 2.5.1. DNA Extraction. Genomic DNA was extracted from 5 Overall survival was deﬁned as the time from the start of to 8 sections of 5 μm thickness of macrodissected formalin- diagnosis until death or until the last follow-up. Relapse-free ﬁxed paraﬃn-embedded (FFPE) tumor blocks, containing at survival was measured from the date of initial diagnosis until least 50% of tumor cells, as determined by an experienced the date of local relapse or regional relapse or last follow-up/ pathologist on H&E-stained paraﬃn slides. &e extraction death (all causes) whichever occurs ﬁrst. was eﬀected using the QIAamp DNA FFPE Tissue Kit RFS and OS rates according to tumor budding, clini- (Invitrogen) and according to the manufacturer’s instruc- copathological factors, and molecular features were deter- tions. DNA concentration (ng/ul) was assessed by Qubit mined using the Kaplan-Meier method, and survival ﬂuorometer. diﬀerences between groups were evaluated by log-rank test. Multivariate analysis was performed using a Cox pro- 2.5.2. PCR and Direct Sequencing. For each sample, mu- portional hazard model to identify independent risk factors tations of KRAS exons 2, 3, and 4 and NRAS exons 2 and 3 for survival. Factors that were signiﬁcant and nearly sig- were ampliﬁed by polymerase chain reaction (PCR). Brieﬂy, niﬁcant in univariate analysis (P< 0.1) were included in 10 ng of template DNA was ampliﬁed using 12× PCR mix multivariate analysis. platinum, 12.5 pmol primers, 50 μmol Mgcl , and 2.5 μl of Data from univariate and multivariate analyses were the corresponding set of PCR primers listed in Table 1. After reported as hazard ratios (HRs) with 95% conﬁdence in- the puriﬁcation of PCR products, the presence of mutations tervals (CIs). All statistics were assessed using 2-sided tests, was detected by direct sequencing using the BigDye Ter- with P values <0.05 considered statistically signiﬁcant. minator V3.1 Cycle Sequencing Kit (ABI Prism) and the Statistical analysis was performed using the IBM SPSS Applied Biosystems 3500Dx Genetic Analyzer (Applied Statistics 21. Biosystem). 3. Results 2.5.3. Pyrosequencing. &e analysis of RAS mutations was performed using the &eraScreen KRAS Pyro Kit (for 3.1. Patient Demographics and Pathological Characteristics. KRAS codons 12 and 13) and the &eraScreen RAS Ex- Patients and tumor characteristics of 100 patients are tension Pyro Kit (for KRAS codons 59/61, 117, and 146 and summarized in Table 2. Among 100 cases, 43 (43.0%) were NRAS codons 12, 13, 59, 61, 117, and 146) (Qiagen, Ger- women and 57 (57.0%) were men with a mean age of 54.9 many), according to the manufacturer’s instructions. As years. Our cohort was characterized by a predominance of described previously [2], 5 µl of template DNA (2–10 ng of the left-sided colon cancers (n � 62, 62.0%), compared to genomic DNA) was ampliﬁed by polymerase chain reaction right-sided CC (n � 38, 38.0%). Histologically, the adeno- (PCR) in a 20 µl volume containing 12.5 µl of PyroMark carcinoma subtype was documented in 86 tumors (86.0%), ® 4 International Journal of Surgical Oncology Table 1: Primer sequences used for PCR. Primer name Primer sequence KRAS-ex 2- F 5′-GGTGGAGTATTTGATAGTGTA- 3′ KRAS-ex 2- R 5′-TGCATATTACTGGTGCAGACC- 3′ KRAS-ex 3- F 5′-AGTAAAAGGTGCACTGTAATAA-3′ KRAS-ex 3- R 5′-ATAATAAGCTGACATTAAGGAG-3′ KRAS-ex 4- F 5′-TGTTACTAATGACTGTGCTATAACTTTT-3′ KRAS-ex 4- R 5′-TATGCTATACTATACTAGGAAATAAAA-3′ NRAS-ex2-F 5′-ATGACTGAGTACAAACTGGTGGTGGTTGGAGCA-3′ NRAS-ex2-R 5′-CACTTTGTAGATGAATATGATCCCACCATAGAG-3′ NRAS-ex3-F 5′-GATTCTTACAGAAAACAAGTGGTTA-3′ NRAS-ex3-R 5′-CATTTGCGGATATTAACCTCTACAG-3′ NRAS-ex4-F 5′-GGAGCAGATTAAGCGAG-3′ NRAS-ex4-R 5′-TCAGCCAAGACCAGACAG-3′ while only 9 (9.0%) tumors were mucinous adenocarcinoma. tumors showed high-grade tumor budding (grade 3). &e 46 (46.0%) tumors were classiﬁed as grade 2 (moderately results are shown in Table 4. diﬀerentiated) and grade 1 (well-diﬀerentiated), and 8 (8.0%) In correlation analysis, we divided the patients into two tumors were classiﬁed as grade 3 (poorly diﬀerentiated). groups, a group with low-grade tumor budding (grade Perineural invasion was observed in 15 (15.3%) tumors. In this 1 + grade 2) that represented 60% of all cases and a group study, the mean number of removed lymph nodes was 20.8 with high-grade tumor budding (grade 3) that represented (range, 1–57). 