MD Athanasios G Kaditis
email: kaditia@hotmail.com

MD Emmanouel I Alexopoulos

MD Konstantinos Evangelopoulos

MD Eleni Kostadima

MD Vasiliki Varlami

MD Evangelos Boultadakis

Nikolaos Liakos

MD Epameinondas Zakynthinos

MSc Elias Zintzaras

MD Konstantinos Gourgoulianis

MSc Nikolaos Liakos Bsc

Correlation of Urinary Excretion of Sodium with Severity of Sleep-Disordered Breathing in Children: A Preliminary Study

Keywords: sleep apnea, blood pressure, fractional urinary excretion of sodium brain natriuretic peptide, natriuresis, obstructive sleep apnea, snoring natriuresis, obstructive sleep apnea, snoring

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.

Introduction

Obstructive sleep apnea-hypopnea is an established risk factor for cardiovascular disease in adults. 1 Elevated blood pressure (BP) and increased BP variability are some of the mechanisms that may contribute to cardiovascular morbidity. [1][2][3][4] Obstructive events during sleep are accompanied by episodically increased sympathetic output, intermittent peripheral vasoconstriction and repeated BP surges. 5 The baroreflex system, which should regularly smooth these BP fluctuations, has reduced sensitivity in sleep apneic adults. 4 Renal excretion of sodium and water is an additional protective mechanism against elevated BP. 6 The rate of nocturnal natriuresis in healthy adults and children is positively associated with BP levels, a phenomenon described by the term "pressure natriuresis". [START_REF] Lurbe | Obesity modifies the relationship between ambulatory blood pressure and natriuresis in children[END_REF][START_REF] Staessen | The relationship between blood pressure and sodium and potassium excretion during the day and at night[END_REF] Indeed, increased nocturnal diuresis and natriuresis have been reported in adults with obstructive sleep apnea-hypopnea compared to healthy controls. [START_REF] Zhang | Alterations in renal function in patients with obstructive sleep apnea syndrome and effects of continuous positive airway pressure[END_REF][START_REF] Gjorup | Increased nocturnal sodium excretion in obstructive sleep apnoea. Relation to nocturnal change in diastolic blood pressure[END_REF][START_REF] Rodenstein | Diurnal and nocturnal diuresis and natriuresis in obstructive sleep apnea. Effects of nasal continuous positive airway pressure therapy[END_REF] Renal sodium excretion correlates with diastolic BP levels and diminishes after treatment of sleep apnea with nasal continuous positive airway pressure (nCPAP). [START_REF] Zhang | Alterations in renal function in patients with obstructive sleep apnea syndrome and effects of continuous positive airway pressure[END_REF][START_REF] Gjorup | Increased nocturnal sodium excretion in obstructive sleep apnoea. Relation to nocturnal change in diastolic blood pressure[END_REF][START_REF] Rodenstein | Diurnal and nocturnal diuresis and natriuresis in obstructive sleep apnea. Effects of nasal continuous positive airway pressure therapy[END_REF] Hence, it has been proposed that increased BP in adults with sleep apnea may be responsible for the enhanced nocturnal natriuresis in these patients. [START_REF] Gjorup | Increased nocturnal sodium excretion in obstructive sleep apnoea. Relation to nocturnal change in diastolic blood pressure[END_REF] Although recent evidence suggests that similar to adults, children with obstructive sleep-disordered breathing (SDB) have high levels of sympathetic activity, increased BP levels and BP variability and reduced sensitivity of the baroreflex system, [START_REF] Amin | Twenty-four-hour ambulatory blood pressure in children with sleepdisordered breathing[END_REF][13][14][15][16] no pediatric studies have explored the potential relationship between severity of intermittent upper airway obstruction during sleep and natriuresis. Thus, in the current investigation

Patients and Methods

Participants and clinical evaluation

The study protocol was approved by the Larissa University Hospital Ethics Committee and parents of participants provided informed consent. Consecutive children with snoring (> 3 nights/week) for the last six months who were referred to the Sleep Disorders Laboratory for polysomnography were recruited in the study. Exclusion criteria for participation were: i) symptoms of respiratory tract infection or acute gastroenteritis; ii) history of renal disease or use of diuretics or antihypertensive medications; and iii) history of cardiovascular, neuromuscular or genetic disorders.

All subjects underwent physical examination. Body mass index (BMI) and BMI z-score were calculated. 17 Obesity was defined as BMI z-score > 1.645. 17 Participants voided in the evening prior to polysomnography and none of them had urine loss or voided overnight. Upon morning arousal from sleep and after the child had been sitting quietly for about 5 minutes, BP was measured three times using the right arm and a cuff of appropriate size (Dinamap Pro 100; Criticon; Tampa, FL, USA). To adjust for the effects of age, gender and height, BP z-scores were determined. 18 Shortly after BP measurement and without having drunk any water, children were instructed to empty their bladder in a container and venipuncture was also performed.

Urine and venous blood samples were collected and processed immediately in the Central Biochemistry Laboratory. Serum and urine sodium and creatinine concentrations were measured using standard laboratory methods.

Polysomnography

Overnight polysomnography in a single night was performed under the supervision of trained personnel. 19 The following parameters were recorded: electroencephalogram (C3/A2, C4/A1, O1/A2); right and left oculogram; submental and tibial electromyogram; body position; electrocardiogram; thoracic and abdominal wall motion; oronasal airflow; and oxygen saturation of hemoglobin.

Obstructive apnea was defined as the presence of chest/abdominal wall motion in the absence of airflow for at least two breaths in duration. 20 Hypopnea was: i) a reduction in the airflow signal amplitude of at least 50% compared to baseline; ii) in the presence of chest/abdominal wall motion; and iii) associated with oxygen desaturation of hemoglobin equal to or greater than 4% or with an arousal. Obstructive apnea-hypopnea index (OAHI) was equal to the number of obstructive and mixed apneas (apneas with both central and obstructive components) and hypopneas per hour of sleep.

Primary outcome measure

Fractional urinary excretion of sodium (FE Na ) was the primary outcome measure. ii) mild SDB (OAHI 1-5 episodes/h); and iii) moderate-to-severe SDB (OAHI > 5 episodes/hour). The three groups of children were compared in terms of age, gender, obesity status, BP and polysomnography indices using χ 2 for categorical characteristics and one-way analysis of variance (ANOVA), followed by post-hoc tests (Bonferroni), for continuous variables. BP measurements were categorized as those: i) in the upper quartile of values for all participants; or ii) in the lower three quartiles of BP values for all participants.

Univariate logistic regression analysis was applied to assess the risk for having systolic or diastolic BP in the upper quartile of measurements, in children with moderate-to-severe or mild SDB compared to those with primary snoring (reference group).

Two general linear models (GLM) were tested to describe the potential effects on FE Na of: i) severity of SDB; ii) systolic or diastolic BP; and iii) the interaction of SDB severity with systolic or diastolic BP, after adjustment for age, gender and obesity (SPSS version 16.0; SPSS, Chicago, IL). In order to assess whether natriuresis was increasing linearly to severity of SDB, linear regression analysis was carried out using FE Na as the dependent variable and OAHI, age, gender and BMI z-score as independent variables.

Results

Subjects' characteristics

A total of 95 children were recruited. Subjects' characteristics and polysomnography findings are summarized in Table 1. There were no significant F o r P e e r R e v i e w 6 differences in terms of age, female-to-male ratio, body mass index z-score or proportion of obese subjects between the three study groups. Children with moderate-to-severe SDB had similar morning systolic BP z-score to those of subjects with mild SDB and higher than subjects with primary snoring (p=0.074 and p=0.046, respectively). A nonsignificant trend for higher diastolic BP with higher OAHI was noted (Table 1).

Effects of SDB severity and BP on FE Na

The cut-off value separating the upper quartile from the lower quartiles for systolic BP z-score was 0.78. The respective cut-off value for diastolic BP z-score was 1.14. Children with moderate-to-severe SDB-but not with mild SDB-were at increased risk for having systolic BP in the upper quartile of measured values compared to subjects with primary snoring [odds ratios (95% confidence intervals): 4.12 (1.12-15.16); p = 0.033 and 1.33 (0.36-4.97); p=0.669]. The association between severity of SDB and diastolic BP was not significant (p>0.05).

SDB and diastolic BP had significant effects on FE Na (p<0.05 and p=0.007, respectively), whereas systolic BP had no effect on FE Na (p=0.715) (Table 2 and Figure 1). More specifically, subjects with moderate-to-severe SDB had similar log-transformed FE Na to that of children with mild SDB (p=0.657) and higher than participants with primary snoring (p=0.003). The interactions between SDB and systolic or diastolic BP were not significant (p=0.246 and p=0.225, respectively). In linear regression analysis, OAHI was significantly associated with FE Na after adjustment for age, gender and BMI zscore (p=0.002) (Table 3). In the present study, it was demonstrated that severity of SDB and morning BP in children have significant effect on renal excretion of sodium. Since sleep apnea in childhood has been related to elevated BP, 15,[START_REF] Amin | Activity-adjusted 24hour ambulatory blood pressure and cardiac remodeling in children with sleep disordered breathing[END_REF] the association between SDB and natriuresis may be mediated in part by BP elevation.

