Michel Daudon
email: michel.daudon@aphp.fr

Paul Jungers

Dominique Bazin

James C Williams

Recurrence rates of urinary calculi according to stone composition and morphology

Keywords: Urinary stone, Stone analysis, Morphology, Recurrence

Few studies have examined the relative risk of recurrence of different stone types. The object of the present study was to evaluate the tendency for stone recurrence as a function of major mineral composition of the stones and morphological characteristics of the stones. This study was carried out using 38,274 stones for which we had data available to specify if the stone was from the first or a subsequent urinary stone episode. Stones were analyzed for morphology by stereomicroscope and for composition by infrared spectroscopy. Overall, 42.7% of stones were from patients who had had a previous stone event, with these being more frequent in men (44.4%) than in women (38.9%, p < 0.0001). Age of first stone occurrence was lowest for dihydroxyadenine (15.7 ± 16.6 years) and highest for anhydrous uric acid (62.5 ± 14.9 years), with the average age of first stones of calcium oxalate falling in the middle (40.7 ± 14.6 years for calcium oxalate dihydrate, and 48.4 ± 15.1 years for calcium oxalate monohydrate, COM). By composition alone, COM was among the least recurrent of stones, with only 38.0% of COM stones coming from patients who had had a previous episode; however, when the different morphological types of COM were considered, type Ic-which displays a light color, budding surface and unorganized section-had a significantly greater rate of recurrence, at 82.4% (p < 0.0001), than did other morphologies of COM. Similarly, for stones composed of apatite, morphological type IVa2-a unique form with cracks visible beneath a glossy surface-had a higher rate of recurrence than other apatite morphologies (78.8 vs. 39-42%, p < 0.0001). Stone mineral type alone is insufficient for identifying the potential of recurrence of the stones. Instead, the addition of stone morphology may allow the diagnosis of highly recurrent stones, even among common mineral types (e.g., COM) that in general are less recurrent.

Introduction

Epidemiological studies on rates of stone occurrence and recurrence in industrialized countries agree on two points: first, urinary stones strike men more often than women and, second, recurrence of at least one more stone occurs in 40-50% of affected persons [START_REF] Ljunghall | Prevalence of renal stones in 60-year-old men. A 10-year follow-up study of a health survey[END_REF][START_REF] Ahlstrand | Recurrences during a 10-year follow-up after first renal stone episode[END_REF][START_REF] Ulmann | Histoire naturelle de la lithiase rénale calcique: données obtenues à partir d'une cohorte de 667 malades[END_REF]. However, few studies have examined the relative risk of recurrence of different stone types. It seems reasonable to state that cystine stone disease, because of its genetic basis, leads to greater stone recurrence than most other types of stone disease [START_REF] Chow | Contemporary urological intervention for cystinuric patients: immediate and long-term impact and implications[END_REF]. Similarly, uric acid stones in older patients are often quite recurrent [START_REF] Millman | Pathogenesis and clinical course of mixed calcium oxalate and uric acid nephrolithiasis[END_REF]. However, it is not so clear what to say about the stones composed of calcium salts. For calcium oxalate, studies from some years ago suggest that weddellite (calcium oxalate dihydrate, COD) stones are more likely to recur than whewellite (calcium oxalate monohydrate, COM) stones [START_REF] Koide | Clinical manifestations of calcium oxalate monohydrate and dihydraté urolithiasis[END_REF], and that stones with a significant component of calcium phosphate are more likely to recur than stones composed of pure calcium oxalate [START_REF] Ohman | Clinical significance of phosphate in calcium oxalate renal stones[END_REF]. The object of the present study was to evaluate the tendency for stone recurrence as a function of the major mineral composition of the stones, but also to take into account the morphological characteristics of the stones and the sex of the patient.

Materials and methods

This study was carried out using 38,274 stones collected from all regions of France for which we have data on both stone recurrence and the patient's age at the first stone episode. The stones were analyzed for morphology and composition (by infrared spectroscopy, as previously described [START_REF] Daudon | Analysis of calculi by infrared spectrophotometry. Advantages and limits of the method[END_REF][START_REF] Daudon | Urinary calculi: review of classification methods and correlations with etiology[END_REF]). Briefly, stones were examined using a stereomicroscope and each was classified according to the established morphological criteria [START_REF] Daudon | Urinary calculi: review of classification methods and correlations with etiology[END_REF][START_REF] Daudon | Stone composition morphology: a window on etiology[END_REF]. These criteria include the appearance of the stone surface but also the appearance of the stone interior after the stone was cleaved one or more times to reveal its presumed nidus. The main characteristics of the stones are summarized in Table 1 and some examples of stone types are given in Fig. 1. Following these visual observations, samples of stone material were dissected away for analysis by Fourier transform infrared spectroscopy. For each stone, patient data were available to specify if this was from the first or a subsequent stone episode. The results are expressed according to the principal constituent, morphological type, and structural characteristics of the stone, and separated according to the sex of the patient.

Statistical comparisons were carried out using ANOVA and Chi-squared tests, as appropriate, using NCSS software (NCSS, Kaysville, Utah, USA).

Results

Of the 38,274 stones, coming from 26,368 males (68.9%) and 11,906 females, [START_REF] Brien | 10,000 analyses of urinary calculi using X-ray diffraction and polarizing microscopy[END_REF],336 (42.7%) were from patients who had had a previous stone event. Of these patients with recurrent stones, 11,702 were male and 4634 female, illustrating the fact that recurrent stones were more frequent in men than in women (44.4 vs 38.9% of the total for each sex, p < 0.0001). The mean age of patients with recurrent stones did not differ between the sexes (45.0 ± 14.3 years old for men, 44.0 ± 14.8 for women).

Mean age at first stone as a function of stone mineral

We first consider the data on patients' initial stone events, looking at the age of first stone occurrence for the different crystalline species. The data are shown in Table 2, where, for each majority stone component, the mean age of the patients is given for each sex, with the stone components listed according to the increasing mean age of first occurrence. It is striking that the first five compositions listed in Table 2that is, the stone types occurring at the earliest ages-are crystalline species that are relatively rare, representing < 5% of all stones [START_REF] Daudon | Stone composition morphology: a window on etiology[END_REF][START_REF] Daudon | Sex and age-related composition of 10617 calculi analyzed by infrared-spectroscopy[END_REF]. We are aware that the small number of samples in these groups is a limiting factor for the significance of the data regarding these stones. However, as expected, due to their inherited origin, the first groups of calculi are dihydroxyadenine and cystine. The crystalline species occurring at the youngest age is dihydroxyadenine. The second species is cystine, followed by octacalcium phosphate and whitlockite, then ammonium urate. Coming next in the table are more common types of stone, the first of which is COD, the calcium-dependant type of calcium oxalate [START_REF] Daudon | Whewellite and weddellite: toward a different etiopathogenesis. The significance of morphological typing of calculi[END_REF][START_REF] Asplin | Metabolic urinary correlates of calcium oxalate dihydrate in renal stones[END_REF][START_REF] Parent | Calcium oxalate stones. Relationship between biochemical risk factors and crystalline phase of the stone[END_REF]. The mean age at which this crystalline species appeared as a stone was 40 years, eight years younger than the mean age for COM stones, the other form of calcium oxalate. The distribution of calcium oxalate stones according to the crystalline phase and the patient's age and gender at the onset of the disease is illustrated in Fig. 2.

Between COD and COM are four forms of phosphate stone. Finally, the species appearing latest in life are the two forms of uric acid. The distribution of uric acid stones according to the patient's age and gender at the onset of the disease is shown in Fig. 3.

Frequency of stone recurrence as a function of principal component

Table 3 shows the proportion of patients having a recurrent stone episode (that is, the second or later stone episode for the patient) for each principal stone component. Within this, the stone components are ranked according to the increasing proportion of recurrent stones. Among the stone mineral types that have the lowest percentage of recurrence, two are phosphate stones, including struvite. However, among the seldom-recurring stones is also COM, the constituent most commonly found in urinary stones [START_REF] Daudon | Stone composition morphology: a window on etiology[END_REF][START_REF] Mandel | Urinary tract stone disease in the United States veteran population. II. Geographical analysis of variations in composition[END_REF][START_REF] Brien | 10,000 analyses of urinary calculi using X-ray diffraction and polarizing microscopy[END_REF]. All the stone compositions that follow struvite in the table-with the exception of COD in men and women, and carbapatite and octacalcium phosphate in women-are more recurrent than the average percentage for all stones. Among these more-recurrent compositions, the two forms of uric acid appear at the head with a percentage of recurrence above 50%, significantly higher than the average percentage for all stones. Finally, the three species at the bottom of Table 3 are particularly recurrent, apparently all forms of stone caused by very active metabolic processes: These are brushite, cystine, and dihydroxyadenine.