85 (87.6%) patients had more than 12 dissected 40% of all cases (Table 4). LN. Positive LNs were identiﬁed in 32 (32.6%) patients (mean � 1.3; rang, 0.1–16). According to the TNM classiﬁca- 3.5. Relationship between Tumor Budding and Clinicopath- tion, 4 (4.2%) of the tumors were stage I, 51 (53.1%) stage II, 25 ological Features. Table 5 shows the results of the association (26.0%) stage III, and 16 (16.7%) IV. In our cohort, 50% of between tumor budding and clinicopathologic factors. Tumor patients have received surgical treatment, and 46% have re- budding grades were signiﬁcantly associated with age, peri- ceived adjuvant chemotherapy. Neoadjuvant treatment was neural invasion, vascular invasion, distant metastases, TNM indicated for only 4% of cases. stage, the occurrence of relapse, and the number of deceased &e mean follow-up time of the patient’s OS was 49.4 cases. Indeed, compared with patients with low-grade tumor months (range, 6–119 months). Among 100 patients, 18 budding (grade 1 + grade 2), patients with high-grade tumor (18.2%) cases of death were recorded. Recurrence was ob- budding (grade 3) had more vascular invasion (28.2% vs. served in 29 (29.3%) patients. &e most frequent site of 15.3%; P � 0.05), more venous invasion (25.6% vs. 8.5%; recurrence was local recurrence (31.0%) followed by peri- P � 0.02), and a higher number of distant metastases (33.3% toneums (24.1%), lung (20.7%), and liver (17.4%). vs. 5.0%; P< 0.001). Also, these patients had a majority of advanced pathologic stage IV tumors (P � 0.001). Interestingly, patients with high-grade tumor budding 3.2. Molecular Features. Concerning molecular character- had signiﬁcantly high relapse rates (P � 0.04). Moreover, istics, the MSI tumors were found in 21 patients (21.0%), peritoneal recurrence was the most frequent recurrence site tumors with KRAS mutations in 31 patients (40.3%), and in tumors with high-grade tumor budding, with a diﬀerence NRAS mutations in 2 patients (2.6%). close to signiﬁcance (P � 0.07). Tumors with high-grade tumor budding were correlated with a higher risk of death (P � 0.02). 3.3. KRAS and NRAS Mutations Classes. All the basic data &ere was no correlation between tumor budding grade are presented in Table 3. Activating KRAS mutations were and gender, tumor localization, histologic subtype, histo- found in 31/77 examined tumor cases (40.3%). Among logic grade, and lymph nodes count. KRAS variants, G12D was the most frequent (11/31, 35.5%), followed by G13D (8/31, 25.8%), G12C (4/31, 12.9%), A146T 3.6. Tumor Budding and Molecular Biomarkers. (3/31, 9.7%), G12V (2/31, 6.5%), G12R (1/31, 3.2%), G12A (1/31, 3.2%), and G13V (1/31, 3.2%). Interestingly, we investigated the relationship between the diﬀerent grades of tumor budding and the molecular Out of 77 cases, two showed NRAS mutations (2/77, 2.6%). One mutation was detected in codon 12 (G12R) (1/2, characteristics of the tumor. &e diﬀerent results are shown in Table 6. According to our results, tumors with high-grade 50%) and the other in codon 61 (Q61L) (1/2, 50%). tumor budding were more likely to be microsatellite stable (MSS) (P � 0.005). &e mutation rate in the KRAS gene was signiﬁcantly 3.4. Incidence of Tumor Budding in Our Population. Among 100 CC cases, 28 (28%) tumors showed low-grade higher in the high-grade TB tumors compared to that in the low-grade TB tumors (P � 0.02). Tumors with KRAS codon tumor budding (grade 1), 32 (32%) tumors showed inter- mediate-grade tumor budding (grade 2), and 40 (40%) 12 mutations tented to have high tumor budding with a International Journal of Surgical Oncology 5 Table 2: Patient demographics, pathological, and molecular Table 2: Continued. features. Characteristics Total (%) Characteristics Total (%) Mortality Age Death cases 18 (18.2%) ≤57 53 (53.0%) Censored cases 81 (81.8%) ≥57 47 (47.0%) MSI status Gender MSS 79 (79.0%) Female 43 (43.0%) MSI 21 (21.0%) Male 57 (57.0%) KRAS mutation Tumor site Presence 31 (40.3%) Right colon 38 (38.0%) Absence 46 (59.7%) Left colon 62 (62.0%) NRAS mutation Histological subtype Presence 2 (2.6%) Adenocarcinoma 86 (86.0%) Absence 75 (97.4%) Mucinous adenocarcinoma 9 (9.0%) Others 5 (5.0%) Histological grade