According to traditional physiology teaching, systemic BP and blood volume are tightly regulated. 6 Any increase in systemic BP is buffered by activation of the baroreflex system (arterial, venous and cardiac baroreceptors, brainstem, sympathetic and parasympathetic neurons) that decreases heart rate, cardiac output and systemic vascular resistance. BP elevation promotes renal excretion of sodium and water both directly, by decreased renal tubular reabsorption of sodium (pressure natriuresis), and indirectly via reduced activity of the renin-angiotensin-aldosterone system. 6-8 Moreover, any blood volume expansion leads to atrial and ventricular distention, and release of ANP and BNP. [START_REF] Dietz | Release of natriuretic factor from rat heart-lung preparation by atrial distension[END_REF][START_REF] Nakagawa | Rapid transcriptional activation and early mRNA turnover of brain natriuretic peptide in cardiocyte hypertrophy. Evidence for brain natriuretic peptide as an "emergency" cardiac hormone against ventricular overload[END_REF] Natriuretic peptides inhibit renal tubular reabsorption of sodium and accentuate renal sodium filtration. [START_REF] Yoshimura | Hemodynamic, renal, and hormonal responses to brain natriuretic peptide infusion in patients with congestive heart failure[END_REF] Studies in adults and children with sleep apnea indicate that at the termination of obstructive events repetitive increases in systemic BP take place. [START_REF] Leuenberger | Surges of muscle sympathetic nerve activity during obstructive apnea are linked to hypoxemia[END_REF][START_REF] Ringler | Hypoxemia alone does not explain blood pressure elevations after obstructive apneas[END_REF][27] Although the baroreflex system should normally attenuate excessive BP variability, both adults and children with obstructive sleep apnea have reduced nighttime baroreflex gain and increased BP variability. 4,16 [START_REF] Zhang | Alterations in renal function in patients with obstructive sleep apnea syndrome and effects of continuous positive airway pressure[END_REF][START_REF] Gjorup | Increased nocturnal sodium excretion in obstructive sleep apnoea. Relation to nocturnal change in diastolic blood pressure[END_REF][START_REF] Rodenstein | Diurnal and nocturnal diuresis and natriuresis in obstructive sleep apnea. Effects of nasal continuous positive airway pressure therapy[END_REF][START_REF] Baruzzi | Atrial natriuretic peptide and catecholamines in obstructive sleep apnea syndrome[END_REF] Results of the present investigation extend the observations from adults to children with obstructive SDB. Of note, children with obstructive sleep apnea or primary snoring have increased prevalence of nocturnal enuresis that frequently resolves after adenotonsillectomy or treatment of adenoidal hypertrophy and SDB with nasal corticosteroids. [START_REF] Alexopoulos | Association between primary nocturnal enuresis and habitual snoring in children[END_REF][START_REF] Capdevila | Increased morning brain natriuretic peptide levels in children with nocturnal enuresis and sleep-disordered breathing: a community-based study[END_REF][START_REF] Weider | Nocturnal enuresis in children with upper airway obstruction[END_REF][START_REF] Alexopoulos | Resolution of nocturnal enuresis in snoring children after treatment with nasal budesonide[END_REF] Increased nocturnal natriuresis and diuresis could conceivably contribute to nocturnal urine loss.

The exact pathophysiologic mechanisms underlying increased renal excretion of sodium in sleep apnea have not been elucidated. Similarly to the current report, Gjorup et al have found that diastolic BP and FE Na in sleep apneic adults were positively correlated. [START_REF] Gjorup | Increased nocturnal sodium excretion in obstructive sleep apnoea. Relation to nocturnal change in diastolic blood pressure[END_REF] Hence, the investigators proposed that this association may be due to pressure natriuresis. Other studies have revealed increased levels of ANP and BNP that could represent the pathophysiologic link between sleep apnea and nocturnal diuresis and natriuresis. [START_REF] Krieger | Effects of treatment with nasal continuous positive airway pressure on atrial natriuretic peptide and arginine vasopressin release during sleep in patients with obstructive sleep apnoea[END_REF]34 A few methodologic issues should be discussed. First, sodium and creatinine concentrations in an early morning urine specimen were used to calculate FE Na . Since the recruited children have not voided overnight, we speculate that the calculated FE Na reflects nocturnal urinary excretion of sodium. Second, hydration status and sodium losses via the sweat glands and the gastrointestinal system and daily sodium intake may influence FE Na . Nevertheless, none of the study participants was dehydrated, was exposed to high temperatures or had symptoms or signs of gastroenteritis. Third, recent studies have documented significant associations between systolic or diastolic BP and severity of SDB using ambulatory BP monitoring. 15,[START_REF] Amin | Activity-adjusted 24hour ambulatory blood pressure and cardiac remodeling in children with sleep disordered breathing[END_REF] A trend for higher morning diastolic BP with increasing OAHI was identified in the present report and the absence of 9 statistical significance may be attributed to the fact that overnight BP monitoring was not performed in the current report. Finally, a healthy control group including children without SDB, who were of normal weight or overweight, was not included. The absence of such a group possibly limited the statistical comparisons and the delineation of an interaction effect between SDB, obesity and BP in natriuresis. Furthermore, the use of a control group with OAHI < 1 episode/hour may be suboptimal, since even primary snoring could be associated with BP elevation. 35 In conclusion, severity of obstructive SDB and diastolic BP are positively associated with urinary excretion of sodium in childhood. Natriuresis may be a defense mechanism to systemic blood pressure fluctuations related to intermittent upper airway obstruction during sleep. We are submitting the second revision of our manuscript PPUL-10-0080 entitled "Correlation of urinary excretion of sodium with severity of sleep-disordered breathing in children: A preliminary study". In preparing our revised manuscript, we have taken under consideration the Reviewer's comments.

F

Along with the marked-up and the clean version of the revised manuscript, we are submitting a point-by-point response to the Reviewer's comments. We will be glad to make any other changes if necessary.

Sincerely

RESPONSE TO REVIEWER'S COMMENTS

1. "In pg 6 para 2, some non-significant results were presented in details with ORs and CI. As these data were non-significant, their ORs are meaningless in a strict sense and their detail reportings are misleading (mild SDB vs primary snoring in systolic BP; and effect of SDB on dBP)".

In the revised manuscript, odds ratios for non-significant results were deleted.

"

Most p-values are reported in 3 digits. This is overkill, probably 2 digits are alright".

This comment is very reasonable. However, few p-values of the present manuscript are close to 0.05 and three digits are necessary to indicate that the respective comparisons are significant (e.g. p=0.046). Thus, for consistency all p-values are presented in 3 digits.

"The conclusive statement on pg 9, 'Elevated FEna…snoring….related to intermittent upper airway obstruction…' is unclear and unrelated"

The conclusion in the revised manuscript has been modified.

"Table 2 is redundant. Either you may combine both tables 2 and 3 or you can drop table 2."

Table 2 was deleted.

"Figure 1 is uninformative. Please drop it. In addition, the description of axis and figure is unclear."

Figure 1 may help the reader to have an overall impression of the numeric data presented in the manuscript. We have made the figure more self-explanatory according to the reviewer's suggestion

Introduction

Obstructive sleep apnea-hypopnea is an established risk factor for cardiovascular disease in adults. 1 Elevated blood pressure (BP) and increased BP variability are some of the mechanisms that may contribute to cardiovascular morbidity. [1][2][3][4] Obstructive events during sleep are accompanied by episodically increased sympathetic output, intermittent peripheral vasoconstriction and repeated BP surges. 5 The baroreflex system, which should regularly smooth these BP fluctuations, has reduced sensitivity in sleep apneic adults. 4 Renal excretion of sodium and water is an additional protective mechanism against elevated BP. 6 The rate of nocturnal natriuresis in healthy adults and children is positively associated with BP levels, a phenomenon described by the term "pressure natriuresis". [START_REF] Lurbe | Obesity modifies the relationship between ambulatory blood pressure and natriuresis in children[END_REF][START_REF] Staessen | The relationship between blood pressure and sodium and potassium excretion during the day and at night[END_REF] Indeed, increased nocturnal diuresis and natriuresis have been reported

in adults with obstructive sleep apnea-hypopnea compared to healthy controls. [START_REF] Zhang | Alterations in renal function in patients with obstructive sleep apnea syndrome and effects of continuous positive airway pressure[END_REF][START_REF] Gjorup | Increased nocturnal sodium excretion in obstructive sleep apnoea. Relation to nocturnal change in diastolic blood pressure[END_REF][START_REF] Rodenstein | Diurnal and nocturnal diuresis and natriuresis in obstructive sleep apnea. Effects of nasal continuous positive airway pressure therapy[END_REF] Renal sodium excretion correlates with diastolic BP levels and diminishes after treatment of sleep apnea with nasal continuous positive airway pressure (nCPAP). [START_REF] Zhang | Alterations in renal function in patients with obstructive sleep apnea syndrome and effects of continuous positive airway pressure[END_REF][START_REF] Gjorup | Increased nocturnal sodium excretion in obstructive sleep apnoea. Relation to nocturnal change in diastolic blood pressure[END_REF][START_REF] Rodenstein | Diurnal and nocturnal diuresis and natriuresis in obstructive sleep apnea. Effects of nasal continuous positive airway pressure therapy[END_REF] Hence, it has been proposed that increased BP in adults with sleep apnea may be responsible for the enhanced nocturnal natriuresis in these patients. [START_REF] Gjorup | Increased nocturnal sodium excretion in obstructive sleep apnoea. Relation to nocturnal change in diastolic blood pressure[END_REF] Although recent evidence suggests that similar to adults, children with obstructive sleep-disordered breathing (SDB) have high levels of sympathetic activity, increased BP levels and BP variability and reduced sensitivity of the baroreflex system, [START_REF] Amin | Twenty-four-hour ambulatory blood pressure in children with sleepdisordered breathing[END_REF][13][14][15][16] no pediatric studies have explored the potential relationship between severity of intermittent upper airway obstruction during sleep and natriuresis. Thus, in the current investigation

Patients and Methods

Participants and clinical evaluation

The study protocol was approved by the Larissa University Hospital Ethics Committee and parents of participants provided informed consent. Consecutive children with snoring (> 3 nights/week) for the last six months who were referred to the Sleep Disorders Laboratory for polysomnography were recruited in the study. Exclusion criteria for participation were: i) symptoms of respiratory tract infection or acute gastroenteritis; ii) history of renal disease or use of diuretics or antihypertensive medications; and iii) history of cardiovascular, neuromuscular or genetic disorders.

