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

Christine Piette

Mathilde Cosson

Gérard Durand

Astrid Lièvre

SWITCHING FROM GUAIAC TO IMMUNOCHEMICAL FAECAL OCCULT BLOOD TEST INCREASES PARTICIPATION AND DIAGNOSTIC YIELD OF COLORECTAL CANCER SCREENING

Keywords: colorectal cancer screening, guaiac faecal occult blood test, immunological faecal occult blood test, colonoscopy, colorectal cancer, adenoma, advanced adenoma

Background

Compared with the guaiac-faecal occult blood test (gFOBT), faecal immunological tests (FIT) are considered to be more effective for colorectal cancer (CRC) screening. However, only scarce research has examined the outcomes of switching to FIT within a mature gFOBT-based CRC screening programme.

Methods

We reported a 15-year experience of biennial FOBT screening in a well-defined population of approximately one million inhabitants, including six gFOBT-based screening rounds and one round with FIT at the 30 µg Hb/g cut-off. The main outcome measures were screening participation, FOBT positivity and advanced neoplasia detection in each round.

Results

In this study, 647 676 screenings were performed in 228 716 different individuals, leading to 17 819 positives and 16 580 follow-up colonoscopies. Compared with the last gFOBT round, switching to FIT led to an increased participation of nearly 20 percentage points, and a fivefold increased detection of CRC and advanced adenoma among invitees (3-fold among attendees). The numbers needed to screen and scope to detect one advanced neoplasia declined from 221 to 66 and from 4.7 to 2.6, respectively.

Conclusions

The present population-based study demonstrated a dramatical increase in the diagnostic yield of advanced neoplasia by switching to FIT within a mature gFOBT-based CRC screening programme.

INTRODUCTION

Randomized controlled studies have shown that colorectal (CRC) screening using a guaiac-based faecal occult blood test (gFOBT) can significantly reduce mortality from CRC. [START_REF] Hewitson | Cochrane systematic review of colorectal cancer screening using the fecal occult blood test (hemoccult): an update[END_REF] Therefore, French National Health Authorities launched CRC screening in France in 2003 using a gFOBT in average-risk people aged 50-74. The programme achieved national coverage in 2008, but mean participation was low compared to other countries. [START_REF] Moutel | Low participation in organized colorectal cancer screening in France: underlying ethical issues[END_REF][START_REF] Klabunde | Participation rates for organized colorectal cancer screening programmes: an international comparison[END_REF] In 2015, French National Health Authorities replaced gFOBT with a faecal immunological test (FIT) because of its higher sensitivity for CRC and cancer precursors [START_REF] Tinmouth | Faecal immunochemical tests versus guaiac faecal occult blood tests: what clinicians and colorectal cancer screening programme organisers need to know[END_REF] but also for its greater acceptance by populations in controlled studies. [START_REF] Van Rossum | Random comparison of guaiac and immunochemical fecal occult blood tests for colorectal cancer in a screening population[END_REF][START_REF] Hol | Screening for colorectal cancer: random comparison of guaiac and immunochemical faecal occult blood testing at different cut-off levels[END_REF] However, in the literature, there is little data in regard to switching to FIT within mature gFOBT-based screening programmes. A pilot study within the national screening programme in England, which replaced the gFOBT with FIT over a 6-month period, reported an increased participation and improved outcomes with FIT compared to gFOBT, which was still running during the same period of time. [START_REF] Moss | Increased uptake and improved outcomes of bowel cancer screening with a faecal immunochemical test: results from a pilot study within the national screening programme in England[END_REF] 'Ille-et-Vilaine' which was one of the first administrative areas in France to implement the national screening programme in 2003, has a 15-year (2003-2017) experience in CRC gFOBT-based screening, as well as the switch to FIT-based screening. Therefore, the aim of this study was to demonstrate increased participation in and improved outcomes of CRC screening following gFOBT replacement by FIT within a mature screening programme in a large population-based study.

POPULATION AND METHODS

Population and screening strategy

This study was conducted in the 'Ille-et-Vilaine' district in France. 'Ille-et-Vilaine' was one of the first administrative areas in France to implement the national CRC screening programme in 2003 using the biennial g-FOBT (Hemoccult II; Beckman Coulter Inc., Villepinte, France). CRC screening was proposed to individuals aged between 50 and 74 years with an average risk of CRC and no contraindications for colonoscopy. Individuals with a personal or family history of CRC or adenoma, those with inflammatory bowel disease, and those who had undergone total colonoscopy in the previous five years were excluded from the mass screening programme. The gFOBT-based national screening programme, which was generalized in France in 2008, ran in France and therefore in 'Illeet-Vilaine' until 2014. The shift from gFOBT to FIT occurred in 'Ille-et-Vilaine' in May 2015; the FIT selected by the French Health Authorities was the OC-Sensor™ test (Eiken, Tokyo, Japan).

Thus, in 'Ille-et-Vilaine', six rounds of gFOBT-based screening and one round of FIT-based screening were performed between 2003 and 2017. Regarding the first six rounds with gFOBT, two particularities should be mentioned. First, following National Health Authorities decisions regarding the replacement of the Hemoccult™ test by the FIT; therefore, the sixth round was shortened, and a gap of 6 months occurred between the end of screening using gFOBT and the start of FIT-based screening. Second, the third round in the district of 'Ille-et-Vilaine' was part of a published randomized controlled study involving three other districts that compared the performance of CRC screening with Hemoccult II™ and OC-Sensor™ tests. [START_REF] Faivre | Comparison between a guaiac and three immunochemical faecal occult blood tests in screening for colorectal cancer[END_REF] In 'Ille-et-Vilaine', therefore, the performance of the third round with gFOBT should be interpreted with the knowledge that 28% of respondents to gFOBT during that round had the FIT performed simultaneously.

The screening strategy was similar for every screening round. An information brochure and an invitation letter were sent to each individual in the target population. People were invited to consult their general practitioners (GPs), who proposed the screening test to eligible subjects seen at their practice. GPs were also asked to state the exclusion criteria, at which point they provided each eligible individual with one screening test. The first 6 months of the screening round corresponded to the medical free-offer phase. A reminder letter including the screening test was sent 8 months later to non-respondents who were not excluded from participation by their GPs. Contrary to national guidelines related to gFOBT, the national FIT-based screening programme decided to discontinue the mailing of the test. However, in 'Ille-et-Vilaine', on the basis of the results of a randomized controlled study that demonstrated the benefit to mailing the FIT test along with the reminder letter, [START_REF] Piette | Additional mailing phase for FIT after a medical offer phase: The best way to improve compliance with colorectal cancer screening in France[END_REF] the screening centre was authorized by the regional health authorities to mail the FIT test with the second reminder letter after the first reminder letter to those in the target population who had not participated during the medical free-offer phase. Thus, we can conclude that the screening strategy and the implication of GPs were similar for gFOBT and FIT delivery in our district.

Participants were instructed to scrape different parts of the surface of their stool with the test probe and to return the test by mail to the central analysis centre in a prepaid envelope as soon as possible. No diet restriction was imposed. Two samples from each of three consecutive stools were required for the gFOBT test, while a single sampling tube was used for the FIT test. Tests were analysed by a single central laboratory without prior rehydration for the gFOBT test. The latter test was defined as positive when one to six square(s) was positive, while a cut-off of 30 µg of haemoglobin per gram of faeces (i.e., 150 ng of haemoglobin per millilitre of buffer) was used for the FIT positivity. The period of time between the accomplishment of the FIT and the dosage by the central laboratory should be less than seven days. The results were sent by mail to each individual, to their general practitioner and to the screening centre. All positives were recommended to undergo colonoscopy. Those with non-analysable tests were invited to redo testing, while those with negative tests were informed to participate again two years later until they were 74 years old.

Outcome measures and statistical analysis

The total population and the population aged from 50 to 74 years in the district were registered at the beginning of each round according to INSEE files. [START_REF]Populations légales 2015. Recensement de la population -régions, départements, arrondissements, cantons et communes[END_REF] The target population of the CRC screening programme was defined as the population aged from 50 to 74 years minus the excluded individuals.

Results of testing were classified as positive, negative or non-analysable. FOBT positivity was defined as the proportion of participants with a positive testing. Compliance to colonoscopy was assessed among individuals with positive testing. Colonoscopies were performed by gastroenterologists. The proportion of completed colonoscopy and the rate of serious complications, such as colon perforation, were prospectively recorded. Colorectal neoplastic lesions (macroscopic features, size, location, and number of lesions) and their treatment were prospectively recorded in the database.

Histopathology records were obtained from pathologists. Colonoscopy findings were classified according to the most advanced lesion: cancer, advanced adenoma, low-risk adenoma. Other colorectal lesions, such as hyperplastic or serrated polyps, were not considered in the current analysis. Advanced adenoma was defined as an adenoma of 10 mm or more in size or with highgrade dysplasia or intra-epithelial carcinoma. The villous component was not taken into account in the definition in accordance with recent French recommendations. 11 Cancers were classified according to the TNM classification system, with Tis corresponding to intra-mucosal carcinoma. 12 Cancers and advanced adenomas were analysed in a pooled group named 'advanced neoplasia', while advanced and low-risk adenomas were analysed in another pooled group named any 'adenoma'.