Table 3: &e frequencies of genetic alteration classes. Well 46 (46.0%) Moderate 46 (46.0%) Mutations Number % Poor 8 (8.0%) KRAS 31 40.3 Venous invasion Codon 12 19 61.3 Presence 20 (20.4%) G12D 11 35.5 Absence 78 (79.6%) G12C 4 12.9 Perineural invasion G12V 2 6.5 Presence 15 (15.3%) G12A 1 3.2 Absence 83 (84.7%) G12R 1 3.2 Number of removed lymph nodes Codon 13 9 29.0 Mean (±SD) 20.8 (±10.8) G13D 8 25.8 <12 12 (12.4%) G13V 1 3.2 ≥12 85 (87.6%) Codon 146 3 9.7 Range 1–57 A146T 3 9.7 Positive lymph node NRAS 2 2.6 Mean (±SD) 1.3 (±2.9) Codon 12 1 50 Presence 32 (8.6%) G12R 1 50 Absence 66 (32.7%) Codon 61 1 50 Average 0.1–16 Q61L 1 50 Lymph node ratio Mean (±SD) 0.1 (±0.14) Range 0.01–0.81 Table 4: Incidence of tumor budding. Distant metastases (M) Tumor budding grades Frequency (%) M0 83 (83.8%) M1 16 (16.2%) Grade 1 (low) 28 (28%) Disease stages Grade 2 (intermediate) 32 (32%) I 4 (4.2%) Grade 3 (high) 40 (40%) II 51 (53.1%) Low-grade tumor budding 60 (60%) III 25 (26.0%) High-grade tumor budding 40 (40%) IV 16 (16.7%) Treatment diﬀerence close to signiﬁcance, as compared with tumors Surgery 50 (50.0%) harboring other KRAS codon mutations (P � 0.05). Surgery + adjuvant chemotherapy 46 (46.0%) Moreover, KRAS G12D mutation was found to be sig- Neoadjuvant therapy 4 (4.0%) niﬁcantly correlated with high-grade TB compared to the Follow-up time (months) other KRAS codon 12 variants (P � 0.007). However, Mean (SD) 49.4 (±29.3) Range 6–119 there was no correlation between KRAS codon 13 variants Recurrence and tumor budding grade. (+) 29 (29.3%) &ere was no signiﬁcant association between tumor (−) 70 (70.7%) budding and NRAS status. Recurrence patterns Liver 5 (17.4%) Lung 6 (20.7%) 3.7. Survival Outcomes according to Tumor Budding. Peritoneum 7 (24.1%) Table 7 shows associations of tumor budding with Local recurrence 9 (31.0%) overall survival and relapse-free survival of CC patients Others 2 (6.8%) when tumor budding is stratiﬁed into three groups 6 International Journal of Surgical Oncology Table 5: Association between tumor budding and clinicopathological features. Variables Low-grade tumor budding High-grade tumor budding P value Age <57 36 (60.0%) 17 (42.5%) 0.03 ≥57 24 (40.0%) 23 (57.5%) Genre Female 28 (46.7%) 15 (37.5%) 0.2 Male 32 (53.3%) 25 (62.5%) Tumor site Right colon 22 (36.7%) 16 (40.0%) 0.4 Left colon 38 (63.3%) 24 (60.0%) Histologic subtype Adenocarcinoma 54 (90.0%) 32 (80.0%) 0.2 Mucinous 3 (5.0%) 6 (15.0%) Others 3 (5.0%) 2 (5.0%) Histologic grade Well 28 (46.7%) 18 (45.0%) 0.5 Moderate 26 (43.3%) 20 (50.0%) Poor 6 (10.0%) 2 (5.0%) Venous invasion Presence 9 (15.3%) 11 (28.2%) 0.05 Absence 50 (84.7%) 28 (71.8%) Perineural invasion Presence 5 (8.5%) 10 (25.6%) 0.02 Absence 54 (91.5%) 29 (74.4%) Number of removed lymph nodes Mean (SD) 21.7 (±8.5) 19.2 (±6.9) 0.2 ˂12 5 (8.6%) 7 (17.9%) 0.1 ˃12 53 (91.4%) 32 (82.1%) Positive lymph node Mean (SD) 1.2 (±0.0) 1.4 (±2.9) 0.8 Presence 19 (32.2%) 13 (33.3%) 0.5 Absence 40 (67.8%) 26 (66.7%) RGL Mean (SD) 0.06 (±0.03) 0.063 (±0.2) 0.9 Synchronous metastasis (M) M0 57 (95.0%) 26 (66.7%) ˂0.001 M1 3 (5.0%) 13 (33.3%) Disease stages I 2 (3.3%) 2 ((5.0%) II 37 (61.7%) 15 (37.5%) 0.001 III 18 (30.0%) 9 (22.5%) IV 3 (5.0%) 14 (35.0%) Follow-up time (months) 27.0 (±19.3) 24.8 (±13.3) 0.7 Recurrence (+) 14 (23.3%) 15 (38.5%) 0.04 (−) 46 (76.7%) 24 (61.5%) Recurrence patterns Liver 2 (14.3%) 3 (20%) 0.07 Lung 3 (21.4%) 3 (20%) Peritoneum 2 (14.3%) 5 (33.3%) Local recurrence 6 (42.9%) 3 (20%) Others 1 (7.1%) 1 (6.7%) Mortality Death cases 7 (11.7%) 11 (28.2%) 0.02 Censored cases 53 (88.3%) 28 (71.8%) (BD1, BD2, BD3) or two groups (low (BD1 + 2), high tumors with high-grade tumor budding were signiﬁcantly (BD3)). correlated with shorter OS (P � 0.04; Figure 2(a)). In the three-tier analysis, tumor budding was not Moreover, these tumors tended to be associated with associated with OS and RFS (BD1 versus BD2 versus shorter RFS with a diﬀerence close to signiﬁcance BD3). In the 2-tier approach (BD1 + 2 versus BD3), (P � 0.09; Figure 2(b)). International Journal of Surgical Oncology 7 Table 6: Association between tumor budding and molecular biomarkers. Variables Low-grade tumor budding High-grade tumor budding P value MSI status MSS 42 (70.0%) 37 (92.5%) 0.005 MSI 18 (30.0%) 3 (7.5%) KRAS status Mutant 14 (23.3%) 17 (42.5%) 0.02 Wild-type 46 (76.7%) 23 (57.5%) KRAS codon types Codon 12 7 (50%) 12 (70.6%) 0.05 Codon 13 5 (35.7%) 4 (23.5%) Codon 146 2 (14.3%) 1 (5.9%) KRAS codon 12 variants G12D 1 (14.3%) 10 (83.3%) 0.007 G12C 2 (28.6%) 2 (16.7%) G12V 2 (28.6%) 0 (0.0%) G12A 1 (14.3%) 0 (0.0%) G12R 1 (14.3%) 0 (0.0%) KRAS codon 13 variants G13D 4 (80%) 4 (100%) 0.3 G13V 1 (20%) 0 (0.0%) NRAS gene status Mutant 1 (1.7%) 1 (2.5%) 0.4 Wild-type 59 (98.3%) 59 (97.5%) Table 7: Analysis of OS and RFS according to tumor budding. Tumor budding Mean OS month (95% CI) P value Mean RFS month (95% CI) P value BD1 85.7 (76.9–94.5) 0.1 88.7 (69.7–107.7) 0.1 BD2 100.5 (84.0–116.9) 108.4 (90.8–126.1) BD3 82.6 (67.2–97.9) 81.4 (62.7–100.1) High 82.5 (67.2–97.8) 0.04 81.4 (62.7–100.1) 0.09 Low 104.2 (94.0–114.4) 99.4 (80.9–103.6) associations have fueled the hypothesis that tumor buds can 4. Discussion pervade the extracellular matrix (ECM) and migrate and &e present study was designed to investigate the rela- disseminate into blood vessels [16]. It was suggested that tionship of tumor budding with the clinicopathological tumor budding harbors the properties of cells undergoing an characteristics and molecular biomarkers of CC. Also, we epithelial-mesenchymal transition (EMT) or a partial-EMT evaluate the prognostic impact of tumor budding on hun- state [16]. In this process, epithelial cells lose intracellular dred CC patients using the ITBCC scoring method of TB on and cell-matrix contacts mediated by E-cadherin, leading to HES slides for the ﬁrst time in the Moroccan population and invasion and metastatic cancer spread [17]. the Middle East and Nord Africa region. Additionally, we observed a signiﬁcant correlation be- In recent years, several reports showed that tumor tween high-grade tumor budding and advanced TNM stage, budding is characterized by diﬀerent clinicopathological and which further supports the results of previous studies as histological features. In this study, we were able to dem- reported by Van et al. [18]. onstrate that high-grade tumor budding (BD3) grade un- Interestingly, our results indicate that patients exhibiting derlines special clinicopathological parameters. In our high-grade tumor budding had a higher rate of recurrence. context, BD3 was signiﬁcantly greater in older age patients In a study conducted on 138 patients, Tanaka et al. [19] and we are the ﬁrst to report this result because none of the reported that tumor budding was signiﬁcantly associated previous studies have found a signiﬁcant association be- with disease recurrence. Another study evaluated 200 pa- tween age and tumor budding grades [5, 12, 13]. tients with CC and reported the same result [20]. Okuyama As reported in many studies [5, 10, 14], we documented a et al. showed a statistically signiﬁcant relationship between positive relationship between high-grade tumor budding, high-grade tumor budding and local recurrence [21]. the presence of vascular invasion, and the presence of &e majority of studies investigating the relationship perineural invasion. Furthermore, we found that tumors between tumor budding and clinicopathological features with high-grade tumor budding were signiﬁcantly charac- have documented its positive correlation with lymph node terized by an increased frequency of distant metastases. &e involvement [22]. Conversely, in our study, we did not ﬁnd same result was reported by Jayasinghe et al. [15]. &ese any association. 8 International Journal of Surgical Oncology 1.0 1.0 0.8 0.8 0.6 0.6 0.4 0.4 0.2 0.2 0.0 0.0 0.0 20.0 40.0 60.0 80.0 100.0 120.0 0.0 25.0 50.0 75.0 100.0 125.0 Time (month) Time (month) Budding Budding Low Low High High (a) (b) Figure 2: Survival curves of CC patients stratiﬁed by budding grades. (a) Overall survival in all patients stratiﬁed by BD1 + 2 (low) versus BD3 (high). (b) Relapse-free survival in all patients stratiﬁed by BD1 + 2 (low) versus BD3 (high). As a point of interest, we also investigated the association According to our results, it can be reported that high between tumor budding and molecular biomarkers, such as grade of tumor budding is generally associated with poor MSI status, KRAS, and NRAS mutations. We observed that prognostic factors (vascular and