All subjects underwent physical examination. Body mass index (BMI) and BMI z-score were calculated. 17 Obesity was defined as BMI z-score > 1.645. 17 Participants voided in the evening prior to polysomnography and none of them had urine loss or voided overnight. Upon morning arousal from sleep and after the child had been sitting quietly for about 5 minutes, BP was measured three times using the right arm and a cuff of appropriate size (Dinamap Pro 100; Criticon; Tampa, FL, USA). To adjust for the effects of age, gender and height, BP z-scores were determined. 18 Shortly after BP measurement and without having drunk any water, children were instructed to empty their bladder in a container and venipuncture was also performed.

Urine and venous blood samples were collected and processed immediately in the Central Biochemistry Laboratory. Serum and urine sodium and creatinine concentrations were measured using standard laboratory methods. Obstructive apnea was defined as the presence of chest/abdominal wall motion in the absence of airflow for at least two breaths in duration. 20 Hypopnea was: i) a reduction in the airflow signal amplitude of at least 50% compared to baseline; ii) in the presence of chest/abdominal wall motion; and iii) associated with oxygen desaturation of hemoglobin equal to or greater than 4% or with an arousal. Obstructive apnea-hypopnea index (OAHI) was equal to the number of obstructive and mixed apneas (apneas with both central and obstructive components) and hypopneas per hour of sleep.

Primary outcome measure

Fractional urinary excretion of sodium (FE Na ) was the primary outcome measure. ii) mild SDB (OAHI 1-5 episodes/h); and iii) moderate-to-severe SDB (OAHI > 5

episodes/hour). The three groups of children were compared in terms of age, gender, obesity status, BP and polysomnography indices using χ 2 for categorical characteristics and one-way analysis of variance (ANOVA), followed by post-hoc tests (Bonferroni), for continuous variables.

BP measurements were categorized as those: i) in the upper quartile of values for all participants; or ii) in the lower three quartiles of BP values for all participants.

Univariate logistic regression analysis was applied to assess the risk for having systolic or diastolic BP in the upper quartile of measurements, in children with moderate-to-severe or mild SDB compared to those with primary snoring (reference group).

Two general linear models (GLM) were tested to describe the potential effects on FE Na of: i) severity of SDB; ii) systolic or diastolic BP; and iii) the interaction of SDB severity with systolic or diastolic BP, after adjustment for age, gender and obesity (SPSS version 16.0; SPSS, Chicago, IL). In order to assess whether natriuresis was increasing linearly to severity of SDB, linear regression analysis was carried out using FE Na as the dependent variable and OAHI, age, gender and BMI z-score as independent variables.

Results

Subjects' characteristics

A total of 95 children were recruited. Subjects' characteristics and polysomnography findings are summarized in Table 1. There were no significant 6 differences in terms of age, female-to-male ratio, body mass index z-score or proportion of obese subjects between the three study groups. Children with moderate-to-severe SDB had similar morning systolic BP z-score to those of subjects with mild SDB and higher than subjects with primary snoring (p=0.074 and p=0.046, respectively). A nonsignificant trend for higher diastolic BP with higher OAHI was noted (Table 1).

Effects of SDB severity and BP on FE Na

The cut-off value separating the upper quartile from the lower quartiles for systolic BP z-score was 0.78. The respective cut-off value for diastolic BP z-score was 1.14. Children with moderate-to-severe SDB-but not with mild SDB-were at increased risk for having systolic BP in the upper quartile of measured values compared to subjects with primary snoring [odds ratios (95% confidence intervals): 4.12 (1.12-15.16); p = 0.033 and 1.33 (0.36-4.97); p=0.669]. The association between severity of SDB and diastolic BP was not significant (p>0.05).

SDB and diastolic BP had significant effects on FE Na (p<0.05 and p=0.007, respectively), whereas systolic BP had no effect on FE Na (p=0.715) (Table 2 and Figure 1). More specifically, subjects with moderate-to-severe SDB had similar log-transformed FE Na to that of children with mild SDB (p=0.657) and higher than participants with primary snoring (p=0.003). The interactions between SDB and systolic or diastolic BP were not significant (p=0.246 and p=0.225, respectively). In linear regression analysis, OAHI was significantly associated with FE Na after adjustment for age, gender and BMI zscore (p=0.002) (Table 3).

Deleted: The respective odds ratios for diastolic BP in the upper quartile of measured values were: 1.51 (0.47-4.82; p = 0.488 and 0.56 (0.17-1.83); p=0.337). In the present study, it was demonstrated that severity of SDB and morning BP in children have significant effect on renal excretion of sodium. Since sleep apnea in childhood has been related to elevated BP, 15,[START_REF] Amin | Activity-adjusted 24hour ambulatory blood pressure and cardiac remodeling in children with sleep disordered breathing[END_REF] the association between SDB and natriuresis may be mediated in part by BP elevation.

Deleted

According to traditional physiology teaching, systemic BP and blood volume are tightly regulated. 6 Any increase in systemic BP is buffered by activation of the baroreflex system (arterial, venous and cardiac baroreceptors, brainstem, sympathetic and parasympathetic neurons) that decreases heart rate, cardiac output and systemic vascular resistance. BP elevation promotes renal excretion of sodium and water both directly, by decreased renal tubular reabsorption of sodium (pressure natriuresis), and indirectly via reduced activity of the renin-angiotensin-aldosterone system. 6-8 Moreover, any blood volume expansion leads to atrial and ventricular distention, and release of ANP and BNP. [START_REF] Dietz | Release of natriuretic factor from rat heart-lung preparation by atrial distension[END_REF][START_REF] Nakagawa | Rapid transcriptional activation and early mRNA turnover of brain natriuretic peptide in cardiocyte hypertrophy. Evidence for brain natriuretic peptide as an "emergency" cardiac hormone against ventricular overload[END_REF] Natriuretic peptides inhibit renal tubular reabsorption of sodium and accentuate renal sodium filtration. [START_REF] Yoshimura | Hemodynamic, renal, and hormonal responses to brain natriuretic peptide infusion in patients with congestive heart failure[END_REF] Studies in adults and children with sleep apnea indicate that at the termination of obstructive events repetitive increases in systemic BP take place. [START_REF] Leuenberger | Surges of muscle sympathetic nerve activity during obstructive apnea are linked to hypoxemia[END_REF][START_REF] Ringler | Hypoxemia alone does not explain blood pressure elevations after obstructive apneas[END_REF][27] [START_REF] Zhang | Alterations in renal function in patients with obstructive sleep apnea syndrome and effects of continuous positive airway pressure[END_REF][START_REF] Gjorup | Increased nocturnal sodium excretion in obstructive sleep apnoea. Relation to nocturnal change in diastolic blood pressure[END_REF][START_REF] Rodenstein | Diurnal and nocturnal diuresis and natriuresis in obstructive sleep apnea. Effects of nasal continuous positive airway pressure therapy[END_REF][START_REF] Baruzzi | Atrial natriuretic peptide and catecholamines in obstructive sleep apnea syndrome[END_REF] Results of the present investigation extend the observations from adults to children with obstructive SDB. Of note, children with obstructive sleep apnea or primary snoring have increased prevalence of nocturnal enuresis that frequently resolves after adenotonsillectomy or treatment of adenoidal hypertrophy and SDB with nasal corticosteroids. [START_REF] Alexopoulos | Association between primary nocturnal enuresis and habitual snoring in children[END_REF][START_REF] Capdevila | Increased morning brain natriuretic peptide levels in children with nocturnal enuresis and sleep-disordered breathing: a community-based study[END_REF][START_REF] Weider | Nocturnal enuresis in children with upper airway obstruction[END_REF][START_REF] Alexopoulos | Resolution of nocturnal enuresis in snoring children after treatment with nasal budesonide[END_REF] Increased nocturnal natriuresis and diuresis could conceivably contribute to nocturnal urine loss.

The exact pathophysiologic mechanisms underlying increased renal excretion of sodium in sleep apnea have not been elucidated. Similarly to the current report, Gjorup et al have found that diastolic BP and FE Na in sleep apneic adults were positively correlated. [START_REF] Gjorup | Increased nocturnal sodium excretion in obstructive sleep apnoea. Relation to nocturnal change in diastolic blood pressure[END_REF] Hence, the investigators proposed that this association may be due to pressure natriuresis. Other studies have revealed increased levels of ANP and BNP that could represent the pathophysiologic link between sleep apnea and nocturnal diuresis and natriuresis. [START_REF] Krieger | Effects of treatment with nasal continuous positive airway pressure on atrial natriuretic peptide and arginine vasopressin release during sleep in patients with obstructive sleep apnoea[END_REF]34 A few methodologic issues should be discussed. First, sodium and creatinine concentrations in an early morning urine specimen were used to calculate FE Na . Since the recruited children have not voided overnight, we speculate that the calculated FE Na reflects nocturnal urinary excretion of sodium. Second, hydration status and sodium losses via the sweat glands and the gastrointestinal system and daily sodium intake may influence FE Na . Nevertheless, none of the study participants was dehydrated, was exposed to high temperatures or had symptoms or signs of gastroenteritis. Third, recent studies have documented significant associations between systolic or diastolic BP and severity of SDB using ambulatory BP monitoring.

  that there is a positive association of urine excretion of sodium with severity of SDB.

FE

  Na was calculated as: FE Na = (urine sodium concentration X serum creatinine concentration)/ (urine creatinine concentration X serum sodium concentration).All concentrations were measured in early morning urine and serum specimens, and were expressed in mg/dL. FE Na values were log-transformed (natural logarithm) to approach a normal distribution. was classified as: i) primary snoring (OAHI < 1 episode/hour);

Figure 1 .