Crude positive predictive values (PPV) for cancer, advanced adenoma, low-risk adenoma, advanced neoplasia and any adenoma were defined as the number of participants with those lesions relative to all participants with positive testing and subsequent colonoscopy. The per-protocol analysis considered the neoplasia detection rates among screening participant (x1000 subjects), while intention-to-treat analysis considered the detection rates among the invited target population (x1000 subjects).

Screen-detected cancers were defined as cancers detected during screening. Interval cancers were cancers diagnosed within a two years-period following a negative test. Non-screen-detected cancers were cancers detected in non-participants within a two-year period following an invitation.

Analyses according to gender and age were performed for FOBT positivity rates and neoplasia detection rates. The number needed to screen (NNScreen) and the number needed to scope (NNScope) to detect one case of advanced neoplasia were calculated with 95% confidence intervals (95% CI). Analyses according the rank of screening (first screening or subsequent screening) were performed for FOBT positivity, PPV for any kind of colorectal lesion, and also for NNScreen and NNScope calculation. Differences in screening outcomes between the FIT-based screening round (seventh round) and the last gFOBT round (sixth round) were calculated using a χ 2 test. Differences in screening outcomes between the first screening group and the subsequent screening group were calculated using a χ 2 test. All P values were two-sided and considered significant if < 0.05. All analyses were carried out with Epi Info™ 7.1.5 software.

RESULTS

Population

From 2003 to 2015, the total population in the district increased from 908 449 to 1 042 884 inhabitants (+ 14.8%, + 1.2% per year), while the population at age 50 to 74 years increased from 218 175 to 269 698 subjects (+ 23.6%, + 2% per year). [START_REF]Populations légales 2015. Recensement de la population -régions, départements, arrondissements, cantons et communes[END_REF] From the latter population, the rate of excluded individuals at each round progressively increased from 13.0% to 17.2% during the same period of time (Table 1). Most people were excluded because they had undergone total colonoscopy in the previous five years. Nevertheless, despite the increased exclusions, the target population for CRC screening continued to increase from 189 812 to 223 374 subjects between 2003 and 2015 (+ 17.7%). The mean age of the target population was 59 years at each round, while the sex ratio (males:females) was 0.91.

Participation rate

The participation rate for gFOBT-based screening was highest in the first round (51.0%) and then progressively decreased to 33.9% by the sixth round. The participation rate for FIT-based screening (7 th round) rebounded to 53.4%. There was a significant increase in participation rate between the last round with gFOBT (round 6) and the first round with FIT (round 7) (+19.5%; p<10 -7 ). As already mentioned, the round 6 was shortened and the population and GPs were waiting for the FIT test, which may explain the lowest participation rate observed in this round. However, the increase in participation rate observed between round 5 and 7 remained highly significant (+12.3%; p < 10 -7 ). In each round, the participation rate was higher in women than men and increased with age (Table 1).

The rebound observed with the switch to FIT was seen in all age groups with a particular benefit for the 50-54 and 70-74 age groups exceeding 50% and reaching almost 70%, respectively (Table 1). We estimated that the participation rate in the seventh round would have been equal to 36.6% without mailing the FIT with the reminder letter along.

gFOBT and FIT results

The gFOBT positivity rate was highest (2.59%) at the first round, and then slightly decreased with the exception of the 3 rd round (3.01%) during which FIT and gFOBT data were pooled for 28% of the gFOBT attendees. Using the 30 µg Hb/g cut-off, the FIT positivity rate was 4.26%. Regardless of the test used or the screening round, the FOBT positivity rate was markedly higher in men than in women. The positivity rate also increased according to age with both tests (Table 1). Most of the non-analysable tests were redone such that the final proportion of non-analysable tests remained low, though notably slightly higher with FIT because of exceeding the expiration date.

Colonoscopy findings

Compliance with colonoscopy following positive testing was constantly superior to 90%. In each round, the number of colonoscopies was higher in men than in women, despite lower participation to screening in men than in women. This difference was related to the higher positivity rate of testing in men and was particularly true for the FIT-based screening round, where 2741 colonoscopies were performed in men vs. 1934 colonoscopies in women (Table 2). In each round, the proportion of complete colonoscopy was superior to 95% (Table 2). Reasons for incomplete colonoscopy have been previously published. 13 Among the 16 580 colonoscopies performed over 14 years, only 10 perforations occurred (perforation rate 0.60%).

The PPV for cancer was the highest (11.1%) in the first round and then progressively declined to 4.9% in the sixth round with gFOBT and then rebounded to 8.5% in the 7 th FIT-based screening round (P<0.001) (Table 2). By contrast, the PPV for advanced adenoma did not change during the six g-FOBT-based screening rounds. However, following the switch to FIT, the PPV for advanced adenoma and advanced neoplasia increased significantly (P<10 -7 ) (Table 2). As a consequence of the concomitant decline of the PPV for cancer and decreased participation rate over the course of the six gFOBT-based screening rounds, the number of screen-detected cancers progressively decreased from 260 to 76 cases per round with gFOBT. By contrast, the number of screen-detected cancers was 395 with FIT screening. A similar increase was observed for the number of subjects with advanced adenoma (Table 2).

Figure 1 depicts the increase of any kind of screen-detected lesions following FIT compared with gFOBT by per-protocol (x1000 screened individuals) or intention-to-treat analysis (x1000 target individuals). The benefit of FIT was already perceptible during the 3 rd round with gFOBT because onequarter attendees simultaneously had both tests performed. Regarding screen-detected advanced neoplasia, which is the major goal of screening, the per-protocol analysis demonstrated that the greater than threefold increase observed between the 6 th and the 7 th round was seen in both sexes and all classes of age (P<10 -7 ) (Fig. 2). By intention-to-treat analysis, a fivefold increased detection of CRC and advanced adenoma was seen among invitees (P<10 -7 ) (Fig. 2).

Screen-and non-screen detected cancers

The proportion of screen-detected, interval and non-screen-detected cancers among cancers diagnosed at each round in the target population is given in Figure 3. The proportion of screen-detected cancers progressively declined over the course of the six gFOBT-based screening rounds, decreasing from 39.9% to 17.6%, whereas the proportion of interval cancers was relatively stable and the proportion of non-screen-detected cancers progressively increased from 42.1% to 67.7%. That tendency radically reversed with the FIT test; the proportion of screen-detected cancers was 61.8% and that of non-screen-detected cancers was 31.8%. With the FIT test, the proportion of interval cancers was only 6.4% (P<10 -7 ).

Screening output measures

Table 3 depicts the estimates of NNScreen and NNScope for detecting one advanced neoplasia. Both NNScreen and NNScope values progressively increased over the course of the six gFOBT-based screening rounds, except for the 3 rd round, while FIT-based screening induced a dramatic decrease in both values for men and women. Indeed, by using FIT, NNScreen was 66 for detecting one advanced neoplasia, which corresponded to a 3.3-fold reduction compared to the value registered at the last gFOBT screening round (P<10 -7 ). NNScope was 2.6 for detecting one advanced neoplasia, which corresponded to a 1.8-fold reduction compared to the value registered at the previous round (P<10-

7

). As expected, both NNScreen and NNScope values were higher in women than in men with any test (Table 3).

Role of screening rank

At each round, except at the first round naturally, the majority of tests that were done, were subsequent tests (70.9 to 83.7%). As expected, the proportion of positive testing was significantly higher for the first screening tests than for subsequent tests, both for gFOBT (P<0.05) and FIT (P<0.01) (Table 1). Regarding gFOBT, the PPV for advanced neoplasia was higher for the first screening test than for subsequent tests (4.4 to 7.3 percentage points, P<0.01). By contrast with the FIT, figures were similar regardless of the screening rank (Table 2). Within the first screening group however, the PPV for advanced neoplasia screened with FIT was significantly lower in individuals who were invited for the first time than in those who had never participated before (30.6% and 40.8%, respectively, P<0.001). Similarly, if NNScreen and NNScope values were higher for the first screening gFOBT than subsequent gFOBT, the values were similar regardless of the rank of screening for the FIT (Table 3). When comparing figures between first tests and subsequent tests among all the gFOBTbased screening rounds, the third round was an exception. As already mentioned, one-quarter of attendees in the third round simultaneously had both tests performed, mostly in people with previous screening.

DISCUSSION

In this study, we presented a population-based experience in biennial FOBT-based CRC screening over a 15-year period, leading to a cumulative 647 676 screenings performed in 228 716 different individuals, 17 819 subjects with positive testing and 16 580 following colonoscopies resulting from a compliance to colonoscopy of 93%. Of course, the population in the district was not fixed during the seven rounds. Indeed, the total population progressively increased by 1.2% per year as the target population for CRC screening increased by 17.5% from 2003 to 2015. The long-term performance of screening programmes, including the switch of gFOBT to FIT in real life, has been not reported in the literature. Six biennial screening rounds with gFOBT were performed in the present study before shifting to FIT for the 7 th round.