For stones of calcium oxalate, brushite and cystine, we have also recorded the number of stone episodes in the patients with recurrent stones. The patients who produced calcium oxalate stones had a mean of 3.5 stone episodes, while that number exceeded 7.5 for both brushite and cystine. This number is probably even higher for patients with uric acid stones, but it is difficult to evaluate with precision, as these patients often repeatedly form sand and/or Whewellite + weddellite + carbapatite very small stones that are passed spontaneously with limited clinical symptoms. Moreover, in contrast to other crystalline species, stones composed of uric acid can be dissolved by urinary alkalization [START_REF] Moran | Uric acid stone disease[END_REF].

Frequency of stone recurrence as a function of stone morphology

The frequency of stone recurrence varied with morphological type (Table 4; Fig. 4). This was especially apparent in calcium oxalate stones where the recurrence rate varied among morphological types from 27 to 82%, and in calcium phosphate stones, where the recurrence rate varied from 39 to 79% among the different morphological types.

Overall, the recurrence rate of COM was relatively low (Table 3), but the morphological types Ie, and especially Ic, had higher rates of recurrence (Fig. 4). Thus, recognition of these morphological types of COM in a patient could alert the physician to the possibility of a greater chance of stone recurrence.

Fig.

A B C D E F G H I J K L M N O
For stones containing mainly COD (type II), the percentage of stones that were recurrent was generally not different from the mean of all stones, except for type Ia + IIb (mixture of COM and COD) which were less recurrent and type IIa + IVa1 (COD and carbapatite) stones which were significantly more recurrent.

Regarding stones composed primarily of forms of calcium phosphate, types IVa1 and IVa2 (both types of carbapatite) were very different, the latter being significantly more recurrent than the mean of all stones from men and women, suggesting the possibility of more active metabolic processes driving this morphological form. Type IVd (brushite) was also significantly more recurrent in both sexes, consistent with the high recurrence rate of this stone type reported in American studies [START_REF] Klee | The clinical implications of brushite calculi[END_REF][START_REF] Parks | Clinical implications of abundant calcium phosphate in routinely analyzed kidney stones[END_REF][START_REF] Evan | Crystal-associated nephropathy in patients with brushite nephrolithiasis[END_REF].

Stones composed of uric acid of type IIIb (not type IIIa, often related to stasis) or cystine were significantly more recurrent than the mean for all stones, consistent with these stones resulting from metabolic processes more active than those that drive the formation of common calcium stones.

Influence of Randall's plaque (RP)

As seen in Table 5, calcium oxalate stones developed from RP actually seem to exhibit a slight tendency to be more recurrent than stones formed by other mechanisms (p = 0.02), with a significant difference according to the gender of patients. In men, 41% of stones with an RP were recurrent by comparison with 38.6% of stones without any RP (p < 0.01). No significant difference was found in stones from women. However, differences exist regarding the stone type. For example, COM stones of type Ia developed from an RP appear significantly more recurrent than COM stones of type Ia without RP in men (38.1 vs 33.5%, p < 0.001) with only a trend in women (32.1 vs 28%, p = 0.07). In addition, stones of mixed types I + II and II + I are significantly more recurrent than type Ia stones in men in both groups of stones with and without RP and in women in the group of stones without RP.

Discussion

Stone disease is a very common finding in industrialized countries and is constantly increasing as revealed by epidemiological studies [START_REF] Stamatelou | Time trends in reported prevalence of kidney stones in the United States: 1976-1994[END_REF][START_REF] Hesse | Study on the prevalence and incidence of urolithiasis in Germany comparing the years 1979 vs. 2000[END_REF][START_REF] Trinchieri | Increase in the prevalence of symptomatic upper urinary tract stones during the last ten years[END_REF]. In most cases, it is a consequence of dietary imbalance and/or chronically insufficient water intake. In general, the correction of these factors should prevent stone recurrence. However, among stone diseases, metabolic forms exist that expose the patient to multiple recurrences of calculi with the possible consequence of altering the kidney function and, sometimes, inducing end-stage renal failure. These may result from inherited or acquired pathologies, the diagnosis of which is of crucial importance to treating them and reducing the risk of stone recurrence and crystallization in the kidney. Stone analysis at the first episode is one method for the early detection of stone diseases which are prone to recur. As shown in the present study, there are great differences in terms of stone recurrence risk according to both stone composition and morphology.

Onset of stones

The mean age at the onset of the disease, as revealed by a stone episode, varied in relation to the stone composition. The earliest component found was dihydroxyadenine, which corresponds to an adenine phosphoribosyltransferase deficiency, an inherited autosomal disease often inducing end-stage renal failure when undiagnosed [START_REF] Gagné | Chronic renal failure secondary to 2,8-dihydroxyadenine deposition: the first report of recurrence in a kidney transplant[END_REF][START_REF] De Jong | 2,8-Dihydroxyadenine stone formation in a renal transplant recipient due to adenine phosphoribosyltransferase deficiency[END_REF][START_REF] Glicklich | 2,8-Dihydroxyadenine urolithiasis: report of a case first diagnosed after renal transplant[END_REF][START_REF] Stratta | Decreased kidney function and crystal deposition in the tubules after kidney transplant[END_REF]. Unexpectedly, there was a strong difference in the onset of the disease according to gender, males being prone to develop stones earlier than females. Perhaps a bias exists due to the small number of cases. Coming next were cystine stones which, despite the fact that they result from a congenital defect in tubular transport, in contrast to dihydroxyadenine stones, are known to make their appearance in the second or third decade of life [START_REF] Singer | Cystinuria: a review of the pathophysiology and management[END_REF][START_REF] Prot-Bertoye | CKD and its risk factors among patients with cystinuria[END_REF]. Indeed, the data here suggest that some cystine stone formers do not present their first stone until the seventh decade of life. The next earliest component was octacalcium phosphate, an instable form of apatite able to convert to it in some months. The next component was whitlockite, an infrequently found form of calcium magnesium phosphate, which, in our experience, is essentially a marker of infection [START_REF] Levillain | Crystalline phase differentiation in urinary calcium phosphate and magnesium phosphate calculi[END_REF].

Ammonium hydrogen urate stones were found significantly earlier in men than in women. A possible explanation is related to stones induced by urea-splitting bacteria. Such stones contain ammonium salts such as struvite and/or ammonium urate. They are especially frequent in very young boys (at the time where uricosuria is still elevated) and more later in life for women.

COD, found as the first stone at the mean age of 40 years, is a rather calcium-dependent form of calcium oxalate stone, mainly observed in hypercalciuric states [START_REF] Daudon | Whewellite and weddellite: toward a different etiopathogenesis. The significance of morphological typing of calculi[END_REF][START_REF] Asplin | Metabolic urinary correlates of calcium oxalate dihydrate in renal stones[END_REF][START_REF] Daudon | Clinical value of crystalluria and quantitative morphoconstitutional analysis of urinary calculi[END_REF]. Four forms of phosphate stone, including amorphous carbonated calcium phosphate, carbapatite, brushite and struvite, also appeared at an average first age in the forties. Interestingly, the average age of onset of the most common form of stone, COM, was later than all of these, at 48 years of age. Of note, in Japan, Koide et al. reported in the eighties that patients who form COD stones are different from those who form Table 4 Frequency of stone recurrence according to the morphological type of the stone (1) Complete description of the stone types is given in Daudon et al. [START_REF] Daudon | Urinary calculi: review of classification methods and correlations with etiology[END_REF][START_REF] Daudon | Stone composition morphology: a window on etiology[END_REF] *p < 0.05; **p < 0.01; ***p < 0.00001 vs females a p < 0.05; b p < 0.01; c p < 0.001; d p < 0.0001; e p < 0.00001 vs mean stone recurrence for a given gender or for total Morphological type (1) Males COM stones and that COD stones were observed earlier than COM stones in Japanese patients [START_REF] Koide | Clinical manifestations of calcium oxalate monohydrate and dihydrate urolithiasis[END_REF]. Finally, the age of first occurrence of uric acid stones was much later, at about 60 years of age, probably related to the increased occurrence at older ages of overweight, metabolic syndrome, and type 2 diabetes [START_REF] Daudon | Influence of body size on urinary stone composition in men and women[END_REF][START_REF] Daudon | Type 2 diabetes increases the risk for uric acid stones[END_REF].