 1 Figure 1. Mean log-transformed fractional urinary excretion of sodium (lnFE Na ) in the

  that there is a positive association of urine excretion of sodium with severity of SDB.

4 Polysomnography

 4 Overnight polysomnography in a single night was performed under the supervision of trained personnel.19 The following parameters were recorded: electroencephalogram (C3/A2, C4/A1, O1/A2); right and left oculogram; submental and tibial electromyogram; body position; electrocardiogram; thoracic and abdominal wall motion; oronasal airflow; and oxygen saturation of hemoglobin.

FE

  Na was calculated as: FE Na = (urine sodium concentration X serum creatinine concentration)/ (urine creatinine concentration X serum sodium concentration).All concentrations were measured in early morning urine and serum specimens, and were expressed in mg/dL. FE Na values were log-transformed (natural logarithm) to approach a normal distribution. was classified as: i) primary snoring (OAHI < 1 episode/hour);

: s Deleted: and 3 Deleted: 4 Page

 34

Figure 1 .

 1 Figure 1. Mean log-transformed fractional urinary excretion of sodium (lnFE Na ) in the three groups of children with SDB according to morning diastolic blood pressure (BP) level (diastolic BP in the upper quartile vs. in the lower quartiles). Less negative lnFE Na values indicate higher excretion of sodium.

F

28 *

 28 p = 0.01 for the effect of severity of SDB on lnFE Na ; the effect of systolic BP and the interaction between SDB and systolic BP were not significant (p=0.715 and p=0.246,

Page 17 of 45 John Wiley & Sons, Inc.

	Pediatric Pulmonology
	Tables
	F o
	r
	P
	e e r
	R e v i e w

Table 1 .

 1 Subjects' characteristics, polysomnography findings and fractional urinary excretion of sodium (FE Na ) values.

	Variables	Primary snoring	Mild SDB	Moderate-to-
					severe SDB
			n = 26	n = 41	n = 28
	Age (years)	6.6 ± 2.8	6.6 ± 2.7	5.7 ± 2.0
	Female gender (%)	9 (34.6)	24 (58.5)	11 (39.3)
	Body mass index z-score	0.8 ± 1.1	0.7 ± 1.4	1.1 ± 1.6
	Obese (%)	7 (26.9)	9 (22)	10 (35.7)
	Systolic BP (mmHg)	97.1 ± 11.0	97.9 ± 12.2	103.1 ± 13.5
	Systolic BP z-score *	-0.02 ± 1.0	0.1 ± 1.0	0.7 ± 1.2
	Diastolic BP (mmHg)	61.4 ± 8.8	62.3 ± 7.3	64.1 ± 7.6
	Diastolic BP z-score	0.5 ± 0.8	0.6 ± 0.7	0.8 ± 0.8
	OAHI index † (episodes/hour)	0.6 ± 0.2	2.5 ± 1.2	15.4 ± 10.0
	Respiratory arousal index †	0.3 ± 0.1	1.4 ± 0.8	5.5 ± 3.6
	(episodes/hour)
	Oxygen desaturation of
	hemoglobin	0.8 ± 0.5	3.0 ± 1.7	17.3 ± 11.9
	( > 4%) index † (episodes/hour)
	SpO 2 nadir † (%)	92.6 ± 1.5	88.9 ± 3.1	81.2 ± 6.6
	FE Na	‡	0.65 ± 0.35	0.81 ± 0.28	1.02 ± 0.61

Table 2 .

 2 Effects of severity of sleep-disordered breathing (SDB) and diastolic blood pressure (BP) on log-transformed fractional urinary excretion of sodium (lnFE Na ) adjusted for age, gender and obesity. Less negative lnFE Na values indicate higher excretion of sodium.

Table 3 .

 3 Multple linear regression analysis model assessing the association of logtransformed fractional urinary excretion of sodium (lnFE Na ) with OAHI adjusted for age, gender and BMI z-score.

	Parameters	β standardized	p values
		coefficient
	Age	0.004	0.964
	Gender	0.002	0.982
	BMI z-score	0.103	0.305
	OAHI	0.329	0.002

  15,[START_REF] Amin | Activity-adjusted 24hour ambulatory blood pressure and cardiac remodeling in children with sleep disordered breathing[END_REF] A trend for higher morning diastolic BP with increasing OAHI was identified in the present report and the absence of

	Page 33 of 45	Pediatric Pulmonology
		statistical significance may be attributed to the fact that overnight BP monitoring was not 34. Kita H, Ohi M, Chin K, Noguchi T, Otsuka N, Tsuboi T, Itoh H, Nakao K, Kuno
			Formatted: Font color: Auto
		performed in the current report. Finally, a healthy control group including children K. The nocturnal secretion of cardiac natriuretic peptides during obstructive sleep
		without SDB, who were of normal weight or overweight, was not included. The absence apnoea and its response to therapy with nasal continuous positive airway pressure.
		of such a group possibly limited the statistical comparisons and the delineation of an J Sleep Res 1998;7:199-207.
		interaction effect between SDB, obesity and BP in natriuresis. Furthermore, the use of a 35. Li AM, Au CT, Ho C, Fok TF, Wing YK. Blood pressure is elevated in children
		control group with OAHI < 1 episode/hour may be suboptimal, since even primary with primary snoring. J Pediatr 2009;155:362-368 e361.
		snoring could be associated with BP elevation. 35
		In conclusion, severity of obstructive SDB and diastolic BP are positively
		F o r associated with urinary excretion of sodium in childhood. Natriuresis may be a defense mechanism to systemic blood pressure fluctuations related to intermittent upper airway obstruction during sleep. F o F o F o F o F o F o r r r r r r	Deleted: Elevated FENa in children with snoring could possibly separate those subjects with hemodynamically significant effects related to i
		P P P P P P P
		e e r e e r e e r e e r e e r e e r e e r
		R e v i e w R e v i e w R e v i e w R e v i e w R e v i e w R e v i e w R e v i e w
		9 15
		John Wiley & Sons, Inc.

Table 1 .

 1 Subjects' characteristics, polysomnography findings and fractional urinary excretion of sodium (FE Na ) values.

	Tables
	Variables	Primary snoring	Mild SDB	Moderate-to-
						severe SDB
			n = 26		n = 41	n = 28
	Age (years)		6.6 ± 2.8		6.6 ± 2.7	5.7 ± 2.0
	Female gender (%)		9 (34.6)		24 (58.5)	11 (39.3)
	Body mass index z-score		0.8 ± 1.1		0.7 ± 1.4	1.1 ± 1.6
	Obese (%)		7 (26.9)		9 (22)	10 (35.7)
	Systolic BP (mmHg)	o	97.1 ± 11.0		97.9 ± 12.2	103.1 ± 13.5
	Systolic BP z-score *	r -0.02 ± 1.0		0.1 ± 1.0	0.7 ± 1.2
	Diastolic BP (mmHg)	P 61.4 ± 8.8		62.3 ± 7.3	64.1 ± 7.6
	Diastolic BP z-score OAHI index † (episodes/hour) Respiratory arousal index †	0.5 ± 0.8 e e 0.6 ± 0.2 r 0.3 ± 0.1	0.6 ± 0.7 2.5 ± 1.2 1.4 ± 0.8	0.8 ± 0.8 15.4 ± 10.0 5.5 ± 3.6
	(episodes/hour) Oxygen desaturation of hemoglobin ( > 4%) index † (episodes/hour) SpO 2 nadir † (%) FE Na ‡	0.8 ± 0.5 92.6 ± 1.5 0.65 ± 0.35	R 3.0 ± 1.7 e v i 88.9 ± 3.1 e w 17.3 ± 11.9 81.2 ± 6.6 0.81 ± 0.28 1.02 ± 0.61

Table 2 .

 2 Effects of severity of sleep-disordered breathing (SDB) and diastolic blood pressure (BP) on log-transformed fractional urinary excretion of sodium (lnFE Na ) adjusted for age, gender and obesity. Less negative lnFE Na values indicate higher excretion of sodium.

	F o
	r
	P
	e e r
	R e v i e w

Table 3 .

 3 Multple linear regression analysis model assessing the association of logtransformed fractional urinary excretion of sodium (lnFE Na ) with OAHI adjusted for age, gender and BMI z-score.

			Deleted: 4
	Parameters	β standardized	p values
		coefficient
	Age	0.004	0.964
	Gender	0.002	0.982
	BMI z-score	0.103	0.305
	OAHI	0.329	0.002
	Abbreviations
	BMI	Body mass index
	OAHI	Obstructive apnea-hypopnea index

Page 43 of 45 John Wiley & Sons, Inc.

	Pediatric Pulmonology
	Page 2: [1] Deleted	kaditia	5/5/2010 11:32:00 PM
	F o
	r
	P
	e e r
		R e v i e w

Table 2 .

 2 Effects of severity of sleep-disordered breathing (SDB) and systolic blood pressure (BP) on log-transformed fractional urinary excretion of sodium (lnFE Na ) adjusted for age, gender and obesity. Less negative lnFE Na values indicate higher excretion of sodium.

John Wiley & Sons, Inc.

John Wiley & Sons, Inc.Pediatric Pulmonology

Abbreviations

* p = 0.043 for the effect of severity of SDB on lnFE Na † p = 0.007 for the effect of diastolic BP on lnFE Na ; the interaction between SDB and diastolic BP was not significant (p=0.225).