Our study demonstrated a slight but regular decline in participation rate during the first six rounds with gFOBT. The lowest rate, recorded in the 6 th round, could be observed because the population and GPs were waiting for the FIT test, the launch of which was expected. The first benefit of the switch to FIT was the increased participation rate of nearly 20 percentage points compared with the last gFOBT-based screening round and 9.8 percentage points compared with the mean value for the 6 gFOBT-based rounds. The benefit of 12 percentage points previously reported in randomized controlled studies, [START_REF] Van Rossum | Random comparison of guaiac and immunochemical fecal occult blood tests for colorectal cancer in a screening population[END_REF][START_REF] Hol | Screening for colorectal cancer: random comparison of guaiac and immunochemical faecal occult blood testing at different cut-off levels[END_REF] was found in our study when comparing participation between the round 7 and the round 5 which best reflects the participation rate with gFOBT given the limitations of the 6th round underlined above. In the present study, the increased participation with FIT was observed for women and men and for all age groups with a notable benefit for the 50-54 and 70-74 age groups (52.5% and 67.6%, respectively). To understand the reasons for this increase in participation, we conducted a survey in previous non-responders to gFOBT who participated in FIT. 14 Among the testrelated major determinants of FIT compliance was the perception that the test was less complicated than the previous test and that a unique stool sample was required. Among the non-test related major determinants of FIT compliance were the feeling of being more concerned and the perception that the GP was more convincing. We could hypothesize that GPs were more motivated to present the new test because it was more reliable but also simpler to realize. GPs were sensitized on these two points during the implementation of the FIT through a new national information and training campaign. As a result of FIT launching in France, 'Ille-et-Vilaine' was the district with the highest participation rate while the mean participation rate in France was only 33% in the 2016-2017 period. 15 However, the participation rate would have been 36% instead of 53% if mailing the test had not been allowed by health authorities as part of a pilot study in our district. Considered together with these results, National Health Authorities have just introduced substantial changes to the screening specifications now authorizing test delivery with the second reminder letter throughout the whole French territory. 16 In order to limit the additional costs however, we chosen for the ongoing 8 th screening round not to mail the FIT to people who were previously non-responders despite invitation to at least three rounds.

The second benefit of the switch to FIT was the rebound in the diagnostic yield of advanced neoplasia, CRC and advanced adenomas. Indeed, the number of screen-detected cancers increased fivefold with FIT screening compared to the last gFOBT-based round, while a similar increase (x 5.5) was observed for the number of subjects with advanced adenoma. Thus, the detection rate of advanced neoplasia per 1000 invitees increased fivefold. That benefit was not solely the consequence of the increased participation rate but also the increased detection rate among attendees as previously shown in controlled studies. [START_REF] Van Rossum | Random comparison of guaiac and immunochemical fecal occult blood tests for colorectal cancer in a screening population[END_REF][START_REF] Hol | Screening for colorectal cancer: random comparison of guaiac and immunochemical faecal occult blood testing at different cut-off levels[END_REF] This result is all the more remarkable given that performance measures of repeat gFOBT screening had markedly decreased, as was also reported in other national gFOBT-based screening programmes. [START_REF] Steele | Results from the first three rounds of the Scottish demonstration pilot of FOBT screening for colorectal cancer[END_REF][START_REF] Kearns | Guaiac faecal occult blood test performance at initial and repeat screens in the English Bowel Cancer Screening Programme[END_REF] The detection rate of advanced neoplasia among attendees increased more than threefold compared with the previous round and was observed in both sexes and all age groups. This benefit was the result of the increased PPV for cancer and advanced adenoma (rate difference 3.6% and 13.7%, respectively) and a testing positivity rate difference of +2.0% (almost twice as high) due to a higher test sensitivity. The benefit of FIT could not be attributed to the important proportion of first tests (28.1%) at the seventh round because the PPV for advanced neoplasia were similar for first and subsequent tests (Table 2).

It is noteworthy that our findings compare favourably with those of a screening-naïve populationbased study using a 10 µg/g cut-off that reported a detection rate of 23 advanced neoplasia per 1000 subjects screened compared to 15.1% in the present study, while the positivity FIT rates were 7.9% and 4.2%, respectively. [START_REF] Grobbee | A randomised comparison of two faecal immunochemical tests in population-based colorectal cancer screening[END_REF] By using the intermediate 20 µg/g cut-off, a pilot study conducted within the mature national gFOBT-based screening programme in England reported a testing positivity rate of 7.8% and a detection rate of cancer and advanced adenomas that had increased twofold and nearly fivefold, respectively. [START_REF] Moss | Increased uptake and improved outcomes of bowel cancer screening with a faecal immunochemical test: results from a pilot study within the national screening programme in England[END_REF] The smaller increase recorded in the present study for advanced adenomas (3.3-fold) could be a natural consequence of the value chosen for the FIT cut-off but could also be related to the inclusive 60-74 age group or a weaker performance of the gFOBT in the English screening programme. [START_REF] Moss | Increased uptake and improved outcomes of bowel cancer screening with a faecal immunochemical test: results from a pilot study within the national screening programme in England[END_REF] In a very large population-based cohort followed for six FIT-based rounds by using the same 20 µg/g cut-off, Zorzi et al. reported crude detection rates in the first screening round that were similar to the values recorded in the present study for cancer (3.3 vs. 3.3 per 1000 subjects screened) and slightly higher for advanced adenoma (15.9 vs. 11.8 per 1000 subjects screened) but the cumulative values over the six consecutive rounds were lower (1.77 for cancer and 10.7 for advanced adenoma per 1000 subjects screened, respectively). [START_REF] Zorzi | Long-term performance of colorectal cancer screening programmes based on the faecal immunochemical test[END_REF] In that Italian study, the number needed to screen and number needed to scope to detect one case of advanced neoplasia were 80.4 and 3.1, respectively (52.0 and 2.7 at the first round), compared with 66 and 2.6 in the present study.

However, the comparability between the two studies is limited by the fact that the analysis was restricted to subjects aged 50 to 64 in the Italian study. [START_REF] Zorzi | Long-term performance of colorectal cancer screening programmes based on the faecal immunochemical test[END_REF] Knowing that an increase in participation and in test positivity rates has implications for colonoscopy workload, it seems, therefore, that the 30 µg Hb/g cut-off chosen by the French Health Authorities is a good compromise between increasing performance outcomes and limiting the number of colonoscopies required. By using Markov models to compare different strategies in a general population of 100,000 individuals aged 50-74 over a 20year period, Lejeune et al. demonstrated that the one-stool sample OC-Sensor with the 30 µg Hb/g cut-off was the most efficient strategy. [START_REF] Lejeune | The cost-effectiveness of immunochemical tests for colorectal cancer screening[END_REF] Goede et al. reported that, compared to the biennial gFOBT programme, switching to FIT at a high 200 ng/ml (40 µg/g) cut-off level could increase the health benefits of the Canadian CRC programme without considerably increasing colonoscopy demand. [START_REF] Goede | benefits and costs of fecal immunochemical testing versus guaiac fecal occult blood testing for colorectal cancer screening[END_REF] The economic analysis of FIT screening in England, using data directly comparing FIT with gFOBT in the NHS screening programme, suggested recently that FIT was highly cost-effective at all thresholds considered, reaching 180 µg/g. [START_REF] Murphy | Cost-effectiveness of the faecal immunochemical test at a range of positivity thresholds compared with the guaiac faecal occult blood test in the NHS Bowel Cancer Screening Programme in England[END_REF] The strengths of the present study are as follows: 1) the long-term experience with FOBT-based screening in a large well-defined population, including the switch to FIT, whose increased performance for the detection of cancers and advanced adenomas compared to gFOBT has been confirmed; 2) the quality metrics of the screening programme, notably for colonoscopy [START_REF] Kaminski | Performance measures for lower gastrointestinal endoscopy: a European Society of Gastrointestinal Endoscopy (ESGE) Quality Improvement Initiative[END_REF] including: a) the high rate for the compliance to colonoscopy following positive testing (93%), b) the high rate for complete colonoscopy (97%), c) the high rates for neoplasms detection despite inter-endoscopist variability demonstrated in organized screening programmes [START_REF] Bretagne | Variations between endoscopists in rates of detection of colorectal neoplasia and their impact on a regional screening program based on colonoscopy after fecal occult blood testing[END_REF][START_REF] Bretagne | Interendoscopist variability in proximal colon polyp detection is twice higher for serrated polyps than adenomas[END_REF][START_REF] Zorzi | Quality of colonoscopy in an organized colorectal cancer screening programme with immunochemical faecal occult blood test: the EQuIPE study (Evaluating Quality Indicators of the Performance of Endoscopy)[END_REF] , and d) the low rate of colonoscopic perforation that was similar to rate of 0.6‰ recently reported in the English NHS bowel screening programme; [START_REF] Derbyshire | Colonoscopic perforations in the English National Health Service Bowel Cancer Screening Programme[END_REF] and 3) the prospective data recording for interval and non-screen-detected CRC, which permitted us to demonstrate the reversal of the ratio between screen-detected and non-screen detected CRC by switching to FIT.