Stone recurrence

A stone recurrence rate around 50% is commonly accepted in the literature, both in children and adults [START_REF] Tasian | Kidney stone recurrence among children and adolescents[END_REF][START_REF] Khan | Kidney stones[END_REF]. However, no data are reported regarding crystalline phases of the stones. Among the stone mineral types seen with the lowest percentages of recurrence (Table 3), three were phosphate stones composed of minerals that have been shown to be related to urinary tract infection (whitlockite, amorphous carbonated calcium phosphate, and struvite) [START_REF] Levillain | Crystalline phase differentiation in urinary calcium phosphate and magnesium phosphate calculi[END_REF]. These results seem reasonable because stones occurring due to infection are not likely to recur if the infection is successfully treated. Within the range of recurrence frequency of infection stones, however, are also stones composed of COM, for which the underlying cause for stone formation is generally a hyperoxaluria of moderate degree [START_REF] Daudon | Whewellite and weddellite: toward a different etiopathogenesis. The significance of morphological typing of calculi[END_REF][START_REF] Normand | Correlations between the cause and the composition of urinary stones -the interest of morphological and infrared analysis[END_REF][START_REF] Daudon | Changes in stone composition according to age and gender of patients: a multivariate epidemiological approach[END_REF]. Thus, it is apparent that COM stones are less likely to be recurrent than are other calcium stones whose origins are also metabolic, but for which the endogenous biochemical abnormalities are more marked. It is also interesting to note that the percentage of recurrent COM stones is less than the mean percentage for all recurrent stones, for both men and women. If we consider that physicians propose for preventing recurrence of all urinary stones adequate dietary advices or medical treatment according to the stone composition (calcium stone, uric acid stone, cystine stone, struvite stone…), it is unlikely that stone recurrence might be a consequence of an inappropriate medical care. Obviously, we cannot exclude that patients were not compliant with physician recommendations. However, there is no evidence that such default of compliance may depend on stone type. Thus, intrinsic characteristics of crystalline phases or of stone morphology appear highly suggestive for different courses of stone disease.

Among the four compositions that are least recurrent, struvite is obviously special. Although in women the percentage of recurrent stones composed of struvite was slightly less than the mean for all stones, struvite stones were considerably more recurrent in men. This difference may occur for a number of reasons. First, anatomical and disease conditions could make the formation of struvite stones more favorable in men, especially prostatic hypertrophy, which often causes incomplete emptying of the urinary bladder. However, in our experience, only one-third of bladder struvite stones associated with prostate hypertrophy were recurrent. Another reason for the higher incidence of recurrent struvite stones in men could be that infection is less expected in men, and thus not recognized and promptly treated as often as it is in women. In any case, when one examines the epidemiologic changes in stones in France over the past 30 years, one can see that the frequency of infection stones has fallen dramatically for women-3.5-fold-while that for men has dropped by only 20% [START_REF] Daudon | Epidemiology of nephrolithiasis in France[END_REF]. Of note, recent epidemiological data suggest that struvite stones could be more frequent in recent decades by comparison to previous reports, especially in men [START_REF] Moses | Changes in stone composition over the two decades: evaluation of over 10,000 stone analyses[END_REF].

Ammonium urate was found to be slightly less recurrent by comparison to the whole series. It was a more recurrent component in women than in men. Although it is always found with hyperuricosuria, other factors are necessary to its formation and it can appear in the context of different pathologies (anorexia and laxative abuse, infection with urea-splitting organisms, poor animal protein diet and chronic diarrhea, etc. [START_REF] Soble | Ammonium acid urate calculi: a reevaluation of risk factors[END_REF][START_REF] Kuruma | Ammonium acid urate urolithiasis in Japan[END_REF]).

Among the more recurrent compositions, octacalcium phosphate and the two forms of uric acid are at levels just slightly above the average percentage for all stones. These stones include a species that is at least suspected of being calcium-dependent (octacalcium phosphate [START_REF] Levillain | Crystalline phase differentiation in urinary calcium phosphate and magnesium phosphate calculi[END_REF][START_REF] Méria | Members of the Urolithiasis Committee of the French Urological Association (2010) Stone formation and pregnancy: pathophysiological insights gained from morphoconstitutional stone analysis[END_REF]), and two species (uric acid, both anhydrous and dihydrate) that are frequently associated with metabolic syndrome and diabetes [START_REF] Daudon | Influence of body size on urinary stone composition in men and women[END_REF][START_REF] Daudon | Type 2 diabetes increases the risk for uric acid stones[END_REF][START_REF] Abate | The metabolic syndrome and uric acid nephrolithiasis: novel features of renal manifestation of insulin resistance[END_REF].

The three most recurrent species were found to be dihydroxyadenine, cystine and, surprisingly, brushite. The first two crystalline species are due to well-defined inherited disorders. In contrast, brushite in stones is not recognized as a marker of any genetically linked pathology; yet the rapid growth of brushite stones leads to recurrence rates similar to those of stones formed as the result of genetic disorders, with 75% of brushite stones coming from patients who have already had at least one stone episode. Moreover, patients with brushite stones very often have had repeated stone episodes and frequently are found to have multiple stones with each incidence and a higher number of urological procedures [START_REF] Klee | The clinical implications of brushite calculi[END_REF][START_REF] Parks | Clinical implications of abundant calcium phosphate in routinely analyzed kidney stones[END_REF][START_REF] Evan | Crystal-associated nephropathy in patients with brushite nephrolithiasis[END_REF]. Brushite stones are also classed, along with those of cystine, as the stones most difficult to break [START_REF] Williams | Variability of renal stone fragility in shock wave lithotripsy[END_REF]. These results suggest the need for more sophisticated studies of the metabolic disorders present in the patients who produce brushite stones. Indeed, the incidence of brushite stones has increased considerably over the past years, from 0.8% of all stones in 1980 to 2.2% today in France [START_REF] Daudon | Composition and morphology of phosphate stones and their relation with etiology[END_REF] and from 1.7 to 4.1% in the United States [START_REF] Mandel | Conversion of calcium oxalate to calcium phosphate with recurrent stone episodes[END_REF].

If one compares the recurrence rate of stones between men and women, one can state that all stone types recur more frequently in men, with the exception of ammonium urate, uric acid dihydrate, brushite, and the stones of an obviously genetic origin (cystine and dihydroxyadenine) for which one observes no significant difference in recurrence rates between the sexes. These data suggest a role for environmental factors, particularly nutritional differences, for those stone types that are more recurrent in men; conversely, endogenous metabolic disorders, congenital or acquired, for which stone occurrence is linked to the nature of the metabolic abnormality rather than to environmental factors, seem to be more important for those stone types for which recurrence does not differ between the sexes.

Relationship between stone recurrence and morphology

A unique aspect of the present study is the examination of relationships between stone recurrence and morphologies of stones, which can differ even within the same compositional class. For example, even though the overall recurrence rate of COM stones was relatively low, the recurrence rates of some morphologies of COM were significantly higher (Fig. 4). We are aware of the relatively small number of stones regarding some morphological types. However, despite this limitation, the significant differences between stones types for the same crystalline species may alert us on special risk factors related to such stone types. Thus, recognition of more recurrent morphologies of COM in a patient could be of benefit in diagnosis and treatment.

Among stones composed mainly of COD (type II), the sub-type IIb was found to be significantly less recurrent than were the sub-types IIa or IIa + IVa1, the latter of which was more recurrent than the mean for all stones, whether just for men or for the entire series. However, these recurrence rates among morphological forms of COD stones-in contrast to what was observed for COM-are relatively small, perhaps reflecting the overall influence of hypercalciuria in the formation of this mineral type [START_REF] Daudon | Sex and age-related composition of 10617 calculi analyzed by infrared-spectroscopy[END_REF][START_REF] Daudon | Whewellite and weddellite: toward a different etiopathogenesis. The significance of morphological typing of calculi[END_REF][START_REF] Asplin | Metabolic urinary correlates of calcium oxalate dihydrate in renal stones[END_REF].

