Thomas Grainville

Jean-François Bretagne
email: jf.bretagne@gmail.com

Christine Piette

Chloé Rousseau

Martin Bordet

Mathilde Cosson

Astrid Lièvre

Jean-François Bretagne

J.-F Bretagne

Management of T1 colorectal cancers detected at screening colonoscopy A study from the French national screening programme

Keywords: Colorectal cancer screening, T1 colorectal cancer, endoscopic therapy, surgical treatment, colorectal cancer prognosis LNM: lymph node metastasis, MDS: multidisciplinary staff, CRC: colorectal cancer, TAE: transanal excision, CI: confidence interval, HR: hazard ratio, OR: odds ratio

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

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1.Introduction

The estimated burden of colorectal cancer (CRC) in the 28 member states of the European Union was 378,400 new cases and 173,200 deaths in 2018, while the corresponding values for 40 countries in Europe were 499,700 and 242,500 cases, respectively [START_REF] Ferlay | Cancer incidence and mortality patterns in Europe: Estimates for 40 countries and 25 major cancers in 2018[END_REF]. There is now robust evidence that population screening can reduce cancer-specific mortality by detecting and treating disease at an early stage [START_REF] Zorzi | Impact on colorectal cancer mortality of screening programmes based on the faecal immunochemical test[END_REF][START_REF] Levin | Effects of Organized Colorectal Cancer Screening on Cancer Incidence and Mortality in a Large Community-Based Population[END_REF]. Fortunately, the implementation of mass screening for CRC is increasing in Europe, with roll-outs ongoing or completed in 17 member states in 2016 compared to only five in 2007 [START_REF] Basu | Status of implementation and organization of cancer screening in The European Union Member States-Summary results from the second European screening report[END_REF]. A national CRC screening programme was implemented in France at the beginning of 2003 and was then generalized to the whole country in 2008; this program includes the biennial faecal blood test Hemoccult II, which was replaced by the OC-Sensor immunochemical test in 2015 [START_REF] Inca | Le Programme de dépistage organisé du cancer colorectal en France[END_REF]. In one district of Brittany, we demonstrated the benefit of the switch from a guaiac method to immunochemical testing in terms of increased participation and detection of CRC and advanced neoplasia [START_REF] Bretagne | Switching from guaiac to immunochemical faecal occult blood test increases participation and diagnostic yield of colorectal cancer screening[END_REF]. If the participation rate, the positivity rate, the detection rate of colorectal lesions and the positive predictive value for adenomas and CRC are well-studied key performance indicators of CRC screening programmes [START_REF] Senore | Performance of colorectal cancer screening in the European Union Member States: data from the second European screening report[END_REF], the management and the prognosis of screen-detected lesions are also quality markers of screening programmes [START_REF] Segnan | European guidelines for quality assurance in colorectal cancer screening and diagnosis[END_REF][START_REF] Steele | European guidelines for quality assurance in colorectal cancer screening and diagnosis. First Edition--Management of lesions detected in colorectal cancer screening[END_REF]; however, these aspects have been studied very little. Based on a population study, we were among the first to report great variations in the management of nonmalignant screen-detected polyps, particularly for those ≥20 mm in size; patients were referred for surgical resection in 20% of cases , with adjusted rates ranging from 0 to 46%, depending on the gastroenterologist performing the screening colonoscopy [START_REF] Roy | Frequency of and risk factors for the surgical resection of nonmalignant colorectal polyps: a population-based study[END_REF]. However, there are few reported data on the management of A c c e p t e d m a n u s c r i p t cancers diagnosed through CRC screening programmes, which are mainly early cancers being the basis for the reduction of CRC-specific mortality in screening programmes [START_REF] Fasoli | The management of malignant polyps in colorectal cancer screening programmes: a retrospective Italian multi-centre study[END_REF][START_REF] Senore | Management of Pt1 tumours removed by endoscopy during colorectal cancer screening: Outcome and treatment quality indicators[END_REF][START_REF] Richards | An evidence-based treatment algorithm for colorectal polyp cancers: results from the Scottish Screen-detected Polyp Cancer Study (SSPoCS)[END_REF].

Therefore, the primary aim of this study was to use a population-based approach to examine the management strategies and the outcomes (survival and recurrence rate) of all patients with pT1 screen-detected CRC in one district of Brittany over a 13-year period. The secondary objectives were i) to identify factors associated with the choice of an initial endoscopic or surgical treatment, ii) to assess whether cases had been the subject of a multidisciplinary staff (MDS) presentation and examine, in case of primary endoscopic resection, if the decision of a complementary surgery or not was taken according to the recommendations and iii) to determine the risk factors associated with disease recurrence.

2.Population & methods

2.1.Study population

This study was a retrospective analysis of a prospective cohort that included all patients with pT1 adenocarcinoma diagnosed by colonoscopy following a positive faecal test through the national screening programme in the 'Ille-et-Vilaine' district from 2003 to 2015. Details on the screening programme were previously reported [START_REF] Bretagne | Switching from guaiac to immunochemical faecal occult blood test increases participation and diagnostic yield of colorectal cancer screening[END_REF]. pT1 carcinomas have invaded the muscularis mucosae into the submucosa but not into the muscularis propria; the endoscopic or surgical treatment of these carcinomas is controversial [START_REF] Roy | Frequency of and risk factors for the surgical resection of nonmalignant colorectal polyps: a population-based study[END_REF]. In this 13-year period, 565 288 faecal blood tests were performed. There were 14 215 (2.5%) positive tests that were followed by 13 245 colonoscopies (93.2%), and 1060 cases of cancer were diagnosed, which included 207 (19.5%) cases of pT1 adenocarcinomas. Among pT1 carcinomas, 181 (87.4%) were detected by guaiac-based faecal testing, while 26 (12.6%) were detected by A c c e p t e d m a n u s c r i p t FIT. The FIT selected by the French Health Authorities was the OC-Sensor™ test (Eiken, Tokyo, Japan) which has been used from May 2015 and was considered to be positive at the cut-off of 30 µg of haemoglobin per gram of faeces.

Demographic and baseline clinical features, colonoscopy results, pathological features of endoscopic biopsy specimens and from endoscopic or surgical resection specimens, therapeutic management and follow-up data were collected. All data related to the screening programme were prospectively collected in the database of the district (ADECI 35), while information on patient follow-up, when missing, was requested from general practitioners and gastroenterologists. Whether cases had been the subject of a MDS presentation was examined in patient medical records.