Our study also has some limitations: a) comparability with literature is limited for colonoscopic findings because we did not take into account villous component for the definition of advanced adenoma according to recent French recommendations. 11 Such an analysis leads to minimizing the rate of advanced adenoma and therefore of advanced neoplasia detected by colonoscopy in the present study. b) some characteristics of the target population, such as levels of deprivation, were not considered for analysis of contributors to screening participation other than age and gender.

To conclude, the present study demonstrated a dramatically increase in participation and improved outcomes of CRC screening with the FIT test introduced into a mature gFOBT-based screening programme. Furthermore, these findings seem to validate at the population level the choice of a 30 µg Hb/g cut-off for the OC-sensor test positivity, even if improving screening participation remains a challenge in France. A) Prevalence of neoplasia detected among individuals (x 1000) invited to participate to the screening programme at each of the seven consecutive screening rounds. B) Prevalence of neoplasia detected among screened individuals (x 1000) at each of the seven consecutive screening rounds. Table 1. Demographic data, participation rates and testing positivity rates at each of the seven screening rounds.

Round 1

2003-2005

Round 2

2005-2007

Round 3

2007-2009

Round 4

2009-2011

Round 5

2010-2012

Round 6

2012-2014

Round 7

2015-2017

Comparison between rounds 6 and 7

P values

  12. Sobin LH, Compton CC. TNM seventh edition: what's new, what's changed: communication from the International Union Against Cancer and the American Joint Committee on Cancer. Cancer 2010;116:5336-9. 13. Bollart AS, Manfredi S, Piette C, et al. Frequency and efficacy of additional investigations following incomplete colonoscopies: A population-based analysis. Dig Liver Dis 2015;47:720-5. 14. Le Pimpec F, Moutel G, Piette C, et al. Faecal immunological blood test is more appealing than the guaiac-based test for colorectal cancer screening. Dig Liver Dis 2017;49:1267-72. 15. Santé publique France. Taux de participation au programme de dépistage organisé du cancer colorectal 2016-2017. http://invs.santepubliquefrance.fr/fr./Dossiers-thematiques/Maladieschroniques-et-traumatismes/Cancers/Evaluation-des-programmes-de-depistage-descancers/Evaluation-du-programme-de-depistage-du-cancer-colorectal/Indicateurs-d-

Figure 1 .

 1 Figure 1.

Figure 2 .Figure 3 .

 23 Figure 2. Detection rates of advanced neoplasia among screened individuals (x 1000) at round 6 and 7.

Acknowledgements

The authors acknowledge the contributions made by the general practitioners and the gastroenterologists in the district of Ille-et-Vilaine. They also acknowledge the staff in charge of the database in the screening centre, particularly Samuel Foucrit, the computer scientist.

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'.

Round 1

2003-2005

Round 2

2005-2007

Round 3

2007-2009

Round 4

2009-2011

Round 5

2010-2012

Round 6

2012-2014

Round 7

2015-2017

Comparison between rounds 6 and 7

P values

Compliance NNScreen: number needed to screen; NNScope: number needed to scope NA: not applicable


Emmanuel Deshayes
email: emmanuel.deshayes@icm.unicancer.fr

Hélène De Forges

Julien Fraisse

Marie-Claude Eberlé

Sophie Guillemard

Anne Fallières

Jean-Pierre Pouget

Raphaël Tetreau

Pierre-Olivier Kotzki

Lore Santoro

Raphael Tetreau

Pierre Senesse

Nicolas Flori

Artificial nutrition in patients with cancer has no impact on tumour glucose metabolism: Results of the PETANC Study

Keywords: Nutritional support, tumour growth, 18 F-FDG PET/CT, cancer, cachexia, supportive care

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

INTRODUCTION

Cachexia is a common problem in patients with advanced cancer, especially oesophageal and head and neck cancer. In these patients, cachexia is correlated with an increase in therapy-related side effects and poorer response to treatment (cancer relapse, lower survival). European and national evidence-based guidelines have been published about nutritional support therapy in patients with cancer [START_REF] Arends | ESPEN guidelines on nutrition in cancer patients[END_REF][START_REF] Senesse | Clinical nutrition guidelines of the French Speaking Society of Clinical Nutrition and Metabolism (SFNEP): Summary of recommendations for adults undergoing non-surgical anticancer treatment[END_REF]. Nutritional support, administered as recommended by these guidelines, has an impact on the patient outcome. However, it is not known whether artificial nutrition could "feed" the tumour and accelerate its growth. According to the European and French learned societies, the available proofs of a positive effect of artificial nutrition on tumour growth are not sufficient to recommend the suppression or delay of this therapy in cachectic patients with cancer. However, these recommendations are based on very few and quite old studies. Specifically, in 1991, Rossi-Fanelli et al. determined the thymidine labelling index in tumour samples collected before and after 14 days of glucose-based or lipid-based parenteral nutrition formula, or isocaloric oral diet (n=27 patients). They did not find a positive effect of glucose (or a negative effect of lipids) on cancer cell proliferation [START_REF] Rossifanelli | Effect of energy substrate manipulation on tumour cell proliferation in parenterally fed cancer patients[END_REF].

Jin et al. assessed tumour cell growth (percentage of cells in S phase and DNA index) and sensitivity to chemotherapy in tumour samples from 91 patients with gastrointestinal cancer and malnutrition after 7 days of various preoperative interventions (parenteral nutrition alone, parenteral nutrition plus chemotherapy, chemotherapy alone, and no treatment) [START_REF] Jin | Effects of parenteral nutrition support and chemotherapy on the phasic composition of tumor cells in gastrointestinal cancer[END_REF]. They found that the nutrition support and chemotherapy combination improved the patients' short-term nutritional status without increasing tumour cell proliferation and prevented some adverse events observed in the group with chemotherapy alone. They also suggested, but without evidence, that parenteral nutrition improves chemotherapy effectiveness.

Recent imaging technologies allows investigating this crucial question in a non-invasive manner, by performing serial 18 F-fluorodeoxyglucose (FDG) positron emission tomography/computed tomography ( 18 F-FDG PET/CT). 18 F-FDG PET/CT imaging data are used to compute static and dynamic FDG parameters. Several studies have shown than dynamic FDG uptake measurements are more accurate than the standardized uptake value (SUV) to assess differences between benign and tumour tissues and to highlight changes in tumour metabolism after treatment [START_REF] Dimitrakopoulou-Strauss | Quantitative approaches of dynamic FDG-PET and PET/CT studies (dPET/CT) for the evaluation of oncological patients[END_REF][START_REF] Epelbaum | Tumor aggressiveness and patient outcome in cancer of the pancreas assessed by dynamic 18F-FDG PET/CT[END_REF]. Moreover, 18 F-FDG PET-based quantification of glucose metabolism is reproducible with a coefficient of variation of approximately 10% (test-retest studies for repeated scans of the same patients) [START_REF] Lodge | Repeatability of SUV in Oncologic 18F-FDG PET[END_REF]. Since glucose transport is the rate-limiting step of glycolysis in tumours, if artificial nutrition would have an effect on tumor activity, it would be by modifying tumor expression of glucose transporters (GLUTs) and hexokinases. Tumour mass changes are of a quite long duration and could not be detected quantitatively using the available methods (CT scan for example) in a short time period, contrary to tumour glucose metabolism which may be modified very quickly after treatment induction.

Therefore, we designed a prospective clinical study to determine whether artificial nutrition has an impact on tumour glucose metabolism in cachectic patients with cancer by comparing 18 F-FDG PET/CT static and dynamic imaging parameters before and after a period of nutrition support.

METHODS

Study design and patients

Inclusion criteria for this prospective study were: patients with histologically-proven head and neck or oesophageal cancer and cachexia (weigh loss >5%) and eligible for artificial (enteral or parenteral) nutrition supplementation; availability of one 18 F-FDG PET/CT exam performed at our centre before inclusion (standard procedure, outside this study); ≥18 years of age, and WHO (ECOG) performance status ≤2. The main exclusion criteria were: administration of a cancer treatment (chemotherapy, targeted therapy or surgery) in the past 2 months, or radiotherapy in the past 4 months; capillary blood glucose >12mmol/L at the first 18 F-FDG PET/CT evaluation; current treatment for diabetes (insulin or oral treatment); and contra-indication to 18 F-FDG PET/CT, such as pregnancy or breastfeeding, or psychological disorders. All patients signed a written informed consent prior to inclusion. The study was conducted in accordance with the Good Clinical Practice requirements (Helsinki Declaration), and was approved by local and national review boards. It was also registered on clinical.trials.gov.