Among stones composed mainly of carbapatite, the morphology of type IVa2 (stones with an irregular but shiny surface, with subsurface cracks visible beneath the translucent surface coat) correlated with a higher incidence of recurrence than for other carbapatite stones. In our experience, these stones are associated with all conditions of distal tubular acidosis, i.e., Sjögren syndrome, inherited renal tubular acidosis, medullary sponge kidney [START_REF] Dessombz | Calcium phosphate stone morphology can reliably predict distal renal tubular acidosis[END_REF]. In contrast, carbapatite morphology IVb (heterogeneous surface composed of incomplete layers with varying texture and color, including bumpy/rough regions) was less recurrent, especially in women. This morphology is frequently associated with infection [START_REF] Daudon | Urinary calculi: review of classification methods and correlations with etiology[END_REF], so the difference in its recurrence between men and women could be related to a poorer detection and/ or a less effective medical surveillance for men in this context than for women.

At last, we found differences in the occurrence rate according to the presence or not of a RP. It was shown since more than twenty years that RP is now the main factor involved in initiating calcium oxalate stone formation [START_REF] Low | Endoscopic mapping of renal papillae for Randall's plaques in patients with urinary stone disease[END_REF][START_REF] Evan | Randall's plaque of patients with nephrolithiasis begins in basement membranes of thin loops of Henle[END_REF][START_REF] Daudon | Randall's plaque as the origin of calcium oxalate kidney stones[END_REF]. Based on visual inspection of papillae thanks to ureteroscopy, it was reported that most calcium stone formers exhibit multiple papillary calcifications [START_REF] Kim | Stone formation is proportional to papillary surface coverage by Randall's plaque[END_REF]. On the other hand, it was reported a higher occurrence of kidney stones related to the papillary coverage by Randall's plaque [START_REF] Matlaga | Endoscopic evidence of calculus attachment to Randall's plaque[END_REF]. Thus, it would be expected that stones developed from RP should be especially at risk to recur. As shown in Table 5, the difference between stones developed from RP and similar stones developed without RP is not so high. However, when stone types are taken into account, calcium oxalate stones made of a combination of COM and COD (mixed types I + II and II + I) are significantly more recurrent than stones exclusively made of COM, suggesting that metabolic differences could exist between patients producing these different types of stones. Further studies are needed to confirm such hypothesis.

In conclusion, recurrence of urolithiasis is quite variable, being correlated with not only the crystalline composition of the stones, but also with morphological types and certain other structural characteristics. Among calcium oxalate stones, COD stones recur considerably more often than do stones composed of COM. Among calcium phosphate stones, octacalcium phosphate and, above all, brushite stones are more recurrent than other phosphate stone compositions. Brushite stones are especially notable in this regard, not only for their high degree of recurrence, but also because they tend to recur as multiple stones and because the underlying causes of brushite stone formation have not yet been clearly established. Our results also show that the morphology of the stones is important. Thus, it would seem appropriate for clinical work not to limit stone analysis to stone composition only, but also to take into consideration the morphology of the stone, as this can help in diagnosis and in pinpointing the factors that are causative for stone formation, so that better measures for prevention of stone recurrence can be prescribed for each patient.

Compliance with ethical standards

Conflict of interest The authors declare no conflict of interest.

Ethical approval All the specimens of our database were de-identified. All procedures performed in the study were in accordance with the ethical standards of the institutional research committee.

Informed consent Informed consent was obtained from all individual participants.

Fig. 2

 2 Fig. 2 Age distribution of patients at the onset of COM or COD stone disease in both genders

Fig. 3

 3 Fig. 3 Age distribution of patients at the onset of uric acid stones in men and women

Table 1

 1 Morphoconstitutional classification of urinary calculi (from[9, 10])

	Morphological type	Subtype Main components	Main morphological characteristics (for more details, see [9, 10])
				Surface	Section
	I	Ia	Whewellite	Mammillary, brown	Concentric, radiating organization,
					brown
		Ib	Whewellite	Rough and mammillary, brown	Unorganized, brown
		Ic	Whewellite	Budding, cream to pale yellow-brown Poorly organized, cream to pale yel-
					low-brown
		Id	Whewellite	Smooth, beige or pale brown	Concentric, compact, beige
		Ie	Whewellite	Locally budding and mammillary,	Locally unorganized, locally radiating
				pale yellow-brown to brown	organization, pale yellow-brown to
					brown
	II	IIa	Weddellite	Bipyramidal crystals aggregated, with	Loose radial organization, pale yellow
				sharp angles and ridges, pale yellow
		IIb	Weddellite ± whewellite Bipyramidal crystals aggregated,	Unorganized, pale yellow
				with blunted angles and ridges, pale
				yellow
		IIc	Weddellite	Rough, grey-beige	Thin layers in periphery, unorganized
					core, grey-beige
	III	IIIa	Uric acid	Smooth, ochre to orange	Concentric, radiating organization,
					orange
		IIIb	Uric acid	Embossed, rough and porous, beige	Unorganized or poorly organized and
				to orange	porous, orange
		IIIc	Various urates	Rough, locally porous, beige to grey-	Unorganized with porous areas, beige
				ish	to greyish
		IIId	Ammonium urate	Embossed, rough and porous, grey-	Alternated layers, thick and brownish,
				brown	thin and greyish
	IV	IVa1	Carbapatite	Rough or slightly embossed, cream	Poorly organized, whitish to cream
				to beige
		IVa2	Carbapatite	Embossed, glazed with cracks, yel-	Alternated layers, thick and yellow-
				low-brown	brown, thin and cream
		IVb	Carbapatite	Heterogenous, both embossed and	Alternated layers, thick and cream, thin
				rough	and yellow-brown
				Heterogenous color, cream to brown
		IVc	Struvite	Amalgamate crystals with blunted	Crude radial crystallization, whitish
				angles and thick ridges, whitish
		IVd	Brushite	Finely rough or aggregated rods,	Concentric, radiating organization,
				cream to beige	cream to beige
	V	Va	Cystine	Rough or grainy, yellowish	Poorly organized, loose radiating
					organization, yellowish
		Vb	Cystine	Smooth, cream to pale yellowish	Concentric in periphery, unorganized
					core, cream to yellow
	VI	VIa	Proteins	Smooth, cream to pale brown	Unorganized, cream to pale brown
		VIb	Proteins	Rough and flaked off, dark brown to	Crude and diffuse foliated structure,
				black	dark brown to black
		VIc	Proteins	Homogenous, smooth, dark brown	Soft concentric dark-brown shell, unor-
					ganized beige core
	Main mixed types
	Ia + IIa and Ia + IIb	Whewellite + weddellite
	IIa + IVa1 and IIb + IVa1 Weddellite + carbapatite
	Ia + IIa + IVa1

Table 2

 2 Mean age (years ± SD) at the first stone episode according to the stone composition *COM = calcium oxalate monohydrate -COD = calcium oxalate dihydrate

	Main component	Males		Females		All		p value
		N	Age	N	Age	N	Age	M vs F
	Dihydroxyadenine	15	7.2 ± 9.3	6	29.0 ± 17.3	21	15.7 ± 16.6	< 0.01
	Cystine	43	26.5 ± 20.1	51	27.7 ± 16.3	94	27.1 ± 18.2	NS
	Octacalcium phosphate pentahydrate	16	25.6 ± 18.4	18	31.0 ± 13.2	34	28.6 ± 15.9	NS
	Whitlockite	21	25.1 ± 19.2	22	37.4 ± 16.3	43	31.4 ± 18.4	< 0.05
	Ammonium hydrogen urate	48	25.4 ± 27.1	35	43.7 ± 19.8	83	33.1 ± 26.1	< 0.001
	Weddellite (COD)*	3440	41.0 ± 14.0	1039	39.7 ± 16.1	4469	40.7 ± 14.6	< 0.05
	Brushite	162	42.8 ± 16.8	59	38.2 ± 16.2	221	41.6 ± 16.9	NS
	Carbapatite	1064	39.4 ± 22.9	1702	43.1 ± 16.9	2766	41.7 ± 19.6	< 0.001
	Amorphous carbonated calcium phosphate	17	37.7 ± 30.1	29	44.4 ± 25.1	46	41.9 ± 27.1	NS
	Struvite	157	44.2 ± 26.5	205	47.2 ± 21.3	362	45.9 ± 23.7	NS
	Whewellite (COM)*	7890	48.8 ± 14.4	3383	47,4 ± 16.5	11,273	48.4 ± 15.1	< 0.01
	Uric acid dihydrate	192	58.3 ± 12.9	40	62.9 ± 14.0	232	59.1 ± 13.3	NS
	Uric acid anhydrous	1080	62.8 ± 14.6	366	61.8 ± 15.1	1446	62.5 ± 14.9	NS