2.2.Colonoscopy data

The following data were collected: the date of colonoscopy, the endoscopy centre where the colonoscopy was performed, the gastroenterologist who performed the colonoscopy and potential complications that occurred during and within 30 days after the colonos copy by using the severity grading system reported by the American Society for Gastrointestinal Endoscopy [START_REF] Cotton | A lexicon for endoscopic adverse events: report of an ASGE workshop[END_REF]. The following characteristics of malignant polyps were collected: polyp morphology (pedunculated or sessile), size when precisely measured or classified into 3 categories (< 20 mm, 20 -29 mm or ≥ 30 mm), polyp location (proximal, distal, or rectal) and modalities for removal (resection technique, en bloc or piecemeal resection), as well as the number of associated polyps. Polyposis was defined as the presence of at least 10 polyps.

The location of the cancer was defined as proximal or distal if it was located proximally or distally to the splenic flexure, respectively.

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When the endoscopic resection of carcinoma was not performed during the initial diagnostic colonoscopy, we investigated whether the patient was referred to another endoscopist for therapeutic colonoscopy.

2.3.Pathology features

For this analysis, pT1 cancers were categorized retrospectively as having a low-risk or highrisk of lymph node metastases (LNM) according to the absence or presence of at least one of the following prognostic pathological criteria notified in the pathological report: poor differentiation, venous and lymphatic invasion, tumour budding, submucosal invasion depth ≥1,000 m for sessile polyps or Haggitt level of invasion ≥3 for pedunculated polyps, and margin involvement with a clearance of 1 mm or less [START_REF] Quirke | Quality assurance in pathology in colorectal cancer screening and diagnosis-European recommendations[END_REF]. We also recorded the width ( or 4,000 m) of submucosal invasion.

2.4.Surgical characteristics

We retrospectively retrieved the following data from the medical records of patients who underwent surgery: date and reason for surgery, type of operation and complications within 30 days of surgery according to the Clavien-Dindo classification [START_REF] Clavien | The Clavien-Dindo classification of surgical complications: five-year experience[END_REF]. In addition to the pathological features that were already described for malignant polyps resected by endoscopy, we collected data on LNM and residual tumour on surgical specimens for those who underwent subsequent surgery after endoscopic resection or an initial operation.

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2.5.Outcomes and statistical analysis

The close follow-up of patients aimed to assess mortality related or not related to tumour progression and local or metastatic recurrence. The primary outcomes included determining the 5-year overall, cancer-specific and recurrence-free survival as well as the long-term CRCspecific mortality and recurrence crude rates for the whole cohort and the subgroups defined by the management strategy. The secondary outcomes were to identify the factors associated with particular management strategies (primary endoscopic or surgical resection) and the risk factors associated with disease recurrence. In patients who had a primary endoscopic resection, the decision to perform a complementary surgery was considered in accordance with recommendations [START_REF] Williams | Management of the malignant colorectal polyp: ACPGBI position statement[END_REF][START_REF] Lecomte | Non-metastatic colonic cancer[END_REF] if the pT1 cancer was at high-risk of LNM as defined above.

Means and standard deviations (SDs) were calculated for continuous variables , and frequencies and percentages were calculated for categorical variables. Groups were compared by the t test or the Wilcoxon rank-sum test for continuous variables and by the χ 2 test or the Fisher exact test for categorical variables.

Recurrence-free survival curves were generated by using the Kaplan-Meier product limit method, and the log-rank test was used to compare survival between the endoscopic therapy group and the surgery groups.

Univariate and multivariate Cox proportional hazards regression models were used to assess the factors associated with cancer recurrence and the treatment modalities. Factors associated with the therapeutic strategy were investigated using univariate logistic regressions. Potential covariates (p≤0.20) were placed into a multivariable logistic regression A c c e p t e d m a n u s c r i p t model that was performed by using a stepwise backward elimination. To investigate the influence of the endoscopist, we compared the results from endoscopists who performed more than 10 colonoscopies that resulted in a pT1 cancer diagnosis to the results of other endoscopists in the univariate and multivariate analysis. Odd ratios (ORs) and the corresponding 95% CIs were used to express the association between the studied features and outcomes. For univariate analysis, the association of each variable with cancer recurrence was tested by a Cox model. To identify the clinical, colonoscopic and pathological features associated with disease recurrence, significant variables with p ≤ 0.20 were included in the multivariable analysis that was performed by using a stepwise backward elimination. Hazard ratios (HRs) and the corresponding 95% confidence intervals (CIs) were used to express the association between the studied features and outcomes.

All analyses were performed using the statistical software program SAS version 9.4 (SAS Institute Inc., Cary, North Carolina, USA). Statistical significance was defined as p ≤ 0.05.

3.Results

3.1.Characteristics of the cohort and the management strategy

The characteristics of 207 patients, malignant polyp and colonoscopy data are given in Table 1. Among the 207 patients, 126 underwent primary endoscopic resection, while 81 underwent primary surgery. Pathological features are given in Table 2 for both groups. In 82 of the 126 endoscopically treated patients, endoscopic resection was followed by subsequent surgery; thus, 44 (21.3%) patients were treated by endoscopic resection alone and 163 (78.7%) were treated by surgery alone or surgery preceded by endoscopic therapy.

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Four patients who underwent complementary surgery had already undergone surgery. Of the 81 patients who underwent primary surgery, 74 had pathological evidence of carcinoma in the endoscopic biopsy specimens.

Among the 126 patients who underwent primary endoscopic resection, polyp-cancer resection was performed during the diagnostic colonoscopy in all patients, except for 8 (6.3%) patients who were either referred to a skilled endoscopist (n=5) or treated by the same endoscopist during an additional colonoscopy (n=3). Endoscopic resection was followed by a complication in 13 (10.3%) patients, with mild, moderate and severe grades in 8, 3 and 2 patients, respectively (Table 3). Patient management was discussed by a MDS in 64 of the 126 (51%) patients. In all patients except for one, the staff's decision was implemented.