Nutritional intervention

Nutrition support therapy was administered in accordance with the French Clinical Nutrition and Metabolism Society guidelines [START_REF] Crenn | SFNEP oncology nutrition guidelines: Place of artificial nutrition in the management of cancer patients[END_REF]. Enteral and parenteral nutrition was adapted to the patient's current oral intake that was deduced from the 30-35cal/kg/day objective. Enteral nutrition was administered with a nasogastric probe. If enteral nutrition was not possible, parenteral nutrition was administered through a central venous catheter.

Assessment

The intra-tumour metabolic glucose activity was evaluated using 18 F-FDG PET/CT imaging at baseline (standard procedure) and after a minimum of 5 days of artificial nutrition. During this time, patients did not receive any cancer treatment. However, treatment initiation (i.e. chemotherapy) could not be delayed by the study. The patient food intake was measured using a visual analogue scale (food intake VAS) at baseline and at the second 18 F-FDG PET/CT exam.

Glucose metabolism

Glucose metabolism differs in normal and cancer cells. Specifically, normal cells produce energy from glucose mainly through the mitochondrial oxidative phosphorylation pathway.

Conversely, cancer cells preferentially produce energy through conversion of glucose into lactate, even in aerobic conditions [START_REF] Warburg | On the origin of cancer cells[END_REF] (i.e., aerobic glycolysis, also known as the "Warburg effect") [START_REF] Vander Heiden | Understanding the Warburg Effect: The Metabolic Requirements of Cell Proliferation[END_REF]. To compensate for the poor energy production yield through the lactate pathway, glycolysis is increased in tumour cells through upregulation of GLUTs and glucose hexokinase. These transporters are not insulin-sensitive (unlike muscles) and therefore are not altered by glycaemia or a fasting period [START_REF] Busing | Impact of blood glucose, diabetes, insulin, and obesity on standardized uptake values in tumors and healthy organs on 18F-FDG PET/CT[END_REF]. 18 F-FDG is a glucose analogue used as a PET radiotracer. Its uptake by tumour cells is directly related to their glucose consumption. Like glucose, it is transported into the cells, phosphorylated to 18 F-FDG-6-phosphate ( 18 F-FDG-6-P), and trapped within the cells. Therefore, 18 F-FDG-PET imaging allows the direct estimation of the cancer cell glucose concentration and glycolytic activity. 18 F-FDG-PET is routinely used in oncology for the initial tumour detection, characterization and staging and for monitoring the therapeutic response in several cancer types [START_REF] Wahl | From RECIST to PERCIST: Evolving Considerations for PET response criteria in solid tumors[END_REF].

Imaging

Both 18 F-FDG-PET and CT exams were performed using the same apparatus (Discovery PET/CT 690 scan, GE Healthcare, Milwaukee, Wisconsin, USA) [START_REF] Bettinardi | Physical performance of the new hybrid PETCT Discovery-690[END_REF], in the same conditions, and approximatively (± 2 hours) at the same time of the day. Patients were asked to strictly fast during the 6 hours before 18 F-FDG injection and artificial nutrition was also stopped. After intravenous injection of 3.5MBq/kg 18 F-FDG, dynamic PET images centred on the tumour were acquired for 40 minutes, followed by static whole-body images at 60 minutes after 18 F-FDG injection. Images were acquired using the List Mode, corrected (for normalization, dead time, activity decay, random coincidence, attenuation and scatter) and then reconstructed in a 256×256 image matrix. The acquired field of view size was 70 cm. Image analysis was performed using the PMOD software. For all patients, the region of interest (ROI) was drawn around the primary tumour by the same experimented nuclear medicine physician to calculate the imaging parameters. SUV is the most commonly used parameter for glucose metabolism quantification, and represents the FDG concentration in a volume of interest (i.e., the tumour or part of the tumour) normalized to the patient's weight and total injected activity: SUV = activity concentration in tissue (injected activity/ body weight)

On static 3D PET images (acquired 60 minutes after 18 F-FDG injection), the following parameters were extracted from the ROI within the tumour:

-SUVmax: the voxel with the highest radioactivity within the ROI; is the most widely used parameters with good inter-observer reproducibility;

-SUVmean: reflects the metabolic activity within the whole tumour; however, it is sensitive to the tumour volume delineation;

-SULpeak (SUL = SUV normalized to the lean body mass): represents the average activity in a small fixed-size ROI that includes the maximum voxel; this is considered to be the best parameter for the assessment of solid cancer response to treatment [START_REF] Wahl | From RECIST to PERCIST: Evolving Considerations for PET response criteria in solid tumors[END_REF].

SUVmean and SUVmax were also assessed in peritumoral healthy tissue.

To reflect variations over time, the dynamic PET (dPET) images acquired just after 18 F-FDG injection were used to calculate the following parameters. The glucose metabolic rate (MRGlu, expressed in µmol.min -1 .100g -1 of tumour tissue) was calculated using the "MRGlu Patlack" mode of the PMOD software, as previously described [START_REF] Patlak | Graphical evaluation of blood-to-brain transfer constants from multiple-time uptake data. Generalizations[END_REF]. The vascular input function was set on an artery found in the view field, and the lump constant (LC) was set to 1. Another PMOD mode ("the two-compartment" mode) allowed calculating the transport constant rates (k) of the two-compartment model (Figure 1): k1, k2, k3, k4 (expressed in h -1 ). These variables are associated with molecular biological processes, such as GLUT (k1-k2) and hexokinase activities (k3-k4). The PET/CT device quantitative accuracy was assessed following the European guidelines for 18 F-FDG PET/CT tumour imaging [START_REF] Boellaard | FDG PET/CT: EANM procedure guidelines for tumour imaging: version 2.0[END_REF],

and the device was accredited for tumour imaging by the European Association of Nuclear Medicine

Research Ltd (EARL).

Figure 1: Two-compartment model of FDG kinetics in tumour cells

Endpoints

The primary endpoint was the difference between pre-and post-artificial nutrition intratumour SUVmax values. Secondary endpoints were the pre-and post-artificial nutrition variations of the static parameters SUVmean and SULpeak, and of the dynamic parameters MRGlu, k1, k2, k3, and k4.

Statistical analysis

Continuous variables were described using medians and ranges, and categorical variables with frequencies and percentages. The Wilcoxon Rank-Sum test was used to compare the distribution of continuous variables between the first and second 18 F-FDG PET/CT exam. All tests were two-sided and a p-value <0.05 was considered as statistically significant. Statistical analyses were performed using STATA 13.0 (StataCorp, College Station, TX, USA).

RESULTS

Patients

Eleven patients were included in our prospective monocentric study (Table 1). Among them, 9

(

Nutritional status

The baseline median food intake VAS score was 3 [0-6]. Artificial nutrition was administered to 10 patients (median energy intake: 21.83 kcal/kg/day [13.16-45.90], with a median protein intake of 0.84 g/kg/day [0.56-1.64]) (Table 1). Artificial nutrition (enteral for eight patients, 80%, and parenteral for two patients, 20%) was used to complement oral nutrition (median oral energy intake:

19.44 kCal/kg/day [5.37-25.95]). On average, patients received artificial nutrition for 7 days ( [START_REF] Dimitrakopoulou-Strauss | Quantitative approaches of dynamic FDG-PET and PET/CT studies (dPET/CT) for the evaluation of oncological patients[END_REF][START_REF] Epelbaum | Tumor aggressiveness and patient outcome in cancer of the pancreas assessed by dynamic 18F-FDG PET/CT[END_REF][START_REF] Lodge | Repeatability of SUV in Oncologic 18F-FDG PET[END_REF][START_REF] Crenn | SFNEP oncology nutrition guidelines: Place of artificial nutrition in the management of cancer patients[END_REF][START_REF] Warburg | On the origin of cancer cells[END_REF]) before the second 18 F-FDG PET/CT exam. The food uptake VAS score was 5 [START_REF] Arends | ESPEN guidelines on nutrition in cancer patients[END_REF][START_REF] Senesse | Clinical nutrition guidelines of the French Speaking Society of Clinical Nutrition and Metabolism (SFNEP): Summary of recommendations for adults undergoing non-surgical anticancer treatment[END_REF][START_REF] Rossifanelli | Effect of energy substrate manipulation on tumour cell proliferation in parenterally fed cancer patients[END_REF][START_REF] Jin | Effects of parenteral nutrition support and chemotherapy on the phasic composition of tumor cells in gastrointestinal cancer[END_REF][START_REF] Dimitrakopoulou-Strauss | Quantitative approaches of dynamic FDG-PET and PET/CT studies (dPET/CT) for the evaluation of oncological patients[END_REF][START_REF] Epelbaum | Tumor aggressiveness and patient outcome in cancer of the pancreas assessed by dynamic 18F-FDG PET/CT[END_REF][START_REF] Lodge | Repeatability of SUV in Oncologic 18F-FDG PET[END_REF][START_REF] Crenn | SFNEP oncology nutrition guidelines: Place of artificial nutrition in the management of cancer patients[END_REF][START_REF] Warburg | On the origin of cancer cells[END_REF][START_REF] Vander Heiden | Understanding the Warburg Effect: The Metabolic Requirements of Cell Proliferation[END_REF] at second PET/CT, higher than that at baseline evaluation, although it was not significant (p=0.13).