Table 3

 3 Frequency of stone recurrence according to the stone composition

	Component	Males		Females		Total
		N	%	N	%	N	%
	ACCP (1)	8	32.0	13	30.2	21	30.9
	Whitlockite	10	32.3	14	38.9	24	35.8
	Whewellite (COM)	5241	39.5*** ,e	1768	34.1 e	7013	38.0 e
	Struvite	143	47.0**	121	36.7	264	41.6
	Presence of struvite	492	44.9***	526	36.5	1018	40.1
	Ammonium Urate	28	36.8	32	47.1	60	41.7
	Weddellite (COD)	2883	45.2***	625	37.2	3508	43.5 a
	Carbapatite	1022	48.8*** ,e	1143	39.9	2165	43.6 a
	Uric acid anhydrous	1193	52.1* ,e	329	46.8 c	1522	50.9 e
	Uric acid dihydrate	198	50.6 a	50	55.6 b	248	51.6 c
	Octacalcium phosphate	28	63.6* ,a	15	45.5	43	55.8
	Brushite	479	75.7 e	143	70.8 e	622	74.5 e
	Cystine	378	90.9 e	293	86.7 e	671	89.0 e
	Dihydroxyadenine	19	95.0 d	7	100 b	26	96.3 e
	All stones	11,702	44.4***	4634	38.9	16,341	42.7

(1)

ACCP = amorphous carbonated calcium phosphate *p < 0.05; **p < 0.01; ***p < 0.00001 vs females a p < 0.05; b p < 0.01; c p < 0.001; d p < 0.0001; e p < 0.00001 vs mean recurrence for a given gender or by comparison to the whole series

Table 5

 5 Comparison between umbilicated stones or notStones of mixed types I + II include stones with various subtypes where COM was the main crystalline phase of calcium oxalate. By contrast, stones of mixed types II + I include stones with various subtypes where COD was the main crystalline phase of calcium oxalate. As shown in the table, the recurrence rate is not the same according to the main crystalline phase of calcium oxalate: when COD is the main crystalline phase, the stones are commonly more recurrent by comparison with type Ia stones used as a reference

				Females		Total
		N	%	N	%	N	%
	Calcium oxalates
	Ia	1735	35.9*** ,e	499	29.8 d	2234	34.3 e
	Ib	58	26.1 c	25	30.9	83	27.4 d
	Ic	29	85.3 e	13	76.5 b	42	82.4 e
	Id	66	40.0	43	46.2	109	42.3
	Ie	44	54.3	26	48.2	70	51.9
	Ia + IIa	424	41.7	147	38.6	571	40.8
	Ia + IIb	1051	39.5** e	306	32.1 b	1359	37.6 e
	IIb + Ia	1433	44.4**	380	38.7	1813	43.1
	IIa	773	44.6**	161	37.6	934	43.2
	IIb	400	39.1 a	86	25.4	486	38.4
	IIa + IVa1	959	48.5 ** ,b	286	40.8	1245	46.5 c
	IIb + IVa1	213	39.1	99	40.4	312	39.5
	Ia + IIa + IVa1	682	46.4**	326	36.8	1008	42.8
	Calcium phosphates
	IVa1 (w/o struvite)	110	47.8** ,a	125	38.0	235	42.0
	IVa2	26	70.3 a	53	84.1 e	79	79.0 e
	IVb	221	48.3***	335	35.2 a	556	39.4 b
	IVd	229	73.6 e	73	68.9 e	302	72.4 e
	Struvite (magnesium ammonium phosphate hexahydrate)
	IVc	29	53.7	17	32.7	46	43.4
	Uric acids
	IIIa	186	45.7	39	50.7 a	225	46.5
	IIIb	840	53.2** ,e	230	47.2 b	1070	51.8 e
	Cystine
	Va	312	90.7	233	84.7 e	545	88.1 e
	Vb	20	100	7	100 b	27	100 e

*p = 0.02; **p < 0.01; ***p < 0.001; ****p < 0.0001 vs non-umbilicated stones † p < 0.05; † † p < 0.01; † † † p < 0.001; † † † † p < 0.0001 vs type Ia stones for a given gender a p < 0.05; b p < 0.01; c p < 0.001; d p < 0.0001 vs females x p < 0.01; xx p < 0.0001 vs mean occurrence of stone recurrence for a given gender

LCMCP, UPMC, CNRS, Collège de France, Paris, France

Laboratoire de Physique des Solides, Université Paris Sud, Orsay, France

Department of Anatomy and Cell Biology, Indiana University, Indianapolis, IN, USA


Anne-Laure Faucon

Michel Daudon

Vincent Frochot

Dominique Bazin

Benoit Terris

Valérie Caudwell

Delayed ileal perforation from sodium polystyrene sulfonate

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

Delayed ileal perforation from sodium polystyrene sulfonate

Anne-Laure Faucon 1 , Michel Daudon 2,3,4 , Vincent Frochot 2,3,4 , Dominique Bazin 3,5,6 , Benoı ˆt Terris 7,8,9 Correspondence: Anne-Laure Faucon, Centre Hospitalier Sud Francilien, Service de Ne ´phrologie-Me ´decine Interne, 116 Blvd Jean Jaure `s, 91100 Corbeil-Essonnes, France. E-mail: anne-laure.faucon@aphp.fr A 66-year-old man, without a significant medical history, was admitted to the hospital for polyarthralgia. He reported hemorrhagic diarrhea and having been treated with amoxicillin and metronidazole a month prior to admission. Reactive arthritis was first suspected. His serum creatinine level was 0.759 mg/dl. He received nonsteroidal anti-inflammatory drugs. After 3 days, he was admitted to our unit with acute kidney failure. His serum creatinine level was 8.932 mg/dl. Proteinase-3 anti-neutrophil cytoplasmic antibodies were strongly positive, and granulomatosis with polyangiitis was confirmed by kidney biopsy. Remission was obtained with plasma exchanges, corticosteroids, and i.v. cyclophosphamide. On day 12, he developed a pneumoperitoneum, and on laparotomy, a localized ileal perforation was noted. A partial ileal resection with ileostomy was performed. Gross examination showed a large ileal ulcer. Histologic analysis showed no sign of vasculitis, but inflammation, with basophilic and purple polygonal crystals in the digestive lumen, in the wall of the ulcer, and at the serosal surface, was noted (Figure 1a, b, andc). Drug-induced crystals were suspected, and a history of sorbitol-free sodium polystyrene sulfonate (SPS), taken orally 20 days before (total dose: 60 g), was obtained. The diagnosis was confirmed using infrared spectroscopy (Figure 2). SPS is a non-absorbable ion-exchange resin widely prescribed for the management of hyperkalemia. SPS-related gastrointestinal adverse events have been reported in isolated case reports, with an incidence of 0.27%-1.8%. These events include diarrhea, bleeding, ischemic colitis, focal and deep ulceration, necrosis, and perforation. The risk factors include chronic kidney disease, hypotension, hypovolemia, immunosuppression, postoperative status, and obstructive bowel disease. The median time from the first SPS dose to the occurrence of symptoms is 2 days (interquartile range: <1-5 d). However, as in our patient, the diagnosis should be suspected, even if SPS has been stopped several days before the adverse event, especially in patients with additional risk factors.