Deleted: 3

Deleted: Table 2. Effects of severity of sleep-disordered breathing (SDB) and systolic blood pressure (BP) on logtransformed fractional urinary excretion of sodium (lnFENa) adjusted for age, gender and obesity. Less negative lnFENa values indicate higher excretion of sodium. ¶ ... [1]


Prof Sergio Siragusa

MD; Sergio Siragusa
email: sergio.siragusa@unipa.it

M.D William Morice

M.D Morie A Gertz

Robert A Kyle

; Philipp

R Greipp

M.D John A Lust

M.D Thomas E Witzig

M.D Martha Q Lacy

M.D Steven R Zeldenrust

Vincent S Rajkumar

Stephen J Russel

M.D Suzanne R Hayman

Francis Baudi

M.D Shaji K Kumar

M.D David Dingli

Angela Dispenzieri

M.D Philip R Greipp

M.D S Vincent Rajkumar

M.D Stephen J Russell

M.D Francis Buadi

Keywords: Amyloid, Multiple Myeloma, Smoldering Myeloma, Asymptomatic, Bone Marrow Biopsy Amyloid, Multiple Myeloma, Smoldering Myeloma, Asymptomatic, Bone Marrow Biopsy

and research institutions in France or abroad, or from public or private research centers.

Asymptomatic immunoglobulin light chain amyloidosis (AL) at the time of diagnostic bone marrow biopsy in newly diagnosed patients with multiple myeloma and smoldering myeloma. A series of 144 cases and a review of the literature

Sergio Siragusa, William Morice, Morie A. Gertz, Robert A. Kyle, Philip R.

Greipp, John A. Lust, Thomas E. Witzig, Martha Q. Lacy, Steven R.

Zeldenrust, S. Vincent Rajkumar, et al.

To cite this version:

Sergio Immunoglobulin light chain amyloidosis may coexist with any of the other PCDs [START_REF] Kyle | Primary systemic amyloidosis: clinical and laboratory features in 474 cases[END_REF][START_REF] Rajkumar | Monoclonal gammopathy of undetermined significance, Waldenström macroglobulinemia, AL amyloidosis, and related plasma cell disorders: diagnosis and treatment[END_REF].

It has been reported that approximately 10% of patients with AL may have MM at the time of diagnosis [START_REF] Kyle | Primary systemic amyloidosis: clinical and laboratory features in 474 cases[END_REF], while only a minority will developed delayed MM (4). Conversely, it has been reported that up to 30% of MM patients may have subclinical amyloid deposits (in subcutaneous fat aspirate, bone marrow biopsy or biopsies of other vital organs) [START_REF] Kyle | Multiple Myeloma and other plasma cell disorders[END_REF][START_REF] Desikan | Incidence and impact of light chain associated (AL) amyloidosis on the prognosis of patients with multiple myeloma treated with autologous transplantation[END_REF]7,[START_REF] Vela-Ojeda | Multiple myelomaassociated amyloidosis is an independent high-risk prognostic factor[END_REF] and in up to 15% of all MM patients a clinically overt AL appear during the course of index MM [START_REF] Rajkumar | Monoclonal gammopathy of undetermined significance, Waldenström macroglobulinemia, AL amyloidosis, and related plasma cell disorders: diagnosis and treatment[END_REF][START_REF] Kyle | Multiple Myeloma and other plasma cell disorders[END_REF]).

The rate of asymtpomatic amyloidosis (asymp-amyloid) in patients with MM or SMM as well as its clinical impact, is uncertain. This topic is significant because unrecognized AL can be associated with significant mortality and morbidity. In the case of patients with SMM, no treatment will be directed against the plasma cell clone, potentially allowing for unbridled progression of the AL. Unrecognized AL in patients diagnosed with MM is also significant because of the potential for higher rates of treatment related morbidity and mortality in patients with occult visceral involvement with AL. However, at the present we deal with discordant information. Some authors found out MM-associated asymp-amyloid in up to 26% of patients at the time of diagnosis [START_REF] Vela-Ojeda | Multiple myelomaassociated amyloidosis is an independent high-risk prognostic factor[END_REF]; this was also an independent high-risk prognostic factor for MM patients, thus suggesting need for searching amyloid and need for aggressive treatment. Others believe that AL deposits, even if not rare, are clinically inconsequential and should not influence MM therapy, so active screening is unnecessary [START_REF] Desikan | Incidence and impact of light chain associated (AL) amyloidosis on the prognosis of patients with multiple myeloma treated with autologous transplantation[END_REF][START_REF] The | Guidelines on the diagnosis and management of AL amyloidosis[END_REF].

Systemic AL can be a devastating disease with an insidious onset and its early recognition may improve clinical outcomes and patients' management. The objective of the present investigation was therefore that of evaluating the rate of asymp-amyloid in MM and SMM patients at the moment of first diagnostic bone marrow (BM) biopsy. mg/dL or myeloma related kidney disease, which was defined as a creatinine greater than 1.5 mg/dL and a 24-hour urinary M-protein to 24-hour urinary total protein ratio of greater than 0.5.

DESIGN AND METHODS

Patients

Diagnosis of asymptomatic AL

Bone marrow biopsies were reviewed and examined for Congo red staining. A patient was considered to have amyloid if the stain was positive and apple green birefringence was found on polarized microscopy. Posterior superior iliac crest was the preferred biopsy site and the specimens were obtained with a modified Jamshidi needle as an outpatient procedure.

Following decalcification the bone marrows were paraffin embedded and H&E stained and Congo red stained sections were reviewed by a single author (WB). Amyloid deposits were detected by examination at 200x under polarized light. Only data from patients with an adequate follow-up period were considered in the final analysis. In addition, serum samples were obtained at diagnosis to determine LDH, β2 microglobulin, albumin, calcium, phosphorus, creatinine, and liver function tests. Serum and urine electrophoresis and immunofixation, as well as bone marrow aspiration and bone X-ray studies were taken at diagnosis.

Statistical analysis

Baseline differences between groups were assessed by the chi-square test (Fisher's Exact Test) for categorical variables and univariate analysis of variance (ANOVA test) or Mann-Whitney U statistic test for parametric and nonparametric analyses. Survival was estimated using the method of Kaplan-and Meier.

RESULTS

Overall characteristics of MM and SMM patients

Of The overall median survival was 70.4 months (figure 1).

At presentation, SMM patients were older, less anemic, had higher creatinine, urine protein, but lower urine M-spikes levels than MM patients. These last patients were more likely to have fatigue and pain, while SMM patients had more frequently paresthesia (Table 1).

Patients with asymptomatic amyloid deposit at presentation

Amyloid was retrospectively detected in the index bone marrow biopsy of 2 of the 144 cases (1.38%, 95% CI -0.6 to 3. He had a history of weight loss and severe chronic obstructive pulmonary disease. At presentation, physical exam did not reveal any abnormalities. The BM biopsy reveled plasmacells up to 25% of the total cellularity; ß2-microglobulin was 1.52 mcg /mL. Bone survey was negative for metastases or myeloma. Clinical and laboratory characteristics are listed in Table 2.

After more than seven years of follow-up, neither of these patients had or developed during their disease signs or symptoms suggestive for organ involvement by amyloid.

Patients without bone marrow amyloid deposition at presentation but subsequent systemic amyloidosis

There was one patient (R.J., male, 51 y.o.) who was diagnosed having MGUS (IgG-λ) in 1992 followed by SMM in 1993. Clinical and laboratory characteristics at presentation are listed in

DISCUSSION

Rate and clinical impact of asymp-amyloid in patients with MM or SMM is uncertain.

While asymptomatic amyloid deposits have been reported to coexist at the time of MM diagnosis in up to 30% of patients (1, 5), less information is available in patients with established MM or SMM who are seen at a facility at which an extensive amyloid directed review of systems and signs is the norm. This issue is topical since unrecognized AL can be associated with significant mortality and morbidity and its early detection could change the management of MM and SMM patients.

We found that only 1.4% of patients with retrospective Congo Red staining of their bone marrow had amyloid present on their presenting bone marrow. This may be a limitation of the present investigation, since it is well known that the sensitivity of bone marrow biopsy is only about 50% in comparison to that of abdominal fat aspirate with a sensitivity of about 80% of confirmed amyloidosis [START_REF] Gertz | Amyloidosis[END_REF]14). It is possible that our approach yielded a low estimate. For example, the one patient, whose initial bone marrow was negative for amyloidosis but who was diagnosed with AL 6 years later, might have had a positive fat aspirate at the time of his earlier bone marrow; if so, he would have had very indolent amyloidosis.

Our data, which may appear to conflict with prior reports, would suggest that a careful amyloid specific review at the time of diagnosis of SMM or MM misses only 1-2% of cases even with a 6.4 person-years of follow-up and therefore abrogates the need to do routine amyloid staining on all cases of SMM or MM. It also supports the Mayo Clinic practice of having a low threshold for amyloidosis screening if non-specific symptoms are present in a patient with a monoclonal gammopathy. no λ predominance was found. The median overall and event free survival of the entire cohort is 66+ and 52 months respectively; the median overall (59+ months vs 66+ months, p=0.9) and event free survival (59+ months vs 52+ months, p=0.9) were similar in cohort with and without amyloid respectively. These authors concluded therefore that, although the incidence of lightchain asymptomatic AL in MM patients is high, it does not influence the outcome of these patients treated with autologous transplantation.

Vela-Ojeda et al evaluated 201 consecutive patients with de novo MM in whom a fat-pad biopsy needle aspiration (stained with Congo red) was performed [START_REF] Vela-Ojeda | Multiple myelomaassociated amyloidosis is an independent high-risk prognostic factor[END_REF]. Overall, 68 patients (34%)

were diagnosed having concomitant AL; 16 (8%) had symptoms consistent with amyloidosis at the time of MM diagnosis while in 52 (25.8%) fat pad aspiration was useful to detect incidental asymptomatic AL. The median follow-up was 36 months (range 12-199). Among patients without AL at the time of diagnostic BM for MM, 69% of them became symptomatic for AL after a median follow-up of 14 months (range 6 to 30). Even excluding 16 patients with obvious symptoms of AL at diagnosis, overall survival was worse in patients who developed late AL (13 vs 64 months of those without AL). Cox regression model for overall survival detected three variables having independent prognostic significance: the presence of AL (RR 3.4, P<0.004), serum albumin levels <3.5 g/dl (RR 3.2, p<0.005) and patients not achieving complete remission or very good partial remission (RR 2.9, p<0.02). The authors then concluded that MM-associated AL represents a poorer prognosis disease even in the absence of symptoms at diagnosis and this specific association may be considered as an independent high-risk prognostic factor. Thus, Authors concluded that routine study of peri-umbilical fat-pad tissue should be mandatory in all patients with MM.