perineural invasion, distant high-grade tumor budding was more common in MSS tu- metastases, MSS status, and KRAS mutations). mors, which is consistent with ﬁndings reported by previous Secondly, we aimed to evaluate the relationship be- studies [12, 23, 24]. In addition, Lugli et al. have recently tween tumor budding and clinical outcome in CC pa- tients, for the ﬁrst time in the Middle East and North showed that tumor buds are infrequently found in colorectal cancers with microsatellite instability (MSI) [7]. It was Africa region. We validated the prognostic eﬀect of tumor budding on overall survival in our cohort using the newly showed that MSS tumors were signiﬁcantly correlated with shorter overall survival of CC patients [25]. established ITBCC criteria for the scoring of tumor We found that tumors with high-grade tumor budding budding on H&E slides. We found that OS was better in had signiﬁcantly more KRAS mutations. &e relationship patients with low-grade tumor budding (BD1/2 versus between KRAS mutation and tumor budding has previously BD3) at all stages; we also observed that high-grade tumor been reported [26, 27]. Jang and colleagues found that 61.8% budding was linked to an increased risk of death. &is of colorectal cancers with high-grade tumor budding harbor method is already included in the Japanese Guidelines for more KRAS mutations [28]. Recently, Trinh et al. [9] and the reporting of CRC. However, many reports suggest that Lugli et al. have reported the same results [7]. Similar to Jang whichever scoring method is utilized, the presence of high-grade tumor budding is correlated with worse et al. [28], we found that the G12D substitution in the KRAS gene was strongly associated with high-grade tumor budding. clinical outcomes [10, 29, 30]. In the literature, several studies are investigating the According to this result, the KRAS G12D mutation could be proposed as a high-grade tumor budding biomarker. impact of tumor budding on CC patient’s survival. Similar to Of note, our group has previously reported KRAS our results, Oh and colleagues pooled results from more mutations as a predictor factor of worse OS [2]. than 4000 Japanese patients from all stages and conﬁrmed We did not ﬁnd any association between NRAS status the positive association of high-grade budding with worse and tumor budding grade in our context. Considering we OS [31]. Trinh et al. also validated the prognostic impact of found only 2 patients harboring NRAS mutations, this tumor budding independent of age, stage, and sex in a cohort ﬁnding does not yet allow us to draw ﬁrm conclusions. including 1320 colorectal cancers [9]. Barresi et al. were also limited by a small number of NRAS Regarding the association between tumor budding and RFS, we observed that patients with high tumor budding had mutated cases (N � 4) and they did not ﬁnd any correlation between NRAS status and tumor budding grade [27]. a shorter RFS but with a diﬀerence close to signiﬁcant Overall survival Relapse free survival International Journal of Surgical Oncology 9 although several reports have conﬁrmed this correlation Authors’ Contributions with signiﬁcant diﬀerences [31, 32]. All authors read and approved the ﬁnal manuscript. A preprint has previously been published [33]. &ere were some limitations to the present study. First is the size of the study cohort. Indeed, the number of cases References included in our study is relatively small, in comparison with some previous reports. Second, our study represents a single [1] H. Sung, J. Ferlay, R. L. Siegel, M. Laversanne, I. Soerjomataram, and A. Jemal, “Global cancer statistics 2020: institution and thus carries the possibility of selection bias GLOBOCAN estimates of incidence and mortality worldwide and does not allow us to generalize our results in the overall for 36 cancers in 185 countries,” CA: A Cancer Journal for population of our country. &ird, it has been demonstrated Clinicians, vol. 71, no. 3, pp. 209–249, 2021. that high-grade tumor budding is signiﬁcantly associated [2] F. El Agy, S. El Bardai, I. El Otmani, Z. Benbrahim, with lymph node metastasis in colon cancer. However, we I. M. H. Karim, and K. Mazaz, “Mutation status and prog- were unable to produce similar results. &is could be likely nostic