DISCLOSURE

All the authors declared no competing interests.

  and Vale ´rie Caudwell 1 1 Department of Nephrology and Internal Medicine, Centre Hospitalier Sud Francilien, Corbeil, France; 2 Department of Physiology, Ho ˆpital Tenon, APHP, Paris, France; 3 Faculty of Medicine, Pierre et Marie Curie University, Paris, France; 4 INSERM UMRS 1155, Paris, France; 5 Laboratoire de Physique des Solides, CNRS UMR 8502, Paris-Sud University, Orsay, France; 6 Laboratoire de Chimie de la Matie `re Condense ´e de Paris, Pierre et Marie Curie University, Colle `ge de France, Paris, France; 7 Department of Pathology, Ho ˆpital Cochin, APHP, Paris, France; 8 Paris-Descartes University, Faculty of Medicine, Paris, France; and 9 INSERM U1016-UMR 8104, Institut Cochin, Paris, France

Figure 1 |Figure 2 |

 12 Figure 1 | Optical microscopy with hematoxylin and eosin staining revealed inflammatory lesions with basophilic and purple polygonal crystals in the ileal ulceration and at the serosal surface (a). Scanning electron microscopy of sorbitol-free sodium polystyrene sulfonate crystals (b,c). To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.

AUTHOR CONTRIBUTIONS

All authors contributed to patient care. A-LF and VC wrote the article. MD, VF, DB, and BT created the figures. All authors have read and approved the submission of the article. Written consent for publication was obtained.


B Pradère

F Poulon

E Compérat

I Lucas

D Bazin

S Doizi

O Cussenot

O Traxer

Darine Abi Haidar
email: abihaidar@imnc.in2p3.fr

Two-photon optical imaging, spectral and fluorescence lifetime analysis to discriminate urothelial carcinoma grades

Keywords: Bladder tumor, two-photon imaging, endomicroscopy, spectroscopy, fluorescence lifetime imaging

In the framework of urologic oncology, mini-invasive procedures have increased in the last few decades particularly for urothelial carcinoma. One of the essential elements in the management of this disease is still the diagnosis, which strongly influences the choice of treatment. The histopathologic evaluation of the tumor grade is a keystone of diagnosis, and tumor characterization is not possible with just a macroscopic evaluation. Even today intraoperative evaluation remains difficult despite the emergence of new technologies which use exogenous fluorophore. This study assessed an optical multimodal technique based on endogenous fluorescence, combining qualitative and quantitative analysis, for the diagnostic of urothelial carcinoma. It was found that the combination of two photon fluorescence, second harmonic generation microscopy, spectral analysis and fluorescence lifetime imaging were all able to discriminate tumor from healthy tissue, and to determine the grade of tumors. Spectral analysis of fluorescence intensity and the redox ratio used as quantitative evaluations showed statistical differences between low grade and high grade tumors. These results showed that multimodal optical analysis is a promising technology for the development of an optical fiber setup designed for an intraoperative diagnosis of urothelial carcinoma in the area of endourology.

Introduction

With more than 79,000 new cases and a projection of 16,390 deaths in 2017 in the US [START_REF] Siegel | Cancer Statistics[END_REF], bladder cancer is one of the deadliest cancer worldwide. For urologists, bladder cancer (BC) and upper urinary tract transitional cell carcinoma (UUT-TCC) are principal issues in urologic oncology. It is therefore critical to diagnose these tumors during endoscopy procedures (cystoscopy or ureteroscopy depending of the tumor localization) in order to confirm lesions macroscopically and to collect a tissue sample to confirm histopathologic characteristics. For specific cases of urothelial carcinoma (BC and UUT-TCC), determination of the histopathological grade of urothelial tumors is one of the keystones in the oncological management. The tumor grade impacts the treatment strategy at various levels, from the type of surgery, to the role of chemotherapy, to the chronological steps in management. In the case of a high grade tumor a total ablation of the organ (radical surgery) may be necessary whereas conservative treatment can be a valid option for low grade tumors associated with bladder cancer and upper urinary tract transitional cell carcinoma (UUT-TCC) [START_REF] Rouprêt | European Association of Urology Guidelines on Upper Urinary Tract Urothelial Cell Carcinoma: 2015 Update[END_REF], [START_REF] Babjuk | EAU guidelines on non-muscle-invasive urothelial carcinoma of the bladder: update 2013[END_REF]. A critical step is proper resection of lesions due to a high risk of recurrence [START_REF] Brausi | Variability in the recurrence rate at first follow-up cystoscopy after TUR in stage Ta T1 transitional cell carcinoma of the bladder: a combined analysis of seven EORTC studies[END_REF] particularly for low grade urothelial carcinoma. This is achievable if both lesion detection and diagnosis are optimal. In this framework, peri-operative screening of tumor characteristics could be a valuable support to the surgeon to adapt the surgery and to plan postoperative cares.

With the development of minimally invasive surgery, the use of miniaturized technology has become a standard procedure, especially for UUT-TCC and BC. For UUT-TCC, flexible ureteroscopy with laser photovaporization allows a reduction in both surgical morbidity and renal function impairment [START_REF] Cornu | Oncologic control obtained after exclusive flexible ureteroscopic management of upper urinary tract urothelial cell carcinoma[END_REF], [START_REF] Daneshmand | Endoscopic management of upper urinary tract transitional cell carcinoma: long-term experience[END_REF]. New technologies have been developed to help the surgeon with intraoperative discrimination of urothelial carcinoma [START_REF] Smith | Inadequacy of biopsy for diagnosis of upper tract urothelial carcinoma: implications for conservative management[END_REF], [START_REF] Keeley | Diagnostic accuracy of ureteroscopic biopsy in upper tract transitional cell carcinoma[END_REF]. Enhanced imaging techniques such as Photodynamic diagnosis (PDD) with 5-ALA [START_REF] Batlle | Porphyrins, porphyrias, cancer and photodynamic therapy--a model for carcinogenesis[END_REF], Narrow Band Imaging system(NBI) [START_REF] Gono | Appearance of enhanced tissue features in narrow-band endoscopic imaging[END_REF], real time optical coherence tomography (OCT) [START_REF] Zagaynova | Contrasting properties of gold nanoparticles for optical coherence tomography: phantom, in vivo studies and Monte Carlo simulation[END_REF] and confocal laser endomicroscopy (CLE) [START_REF] Bonnal | Confocal laser endomicroscopy of bladder tumors associated with photodynamic diagnosis: an ex vivo pilot study[END_REF] are now proposed for diagnosis. However, all of these technologies have limitations including low specificity (NBI), or difficulty of interpretation by the doctors (OCT, CLE), compared to the gold standard histological diagnosis, due to the lack of multimodality and database images.

After two decades of becoming increasingly popular, two photon microscopy (TPM) has now become an important technique for biological tissue imaging. The development of miniaturized high-numerical aperture optics [START_REF] Hamzeh | Optimization and characterization of nonlinear excitation and collection through a gradient-index lens for high-resolution nonlinear endomicroscopy[END_REF], lasers with optimized pulse delivery [START_REF] Ibrahim | Characterization of fiber ultrashort pulse delivery for nonlinear endomicroscopy[END_REF], and miniaturized scanning system [START_REF]Development of a real-time flexible multiphoton microendoscope for label-free imaging in a live animal[END_REF], [START_REF] Duan | Optical coherence tomography endoscopic probe based on a tilted MEMS mirror[END_REF] has opened the way for clinical applications. Nonlinear microscopy, as classical confocal imaging, can provide an analysis of tissue using only endogenous labels, and thus eliminates the need for exogenous fluorescence labeling. Second Harmonic Generation (SHG) can access characteristics of tissue structure which have a non-centrosymmetric geometry, such as extra-cellular matrix, muscle fibers, walls of blood vessels, while two photon endogenous fluorescence (TPEF) measurements can detect and show the presence of endogenous fluorophores associated with cellular metabolic activity (reduced nicotinamide adenine dinucleotide: NADH and flavin adenine dinucleotide: FAD).