Petruzziello et al (2009) evaluated retrospectively the bone marrow smears of 166 unselected patients affected by MM (126 at diagnosis and 40 after treatment); no data were reported about the presence of signs or symptoms suggestive for concomitant systemic AL [START_REF] Petruzziello | Amyloid in bone marrow smears of patients affected by multiple myeloma[END_REF].

All samples were stained with Congo red and studied by transmission and birefringence microscopy. Both focal and diffuse involvement were considered positive. Overall, 67 patients were positive for AL and 99 were negative to Congo red and apple-green birefringence. In particular, 51 of the 126 patients studied at diagnosis and 16 of the 40 patients with advanced disease were positive. Authors failed to find correlations between bone marrow amyloid deposits and immunoglobulin type, disease stage, plasma cells percentage, hemoglobin, calcium, creatinine, albumin, or ß2-microglobulin.

Although the main objective of our investigation was that of evaluate the rate of asymamyloid at the time of first BM, we were able to look at the clinical outcome of our cohort who had a median follow up of 6 years. Neither of the patients who were retrospectively found to have amyloid in their index bone marrows developed symptomatic AL, and their survival did not differ from the other patients. Among Congo Red negative patients, we detected one (SMM patient) who developed symptomatic AL during a follow up of 72.6 months. He had serum λ light-chain, an isotype frequently reported in patients with AL [START_REF] Solomon | Nephrotoxic potential of Bence Jones proteins[END_REF].

One potential caveat in studies looking at the detection of asymp-amyloid relies on patients' selection. In fact, AL requires a high index of suspicion and most of AL cases are often misdiagnosed with other PCDs, mainly with MM (The UK Myeloma Forum AL Amyloidosis Guidelines Working Group, 2004). It is not unlikely that the true occurrence of asymptomatic AL has been previously overestimated. Because of the high level of suspicion for amyloid at our Amyloid Center, we are confident that all patients evaluated did not have (even sub-clinical) AL at the time of diagnostic BM for MM or SM. Therefore, evaluation of adequate specimens in experienced laboratories is necessary to maintain high diagnostic sensitivity and specificity.

In conclusion, we evaluated rates and clinical significance of amyloid deposit at the time of first diagnostic BM biopsy in newly diagnosed MM and SMM. Our findings show that fewer than 2% of patients in both groups have asymp-amyloid thus discouraging any specific search for amyloid deposit when careful evaluation of clinical presentation and patient's history are not suggestive for systemic AL.

  Siragusa, William Morice, Morie A. Gertz, Robert A. Kyle, Philip R. Greipp, et al.. Asymptomatic immunoglobulin light chain amyloidosis (AL) at the time of diagnostic bone marrow biopsy in newly diagnosed patients with multiple myeloma and smoldering myeloma. A series of 144 cases and a review of the literature. Annals of Hematology, 2010, 90 (1), pp.101-106. 10.1007/s00277-010-1028-8. hal-00557757 INTRODUCTION Multiple myeloma (MM), smoldering multiple myeloma (SMM), immunoglobulin light chain amyloidosis (AL) and monoclonal gammopathy of undetermined significance (MGUS) represent a spectrum of plasma cells disorders (PCDs). AL is a rare and serious disorder characterized by the deposition of amyloid fibrils in different tissues with more than 80% of patients dead within 2 years of diagnosis (1, 2)

  the 1180 patients with a diagnosis of MM (959) or SMM (221) seen at Mayo Clinic

within 30 days of their diagnosis, biopsies were available from 144 (F 59, M 84). Among patients evaluated 77 had a diagnosis of MM and 67 of SM. Characteristics of MM and SM patients at time of diagnosis are reported in Table

1

. One hundred-thirty-six (95.8%) patients had a positive serum M-spike before diagnostic BM. Median serum M-spike at the time of diagnostic BM biopsy was 2.93 (range 0.6-9.5). At a median follow-up of living patients of 76.2 months (range 0-215.9), 46 (31.9%) patients were alive, 93 (64.5%) dead and 5 (3.4%) lost to follow-up.

  , 66 y.o.) was diagnosed having MM; at presentation, she had moderate anemia (Hg 9.4 g/l), pain, multiple fractures but not hypercalcemia, renal or liver tests alteration. No measurable M-spike was identified on electrophoresis, but there was a monoclonal kappa light chain detectable in the serum by immunofixation. Urine protein electrophoresis showed a total urine protein of 280 mg with an M-spike of 249 mg/24h that was confirmed to be kappa light-chain.Quantitative immunoglobulins were not done. The bone marrow biopsy revealed plasma-cells comprising 30% of the total cellularity. Beta-2-microglobulin was elevated at 2.28 mcg/mL. Cytogenetic studies by FISH did not show IGH translocation, monosomy or deletion 13, deletion of p53 or trisomy 3, 7, 9, 11, 13, 15 or 17. Cytogenetics studies of BM showed a normal karyotype (46, XX). The second patient (RW, male, 77 y.o.) was diagnosed with SMM (IgG-κ).

2), 1 in MM and 1 in SMM group, respectively. The first (OE, female

Table 2 .

 2 At the presentation physical examination was normal. He did not have weakness, fatigue, bleeding or increased bruising, not bone pain, change in his voice or tongue, steatorrhea, paresthesia, claudication or weight loss. He developed symptomatic AL after 72.6 months (February 1999) from initial diagnosis of SMM. He was treated with melphalan/prednisone. At the time of last contact in 2002, he considered himself to be in good health.

Table 3

 3 puts our findings into the context of prior reports. One of the first reports addressing this question was by Desikan et al[START_REF] Desikan | Incidence and impact of light chain associated (AL) amyloidosis on the prognosis of patients with multiple myeloma treated with autologous transplantation[END_REF], who prospectively evaluated fat pad aspirate and BM biopsy for amyloid deposit in a cohort of 84 MM patients. Amyloid was detected in 38% of patients; fat pad aspirate was positive in 25% and BM in 10% of cases and other organ sites in 7 patients. MM or SMM patients did not differ in disease characteristics and, particularly,

Table 1 . Baseline patients' characteristics

 1

	Characteristics	MM population	SM population
		(n=77)	(n=67)
	Median months from biopsy to last follow up (range)	65 (10.4-169.2)	64.6 (0-210)
	Such Fatigue, %	13 (16.8)	5 (7.4)
	Pain, %	42 (54.5)	1 (1.5)
	Paresthesia, %	1 (1.3)	3 (4.4)
	Bleeding, %	0	1 (1.5)
	Edema, %	0	1 (1.5)
	Dyspnea, %	6 (7.8)	1 (1.5)

Table 2 . Characteristics of two patients with detection of amyloid deposit at presentation and one patient without amyloid deposit but subsequent systemic amyloidosis

 2

	Clinical/laboratory characteristics	Patients with amyloid deposit at	Patient without
	at diagnosis	presentation	amyloid deposit at
		MM patient	SM patient	presentation but
				subsequent AL
	Months from biopsy to last follow-	86.4	87.6	72.6
	up
	Age (y)	66	77	51
	Hg (g/dl)	9.4	13	12.3
	Platelets (n. x 10 9 /l)	328	201	446
	Pro-thrombin time (sec.)	10.1	NA	NA
	Serum protein (g/dl)	NA	7.4	6.9
	Urine protein (mg/dl)	280	8	13
	Serum Albumin (g/dl)	2.9	3.6	3.5
	Alkaline Phosphate (U/l)	NA	145	152
	Creatinine (mg/100 ml)	0.8	1.1	0.9
	Β2-microglobulin (µg/ml)	2.28	1.56	1.5
	C-reactive protein level (mg/dl)	NA	NA	0.59
	Serum light-chain isotype	kappa	kappa	lambda
	NA: Not Available

Table 3 . Studies considering relationship between asymptomatic AL in myeloma patients MM cases with incidental amyloidosis

 3

	Author ref	MM cases,	AL cases,	Tissues stained	Prognostic
		n	n (%)	for amyloid	factor?
	Desikan 6	84	32 (30.8)	Subcutaneous	No
				abdominal fat
				aspirate
				(SAFA) in 25,
				bone marrow in
				8 and other
				organ sites in 7
				patients
	Vela-Ojeda 8	201	68 (34)*	SAFA	Yes
	Petruziello 12	166	67 (40.3)	Cytological	No
				techniques in
				bone marrow
				smears
	Present study MM	77	1 (1.3)	Bone marrow	No
	Present study SMM	67	1 (1.5)	Bone marrow	No
		AL cases with asymptomatic MM
		AL cases,	MM case,		Comments

*Conflict

of interest Click here to download Conflict of interest: Conflict of Interest Disclosure Form.doc


L N Van Steenbergen

MSc G Van Lijnschoten

MD Hjt J T Rutten

MD Vepp E P P Lemmens

PhD Jww W W Coebergh

Improving lymph node detection in colon cancer in community hospitals and their pathology department in southern Netherlands

Keywords: colon cancer, cost-effectiveness, lymph node detection, Patent blue V, trend

Improving lymph node detection in colon cancer in community hospitals and their pathology department in southern Netherlands