Among the 81 patients who underwent primary surgery, 72 (88.9%) patients underwent surgery because endoscopic resection was considered unsuitable by the endoscopist, 7 (8.6%) patients underwent surgery after an attempt of the endoscopic resection, and 2 (2.5%) underwent surgery because of synchronous polyposis. The operations that were performed and the post-operative complications are given in Table 3. A mean of 13.1 ± 9.4 lymph nodes were resected, but fewer than 12 lymph nodes were harvested in 31 (38.2%) patients, and 7 patients did not undergo lymph node dissection. Among the 74 patients who underwent lymph node dissection, LNM was present in 7 (9.5%) patients. The patient management of 38 (47%) patients was discussed by an MDS.

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Finally, 86 patients underwent subsequent surgery: 81 (94.2%) after primary endoscopic resection and 4 (4.7%) after primary surgery. The operations performed and post-operative complications are given in Table 3. Residual adenoma or adenocarcinoma were observed on surgical specimens in 5 of the 82 patients with endoscopic resection and in 2 of the 4 patients who had already undergone surgery. A mean of 13.2 ± 8.9 lymph nodes were resected, but fewer than 12 lymph nodes were harvested in 31 (38.7%) patients and 4 patients did not undergo lymph node dissection, which included 2 patients who had already undergone surgery. Among the 82 patients who underwent lymph node dissection, 9 (11%) had LNM.

3.2.Outcomes

3.2.1.Main outcomes

Follow-up data were available for 198 patients, and the median follow-up was 6.7±3.9 years. Thirty-six (18.2%) patients died during the 13-year period of follow-up, and 5

(2.4%) of these deaths were related to cancer recurrence. The 5-year overall and cancerspecific survival rates were 95.5% (95% CI, 90.8; 97.9) and 98.8% (95% CI, 95.4; 99.7%), respectively. Tumour recurrence occurred in 11 patients (5.6%) (8, 2 and 1 patients in the groups treated by primary surgery, endoscopic resection only and primary endoscopic resection followed by subsequent surgery, respectively), which included 4 local and 7 metastatic recurrences (Table 4). Among the 8 patients who recurred after primary surgery, 3 patients underwent TAE and did not undergo subsequent surgery despite being at high-risk for LNM. The two patients who had disease recurrence after receiving only endoscopic therapy did not undergo subsequent surgery despite having unsafe or invaluable margins on A c c e p t e d m a n u s c r i p t the resection specimen. The only patient who had recurrence following subsequent surgical resection after endoscopic therapy had LNM based on the surgical resection specimen. The time to recurrence was less than 2.5 years in 6 patients. Among the 11 patients who relapsed, 6 died, including 5 cancer-related deaths and one death due to heart failure. The 5year recurrence-free survival rate was 96.1% (95% CI, 91.8; 98.1%) (Fig. 1A), and the 5-year recurrence-free survival rate was not significantly different between patients treated by primary endoscopic resection (96.9% [95% CI, 90.4; 99%]) and treated with primary surgery (94.9% [95% CI, 87; 98.1%]) (p=0.059) (Fig. 1B). The 5-year recurrence-free survival also was not significantly different among the 3 subgroups defined by the treatment strategy: endoscopic resection alone (93.8% [95% CI, 76.7; 98.5%], endoscopic resection with subsequent surgery (98.5% [95% CI, 90; 99.8%] and primary surgery (94.9% [95% CI, 87; 98.1%] (log rank, p=0.12) (Fig. 1C). The distribution of recurrences and cancer-related deaths according to cancer site are given in Table 4.

3.2.2.Secondary outcomes

3.2.2.1.Factors associated with either primary endoscopic or surgical treatment

In multivariable analysis, the three factors independently associated with surgical resection were polyp size >20 mm, sessile morphology of the polyp-cancer and a date of diagnosis before 2007 (Table 5).

3.2.2.2.Factors associated with subsequent surgery following primary endoscopic resection

Among the different features considered when deciding whether to perform subsequent surgery, involved, unsafe or invaluable resection margins were the only risk A c c e p t e d m a n u s c r i p t factor significantly (p<0.0001) associated with surgery in univariate and multivariate analysis.

Among the 44 patients who underwent only endoscopic resection, 7 (15.9%) patients did not undergo subsequent surgery, although they were at high risk for LNM. Among the 82 patients who underwent complementary surgery after endoscopic resection, 15 (18.3%) were at low risk for LNM.

Factors associated with disease recurrence

In univariate analysis, the Cox model showed that piecemeal endoscopic resection, involved or unsafe resection margins and sessile morphology of the polyp cancer were significantly associated with disease recurrence. In multivariate analysis, involved or unsafe resection margins (OR=15.3 [95% CI, 2.8; 83.7], p=0.0018) and primary surgery (OR=12.2 [95% CI, 2.4; 63.6], p=0.0029) were the only factors that were independently associated with recurrence. When the 6 patients who underwent primary transanal excision (TAE) were considered to have undergone endoscopic resection (as it does not comprise any lymph node dissection), primary surgery was no longer found to be a significant risk factor for disease recurrence (data not shown).

4.Discussion

The primary aim of the present population-based study was to examine the management of pT1 screen-detected colorectal carcinoma and to assess the long-term outcomes of 207 cases of pT1 screen-detected colorectal carcinoma over a 13-year period. The present study emphasizes the good prognosis of pT1 screen-detected colorectal carcinoma patients. Thus, the 5-year overall, cancer-specific and recurrence-free survival of the cohort was 95.5%, A c c e p t e d m a n u s c r i p t 98.8% and 96%, respectively, while the long-term cancer-specific mortality and recurrence crude rates were 2.4% and 5.6%, respectively; these findings were in perfect agreement with those from a Scottish study (2% and 6%, respectively) [START_REF] Richards | An evidence-based treatment algorithm for colorectal polyp cancers: results from the Scottish Screen-detected Polyp Cancer Study (SSPoCS)[END_REF]. The excellent prognosis of screen-detected early carcinoma is an exciting outcome, highlighting the beneficial effect of screening programmes in reducing cancer-specific mortality [START_REF] Zorzi | Impact on colorectal cancer mortality of screening programmes based on the faecal immunochemical test[END_REF][START_REF] Levin | Effects of Organized Colorectal Cancer Screening on Cancer Incidence and Mortality in a Large Community-Based Population[END_REF]. Additionally, we demonstrated that 5-year recurrence-free survival did not differ significantly among the three subgroups defined by the treatment strategy: endoscopic therapy alone, endoscopic therapy with subsequent surgery or primary surgery (93.8%, 98.5% and 94.9%, respectively).