value of KRAS and NRAS mutations in Moroccan attributable to the size and the characteristics of our sample. colon cancer patients: a ﬁrst report,” PLoS One, vol. 16, no. 3, Above all, our study is the ﬁrst report investigating the prognostic impact on colon cancer patients in the Middle [3] C. Zhang, Z. Mei, J. Pei, M. Abe, X. Zeng, and Q. Huang, “A East and Nord Africa region. modiﬁed tumor-node-metastasis classiﬁcation for primary operable colorectal cancer,” JNCI Cancer Spectrum, vol. 5, 5. Conclusions [4] A. Lugli, R. Kirsch, Y. Ajioka et al., “Recommendations for reporting tumor budding in colorectal cancer based on the In this study, we concluded that high-grade tumor budding International Tumor Budding Consensus Conference was strongly associated with unfavorable clinicopathological (ITBCC) 2016,” Modern Pathology, vol. 30, no. 9, features, like a perineural invasion, venous invasion, and pp. 1299–1311, 2017. distant metastases, and special molecular biomarkers which [5] H. C. van Wyk, J. H. Park, J. Edwards, P. G. Horgan, are MSS status and KRAS mutations. We deﬁned KRAS D. C. McMillan, and J. J. Going, “&e relationship between G12D mutation as a biomarker of high-grade tumor tumour budding, the tumour microenvironment and survival budding. in patients with primary operable colorectal cancer,” British Our results also indicate that high-grade tumor budding Journal of Cancer, vol. 115, no. 2, pp. 156–163, 2016. eﬀectively aﬀects the overall survival of CC patients. Based [6] I. Centeno, A. Paasinen Sohns, M. Flury et al., “DNA proﬁling of tumor buds in colorectal cancer indicates that they have the on these ﬁndings and the ITBCC group recommendations, same mutation proﬁle as the tumor from which they derive,” tumor budding should be taken into account along with Virchows Archiv, vol. 470, no. 3, pp. 341–346, 2017. other clinicopathologic factors in the risk assessment of [7] A. Lugli, I. Zlobec, M. D. Berger, R. Kirsch, and colorectal cancer. I. D. Nagtegaal, “Tumour budding in solid cancers,” Nature Reviews Clinical Oncology, vol. 18, no. 2, pp. 101–115, 2021. Data Availability [8] G. Sammarco, G. Gallo, G. Vescio et al., “Mast cells, microRNAs and others: the role of translational research on &e datasets used/or analyzed during the current study are colorectal cancer in the forthcoming era of precision medi- available from the corresponding author on reasonable cine,” Journal of Clinical Medicine, vol. 3, no. 9, p. 2852, 2020. request. [9] A. Trinh, C. Ladrach, ¨ H. E. Dawson et al., “Tumour budding is associated with the mesenchymal colon cancer subtype and Ethical Approval RAS/RAF mutations: a study of 1320 colorectal cancers with Consensus Molecular Subgroup (CMS) data,” British Journal &is study protocol was reviewed and approved by Hassan II of Cancer, vol. 119, no. 10, pp. 1244–1251, 2018. [10] A. C. Rogers, D. C. Winter, A. Heeney et al., “Systematic University Hospital Ethics Committee of Fez, Morocco, review and meta-analysis of the impact of tumour budding in under reference no. 13/18 according to the World Medical colorectal cancer,” British Journal of Cancer, vol. 115, no. 7, Association Declaration of Helsinki. pp. 831–840, 2016. [11] I. Zlobec and A. Lugli, “Tumour budding in colorectal cancer: Consent molecular rationale for clinical translation,” Nature Reviews Cancer, vol. 18, no. 4, pp. 203-204, 2018. All patients gave verbal informed consent before the start of [12] R. P. Graham, R. A. Vierkant, L. S. Tillmans et al., “Tumor the study. budding in colorectal carcinoma: conﬁrmation of prognostic signiﬁcance and histologic cutoﬀ in a population-based co- Disclosure hort,” =e American Journal of Surgical Pathology, vol. 39, no. 10, pp. 1340–1346, 2015. &e authors alone are responsible for the content and [13] M. Karlberg, K. Stenstedt, M. Hallstrom, ¨ P. Ragnhammar, writing of the paper. C. Lenander, and D. Edler, “Tumor budding versus mismatch repair status in colorectal cancer - an exploratory analysis,” Anticancer Research, vol. 38, no. 8, pp. 4713–4721, 2018. Conflicts of Interest [14] H. Dawson, F. Galuppini, P. Trager ¨ et al., “Validation of the &e authors declare no conﬂicts of interest. International Tumor Budding Consensus Conference 2016 10 International Journal of Surgical