Metabolic 18 F-FDG PET/CT data

The values of all the tumour glucose metabolism parameters (static and dynamic) were not significantly different between first and second 18 F-FDG PET/CT (Table 2 and Figure 2). Specifically, the pre-and post-artificial nutrition mean SUVmax scores (primary outcome) were comparable (11.0 [7.8-22.2] and 10.2 [7.3-20.6]). There was also no significant difference in SUVmean and SUVmax in peritumoral tissue between the first and second 18 F-FDG PET/CT (

DISCUSSION

In this study we show that artificial nutrition in patients with head and neck and oesophageal cancer has no impact on tumour glucose metabolism, assessed by 18 F-FDG PET/CT (both static dynamic PET parameters). This is, to our knowledge, the first clinical study that studied the impact of standard nutritional support (for a mean of 7 days) using non-invasive visualization of tumour glucose metabolism by 18 F-FDG PET.

In 2004, Bozetti et al. performed a study with 18 F-FDG PET/CT in 12 patients with liver metastases from colorectal cancer [START_REF] Bozzetti | Glucose-based total parenteral nutrition does not stimulate glucose uptake by humans tumours[END_REF]. They investigated the effect of the administration of glucoseor lipid-based total parenteral nutrition (4 mg/kg/min glucose, or 2 mg/kg/min lipids and 0.7 mg/kg/min amino acids) for three hours before 18 F-FDG PET/CT, compared with control (fasting without glucose/lipid infusion). They did not find any significant change in the SUV score of liver metastases. However, this study assessed the immediate effect of a short load of glucose or lipid [START_REF] Bozzetti | Glucose-based total parenteral nutrition does not stimulate glucose uptake by humans tumours[END_REF],

whereas our present work evaluated the impact of a longer nutritional support (at least 5 days). The reviews by Bozetti et al. and by Bossola et al. [START_REF] Bossola | Does nutrition support stimulate tumor growth in humans?[END_REF][START_REF] Bozzetti | Nutritional support and tumour growth in humans: a narrative review of the literature[END_REF] identified only few studies (controlled or not) on the impact of nutritional interventions on tumour growth. In all these studies, the number of patients was small (n=10 to 20), and the definition of malnutrition and of cachexia heterogeneous.

Moreover, in most of them, tumour growth was assessed based on cancer cell cycle kinetic parameters (DNA index, DNA distribution by flow cytometry, labelling index with tritiated thymidine or bromodeoxyuridine). Among the five controlled and randomized studies (nutritional support for 6 to 18 days) [START_REF] Jin | Effects of parenteral nutrition support and chemotherapy on the phasic composition of tumor cells in gastrointestinal cancer[END_REF][START_REF] Bozzetti | Total parenteral nutrition and tumor growth in malnourished patients with gastric cancer[END_REF][START_REF] Dionigi | Pre-operative nutritional support and tumour cell kinetics in malnourished patients with gastric cancer[END_REF][START_REF] Edstrom | Cell cycle distribution and ornithine decarboxylase activity in head and neck cancer in response to enteral nutrition[END_REF][START_REF] Pacelli | Parenteral nutrition does not stimulate tumor proliferation in malnourished gastric cancer patients[END_REF], only two reported an increase in tumour cell proliferation following artificial nutrition [START_REF] Jin | Effects of parenteral nutrition support and chemotherapy on the phasic composition of tumor cells in gastrointestinal cancer[END_REF][START_REF] Bozzetti | Total parenteral nutrition and tumor growth in malnourished patients with gastric cancer[END_REF]. These reviews concluded that there is no evidence for an effect of nutritional support on tumour growth. In agreement, the European guidelines state that "theoretical arguments that nutrients "feed the tumour" are not supported by evidence related to clinical outcome and should not be used to refuse, diminish or stop feeding". Since the studies by Rossi-Fanelli [START_REF] Rossifanelli | Effect of energy substrate manipulation on tumour cell proliferation in parenterally fed cancer patients[END_REF] and Jin [START_REF] Jin | Effects of parenteral nutrition support and chemotherapy on the phasic composition of tumor cells in gastrointestinal cancer[END_REF] quoted in the European guidelines, imaging techniques and nutritional support guidelines have changed.

Nevertheless, we found surprising that studies on this topic are quite rare, especially now when dieticians and nutritionists are involved in the management of patients with cancer. On the other hand, recent publications have evaluated the link between fasting and tumour cell sensitivity to chemotherapy [START_REF] Laviano | Toxicity in chemotherapy--when less is more[END_REF]. A recent comprehensive review of all literature data (many animal studies and few epidemiological and clinical studies) (https://www6.inra.fr/nacre/Le-reseau-NACRe/Publications/ Rapport-NACRe -jeune-regimes-restrictifs-cancer-2017) concluded that there is no evidence that fasting and restrictive diets (i.e., intermittent fasting, caloric restriction and ketogenic diets) have any effect (beneficial or deleterious) on cancer prevention and treatment. FDG tumour uptake may be influenced by lactate levels in tumours ( [START_REF] Zhou | Relationship between 18F-FDG accumulation and lactate dehydrogenase A expression in lung adenocarcinomas[END_REF]), and Schroeder et al. showed that a ketogenic diet (which differs in terms of carbohydrate intake from the artificial nutrition delivered in our study) decreased tumour lactate levels in patients with head and neck cancer [START_REF] Schroeder | Decline of lactate in tumor tissue after ketogenic diet: in vivo microdialysis study in patients with head and neck cancer[END_REF]. A difference in our outcome parameters could have been linked to the tumour lactate levels. Plasma glucose levels were identical for PET1 and PET2. However, as tumours are not insulin-dependent tissues, a difference in plasma glucose level at PET1 and PET2 would probably have no impact on our results.

The small number of patients, although in the range of the previously published studies, is a limitation of our study. The lack of significant differences between PET1 and PET2 may be caused by the small size of the studied cohort. We had planned to include 20 patients, but accrual was difficult mainly due to the protocol requirements: (i) organizing in a very short time both nutritional support and a second 18 F-FDG PET/CT to ensure no delay in treatment initiation; and (ii) most potentially eligible patients had already undergone 18 F-FDG PET/CT imaging before arrival in our centre.

Performing three 18 F-FDG PET/CT scans, which would have allowed patient inclusion, was not acceptable. Other limitations are the relatively short period of nutritional support (median = 7 days) before the second 18 F-FDG PET/CT (due to the organizational constraints described above and because it was not acceptable to delay patients cancer therapy initiation), and the inclusion only of patients with head and neck or oesophageal cancer only because 18 F-FDG PET/CT is performed at diagnosis as standard practice.

In conclusion, our clinical study did not find any effect of artificial nutrition on tumour metabolism assessed with 18 F-FDG PET/CT in cachectic patients with head and neck or oesophageal cancer. Patients all received artificial nutrition according to the current guidelines in order to ensure the best support to reduce side-effects, and to satisfy the nutritional requirements of patients with cancer. showing the absence of visual differences in 18 F-FDG uptake (images acquired 60 minutes after 18 F-FDG injection)

FIGURE AND TABLE LEGENDS

Figure 2 :

 2 Figure 2: Representative 18 F-FDG PET/CT images centred on the oesophageal cancer at baseline

Figure 2 :

 2 Figure 2: Representative 18 F-FDG PET/CT images centred on the oesophageal cancer at baseline

Table 1 :

 1 Patients' characteristics at baseline and nutritional status 190

	The patients' median weight loss was 4.05% (0.6-6.1) at 1 month and 10.8% (7.3-34) at 6
	months post-diagnosis. One patient was excluded from the study after the first 18 F-FDG PET/CT
	because he required oesophageal dilatation and stent that could induce inflammation and, thus, bias the
	analysis results.

81.8%) were men. The median age was 61 years [range: 52-74], and the WHO (ECOG) performance status score ranged between 0 (n=4, 36.4%) and 1 (n=7, 63.6%). Three patients (27.3%) had head and neck, and eight (72.7%) oesophageal cancers. None of the patients received any cancer treatment prior to inclusion in this study, but all patients were intended to be curatively treated. The median duration between cancer diagnosis and inclusion in the trial was 19.5 days

. WHO: World Health Organization; VAS: Visual Analogue Scale; kcal: kilocalories

Table 2

 2

	).
	PET/CT 1	PET/CT 2	p-value

FDG: 18 F-Fluorodeoxyglucose, SUV: Standardized Uptake Value; SUL: Standardized Uptake Value corrected for Lean Body Mass, MRGlu: Metabolic Rate of Glucose

Table 2 :

 2 Glucose metabolism parameters at the first and second18 F-FDG PET/CT

Table 1 :

 1 Patients' characteristics at baseline and nutritional status

Table 2 :

 2 Glucose metabolism parameters at the first and second18 F-FDG PET/CT Figure 1: Two-compartment model of FDG kinetics in tumour cells

ACKNOWLEDGEMENTS

The authors thank the patients and their families for their participation in the study. They also thank Lobna Rifai and Caroline Constant for their work as project manager and clinical research assistant,

FUNDING SOURCES

This investigator-initiated trial was funded by the APARD foundation. The study funder had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; or decision to submit the manuscript for publication.