These results confirmed those of a North-Western Italian study that also examined a screening programme [START_REF] Senore | Management of Pt1 tumours removed by endoscopy during colorectal cancer screening: Outcome and treatment quality indicators[END_REF]. In contrast, in a large American population-based study conducted outside of the screening setting, a better recurrence-free survival was reported in the group of surgical patients, a result that could be explained by the fact that among the surgical patients with pT1 carcinoma, those with LMN were not included [START_REF] Mounzer | Endoscopic and surgical treatment of malignant colorectal polyps: a population-based comparative study[END_REF]. Although there was no significant difference in 5-year recurrence-free survival of surgical and nonsurgical patients found in our study, logistic regression analysis initially found that primary surgery was an independent risk factor for disease recurrence, in addition to an involved, unsafe or invaluable resection margin. However, it was not confirmed by a new analysis in which the 6 patients who underwent TAE were moved to the group of patients who have undergone endoscopic resection.

Interestingly, the 5 patients who had disease recurrence after receiving only endoscopic therapy (including TAE) did not undergo subsequent surgery despite having unsafe or invaluable margins on the resection specimen (n=3) or other risk factor of LNM (vascular emboli and submucosal invasion depth ≥1,000 m) (n=2). Moreover, 4 of the 6 patients who had disease recurrence after surgery (including one subsequent surgery after primary A c c e p t e d m a n u s c r i p t endoscopic resection) had LNM or fewer than 12 lymph nodes harvested on the surgical resection specimens. These data highlight once more the prognostic value of pathological features, especially a positive resection margin [START_REF] Belderbos | Long-term Recurrence-free Survival After Standard Endoscopic Resection Versus Surgical Resection of Submucosal Invasive Colorectal Cancer: A Population-based Study[END_REF], which was an independent risk factor for disease recurrence in our study, but also a poor differentiation, vascular invasion, tumour budding, a submucosal invasion depth ≥1,000 m for sessile polyps or Haggitt level of invasion ≥3 for pedunculated polyps as well as lymph node involvement for patients undergoing surgery. These histological parameters were not found as prognostic factors in the present study, which may be explained by the large proportion of missing data, particularly concerning tumour budding (90%), Haggitt classification (66%) and submucosal invasion depth (58%). Concerning tumour budding, however, no authoritative endorsements recommended its inclusion in the pathology report at the time of the present study because of the heterogeneity of definitions and measurement methods as well as its poor reproducibility and controversial prognostic impact [START_REF] Quirke | Quality assurance in pathology in colorectal cancer screening and diagnosis-European recommendations[END_REF]. A recent meta-analysis has since demonstrated its role as a risk factor for nodal metastasis specifically in pT1 colorectal cancer [START_REF] Cappellesso | Tumor budding as a risk factor for nodal metastasis in pT1 colorectal cancers: a meta-analysis[END_REF]. A revision of histological specimens by a gastrointestinal pathologist, perf orming a re-evaluation of all risk factors for lymph node metastasis, would have been interesting in our study as it has been shown to significantly modify risk assessment of pT1 endoscopically resected colorectal cancer and their clinical management [START_REF] Vinciguerra | Pathologist second opinion significantly alters clinical management of pT1 endoscopically resected colorectal cancer[END_REF].

In our study, it should be added that lymph node involvement is most likely underestimated in patients who have had a surgery because 38% of patients had less than 12 lymph nodes removed in the primary and subsequent surgery groups and 9% and 5% of them respectively, did not have any lymph node dissection. However, lymph node invasion is one of the factors, if not the most important prognostic factor in resected colorectal cancers [START_REF] André | Multicenter International Study of Oxaliplatin/5-Fluorouracil/Leucovorin in the Adjuvant Treatment of Colon Cancer (MOSAIC) Investigators. Oxaliplatin, fluorouracil, and leucovorin as adjuvant treatment for colon cancer[END_REF]. Taking into account these findings, which reflect real-life practices, we can easily A c c e p t e d m a n u s c r i p t consider that the proportion of high-risk pT1 cancers was underestimated in our study and that the incidence of disease recurrence would have been much lower if the recommendations have been implemented. In view of these limitations, it is difficult to draw any robust conclusion about the best therapeutic strategy to offer in the case of pT1 cancer.

Compared to similar studies, the direct surgical referral rate (39%) in this study was slightly higher than that in Italian screening programmes (24-30%) [START_REF] Fasoli | The management of malignant polyps in colorectal cancer screening programmes: a retrospective Italian multi-centre study[END_REF][START_REF] Senore | Management of Pt1 tumours removed by endoscopy during colorectal cancer screening: Outcome and treatment quality indicators[END_REF] but was slightly lower than that in the UK (43%) [START_REF] Gill | Management and short-term outcome of malignant colorectal polyps in the north of England[END_REF], with a slightly lower referral rate for radical surgery (57-69% and 53%, respectively, compared to 64%). Nevertheless, the overall surgical rate (78.7%) that we found was similar to that of an Italian study [START_REF] Senore | Management of Pt1 tumours removed by endoscopy during colorectal cancer screening: Outcome and treatment quality indicators[END_REF]. To investigate which variables were most relevant to surgical decision making, the multivariate analysis highlighted the role of polyp-cancer morphology and the date of colonoscopy. Thus, 75% of patients who underwent primary surgery had sessile polyps, 49.4% of malignant polyps were 30 mm, while the patients screened after 2007 were almost 3 times more likely to undergo primary endoscopy than surgical resection. This result could be related to the introduction of new and better trained gastroenterologists and/or to the improvement of gastroenterologists through post-graduate training. However, the gastroenterologist who performed the screening colonoscopy was not independently associated with the choice of treatment based on multivariate analysis, although some gastroenterologists had significantly lower odds ratios for endoscopic resection rates than the gastroenterologist with the highest endoscopic resection rate based on univariate logistic regression analysis.