Oncology recommendations on tumor budding in stage I-IV colorectal [30] R. Cappellesso, C. Luchini, N. Veronese et al., “Tumor cancer,” Human Pathology, vol. 85, pp. 145–151, 2019. budding as a risk factor for nodal metastasis in pT1 colorectal [15] C. Jayasinghe, N. Simiantonaki, and C. J. Kirkpatrick, “His- cancers: a meta-analysis,” Human Pathology, vol. 65, pp. 62–70, 2017. topathological features predict metastatic potential in locally advanced colon carcinomas,” BMC Cancer, vol. 15, no. 1, p. 14, [31] B. Y. Oh, Y. A. Park, J. W. Huh et al., “Prognostic impact of 2015. tumor-budding grade in stages 1-3 colon cancer: a retro- [16] A. D. Grigore, M. K. Jolly, D. Jia, M. C. Farach-Carson, and spective cohort study,” Annals of Surgical Oncology, vol. 25, H. Levine, “Tumor budding: the name is EMT. Partial EMT,” no. 1, pp. 204–211, 2018. Journal of Clinical Medicine, vol. 5, no. 5, 2021. [32] F. Petrelli, E. Pezzica, M. Cabiddu et al., “Tumour budding [17] A. Lugli, E. Karamitopoulou, and I. Zlobec, “Tumour bud- and survival in stage II colorectal cancer: a systematic review ding: a promising parameter in colorectal cancer,” British and pooled analysis,” Journal of Gastrointestinal Cancer, Journal of Cancer, vol. 106, no. 11, pp. 1713–1717, 2012. vol. 46, no. 3, pp. 212–218, 2015. [18] H. C. van Wyk, J. Park, C. Roxburgh, P. Horgan, A. Foulis, [33] F. E. agy, S. E. bardai, L. Bouguenouch, N. Lahmidani, and D. C. McMillan, “&e role of tumour budding in pre- M. E. abkari, and B. E. bachir, “Prognostic impact of tumor dicting survival in patients with primary operable colorectal budding on moroccan colon cancer patients,” 2021, https:// cancer: a systematic review,” Cancer Treatment Reviews, www.researchsquare.com/article/rs-546470/v1. vol. 41, no. 2, pp. 151–159, 2015. [19] M. Tanaka, Y. Hashiguchi, H. Ueno, K. Hase, and H. Mochizuki, “Tumor budding at the invasive margin can predict patients at high risk of recurrence after curative surgery for stage II, T3 colon cancer,” Diseases of the Colon & Rectum, vol. 46, no. 8, pp. 1054–1059, 2003. [20] T. Nakamura, H. Mitomi, H. Kanazawa, Y. Ohkura, and M. Watanabe, “Tumor budding as an index to identify high- risk patients with stage II colon cancer,” Diseases of the Colon & Rectum, vol. 51, no. 5, pp. 568–572, 2008. [21] T. Okuyama, M. Oya, and H. Ishikawa, “Budding as a useful prognostic marker in pT3 well- or moderately-diﬀerentiated rectal adenocarcinoma,” Journal of Surgical Oncology, vol. 83, no. 1, pp. 42–47, 2003. [22] A. Mehta, M. Goswami, R. Sinha, and A. Dogra, “Histo- pathological signiﬁcance and prognostic impact of tumor budding in colorectal cancer,” Asian Paciﬁc Journal of Cancer Prevention: Asian Paciﬁc Journal of Cancer Prevention, vol. 19, no. 9, pp. 2447–2453, 2018. [23] J. R. Jass, M. Barker, L. Fraser, M. D. Walsh, V. L. J. Whitehall, and B. Gabrielli, “APC mutation and tumour budding in colorectal cancer,” Journal of Clinical Pathology, vol. 56, no. 1, pp. 69–73, 2003. [24] I. Zlobec, M. P. Bihl, A. Foerster, A. Ruﬂe, and A. Lugli, “&e impact of CpG island methylator phenotype and micro- satellite instability on tumour budding in colorectal cancer,” Histopathology, vol. 61, no. 5, pp. 777–787, 2012. [25] F. El Agy, I. E. Otmani, A. Mazti, N. Lahmidani, A. Oussaden, and M. El Abkari, “Implication of microsatellite instability pathway in outcome of colon cancer in Moroccan pop- ulation,” Disease Markers, vol. 2019, Article ID 3210710, 10 pages, 2019. [26] F. Prall and C. Ostwald, “High-degree tumor budding and podia-formation in sporadic colorectal carcinomas with K-ras gene mutations,” Human Pathology, vol. 38, no. 11, pp. 1696–1702, 2007. [27] V. Barresi, L. Reggiani Bonetti, and S. Bettelli, “KRAS, NRAS, BRAF mutations and high counts of poorly diﬀerentiated clusters of neoplastic cells in colorectal cancer: observational analysis of 175 cases,” Pathology, vol. 47, no. 6, pp. 551–556, [28] S. Jang, M. Hong, M. K. Shin et al., “KRAS and PIK3CA mutations in colorectal adenocarcinomas correlate with ag- gressive histological features and behavior,” Human Pathol- ogy, vol. 65, pp. 21–30, 2017. [29] V. H. Koelzer, I. Zlobec, and A. Lugli, “Tumor budding in colorectal cancer-ready for diagnostic practice?” Human Pathology, vol. 47, no. 1, pp. 4–19, 2016.