COMPETING INTERESTS STATEMENT

The authors have no conflict of interest to disclose regarding the present study.


Papon

Emilie Bequignon
email: emilie.bequignon@gmail.com

Huong Dang

MD Françoise Zerah-Lancner

MD-PhD André Coste

MD Laurent Boyer

MD-PhD Jean François Papon

Dr Emilie Béquignon

"Unilateral recurrent laryngeal nerve palsy post-thyroidectomy: looking for hyperventilation syndrome"

Keywords: Unilateral recurrent laryngeal nerve palsy post-thyroidectomy: Looking for hyperventilation syndrome Recurrent Laryngeal Nerve palsy, Unilateral vocal fold paralysis, hyperventilation syndrome, dyspnea

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

Introduction

The main symptom of unilateral Recurrent Laryngeal Nerve (RLN) palsy is hoarseness because of a remaining glottic gap during phonation. Patients with bilateral vocal fold paralysis typically have no impairment of the voice but inspiratory dyspnea. However, in 5-63% of cases, dyspnea also occur in unilateral RLN palsy [START_REF] Brunner | Subjective breathing impairment in unilateral vocal fold paralysis[END_REF][START_REF] Laccourreye | Unilateral laryngeal nerve paralysis in the adult: Epidemiology, symptoms, physiopathology and treatment[END_REF]. The relationship between unilateral RLN palsy after surgery and dyspnea has been poorly studied. In patients with unilateral RLN palsy, the air leak could be responsible of frequent inspirations favoring the development of hyperventilation syndrome (HVS).

The HVS is defined as chronic or recurrent changes in breathing pattern, causing respiratory and non respiratory complaints of variable severity induced by physiologically inappropriate and reproducible hyperventilation [START_REF] Christophers | The hyperventilation syndrome[END_REF][START_REF] Lewis | Mechanism and Management of Hyperventilation Syndromes[END_REF]. HVS is characterized by a variety of somatic symptoms (e.g. respiratory symptoms including short of breath, tight feeling in chest, chest pain, unable to breathe deeply, and also non respiratory symptoms such as fatigue, heart palpitations, tachycardia, anxiety, feeling tense, light-headedness, dysphagia, dizziness, tetany, myalgias, cramping and paraesthesias) [START_REF] Christophers | The hyperventilation syndrome[END_REF][START_REF] Lewis | Mechanism and Management of Hyperventilation Syndromes[END_REF]. The diagnosis of HVS is oriented by the Nijmegen score [START_REF] Van Doorn | Control of the end-tidal PCO2 in the hyperventilation syndrome: effects of biofeedback and breathing instructions compared[END_REF]. Other suggestive parameters for HVS are hypocapnia and respiratory alkalosis on the arterial blood gas. On the other hand, normality of Nijmegen score and arterial blood gas doesn't exclude the diagnosis of HVS. The hyperventilation test (HPVT) is required for HVS diagnosis. The aim of this study was to determine if dyspnea in patients with RLN palsy could be associated with HVS.

Material and methods

We conducted a preliminary observational prospective study over a 12-month period in consecutive patients who exhibited a definitive unilateral RLN palsy after thyroidectomy for benign disease and who complained of unexplained chronic dyspnea (i.e. cases of unilateral RLN palsy lasting longer than 6 months despite speech therapy). Dyspnea was considered as unexplained since lung function testing, electrocardiography, echocardiography were normal.

We confirmed unilateral RLN palsy by videolaryngoscopy in all patients and the position of paralyzed vocal fold was systematically reported. The otolaryngologists screened all patients for the presence or absence of unilateral RLN palsy by laryngoscopy, both pre-and postoperatively. This study was approved by the local research ethics committee (IRB).

Informed consent was obtained from all individual participants included in the study.

For each patient we performed the Nijmegen score, arterial blood gas and hyperventilation test. Hyperventilation test was positive according to already published criteria [START_REF] Gardner | Controlled study of respiratory responses during prolonged measurement in patients with chronic hyperventilation[END_REF][START_REF] Vansteenkiste | Diagnostic tests of hyperventilation syndrome[END_REF] (VMAX SensorMedics, SPECTRA 29 S). The Nijmegen questionnaire, comprising 16 items corresponding to the most frequent symptoms of HVS, can be used as a screening tool. Each item of the questionnaire is scored from 0 to 4 giving a maximum score of 64 and a score of >23 is commonly used as a cut-off [START_REF] Van Doorn | Control of the end-tidal PCO2 in the hyperventilation syndrome: effects of biofeedback and breathing instructions compared[END_REF]. The diagnosis of HVS was defined if at least two criteria were present among: Nijmegen score >23; reproduction of at least 2 usual symptoms during HVPT; an expirated pressure of CO2 (EpCO2) < 30mmHg or < 90% of the initial EpCO2 after a 5 minutes recovery period following a 3 minutes voluntary hyperventilation [START_REF] Chenivesse | Severely impaired health-related quality of life in chronic hyperventilation patients: exploratory data[END_REF].

Results

Three hundred and sixty-six thyroidectomies were performed during the study period.

Twenty-three unilateral Recurrent Laryngeal Nerve (RLN) palsies occurred in this period (6.3%). No patient exhibited a bilateral RLN palsy. Eleven patients had a transient unilateral RLN palsy that lasted less than three months. In two patients, a permanent unilateral RLN palsy occurred but was not associated with dyspnea. Ten non smokers patients (9 women and 1 man) were considered the study population (10%) i.e. including permanent unilateral RLN palsy (lasted more than six months) and dyspnea criteria. Mean (range) age of the patients was 59 years (44-76) (table 1). None of them reported dyspnea before the surgery. The postoperative hormonal balance (thyroid stimulating hormone (TSH) and calcium and phosphorus homeostasis) was normal in all patients of the study population. The laryngeal examination showed a paramedian position of the vocal fold in six patients (60%) and an intermediate position in 4 patients (40%) (table 2). No laryngeal oedema was described. An amyotrophy of the paralyzed vocal fold was reported in 5 patients (50%) with an induced sub-offset of the vocal fold in all cases. A glottic air leak was observed in 5 patients (50%) (table 2). Spirometry was normal in all patients (table 1). HVS was diagnosed in 8 patients (80%) (table 1). The diagnosis of HVS was defined if at least two criteria were present among: Nijmegen score >23; reproduction of at least 2 usual symptoms during HVPT; an expirated pressure of CO2 (EpCO2) < 30mmHg or < 90% of the initial EpCO2 after a 5 minutes recovery period following a 3 minutes voluntary hyperventilation [START_REF] Chenivesse | Severely impaired health-related quality of life in chronic hyperventilation patients: exploratory data[END_REF]. In our series, 5 patients have suggestive parameters for HVS on the arterial blood gas with hypocapnia (<35 mmHg) and/or respiratory alkalosis (Ph >7.42). Two patients presented a respiratory alkalosis on the arterial blood gas, but this criterion was insufficient to set the diagnosis of HVS (table 1).

Among the 5 patients with hypocapnia (PaCO2 <35 mmHg), 3 were diagnosed with HVS.

Out of 8 patients with confirmed diagnosis of HVS, 6 patients had positive Nijmegen score (75%). In Nijmegen, the main associated symptoms reported were feeling of anxiety (7/8 patients), feeling tense (6/8 patients), short of breath (8/8 patients). In 7/8 patients with confirmed HVS, HPVT was abnormal based on subjective criteria (reproduction of at least 2 usual symptoms) and on objective criteria (an expirated pressure of CO2 (EpCO2) < 30mmHg after a 5 minutes recovery period following a 3 minutes voluntary hyperventilation).