Given that only 30% of patients who underwent primary surgery had high-risk pathological features of LNM or had LNM on surgical specimens, one may wonder why the other patients A c c e p t e d m a n u s c r i p t were directly referred to a surgeon without a referral to expert endoscopists. There was no clear, determinable reason why endoscopic resection was not considered suitable based on the diagnostic colonoscopy. One could speculate that the proportion of primary surgery would have decreased if patients had been referred to endoscopists with more expertise for the characterization and treatment of complex polyps. Indeed, it has been shown in the UK that the rate of primary surgical resection was lower for T1-CRC identified from screening than for those resulting from diagnosis [START_REF] Dattani | Variations in the management of significant polyps and early colorectal cancer: results from a multicentre observational study of 383 patients[END_REF]. In the UK but not in France, endoscopists are accredited for screening colonoscopy, and polypectomy skills are a part of the national accreditation process for colonoscopy certification [START_REF] Subramaniam | Competency in polypectomy: when desirable becomes essential[END_REF]. The relatively high rate of postendoscopic complications observed in the present study, compared to the rates reported in the English National Health Service Bowel Cancer Screening Programme [START_REF] Derbyshire | Post-polypectomy bleeding in the English National Health Service Bowel Cancer Screening Programme[END_REF][START_REF] Derbyshire | Colonoscopic perforations in the English National Health Service Bowel Cancer Screening Programme[END_REF], could be regarded as a marker of less experience in polypectomy. Similarly, in their meta-analysis of 21 population-based studies, Reumkens et al. reported low rates of post-polypectomy perforation and haemorrhage, equal to 0.8 and 9.8 p.1000 colonoscopies including all types of polyps [START_REF] Reumkens | Post-colonoscopy complications: a systematic review, time-trends, and meta-analysis of population-based studies[END_REF]. However, in their meta-analysis assessing the efficacy and safety of endoscopic resection for large colorectal polyps (≥20 mm), 58% of which being invasive cancers, Hassan et al. reported much higher values for perforation and bleeding (1.5%, 6.5%, respectively) [START_REF] Hassan | Efficacy and safety of endoscopic resection of large colorectal polyps: a systematic review and meta-analysis[END_REF]. In their study specifically devoted to endoscopic resection of colorectal cancers T1, Overwater et al. reported post-endoscopic complication rates of 6.5%, including 4.1% for bleeding, in a series of 339 high-risk T1 cancer cases requiring secondary surgery [START_REF] Overwater | Endoscopic resection of high-risk T1 colorectal carcinoma prior to surgical resection has no adverse effect on long -term outcomes[END_REF]. In another recent multicentric study from Netherlands also devoted to T1 carcinomas, the rate of post-endoscopic adverse events was 5.5%, including 3.7% for bleeding [START_REF] Van De Ven | Periprocedural adverse events after endoscopic resection of T1 colorectal carcinomas[END_REF]. Even if the authors stated that the complication rate was similar to that observed for adenomas, we argue that the safety of endoscopic resection remains to be proven for early, but invasive A c c e p t e d m a n u s c r i p t

CRCs. One could also speculate from our findings that systematically discussing the medical records of polyp patients with a MDS could also help to reduce surgical referrals. The choice between endoscopic or surgical strategies is not insignificant since surgery leads to an increase in costs for screening programme managers and in complications for tested patients [START_REF] Law | Endoscopic resection is cost-effective compared with laparoscopic resection in the management of complex colon polyps: an economic analysis[END_REF]. Furthermore, from the patient's perspective, endoscopic treatment provides a quicker recovery than surgery and does not provoke more fear of cancer recurrence [START_REF] Dang | Quality of life and fear of cancer recurrence in T1 colorectal cancer patients treated with endoscopic or surgical tumor resection[END_REF].

Another argument supporting primary endoscopic resection is that even in cases of high-risk T1-CRC, there was not an increased risk of disease recurrence or LNM reported after secondary surgery compared with primary surgery [START_REF] Overwater | Endoscopic resection of high-risk T1 colorectal carcinoma prior to surgical resection has no adverse effect on long -term outcomes[END_REF][START_REF] Rickert | Oncologic colorectal resection after endoscopic treatment of malignant polyps: does endoscopy have an adverse effect on oncologic and surgical outcomes?[END_REF]. The only risk factor associated with subsequent surgery following endoscopic therapy identified in the present study was involved, unsafe or invaluable resection margins, which was in agreement with other studies [START_REF] Fasoli | The management of malignant polyps in colorectal cancer screening programmes: a retrospective Italian multi-centre study[END_REF]. However, it is noteworthy that 18.3% of the 82 patients did not have any pathological risk factors. Among them, none had been the subject of a MDS presentation.

The main limitations of the present study were its retrospective nature and the suboptimal collection of data regarding some histopathology items as previously detailed. These limitations could explain why some pathological features, such as budding, were not found to be significantly associated with disease recurrence. However, progress has been noted over time; for example, the depth of submucosal invasion was not recorded before 2007 in 92.3% of cases but was recorded in 89.7% of cases after 2007. The use of standardized forms that are now recommended for pathology reports of endoscopically resected pTis and pT1 colorectal polyps by French guidelines [START_REF] Selves | Guidelines for the management of malignant colo-rectal polyps (pTis and pT1) treated by endoscopic resection[END_REF] should improve the quality of pathology reports in the future. The reassessment of pathology that we were unable to perform for this study could have modified the risk assessment, as already mentioned [START_REF] Vinciguerra | Pathologist second opinion significantly alters clinical management of pT1 endoscopically resected colorectal cancer[END_REF]. Because deviant cases A c c e p t e d m a n u s c r i p t had not been the subject of a MDS presentation, we cannot tell from the records review whether the absence of additional surgery for some high-risk patients or the occurrence of surgery for some low-risk patients was a patient's decision or that of the gastroenterologist.

It is also possible that the existence of co-morbidity factors, not identified in our study, may have influenced the decision not to operate or to reoperate on some high-risk patients.

Finally, we cannot extrapolate our findings to the whole country, even if we know that our previous findings regarding nonmalignant polyps were replicated in other districts in France [START_REF] Denis | Management of nonmalignant colorectal polyps[END_REF]. This study also had several strengths, particularly the completeness of the data collected concerning colonoscopy and surgical findings and the median length of 6.5 years for follow-up, never reported so far, which may explain the observed rate of disease recurrence. Of the 11 recurrences, 5 were diagnosed after 54 months.