Journal

International Journal of Surgical Oncology – Hindawi Publishing Corporation

Published: Jan 21, 2022

References

Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries,

H. Sung, J. Ferlay, R. L. Siegel, M. Laversanne, I. Soerjomataram, and A. Jemal
Mutation status and prognostic value of KRAS and NRAS mutations in Moroccan colon cancer patients: a first report,

F. El Agy, S. El Bardai, I. El Otmani, Z. Benbrahim, I. M. H. Karim, and K. Mazaz
A modified tumor-node-metastasis classification for primary operable colorectal cancer,

C. Zhang, Z. Mei, J. Pei, M. Abe, X. Zeng, and Q. Huang
Recommendations for reporting tumor budding in colorectal cancer based on the International Tumor Budding Consensus Conference (ITBCC) 2016,

A. Lugli, R. Kirsch, Y. Ajioka et al.
The relationship between tumour budding, the tumour microenvironment and survival in patients with primary operable colorectal cancer,

H. C. van Wyk, J. H. Park, J. Edwards, P. G. Horgan, D. C. McMillan, and J. J. Going
DNA profiling of tumor buds in colorectal cancer indicates that they have the same mutation profile as the tumor from which they derive,

I. Centeno, A. Paasinen Sohns, M. Flury et al.
Tumour budding in solid cancers,

A. Lugli, I. Zlobec, M. D. Berger, R. Kirsch, and I. D. Nagtegaal
Mast cells, microRNAs and others: the role of translational research on colorectal cancer in the forthcoming era of precision medicine,

G. Sammarco, G. Gallo, G. Vescio et al.
Tumour budding is associated with the mesenchymal colon cancer subtype and RAS/RAF mutations: a study of 1320 colorectal cancers with Consensus Molecular Subgroup (CMS) data,

A. Trinh, C. Lädrach, H. E. Dawson et al.
Systematic review and meta-analysis of the impact of tumour budding in colorectal cancer,

A. C. Rogers, D. C. Winter, A. Heeney et al.
Tumour budding in colorectal cancer: molecular rationale for clinical translation,

I. Zlobec and A. Lugli
Tumor budding in colorectal carcinoma: confirmation of prognostic significance and histologic cutoff in a population-based cohort,

R. P. Graham, R. A. Vierkant, L. S. Tillmans et al.
Tumor budding versus mismatch repair status in colorectal cancer - an exploratory analysis,

M. Karlberg, K. Stenstedt, M. Hallström, P. Ragnhammar, C. Lenander, and D. Edler
Validation of the International Tumor Budding Consensus Conference 2016 recommendations on tumor budding in stage I-IV colorectal cancer,

H. Dawson, F. Galuppini, P. Träger et al.
Histopathological features predict metastatic potential in locally advanced colon carcinomas,

C. Jayasinghe, N. Simiantonaki, and C. J. Kirkpatrick
Tumor budding: the name is EMT. Partial EMT,

A. D. Grigore, M. K. Jolly, D. Jia, M. C. Farach-Carson, and H. Levine
Tumour budding: a promising parameter in colorectal cancer,

A. Lugli, E. Karamitopoulou, and I. Zlobec
The role of tumour budding in predicting survival in patients with primary operable colorectal cancer: a systematic review,

H. C. van Wyk, J. Park, C. Roxburgh, P. Horgan, A. Foulis, and D. C. McMillan
Tumor budding at the invasive margin can predict patients at high risk of recurrence after curative surgery for stage II, T3 colon cancer,

M. Tanaka, Y. Hashiguchi, H. Ueno, K. Hase, and H. Mochizuki
Tumor budding as an index to identify high-risk patients with stage II colon cancer,

T. Nakamura, H. Mitomi, H. Kanazawa, Y. Ohkura, and M. Watanabe
Budding as a useful prognostic marker in pT3 well- or moderately-differentiated rectal adenocarcinoma,

T. Okuyama, M. Oya, and H. Ishikawa
Histopathological significance and prognostic impact of tumor budding in colorectal cancer,

A. Mehta, M. Goswami, R. Sinha, and A. Dogra
APC mutation and tumour budding in colorectal cancer,

J. R. Jass, M. Barker, L. Fraser, M. D. Walsh, V. L. J. Whitehall, and B. Gabrielli
The impact of CpG island methylator phenotype and microsatellite instability on tumour budding in colorectal cancer,

I. Zlobec, M. P. Bihl, A. Foerster, A. Rufle, and A. Lugli
Implication of microsatellite instability pathway in outcome of colon cancer in Moroccan population,

F. El Agy, I. E. Otmani, A. Mazti, N. Lahmidani, A. Oussaden, and M. El Abkari
High-degree tumor budding and podia-formation in sporadic colorectal carcinomas with K-ras gene mutations,

F. Prall and C. Ostwald
KRAS, NRAS, BRAF mutations and high counts of poorly differentiated clusters of neoplastic cells in colorectal cancer: observational analysis of 175 cases,

V. Barresi, L. Reggiani Bonetti, and S. Bettelli
KRAS and PIK3CA mutations in colorectal adenocarcinomas correlate with aggressive histological features and behavior,

S. Jang, M. Hong, M. K. Shin et al.
Tumor budding in colorectal cancer-ready for diagnostic practice?

V. H. Koelzer, I. Zlobec, and A. Lugli
Tumor budding as a risk factor for nodal metastasis in pT1 colorectal cancers: a meta-analysis,

R. Cappellesso, C. Luchini, N. Veronese et al.
Prognostic impact of tumor-budding grade in stages 1-3 colon cancer: a retrospective cohort study,

B. Y. Oh, Y. A. Park, J. W. Huh et al.
Tumour budding and survival in stage II colorectal cancer: a systematic review and pooled analysis,

F. Petrelli, E. Pezzica, M. Cabiddu et al.
Prognostic impact of tumor budding on moroccan colon cancer patients,

F. E. agy, S. E. bardai, L. Bouguenouch, N. Lahmidani, M. E. abkari, and B. E. bachir

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Prognostic Impact of Tumor Budding on Moroccan Colon Cancer Patients

Prognostic Impact of Tumor Budding on Moroccan Colon Cancer Patients

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Prognostic Impact of Tumor Budding on Moroccan Colon Cancer Patients

Prognostic Impact of Tumor Budding on Moroccan Colon Cancer Patients

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