INTRODUCTION

Colon cancer is one of the most frequent cancers in the Netherlands with over 6,000 new cases annually. [1] In 2007 over 3,800 patients died of colon cancer [2]. Resection of the tumour with adequate margins and the associated mesentery, including draining lymph nodes, is the primary modality of treatment of colon cancer. Generally, only patients with positive lymph nodes benefit from adjuvant chemotherapy. [START_REF] Gill | Pooled analysis of fluorouracil-based adjuvant therapy for stage II and III colon cancer: who benefits and by how much?[END_REF] Therefore, lymph node analysis is one of the critical factors for therapeutic decision-making. A minimum number of 12 identified lymph nodes is defined as adequate assessment by the International Union Against Cancer (UICC). Recently, in the Netherlands the minimum number of lymph nodes that should be examined was reduced to 10.[4] The 5-year survival for patients with pT 3-4 N0 M0 (stage II) is approximately 59%, versus only 42% for pT any N+ M0 (stage III) patients. [5] For obvious reasons, high-risk nodenegative patients often receive adjuvant chemotherapy. The Dutch guidelines define high risk as stage II patients with pT4 or poorly differentiated tumours, tumours with angio-invasion, or patients with fewer than 10 lymph nodes evaluated. [4] Recently, the QUASAR trial showed a small survival benefit for these patients compared to high-risk node-negative patients who were not treated with adjuvant chemotherapy. [START_REF] Collaborative | Adjuvant chemotherapy versus observation in patients with colorectal cancer: a randomised study[END_REF] However, a proportion of patients with an insufficient number of lymph nodes examined truly has negative lymph nodes and has received adjuvant chemotherapy unnecessarily. Besides avoiding the potential burden of this treatment for the individual patient, large savings could be made by reducing the proportion of patients eligible for chemotherapy.

The number of lymph nodes examined in colon cancer patients in southern Netherlands is often inadequate, since 70% of the patients had fewer than 12 lymph nodes examined. [START_REF] Lemmens | Pathology practice patterns affect lymph node evaluation and outcome of colon cancer: a population-based study[END_REF] This was communicated to these departments by means of individual feedback, discussions in multidisciplinary working groups and educational presentations, which increased awareness and resulted in an increased fixation time which has shown to improve lymph node detection. [START_REF] Poller | Method of specimen fixation and pathological dissection of colorectal cancer influences retrieval of lymph nodes and tumour nodal stage[END_REF] Besides, a subset of colon specimens were injected ex-vivo with Patent blue V dye in the mesocolon next to the tumour. The purpose of this study was to investigate whether this set of measures directed at increasing lymph node detection among colon cancer patients since 1999 led to clinically relevant changes in lymph node detection rate, and to evaluate the costs saved by these interventions.

M A N U S C R I P T A C C E P T E D ARTICLE IN PRESS

4

MATERIALS AND METHODS

Data collection and patients

Population-based data from the Eindhoven Cancer Registry (ECR), which is maintained by the Comprehensive Cancer Centre South, was used to investigate the trend in lymph nodes examined and to select a control group for the comparison with the Patent blue stained group.

The ECR collects data on all patients newly diagnosed with cancer in the southern part of the Netherlands. Information about patient characteristics, such as gender and date of birth, and tumour characteristics, such as date of diagnosis, subsite (International Classification of Diseases for Oncology (ICD-O-3)), grade, pathological stage, and number of lymph nodes examined were recorded. Tumour subsite was categorized into two sites; proximal colon, consisting of tumours in the coecum, appendix, ascending colon, hepatic flexure, transverse colon, and splenic flexure (C18.0-C18.5); and distal colon consisting of tumours in the descending colon and sigmoid colon (C18.6-C18.7). This information is obtained routinely from the medical records. The quality of the data is high, due to thorough training of the registration team and computerized consistency checks at regional and national levels.

Completeness has been estimated to be at least 95%. [START_REF] Schouten | Completeness of cancer registration in Limburg, The Netherlands[END_REF] All cases of primary colon cancer stage I to III (pT any N any M0) registered between 1999 and 2007 treated in one of the hospitals served by the regional department of pathology in Eindhoven were included in the lymph node detection trend analysis (n=1,501).

Lymph node detection technique

M A N U S C R I P T A C C E P T E D ARTICLE IN PRESS 5
Immediately after resection, the surgeon injected 0.25-1.0 ml of Patent blue dye V in the mesocolon neighbourhood of the tumour. The injection site was gently massaged for 30 seconds. After 42-48 hours in 4% buffered formalin, the specimen was examined by a pathologist in the routine setting. Afterwards, the colon is cut transversally into slices 0.5 to 1.0 cm thick. All slices of the specimen were examined by the pathologist for lymph nodes, which were routinely processed for histological examination using conventional methods.

Statistical analysis

The median number of lymph nodes examined from 1999 to 2007 was assessed in order to determine the percentage of cases with insufficient lymph nodes examined. A Cochrane-Armitage trend test was used to investigate the trend in the proportion of patients with insufficient lymph nodes examined. Differences between the group who underwent the new lymph node technique and a control group with standard pathology examination were described, focusing mainly on the number of lymph nodes examined and tumour characteristics. Chi-square tests were conducted to test the differences in percentages of patients with insufficient lymph nodes evaluated. A Mann-Whitney test was conducted to test the difference in the number of lymph nodes examined between the Patent blue dye and the control group. SAS/STAT® statistical software (SAS system 9.1, SAS Institute, Cary, NC) was used for the analyses.

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RESULTS

Trend in lymph node examination

The proportion of colon cancer patients with an insufficient ( <12) number of lymph nodes examined decreased (p trend <0.0001) (Fig 1). The proportion of patients with less than 10 lymph nodes examined decreased from 79% in 1999 to 35% in 2007. Considering patients with N0 stage only, resulted in a similar proportion of patients with insufficient lymph nodes examined.

Patent blue-staining

The median age of the Patent blue-stained group was 73 years and 72 years for the control group. The control group contained slightly more patients with stage I disease and less stage II. In the Patent blue-stained group less tumours were located in the proximal colon compared to the control group. However, these differences were not found significant (Table 1).

The median number of lymph nodes examined in the Patent blue-stained group was significantly higher compared to the control group (Fig 2). In the Patent blue-stained group 37% of the patients had fewer than 12 lymph nodes examined, while this was significantly higher in the control group (Table 2). After excluding patients with a stage I (T 1-2 N0 M0) tumour, the percentage of patients with fewer than [START_REF] Gagliardi | Multiple factors influence compliance with colorectal cancer staging recommendations: an exploratory study[END_REF]

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7 Almost 70% of the lymph nodes examined coloured blue, whereas only 9% of the blue lymph nodes being positive in the total study population. In 69% of the N+ patient group non-stained positive lymph nodes were found; however the large majority (78%) of these patients also had blue-stained positive lymph nodes.

Cost-effectiveness

In 1999, 79% of the patients with stage II disease were N0 with fewer than 10 lymph nodes examined compared to 55% in 2007. According to the Dutch guidelines these patients are considered high-risk and should receive adjuvant chemotherapy at the cost of approximately €35,000 per person [START_REF] Ferro | Variation in the cost of medications for the treatment of colorectal cancer[END_REF][START_REF] Meropol | Cost of cancer care: issues and implications[END_REF]. Extrapolating these results to all patients diagnosed with colon cancer stage II in the department of pathology in Eindhoven in 2007 (n=92), 22 patients would have been rendered ineligible for adjuvant chemotherapy, because of a sufficient lymph node detection. This would have saved about 22*€35,000 = €770,000 per 92 patients.

One third of this saving can be attributed to the Patent blue staining. However, some patients might be upgraded from node negative to node positive and should receive chemotherapy anyway.

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8

DISCUSSION

We demonstrated a significant increase in the number of lymph nodes examined in colon cancer patients by a regional pathology department between 1999 and 2007. Staining the resection specimen ex-vivo with Patent blue V dye clearly increased lymph node detection in colon cancer patients, in addition to the gradual increase observed in the number of lymph nodes examined over time, especially after 2006 when fixation time was increased. In the Patent blue-stained group the median number of lymph nodes examined was higher compared to the control group. The proportion of patients with fewer than 12 lymph nodes decreased from 56% in the unstained control group to 37% in the Patent blue-stained group. Enormous chemotherapy-related savings could be attained by better lymph node detection, and therefore reducing the proportion of high-risk node-negative colon cancer patients. The Patent blue intervention was aimed at increasing the number of lymph nodes examined. The detection of a possible sentinel node was therefore not the aim of this study.

Improvement of lymph node detection

Lymph node detection in a population-based study of colon cancer patients diagnosed in 1999-2002 in southern Netherlands was poor with a median of 6 lymph nodes examined in the ECR region, with the median for the department of pathology in Eindhoven being 8. [START_REF] Lemmens | Pathology practice patterns affect lymph node evaluation and outcome of colon cancer: a population-based study[END_REF] This result was communicated to the departments of pathology in October 2005 in the region by means of individual feedback and discussions in multidisciplinary working groups.