TPM offers several advantages such as intrinsic sectioning, lower photobleaching, lower and localized phototoxicity, higher penetration depth into the tissues, and an absence of overlap between excitation and emission [START_REF] Zanello | Multimodal optical analysis discriminates freshly extracted human sample of gliomas, metastases and meningiomas from their appropriate controls[END_REF]- [START_REF] Ibrahim | Spectral and fluorescence lifetime endoscopic system using a double-clad photonic crystal fiber[END_REF]. Above all, it provides a different endogenous contrast analysis compared to standard microscopy and confocal endomicroscopy. Although the heterogeneity of tissue structure can lead to complex signal interpretation, endogenous fluorescence analysis of urothelial tissue could be a promising way to avoid metabolic alteration related to external markers [START_REF] Palmer | Detection of urinary bladder cancer cells using redox ratio and double excitation wavelengths autofluorescence[END_REF], [START_REF] Schäfauer | Detection of bladder urothelial carcinoma using in vivo noncontact, ultraviolet excited autofluorescence measurements converted into simple color coded images: a feasibility study[END_REF]. The multimodal technique is an improved alternative to the unimodal one since it highlights tumors characteristics more specifically and precisely.

Nevertheless, non-linear flexible endoscopy imaging development remains a challenge. Although some modalities such as SHG have already been evaluated in-vivo [START_REF] Llewellyn | Minimally invasive high-speed imaging of sarcomere contractile dynamics in mice and humans[END_REF] and multiphoton microscopy evaluated on urothelial and prostate samples [START_REF] Zhuo | Multiphoton microscopy of unstained bladder mucosa based on two-photon excited autofluorescence and second harmonic generation[END_REF], [START_REF] Yadav | Multiphoton Microscopy of Prostate and Periprostatic Neural Tissue: A Promising Imaging Technique for Improving Nerve-Sparing Prostatectomy[END_REF], only one study so far has explored the potential of multimodal optical analysis in the area of uro-oncology [START_REF] Cicchi | Time-and Spectral-resolved two-photon imaging of healthy bladder mucosa and carcinoma in situ[END_REF].

This preliminary study was conducted to establish the efficiency of a multimodal imaging process based on endogenous fluorescence analysis to discriminate grades of urothelial tumours as done in histopathologic grading. For that reason, we conducted a qualitative and quantitative study, using different contrasts to define optical markers of tumours grade. This study show that optical imaging and analysis could provide reproducible and reliable clinical results.

To the best of our knowledge, this preliminary study assesses for the first time the ability of multimodal optical analyses to identify pathological features and histopathological grades associated with urothelial carcinoma, in a manner consistent with classical histopathological evaluation.

Material and Methods

Sample management

This prospective monocentric study had included a collection of samples from patients suspected to have an urothelial carcinoma at fibroscopy or on imagery. The institutional review board (IRB) of the Tenon Hospital center -University Pierre-Marie Curie approved the study (IRB-00003835), all the following methods were performed in accordance with the relevant guidelines and regulations issue in this protocol and informed consent were signed by the patients.

After endoscopic biopsy or resection, urothelial samples were fixed in a formalin zinc solution.

Immediately after, the samples were transferred on a specific nonlinear multimodal set-up to perform the acquisitions. After the multimodal optical analysis, the samples were conditioned and analyzed by the pathologist. Detailed clinical data including age at diagnosis, past medical history including urothelial carcinoma and previous treatment were recorded, such as histopathological data.

Confocal, two-photon microscopy and fluorescence lifetime imaging on a microscopic multimodal setup

A Mai Tai DeepSee Ti:sapphire laser with automated dispersion compensation (Spectra-Physics, Santa Clara, USA) was used as the source for a TCS SP8 MP microscope (Leica Microsystems, A hybrid detector was used to perform the spectral acquisition. A prism dispersed the fluorescence and the spectral detected band was selected with a specific motorized split mirror placed before the hybrid detector. The spectral resolution was 10 nm, covering the range from 380 nm to 780 nm. Spectral acquisition and single-photon counting technology fluorescence lifetime imaging were couples by a unique system which integrated hardware and software (PicoQuant GmbH, Berlin, Germany) with the high-end confocal system Leica TCS SP8 MP.

A spectral mosaic was acquired on a 3x3 images area selected with the microscope software: a spectral measurement was made for each image of the mosaic then the software merged the information to give a mean spectrum of the mosaic. For fluorescence lifetime imaging measurements, a mosaic of 9 images per sample were analyzed using appropriate excitation wavelengths. The image size was 512×

512 pixels, at 100 Hz and represented the average of 10 frames.

Four different optical signals on the region of interest (ROI) were recorded with our multimodal setup:

1) one and two-photon spectral analysis, 2) two-photon fluorescence lifetime imaging measurement,

3) SHG imaging, and 4) fluorescence imaging under one-and two-photon excitation.

Histopathology analysis

All the samples were analyzed by the same senior uropathologist using hematoxylin-eosin-saffran (HE) staining without prior knowledge of the two-photon analysis status. Results were given according to the WHO 2016 classification for urothelial carcinoma [START_REF] Humphrey | The 2016 WHO Classification of Tumours of the Urinary System and Male Genital Organs-Part B: Prostate and Bladder Tumours[END_REF]. Correlation between point-topoints optical indexes, imaging abnormalities and pathological examination of biopsy samples in each particular tumor type and grade was performed. All samples were categorized and classified as healthy urothelium or high grade or low grade tumor by the pathologist.

Data Analysis

Spectral analysis

Five circular Regions of interest (ROI) of 200μm diameter were placed on each spectral mosaic stack at the 870nm excitation wavelength. A spectrum was generated for each ROI using Image J to calculate the mean intensity of each image of the stack. This spectrum was then fitted using a Matlab script developed in the lab and previously used in rat and human brain tumors [START_REF] Haidar | Spectral and lifetime domain measurements of rat brain tumors[END_REF], [START_REF] Zanello | Spectral and Lifetime Measurements of the Endogenous Fluorescence Variation of Freshly Resected Human Samples over Time -Measuring Endogenous Fluorescence Changes at Different Moment after Tumor or Epileptic Cortex Excision[END_REF]. The script was readapted to urothelial tissue, two components where identify in the emitted response: the nicotinamide adenine dinucleotide (NADH) and the flavin adenines dinucleotide (FAD). Indeed, the absence of oxygen or a need to increase glucose catabolism cause an increase in NADH fluorescence.

Hence, the redox ratio is already well-known to be associated with malignant transformation [START_REF] Skala | In vivo multiphoton microscopy of NADH and FAD redox states, fluorescence lifetimes, and cellular morphology in precancerous epithelia[END_REF]- [START_REF] Varone | Endogenous two-photon fluorescence imaging elucidates metabolic changes related to enhanced glycolysis and glutamine consumption in precancerous epithelial tissues[END_REF].

Different excitation wavelengths were used. For each excitation wavelength a fit was applied to extract the contribution of the different molecules.

Results

Samples characteristics:

In our study, fourteen patients were enrolled prospectively, including sixteen samples of normal and tumoral urothelium. Among them, fifteen samples were of bladder origin and one from the upperurinary tract. Amongst them, ten represented urothelial carcinoma (five reported as low grade tumors and five as high grade tumors). Six samples of non tumoral urothelium were also examined.

Correlation between two-photon fluorescence images and Hematoxylin and Eosin staining

To highlight the performances of non-linear microscopy in tissue diagnosis, we compared our images with the histological analysis, considered as gold standard. Results are shown in Figure 2.

Three major urothelial aspects were identified by fluorescence and compared with the histopathological morphology. First a healthy layer of urothelial cells, second low grade tumor architecture displaying exophytic growth with a fibrovascular core and increased layers of urothelial cells without major atypia or necrosis, and third an invasive high grade tumor, with major atypia and also with exophytic features and a fibrovascular core, typical for pTa tumours (pathologically staged non invasive papillary carcinoma). Moreover, the SHG signal gave us access to the architecture and characteristics of the stroma and the lamina propria and could also be compared with the histological features.

The TPF images were collected through the whole sample, from the epithelial surface to the deepest part, mostly to the detrusor muscle. After the determination of the grade by the multimodal optical analysis, the histopathological images (see Figure 2) were chosen by the pathologist for a better correlation to the TPF images, in order to highlight the specific structures.

Discussion and conclusion

The multimodal optical analysis of urothelial samples from bladder and urinary upper tract has proven its capacity to characterize the urothelium and discriminate healthy tissue, low grade and high grade tumor. This characterization was achieved by a qualitative and a quantitative analysis. These preliminary results are promising for the development of a peri-operative diagnostic tool.