In conclusion, the present study shows that the prognosis of early (pT1) screen-detected CRC is excellent, regardless of the management strategy used. This study demonstrates that a large proportion of patients with pT1-CRC underwent direct or subsequent surgery, while surgery could probably have been avoided to a certain extent by referring the patient to a skilled endoscopist. The proportion of patients who underwent primary surgery decreased with time, suggesting an improvement in the training of gastroenterologists in endoscopic resection techniques. Our study also shows that there is room for improvement in the quality of pathology reports and for systematically discussing patient records with a MDS to define the best treatment strategy for each patient.

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5.Acknowledgements

The authors acknowledge the contributions of the general practitioners, gastroenterologists and surgeons in the district of Ille-et-Vilaine. They also acknowledge the staff in charge of the database at the screening centre, particularly Samuel Foucrit, a computer scientist. Long-term recurrence-free survival of the whole cohort Fig. 1B Long-term recurrence-free survival in patients primarily treated by endoscopic resection or surgical resection

Fig. 1C

Long-term recurrence-free survival in patients treated by endoscopic resection alone, endoscopic resection followed by subsequent surgery, or primary surgery

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Funding

None

Ethics approval and consent to participate

The colorectal cancer screening programme was declared and approved by the CNIL (Commission Nationale de l'Informatique et des Libertés) on August 30 th , 2002 (n° 812571). This research was approved by the CCTIRS (Comité Consultatif pour le Traitement de l'Information en Matière de Recherche dans le Domaine de la Santé).


Malgorzata Kloc
email: mkloc@houstonmethodist.org

Rafik M Ghobrial

Ernest Kuchar

Slawomir Lewicki

Jacek Z Kubiak
email: jacek.kubiak@univ-rennes1.fr

Małgorzata Kloc

Sławomir Lewicki

Malgorzata Kloc

Development of child immunity in the context of COVID-19 pandemic

Keywords: child immunity, COVID-19, immune system development, macrophages

niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Development of the immune system

During pregnancy, the developing fetus has to be tolerant of the maternal antigens, and the maternal immune system has to be fetotolerant. The reshaping of mother/fetus immune response, still not fully understood, relies on multiple mechanisms, including the emerging role of T regulatory cells (Tregs) in the achievement and maintenance of this tolerance [START_REF] Guleria | Maternal Acceptance of the Fetus: True Human Tolerance[END_REF]. After birth, the newborn becomes exposed to the enormous number of foreign antigens that require swift immune response. However, the immune system of the newborns is underdeveloped and subdued, fully maturing during the first 7-8 years of life. The first line immune responders present already in the fetus and newborn are the innate immune cells: monocytes, macrophages, dendritic cells, and neutrophils. The monocytes and macrophages in the newborn are immature and have lower cytokine response than in the adult. The mature neutrophils are already present at the 13 th week of pregnancy and increase in number during consecutive weeks. After birth, the number of neutrophils stabilizes but they have only weak bactericidal activity [START_REF] Simon | Evolution of the immune system in humans from infancy to old age[END_REF]. Interestingly, the newborns' plasmacytoid dendritic cells (pDC) show very low production of alpha and beta interferon in response to different viruses, such as respiratory syncytial virus, herpes simplex virus and cytomegalovirus [START_REF] Simon | Evolution of the immune system in humans from infancy to old age[END_REF][START_REF] Schüller | Preterm neonates display altered plasmacytoid dendritic cell function and morphology[END_REF]. The NK cells of the fetus have low cytolytic activity, are hyperresponsive to the immunosuppressive activity of transforming growth factor-beta (TGF-β), and are hyporesponsive against cells lacking major histocompatibility complex (MHC) class I.

At birth, the cytolytic activity of NK cells is only 50% of that in the adults, and they are hyporesponsive to IL-2 and IL-15 activation (2). Such a muted innate immune system in newborns results in a higher than in the adults, susceptibility to bacterial and viral infection.

Studies are indicating that in older children the innate and adaptive immune system develops the immunological (trained) memory of hemopoietic progenitors and NK cells, after the repeated exposure to the antigens and vaccinations, which may give cross-protection against reinfections (4 -10).

Besides the innate immunity, the fetus, newborn, and developing child also have adaptive immunity. One of the cellular components of adaptive immunity are the T cells. Although the CD4-and CD8-positive T cells are established around the 15 th week of pregnancy, and the mature T cells are already present in the newborn, they differ from the adult T cells by being more tolerogenic, and hyporesponsive to the antigens [START_REF] Simon | Evolution of the immune system in humans from infancy to old age[END_REF]. The newborns also have a special, recently discovered, a subpopulation of interleukin-8 (CXCL8)-producing T cells, which activate A c c e p t e d m a n u s c r i p t antimicrobial neutrophils (see above), and unconventional, γδT cells, which number decreases with age [START_REF] Simon | Evolution of the immune system in humans from infancy to old age[END_REF][START_REF] Gibbons | Neonates harbour highly active gamma delta T cells with selective impairments in preterm infants[END_REF][START_REF] Silva-Santos | Gamma delta T-cell conference 2012: close encounters for the fifth time[END_REF]. Other cells of the adaptive immunity are the B cells. In the newborn, and through the first few months of life, around 40% of the circulating B cells are the B1 cells that only produce low-affinity IgM, and later in life, they become replaced by the conventional B2 cells [START_REF] Simon | Evolution of the immune system in humans from infancy to old age[END_REF][START_REF] Hannet | Developmental and maturational changes in human blood lymphocyte subpopulations[END_REF]. Because the newborns and young children have an underdeveloped and immature immune system they have to, at least partially, rely on the immune factors supplied by the mother. They are also theories that the immune system of the fetus is trained by maternal inflammation, which influences, and establishes a long-lasting immunologic memory in the fetal hematopoietic stem cells [START_REF] Apostol | Training the Fetal Immune System Through Maternal Inflammation-A Layered Hygiene Hypothesis[END_REF]. Below we summarize the components and routs of maternally derived immunity.