Educational presentations create awareness among pathologists and surgeons, which result in an improvement in lymph node staging practice. To further increase the number of lymph nodes examined, several steps were taken by various regional pathology departments involved. In 2006 they increased fixation time to 42-48 hours, which has been reported by several studies to lead to an increase in lymph node detection. [START_REF] Poller | Method of specimen fixation and pathological dissection of colorectal cancer influences retrieval of lymph nodes and tumour nodal stage[END_REF][START_REF] Burroughs | ACP Best practice no 159. Examination of large intestine resection specimens[END_REF] The closer collaboration between surgeons and pathologists was expressed in 2007 with the start of the Patent blue staining method in the department of pathology in Eindhoven described in this study. Other pathology departments in the ECR region also studied comparable methods to increase lymph node yield. [START_REF] Van Schaik | Ex vivo sentinel lymph node "mapping" in colorectal cancer[END_REF] Population-based studies reported a median number of examined lymph nodes of 6 to 12 in the period between 1990 and 2005, [START_REF] Lemmens | Pathology practice patterns affect lymph node evaluation and outcome of colon cancer: a population-based study[END_REF][START_REF] Bilimoria | Lymph node evaluation as a colon cancer quality measure: a national hospital report card[END_REF][START_REF] Lemmens | Mixed adherence to clinical practice guidelines for colorectal cancer in the Southern Netherlands in 2002[END_REF][START_REF] Baxter | Lymph node evaluation in colorectal cancer patients: a population-based study[END_REF][START_REF] Jestin | Cancer staging and survival in colon cancer is dependent on the quality of the pathologists' specimen examination[END_REF][START_REF] Maurel | Lymph node harvest reporting in patients with carcinoma of the large bowel: a French population-based study[END_REF][START_REF] Wright | Lymph node retrieval and assessment in stage II colorectal cancer: a population-based study[END_REF] while singlehospital studies reported median numbers up to 18. [START_REF] Ostadi | Factors affecting the number of lymph nodes retrieved in colorectal cancer specimens[END_REF]22,[START_REF] Goldstein | Lymph node recoveries from 2427 pT3 colorectal resection specimens spanning 45 years: recommendations for a minimum number of recovered lymph nodes based on predictive probabilities[END_REF]

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The experiences of the pathologists with the new staining technique varied and there was some resistance initially. However, this decreased when the technique was well implemented and the surgeons got more experience with the injection of Patent Blue V dye. Some pathologists emphasized the simplified detection of lymph nodes, due to the increased solidness and colouring of the lymph nodes. Whereas other pathologists preferred the traditional method with a more extensive search for lymph nodes to increase the detection rate. To reduce the workload for pathologists and to ensure adequate lymph node detection in the future, technicians will be trained to search for lymph nodes in the specimen.

There are several ways to increase the lymph node detection rate, including fat clearance methods [START_REF] Hyder | A critical review of chemical lymph node clearance and staging of colon and rectal cancer at Ferguson Hospital, 1977 to 1982[END_REF] which are rather labour-intensive and hazardous chemicals are needed. [START_REF] Brown | Efficacy of manual dissection of lymph nodes in colon cancer resections[END_REF] Refixation of a specimen in a lymph node-revealing solution resulted in a higher detection rate, [START_REF] Svec | Re-fixation in a lymph node revealing solution is a powerful method for identifying lymph nodes in colorectal resection specimens[END_REF] although it is an extra step for the pathologist. The median number of lymph nodes increased from 5 to 13 [START_REF] Kelder | Effect of modified Davidson's fixative on examined number of lymph nodes and TNM-stage in colon carcinoma[END_REF] and from 10 to 17 [START_REF] Iversen | Increased lymph node harvest from colorectal cancer resections using GEWF solution A randomized study[END_REF] with different modified fixatives. Another research group used a blue marking liquid injected into the rectal artery in 24 patients with rectal cancer with 27 lymph nodes examined in the stained group versus 14 in the controls. [START_REF] Markl | Methylene blue injection into the rectal artery as a simple method to improve lymph node harvest in rectal cancer[END_REF] The Patent blue staining method did not change the stage distribution significantly. However, a larger proportion of patients had sufficient lymph nodes examined. Three-year overall survival for stage II patients with sufficient compared to stage II patients with insufficient lymph nodes examined in 1999-2007 in the ECR region was 83% vs. 69%. At hospital level, number of lymph nodes examined was not associated with stage distribution or use of adjuvant chemotherapy. [22,[START_REF] Bui | Lymph node counts, rates of positive lymph nodes, and patient survival for colon cancer surgery in Ontario, Canada: a population-based study[END_REF] However, at the patient level, higher lymph node count was associated with improved survival, relative to fewer than 12 nodes. [START_REF] Chang | Lymph node evaluation and survival after curative resection of colon cancer: systematic review[END_REF] The lymph node detection rate is not only affected by the thoroughness of the surgeon during the lymphadenectomy to remove all potential lymph node metastases, but also to the extent and diligence of the pathologists' examination which is provoked by medical oncologists. The inter-individual differences in biological behaviour of the tumour and/or host also affect the lymph node detection rate. [START_REF] Baxter | Lymph node evaluation in colorectal cancer patients: a population-based study[END_REF][START_REF] Sarli | Number of lymph nodes examined and prognosis of TNM stage II colorectal cancer[END_REF] Furthermore, more lymph nodes were examined during right than left colectomies, [START_REF] Baxter | Lymph node evaluation in colorectal cancer patients: a population-based study[END_REF][START_REF] Bilimoria | Impact of tumor location on nodal evaluation for colon cancer[END_REF] which was in line with our results. We calculated a saving of almost €1,000,000 per year for one department of pathology comparing the number of node-negative patients with less than 10 lymph nodes examined in 1999 with 2007. One third of this saving can be attributed to the Patent blue staining.

Cost-effectiveness

However, some patients might be upgraded from node negative to node positive, and should receive chemotherapy anyway, reducing the saving. No previous studies are known in which chemotherapy-related savings are shown like in our study. Thus, the set of measures as described in this study can be considered highly cost-effective.

In conclusion, a diverse set of measures including increased awareness of pathologists and surgeons, improved communication between pathologists and surgeons, increased fixation time, and Patent blue V dye staining increased the number of examined lymph nodes among patients with colon cancer. This would reduce the proportion of node-negative patients who would otherwise have received unnecessary adjuvant chemotherapy. Besides avoiding the potential burden of this treatment for the individual patient, large savings can be made due to the reduced proportion of high-risk node-negative patients.

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  For the Patent blue-staining study 86 consecutive patients who underwent elective or nonelective curative surgical resection for colon cancer between May 2007 and February 2008 were included as cases. A subset of 84 patients from the ECR evaluated at the Department of Pathology in Eindhoven and diagnosed in 2007, whose specimen were not stained, was used as the control group for the Patent blue staining study. These were patients who underwent surgery in 2007 before the start of the study and patients who were missed due to logistic reasons. All patients included in the Patent blue staining study, both cases and controls, underwent a standard surgical resection and lymphadenectomy according to the location of the tumour. Patients with rectal cancer were not included, since lymph node detection in rectal cancer is hindered by neoadjuvant treatment.

  lymph nodes examined in the Patent blue-stained group was 37% and in the control group 47% (p=0.40). With 10 lymph nodes as a cut-off point,[4] 21% of the patients in the Patent blue-stained group and 45% of the control patients had an insufficient number of lymph nodes examined (Table 2). No significant difference was found between the proportion of N+ patients in the Patent Blue and the control group. Considering only patients with a pT3 colon tumour, 40% of patients in the Patent bluestained group versus 34% of patients in the control group had fewer than 12 lymph nodes examined (p=0.66) A median number of 15 (range 2-45) lymph nodes were examined for patients with a tumour in the proximal colon in the Patent blue-stained group and 13 for patients with a tumour in the distal colon (results not shown). In the Patent blue-stained group as well as the unstained group, the majority of patients with insufficient lymph nodes examined had stage N0, especially in the categories of 6-8 and 9-11 lymph nodes examined (Fig 3).

  lymph nodes examined are considered high risk patients who should receive chemotherapy.[4] Therefore, a large reduction in chemotherapy costs can be achieved by increasing the proportion of patients with a sufficient number of lymph nodes examined.

Figure 1 :Figure 2 :Figure 3 :

 123 Figure 1: Colon cancer patients with an insufficient number of lymph nodes examined in the region of the Department of Pathology in Eindhoven since 1999 (n=1,501)

Table 1 :

 1 General characteristics for patients in the Patent blue stained study group and the

	control group
		Patent blue V stained	Control group	p-value
		group (n=86 (%))	(n=84 (%))
	Mean age (years)	71 (SD: 10)	70 (SD: 11)	0.6
	Gender
	-male	44 (51)	40 (48)	0.7
	-female	42 (49)	44 (52)
	Depth of penetration			0.1
	-pT1	4 (5)	9 (11)
	-pT2	13 (15)	8 (9)
	-pT3	58 (67)	46 (55)
	-pT4	11 (13)	20 (24)
	Stage			0.7
	-I (pT1/2 N0 M0)	13 (15)	16 (19)
	-II (pT3/4 N0 M0)	38 (44)	33 (39)
	-III (pT any N1/2 M0)	35 (41)	35 (42)
	Tumour site			0.3
	-Proximal colon	44 (53) 1	51 (61)
	-Distal colon	39 (47)	33 (39)

1 For 3 patients, tumour site was missing

Table 2 :

 2 Lymph node involvement in newly diagnosed colon cancer patients in the Patent blue stained study group (n=86) and the control group (n=84)

		Patent blue V stained	Control group (n=84)	p-value
		group (n=86)
	Median number of	14 (2-45)	11 (0-39)*	<0.0001
	examined lymph nodes
	(range)
	N0	51 (59%)	45 1 (56%)	0.7
	N+	35 (41%)	35 (44%)
	<10 lymph nodes	18 (21%)	38 (45%)*	<0.0001
	examined
	<12 lymph nodes	32 (37%)	47 (56%)**	0.014
	examined
	1 In the control group 4 patients had Nx stage

ACKNOWLEDGEMENTS

The authors thank the registration team of the Eindhoven Cancer Registry for their dedicated data collection. We would like to thank the pathologists of the Institute for Pathology and Medical Microbiology in Eindhoven and the following hospitals for their cooperation: Catharina Hospital Eindhoven, St Anna Zorggroep Geldrop, and Maxima Medical Centre.

This work was carried out with a grant from the Dutch Cancer Society (IKZ 2006-3588).

A C C E P T E D

CONFLICT OF INTEREST

The authors have no conflict of interest.

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Conflict of interest

The authors state that they have no conflict of interest.