Multiphoton microscopy is a rising technology in the field of tissue analysis. Its intrinsic characteristics make it an increasingly studied technique even in urology [START_REF] Yadav | Multiphoton Microscopy of Prostate and Periprostatic Neural Tissue: A Promising Imaging Technique for Improving Nerve-Sparing Prostatectomy[END_REF], [START_REF] Jain | Multiphoton microscopy: a potential intraoperative tool for the detection of carcinoma in situ in human bladder[END_REF]. Indeed, in urology, some studies have already shown a good correlation between multi-photonic microscopy and histopathologic imaging. Yadav et al. have demonstrated the discrimination of periprostatic neural tissue on a rat model with using 780 nm as excitation wavelength excitation [START_REF] Yadav | Multiphoton Microscopy of Prostate and Periprostatic Neural Tissue: A Promising Imaging Technique for Improving Nerve-Sparing Prostatectomy[END_REF]. For urothelial carcinoma, Jain et al. [START_REF] Jain | Multiphoton microscopy: a potential intraoperative tool for the detection of carcinoma in situ in human bladder[END_REF] have assessed the diagnostic potential of this technique in identifying and differentiating benign from malignant flat bladder lesion, especially carcinoma in situ (CIS). These studies have shown a good correlation between multi-photonic microscopy and histopathology. However, none of them have performed a quantitative analysis such as spectral analysis or FLIM analysis. Multimodality as quantitative and qualitative analysis is important for reliable and precise When multiphoton microscopy is used alone, it is still limited by the difficulty of easily recognizing abnormal aspects such as intranuclear modifications. In our study, multiphonic imaging also lead us to recognize and discriminate structures as seen in histopathology. In particular, it was possible to discern healthy urothelium from low and high grade tumors. It is very interesting and promising that optical imaging could be used for live peri-operative histology. It offers high resolved, rapid images comparable to histological images. But because of the learning curve for the tissue recognition and potential intraoperative variability, imaging alone is not sufficient for a reproducible diagnostic tool. This is why, multimodality by adding quantitative analysis to qualitative makes major contribution to clinical applicability in endo-urology.

Our study has shown that fluorescent spectral analysis can discriminate among different types of urothelial tissue from healthy to high grade tumors. Likewise, the redox ratio and FLIM analysis were able to significantly distinguish tissues characteristics. To date, only one study has used the multimodality for urothelial evaluation. Cicchi et al. have assessed TPM for healthy bladder mucosa and CIS. Their results shown a potential difference between both groups even with the redox ratio analysis. But again, the limitation to the diagnosis of CIS is a limitation for a real applicability comparatively to our study which was focused on the grade. To evaluate the redox ratio, different formula are proposed in the literature but we decided to take the most used [START_REF] Palmer | Detection of urinary bladder cancer cells using redox ratio and double excitation wavelengths autofluorescence[END_REF], [START_REF] Cicchi | Time-and Spectral-resolved two-photon imaging of healthy bladder mucosa and carcinoma in situ[END_REF], [START_REF] Palmer | Optical redox ratio and endogenous porphyrins in the detection of urinary bladder cancer: A patient biopsy analysis[END_REF].

Discrimination was also obtained by using the FLIM analysis. The FLIM data are bi-exponential fitted, each component fitted will be associated to one of the form of FAD in tissue, either proteinbound or free. The average amplitude lifetime translates the distribution between free and bound FAD, having longer average lifetime In tumoral tissue means that the free FAD is in higher concentration.

This change in the ratio of protein-bound FAD is a consequence of the impact of the Warburg effect.

In tumor, an increased metabolizing of glucose to lactate by neoplastic tissue compared with healthy tissue [START_REF] Warburg | The metabolism of tumors in the body[END_REF]. Indeed the major co-enzyme in this reaction are NADH and FAD. This is in correlation with previous results shown on healthy bladder mucosa compared to in situ carcinoma [START_REF] Jain | Multiphoton microscopy: a potential intraoperative tool for the detection of carcinoma in situ in human bladder[END_REF].

There are some limitations to discuss. First, we analyzed fixed samples. This could lead to some modifications of the fluorescence and the redox ratio analysis compared to an in-vivo analysis. Indeed, the NADH and FAD concentration decrease after the excision of the tissue and that could affect the performance of redox ratio analysis. Nevertheless, our analysis has been able to discriminate the different tissue as described in the previous literature for the redox ratio in healthy and tumoral tissues [START_REF] Palmer | Detection of urinary bladder cancer cells using redox ratio and double excitation wavelengths autofluorescence[END_REF], [START_REF] Cicchi | Time-and Spectral-resolved two-photon imaging of healthy bladder mucosa and carcinoma in situ[END_REF], [START_REF] Palmer | Optical redox ratio and endogenous porphyrins in the detection of urinary bladder cancer: A patient biopsy analysis[END_REF], [START_REF] Zipfel | Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation[END_REF]- [START_REF] Alhallak | Optical redox ratio identifies metastatic potential-dependent changes in breast cancer cell metabolism[END_REF]. This means that the redox ratio is also a promising tool for an invivo evaluation. The other limitation of our study is the low number of samples included. To confirm our results, we need to increase the number of samples and work on fresh biopsy analysis.

These preliminary results confirm the potential of multimodal optical analysis and represent the first step for the development of a laser fiber optical tool for peri-operative diagnosis. The possibility of discriminating the tumor's grade during the surgery is an essential tool for the oncologic management.

  Wetzlar, Germany) to perform two-photon microscopy. The set-up allows us to record imaging, emission spectra and lifetime fluorescence. The laser cavity had over 2.4 W of average power at 800 nm and was tunable from 690 nm to 1040 nm. Two supersensitive Leica hybrid nondescanned (HyD NDD) detectors recorded the faintest structures from deep-tissue sections. For two-photon imaging experiments, fluorescence was collected after the microscope objective via a dichroic beamsplitter, transparent to wavelengths greater than 680 nm. The collected visible spectrum was split by another dichroic cube (FF495-Di03-25 × 36, Semrock, New York, USA) into two channels toward the HyD NDD detectors. This configuration minimized loss in the fluorescence collection. The presence of two additional filters (FF01-448/20-25 and FF01-520/35-25, Semrock, New York, USA) on the dichroic cube set the detection band, respectively one to collect Second Harmonic Generation(SHG) signal and the other one to collect the endogeneous fluorescence.

  Figures 1.a.b. show the response of endogenous molecules using two different excitation wavelengths. As shown, 810 nm excitation wavelength don'tgive access to the SHG. Exciting with 870 nm could be reasonable to collect the metabolic molecules as well as SHG. Consequently, to avoid experimental bias and propose the fastest method to analyze tissue only the 870nm wavelength was used in all our analysis. We extracted from this fit the maximum intensity at the max emission wavelength of each component (I max-NADH , I max-FAD ) to then calculate a redox ratio, see Figure1.c.

  Figure 1 waveleng intensity

  Normal urothelium (Figure2; 2a,2d) showed multilayered cells without disorganized architecture, the cells were identify in histological images by dark violet spots and in TPEF images by dark hole in the homogeneous red fluorescent signal, highlighting easily the organization of the layers. There was no atypia

Figure 2

 2 Figure 2. images at (d,e,f) of Fibrovasc

  Figure 3 detector.

  Figure 4 normal ur region of including

Figure 5 .

 5 Figure 5. Fluorescence Lifetime analysis at 870nm excitation wavelength. FLIM images of (a) healthy, (b) low and (c)high grade tumor cells, (d) respective average lifetime histogram of each image.(e) Variation of mean amplitude lifetime between healthy urothelium, low grade and high grade tumor compared in fifteen ROIs in each type of tissue.

  , Jain et al. have focused only on the capacity to detect CIS but have not sought to determine papillary tumors characteristics especially the grade. As PDD and NBI are performant tools for the CIS diagnosis, it is more interesting to develop a specific peri-operative tool able to help determine the histologic grade in order to improve oncologic management.

Acknowledgements

This work is supported by PhysiCancer in Plan Cancer with "IMOP" grant, by CNRS with "Défi instrumental" grant, and the Institut National de Physique Nucléaire et de Physique des Particules (IN2P3). This work was done in the PIMPA Platform partly funded by the French program "Investissement d'Avenir" run by the "Agence Nationale pour la Recherche" (grant "Infrastructure d'avenir en Biologie Santé -ANR -11-INBS-0006"). This work is supported by the French association of Urologist (AFU) with a research grant delivered to B.P. The authors declare no competing financial interests.