The last patient with confirmed HVS (the 6 th patient in the table 1) was a 49-year old woman with a medical history of arterial hypertension and a surgery of total thyroidectomy for benign multinodular disease. She had no dyspnea, no allergy, no cardiac dysfunction and no smoking habit before surgery. Pre-operative physical exam including indirect laryngoscopy showed normal vocal fold and symmetric arytenoid mobility. One day after surgery, a dysphonia without dyspnea was reported. Indirect laryngoscopy objectivized immobility of

Discussion

Breathing problems are not usually described in unilateral RLN palsy but in clinical practice, several patients complain of dyspnea. In a series of 63 patients with unilateral RLN palsy, 75% of them reported breathing problems in everyday life, half of them with considerable to severe problems [START_REF] Brunner | Subjective breathing impairment in unilateral vocal fold paralysis[END_REF]. One limitation of our study is the selection of a group on basis of dyspnea. This criterion is subjective but no dyspnea was reported before surgery. An organic etiology must be firstly explored in case of dyspnea with RLN palsy: a cardiorespiratory insufficiency or a hypopharyngeal and laryngeal local cause. In an uncommon reported case of dyspnea with unilateral vocal fold paralysis in a patient with acromegaly, the etiology of dyspnea was due to hypopharyngeal and laryngeal soft tissue swelling [START_REF] Lerat | An uncommon case of dyspnea with unilateral laryngeal paralysis in acromegaly[END_REF]. Nasofibroscopy revealed a global and major oedema of the glottis and supraglottis and also a paralysis of the left vocal fold. In our series, no cardiac or lung dysfunction was found to explain dyspnea in our patient and no oedema was reported in nasofibroscopy. Another cause of dyspnea after surgery after thyroidectomy could be hypothyroidism [START_REF] Sadek | Pulmonary consequences of hypothyroidism[END_REF]. Although hypothyroidism has an insidious onset and relatively asymptomatic, exertional dyspnea and fatigue can be the presenting complaints [START_REF] Sadek | Pulmonary consequences of hypothyroidism[END_REF]. In a recent article about pulmonary consequences of hypothyroidism DLCO and exercise testing parameters were significantly reduced (Even with the presence of normal chest X-ray, arterial blood gases, and spirometry) in 42 patients with hypothyroidism versus 12 control subjects [START_REF] Sadek | Pulmonary consequences of hypothyroidism[END_REF]. In our series, all patients with dyspnea and UVFP had normal value of thyroid stimulating hormone (TSH). Dyspnea caused by UVFP is generally not highlighted in the literature. Several hypotheses have been proposed in literature to explain the pathophysiology of dyspnea in RLN palsy. In a recent series of 10 patients with dyspnea and UVFP with no significant cardiorespiratory insufficiency, the etiology of dyspnea is variable according to the author: in six patients the glottic opening was insufficient for effortless breathing. In four cases, the respiratory insufficiency was more a manifestation of limits on exertion relative to their very active lifestyle (e.g., hunting or sport activity) [START_REF] Matievics | Functional outcomes of endoscopic arytenoid abduction lateropexy for unilateral vocal cord paralysis with dyspnea[END_REF]. However, our results tend to confirm an absence of correlation between the position of the paralyzed vocal fold and the severity of the dyspnea in adults. In the series of Brunner et Al., no correlation between grade of dyspnea and position of the paralyzed vocal fold or reduced glottic space was observed [START_REF] Brunner | Subjective breathing impairment in unilateral vocal fold paralysis[END_REF]. In the same way, in a series of thirteen patients with benign glottic or subglottic stenosis, there was no correlation between glottic resistance values and dyspnea score [START_REF] Wassermann | Measuring in situ central airway resistance in patients with laryngotracheal stenosis[END_REF].

For Laccourreye et al. 2 , dyspnea could be explained by synkinesis that reflects a process of aberrant re-innervation of paralyzed laryngeal muscles (nerve fibers to re-innervate adductor laryngeal muscles whose function is abductor and vice -versa) [START_REF] Laccourreye | Unilateral laryngeal nerve paralysis in the adult: Epidemiology, symptoms, physiopathology and treatment[END_REF]. For Brunner et al. [START_REF] Brunner | Subjective breathing impairment in unilateral vocal fold paralysis[END_REF], dyspnea could be explained by neurosensorial deficits of the laryngeal mucosa or by discrepancy between effort and output of breathing.

The HVS is a psychosomatic stress-related disorder. On a psychopathological level, the HVS begins with an initial stress that creates increasing ventilation leading to a sensation of dyspnea due to an inappropriate muscular effort. Then a vicious circle appears since dyspnea becomes an anxious stimulus that maintains a chronic hyperventilation. In unilateral RLN palsy, the intense emotional stress due to the surgical complication and the resulting dyspnea could initiate the process of HVS. According to this theory, we showed in our series that all patients complained dyspnea and short of breath and an anxiety was reported in almost patients (7/8 patients). According to Boulding et al., dyspnea is unlikely to be caused solely by inadequate or inefficient ventilation either due to hyperventilation or an abnormal breathing pattern and additional factors, such as poor subjective awareness of breathing by patients, are thought to play a role. HVS could occur after surgery because of a change in breathing sensation or breathlessness [START_REF] Boulding | Dysfunctional breathing: a review of the literature and proposal for classification[END_REF]. Many patients after thyroidectomy complain of tightness and pressure and a sensation of constriction which may be a contributory factor to HVS. In a recent study, frustration, isolation, fear, and altered self-identity were primary themes reporting by patients with RLN [START_REF] Francis | Life Experience of Patients With Unilateral Vocal Fold Paralysis[END_REF]. Moreover, HVS could explain the decreased quality of life and general health status and the negative impact on social functions of patients with RLN palsy [START_REF] Spector | Quality-of-life assessment in patients with unilateral vocal cord paralysis. Otolaryngology--head and neck surgery[END_REF][START_REF] Fang | Quality of life measures and predictors for adults with unilateral vocal cord paralysis[END_REF] In order to prove a causal relationship between unilateral RLN palsy and HVS, a prospective study is necessary to compare the pre-and post-thyroidectomy values in patients with and without palsy (i.e. a control group without palsy).

Conclusion

In summary, this preliminary study is a proof of concept that HVS might explain chronic dyspnea among patients with unilateral RLN palsy. Thus a global clinical evaluation, combining ENT exam, Nijmegen score and HPVT needs to be considered in the diagnostic and therapy strategies in patients with unilateral RLN palsy and dyspnea. Further data are required to determine the incidence of respiratory impairment and HVS among patients with unilateral RLN palsy. In case of HSV diagnosis, a specific rehabilitation therapy (i.e. hypoventilation therapy) can be proposed in order to improve symptoms and quality of life of patients with unilateral RLN palsy.

Disclosure of interest:

  left arytenoid and a left vocal fold in intermediate position with no oedema. At one month follow-up, she complained about progressive breathing difficulties on exertion (shopping and climbing stairs) and shortness of breath. Clinical examination confirmed immobility of left arytenoid and a left vocal fold in intermediate position with no edema and a supraglottic compression during phonation. Pulmonary auscultation and chest radiograph were normal. She had voice therapy during 3 months. At 6 months follow-up, she complained about worsened dyspnea at rest, shortness of breath and anxiety. Physical exam showed no improvement of laryngeal mobility (with an amyotrophy of the left vocal fold) except improvement of the supraglottic compression. Nijmegen score was very high (39) and HVS was confirmed by an abnormal EpCO2 without reproduction of symptoms during HPVT.

  figures have been or will be published or submitted to another scientific journal or are being

Table 2 : Description of laryngeal examination in the study population n=10

 2

	Patient Sex	Age (years)	Body mass index (kg/m2)	vocal fold position	PaCO2** (mmHg)	pH	FEV1 (%pred)	FVC (% pred)	FEV1/FVC	Nijmegen score***		HYPERVENTILATION TEST		HVS diagnosis
												Basal EpCO2 (mmHg)	EpCO2 after 3 minutes of hyperventilation (mmHg)	EpCO2 after 5 minutes recovery (mmHg)****	Reproduction of at least 2 symptoms
	1	F	67	25	paramedian	45.2	7.4	126	121	86.15	25*	25.1	12.3	25.8*	Yes*	Positive*
	2	F	44	23	paramedian	32.3*	7.47*	108	115	81.2	15	33.7	19.5	34	no	Negative
	3	F	76	30	paramedian	39.2	7.46*	90	92	81	3	39.9	18.1	28*	Yes*	Positive*
	4	M	72	29	intermediate	30*	7.39	104	104	76	13	26.7	12	26*	Yes*	Positive*
	5	F	62	19	intermediate	21.6*	7.57*	91	NA	NA	30*	22.7	14.6	16.5*	Yes*	Positive*
	6	F	49	23	intermediate	41	7.42	95	NA	NA	39*	29	16.7	25.4*	no	Positive*
	7	F	76	26	paramedian	37	7.4	105	120	70	34*	37	17	25*	Yes*	Positive*
	8	F	66	26	paramedian	34.8*	7.44*	81	85	80	14	37.3	25.9	33.7	no	Negative
	9	F	25	28	paramedian	33*	7.4	89	99	93	34*	27.9	15.6	25*	Yes*	Positive*
	10	F	53	24	intermediate	41	7.4	85	90	88	45*	35	19.5	24*	Yes*	Positive*
	Abbreviations: F: female, M: Male, pred: predicted, FEV1:forced expiratory volume after one
	second, FVC : forced vital capacity, EpCO2: expirated pressure of CO2
	*=abnormal values contributing to establish the diagnosis of HVS
	**Normal values of PaCO2 : between 35 à 45 mmHg
	***Nijmegen score: abnormal value>23
	****EpCO2 after 5 minutes recovery (mmHg): Abnormal value of <30mmHg
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Acknowledgements:

The authors would like to acknowledge Emmanuel Lanaspre for their support of this work.