2.Maternal transmission of immunity 2a. Placental transmission of immunity

The placenta develops from the trophectoderm of the blastocyst and serves as a protective barrier, and promotes the growth of the fetus. During pregnancy the placenta reciprocally exchanges gases, nutrients, and waste products between the mother and fetus [START_REF] Maltepe | Placenta: The Forgotten Organ[END_REF]. The placenta is also crucial for providing fetal and newborn immunity. Because the placenta is continuously exposed to the pathogens present in the mother's blood, it has several mechanisms protecting the fetus from the infection. The surface of the placenta is built of a continuous layer of cells without cell junctions, and the brush border of the placental surface has a network of very dense actin filaments. These two biophysical barriers prevent the entry of pathogens from the mothernal blood. In addition, the unique composition of the membrane of placental cells prevents the attachment and penetration of some of the pathogens [START_REF] Ander | Human Placental Syncytiotrophoblasts Restrict Toxoplasma gondii Attachment and Replication and Respond to Infection by Producing Immunomodulatory Chemokines[END_REF][START_REF] Ander | Immune responses at the maternal-fetal interface[END_REF]. The placenta also secrets the antiviral compounds such as type III interferons, and, the enclosed within exosomes unique miRNAs, the trophomiRs, which prevent viral infection [START_REF] Bayer | Human trophoblasts confer resistance to viruses implicated in perinatal infection[END_REF][START_REF] Bayer | Type III Interferons Produced by Human Placental Trophoblasts Confer Protection Against Zika Virus Infection[END_REF][START_REF] Sadovsky | The Function of TrophomiRs and Other MicroRNAs in the Human Placenta[END_REF].

In humans, starting from the 16 th week of gestation, until birth, the placenta is also involved in the continuous transfer of passive immunity from the mother to the fetus. The surface cells of the placenta express neonatal receptors for immunoglobulin G (IgG), which bind and pass maternal antibodies to the fetus. In consequence, at birth, the fetus has more maternal IgGs than the mother [START_REF] Maltepe | Placenta: The Forgotten Organ[END_REF][START_REF] Ander | Immune responses at the maternal-fetal interface[END_REF][START_REF] Fu | Placental antibody transfer efficiency and maternal levels: specific for measles, coxsackievirus A16, enterovir us 71, poliomyelitis I-III and HIV-1 antibodies[END_REF], which can protect infant during first months of life, until the maturation of their own immune system. Thus, the preterm infants, with a lower level of maternally derived antibodies, are more vulnerable to the infection.

A c c e p t e d m a n u s c r i p t 2b. Vaginal delivery shapes infant's immunity

The vaginal delivery shapes, through the contact with mother's microorganisms, the composition of a child's gut microbiota, which, in turn, affects the development of the newborn's immune system [START_REF] Wampach | Birth mode is associated with earliest strain-conferred gut microbiome functions and immunostimulatory potential[END_REF]. The recent analysis, using the 16S rRNA gene amplicon sequencing and highresolution metagenomics of the microorganisms present in the neonatal gut after vaginal and caesarian section delivery, indicates that the vaginal birth enriches the gram-negative bacteria, including 23 enteric taxa of Bacteroidetes and Actinobacteria, in the newborns. This stimulates, via lipopolysaccharide (LPS) pathway, the production of pro-inflammatory cytokines TNF-a and IL-18, increases the immunostimulatory potentials of the gut microbiome, and primes immune system of neonates born by vaginal delivery [START_REF] Wampach | Birth mode is associated with earliest strain-conferred gut microbiome functions and immunostimulatory potential[END_REF].

2c. Breast milk immune factors

Breast milk contains not only the basic nutrients (proteins, carbohydrates, and fats) but also a multitude of factors that drive development and maturation of the immune system and protect newborns from the environmental pathogens. Milk contains not only antibacterial compounds, chemokines, cytokines, immunoglobulins, hormones, and growth factors [START_REF] Ballard | Human Milk Composition: Nutrients and Bioactive Factors[END_REF] but also the immune cells including lymphocytes, neutrophils, and macrophages.

The composition and the level of these compounds change during the different phases of lactation in response to the changing needs of the growing and developing infant. Recent studies of the milk proteome [START_REF] Zhu | The Functional Power of the Human Milk Proteome[END_REF] showed that between the first 72 hours and 3 days postpartum the colostrum milk has the highest level of the immunoregulatory factors. The transitional milk produced between the 3 rd and 15 th day is very rich in proteins, and the mature, produced from day 16 th , milk is enriched in fatty acids, and its overall composition becomes less variable. The antimicrobial compounds in the milk include among others: lactoferrin, haptocorrin, lysozyme, defensins, cathelicidins, proteins of the complement system, components of the lactoperoxidase system (LP-s), and various glycans. The lactoferrin and haptocorrin are bacteriostatic through the binding of free ions, and vitamin B12, respectively, which are required for bacterial growth [START_REF] Lönnerdal | Human Milk Proteins: Key Components for the Biological Activity of Human Milk[END_REF].

The lysozyme, defensins, and cathelicidins disrupt or puncture the wall of the bacteria, leading to their lysis [START_REF] Lönnerdal | Bioactive Proteins in Breast Milk[END_REF]. The proteins of the complement system opsonize pathogen to promote phagocytosis, act as the chemoattractants recruiting and activating phagocytes, and /or puncture bacterial membrane [START_REF] Ogundele | Complement-mediated Bactericidal Activity of Human Milk to a Serum-Susceptible Strain of E. Coli 0111[END_REF]. The lactoperoxidase catalyzes (from the thiocyanate ions in the A c c e p t e d m a n u s c r i p t presence of hydrogen peroxide) production of the antibacterial substance, and glycans reduce proliferation and virulence of some pathogens [START_REF] Koksal | An Important Milk Enzyme: Lactoperoxidase. Milk Proteins -From Structure to Biological Properties and Health Aspects[END_REF].

Human early colostrum milk contains around 5 million leukocytes/ml. Among them, 10% are B cells, T cells, and NK cells. The remaining leukocytes are mainly neutrophils and macrophages.

The number of leukocytes decreases with time, and mature milk has approximately 10 times fewer leukocytes than the colostrum [START_REF] Jackson | Breastfeeding, the Immune Response, and Long-term Health[END_REF][START_REF] Palmeira | Immunology of breast milk[END_REF]. The breast milk also contains a high number of recently discovered innate lymphoid cells (ILCs), which had been divided into 3 subgroups: ILC1s, ILC2s, and ILC3s, with different cytokine and gene expression profiles [START_REF] Baban | Presence and Profile of Innate Lymphoid Cells in Human Breast Milk[END_REF][START_REF] Eberl | Innate lymphoid cells: A new paradigm in immunology[END_REF]. The ILCs do not have receptors for the antigens but, instead, rapidly respond to infection and produce the same array of inflammatory mediators like the T cells [START_REF] Eberl | Innate lymphoid cells: A new paradigm in immunology[END_REF]. The immune cells present in breast milk are resistant to the child digestive enzymes and can mount a vigorous immune response by directly destroying pathogens they encounter and shape the infant's gut microbiota, and immunity [START_REF] Jackson | Breastfeeding, the Immune Response, and Long-term Health[END_REF][START_REF] Palmeira | Immunology of breast milk[END_REF][START_REF] Baban | Presence and Profile of Innate Lymphoid Cells in Human Breast Milk[END_REF][START_REF] Eberl | Innate lymphoid cells: A new paradigm in immunology[END_REF][START_REF] Doare | Mother's Milk: A Purposeful Contribution to the Development of the Infant Microbiota and Immunity[END_REF].

Incidence rate and severity of COVID-19 in children

Over the years, the research and clinical data have shown that because children have an immature immune system, they are more prone than the adults to microbial infections, and have higher severity of symptoms, which is especially true for the newborns and very young children.

The analyses of clinical data from the current COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are conflicting in this respect. Some data indicate that children are rarely infected and have less severe symptoms, and other show otherwise (4). Analysis of clinical data of 2135, 2-13 years old children in China, between January 16, 2020, and February 8, 2020, showed that more than 90% of children were either asymptomatic or had less severe symptoms than the adults and that children are no less susceptible than adults to SARS-CoV-2 infection, and the infants are more vulnerable (10.6%) to the infection than older children [START_REF] Dong | Epidemiology of COVID-19 Among Children in China[END_REF]. Some studies showed that the frequency and severity of infection in 0-19 years old children are much lower than in adults. Analysis from China showed 0.9% infection in children 0-9 years old, 1.2% in children 10-19 years old, and 0.2% fatality [START_REF] Liu | The epidemiological characteristics of an outbreak of 2019 novel coronavirus diseases (COVID-19) in China Novel Coronavirus Pneumonia Emergency Response[END_REF]. Similar results were reported from Italy with 0.5% cases in 0-9 years old children, 0.7% in 10-19 years old children, and no fatalities (www.epicentro.iss.it/coronavirus/bollettino/Bollettino-sorveglianza-integrata-COVID-19_23-A c c e p t e d m a n u s c r i p t marzo%202020.pdf, cited in 4). If the older children are really less vulnerable to COVID-19, what would be a plausible explanation for this phenomenon? One explanation is that the child's immune system has been trained by the maternal respiratory inflammations to develop a longlasting immunologic memory against respiratory viruses [START_REF] Cristiani | Will children reveal their secret? The coronavirus dilemma[END_REF][START_REF] Apostol | Training the Fetal Immune System Through Maternal Inflammation-A Layered Hygiene Hypothesis[END_REF]. Also, frequent exposure of children to respiratory viruses in daycare, kindergarten, and school may lead to the development of partial protection against the SARS-CoV-2 virus. Another explanation may lie in the difference between children and adults in the expression level of the SARS-CoV-2 receptor.

The entry of the SARS-CoV-2 to the host cells relies on the angiotensin-converting enzyme 2 (ACE2) receptors present in various tissues, and also in lung epithelial cells, and alveolar macrophages [START_REF] Yan | Structural basis for the recognition of SARS-CoV-2 by full-length human ACE2[END_REF][START_REF] Verdecchia | The pivotal link between ACE2 deficiency and SARS-CoV-2 infection[END_REF]. Recently, Bunyavanich et al. [START_REF] Bunyavanich | Nasal Gene Expression of Angiotensin-Converting Enzyme 2 in Children and Adults[END_REF] showed that in the cohort of 305 individuals (4 to 60 years old) the expression of ACE2 in the nasal epithelium, which serves as a virus' point of entry, increased with age: ACE2 expression was lowest in younger children (below 10 years old), and successively higher in older children (10 -17 (Ang1-7), and balancing the ratio and level of Ang II/Ang1-7, which affects inflammation, the higher number of ACE2 receptors in children lung pneumocytes would protect against COVID-19 lung inflammation. Theoretically, both of these contradictory theories may be valid. It is possible that the lower number of ACE2 receptors in the children's nasal epithelium limits the virus entry, while the higher level of ACE2 receptors in the children's lungs protects against the virus. Another issue worth of pursuing would be the difference, if any, between children and adult lung macrophage response to SARS-CoV-2 infection. One of the reasons for the severity of COVID-19 infection is the overdrive of the inflammatory response in alveolar macrophages, which also have ACE2 receptors, become infected with SARS-CoV-2, and drive the so-called cytokine storm [START_REF] Yan | Structural basis for the recognition of SARS-CoV-2 by full-length human ACE2[END_REF][START_REF] Ye | Cytokine Storm in COVID-19 and Treatment[END_REF]. The ongoing clinical trrials with the Anakinra, the drug which inhibits pro-inflammatory cytokines interleukin (IL)-1α and IL-1β, which act in the monocyte/macrophage-driven cytokine storm in the COVID-19 patients, can be a novel therapy

  years old), young adults (1-24 years old), and highest in the adults (above 25 years old). The authors suggest that the lower expression of ACE2 receptor in children may explain why children are less prone to SARS-CoV-2 infection. However, Cristiani et al. (4) and Chen et al. (39) present a completely opposite, and more convoluted, theory of the effect of ACE2 expression on the vulnerability to and severity of the infection. They argue that because the ACE2 has also a role, especially prominent in the lungs, in the conversion of the angiotensin II to angiotensin-(1-7)

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  p t e d m a n u s c r i p t for the ARDS[START_REF] King | Anakinra in COVID-19: important considerations for clinical trials[END_REF].Thus, it would be very interesting to study if the less severe symptoms of COVID-19 in children are related to the decreased response and downregulation of cytokine storm of their lung macrophages.

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Acknowledgements

We acknowledge the funding from the Polish Ministry of National Defense "Kościuszko" # 5508/2017/DA grant to JZK.