- DESCRIPTION

THE DETECTION OF BLADDER TUMOR FROM THE BLADDER WASH SAMPLE

(BLADDER INSTILLATION) BY USING FT-IR SPECTROSCOPY

Field of Invention

The present invention relates to a method for the detection of bladder tumor from the bladder wash sample (bladder instillation) by using FT-IR spectroscopy.

Prior Art

Bladder cancer is one of the most common urogenital cancers worldwide. In prior art, two techniques are commonly used for bladder cancer diagnosis: urine cytology and cystoscopy. Among the other methods used in bladder cancer diagnosis, urine markers are also popular. The performance of the methods used in the diagnosis of the disease can be described with the concepts of sensitivity ad specificity. The sensitivity measures the proportion of actual positives, which are correctly identified, e.g., the percentage of sick people who are identified as having the condition; and the specificity measures the proportion of negatives, which are correctly identified, e.g. the percentage of well people who are identified as not having the condition. A theoretical, optimal prediction can achieve 100% sensitivity (i.e., predict all people from the sick group as sick) and 100% specificity (i.e., not predict anyone as a sick from the healthy group).

Urine cytology is a test used to help detect cancer of the urinary tract system, including cancer of the bladder, urethra, ureters and kidneys. It investigates the malignant cells in the bladder wash or urine sediment. Since the adhesion is weak between malignant cells, they generally flow into the urine or into the bladder wash. The investigation of the cancer cells in the urine or in the bladder wash is useful in determination of high grade malignancies and CIS. Although the urine cytology has been a diagnostic tool commonly used in the bladder tumor diagnosis, it has low sensitivity (range 13-75 %) especially in low grade tumors (sensitivity 17 %), with good specificity (range 85-100 %) when all cases were considered. In urine molecular markers, sensitivity and specificity are also low in low grade tumors (sensitivity 45-79 %, specificity 55-87 %) [van Rhijn et al., 2009]. Since the cells of low grade urothelial tumors lack many features associated with malignancy, such as nuclear pleomorphism, coarsely clumped chromatin, and large nucleoli, it is difficult to make diognosis for low grade tumors using urine cytology. Therefore, while positive cytology is a contributory investigation in the diagnosis of bladder tumor, negative cytology does not exclude the possibility of low-grade bladder tumor. In addition, factors such as differences in urine collection, differences in sample processing of laboratories, urinary infection existence, urinary stone existence, intracavitary therapy and long-term catheter usage can affect the reliability of the cytology [Nabi eta/., 2003].

Cystoscopy, on the other hand, is an invasive technique that disturbs patient comfort, which sometimes may lead the patient to discontinue to follow-up. Also, it is relatively expensive [Lotan et al., 2002; Botteman et al., 2003]. In fact, because of the lifelong need for monitoring for recurrence with cystoscopy and for treatment of recurrent tumors, the cost per patient with bladder cancer from diagnosis to death is the highest among all cancers ($96,000 to $187,000 per patient in the United States) [Botteman et al., 2003]. Another problem is that the interpretation of the biopsy collected during cystoscopy can be highly time consuming, subjective and there are inconsistencies between the results of different pathologists and even between the different results of the same pathologist. Also, interpretations of biopsies can be confounded by sampling problems such as the absence of the muscular layer in the specimen, or the exclusion of the bladder wall in biopsies of large tumours growing exophytically that can affect the staging. Most significantly, the basic tools available to determine tumour behaviour, malignant potential and chance for reoccurrence provided by the current pathological staging methods can be highly subjective. A new approach which is automated, not costly, and that does not disturb patient comfort is highly needed.

There are many other different tests used for the diagnosis of the bladder tumor. These techniques are imaging techniques (such as intravenous pyelogram), urine molecular test methods as mentioned before (such as NMP22, BTA) and cell based techniques (such as urovision, quanticyt). The investigation of new tumor markers is an attractive subject in the early bladder tumor detection, follow-up and in the detection of the prognosis. These methods also have some advantages and disadvantages. For example, urine markers are also affected by the environmental factors, such as urinary infections, instrumentation and urinary calculus disease. Moreover, benign inflammatory conditions, such as hematuria and pyuria may give false positive results for BTA stat and NMP22 in more than 25% of cases. Although the combined usage of some tests with urine cytology can be useful, none of the tests is 100% sensitive and specific. Because of the problems faced in the standardization of these tests and the limitations in their productions, their routine usage is still not suitable. Therefore, until a 100% sensitive and specific test is found, none of these tests is accepted as an alternative to the cystoscopy and cytology by the clinicans and pathologists. Therefore, cystoscopy combined with urine cytology still retains its status as the gold standard in the screening and detection of the bladder tumor (Ekici et a/., 2005).

Because of the reasons mentioned above, the results of urine cytology and urine molecules test methods are not 100 percent accurate. For the detection of the bladder tumor urine cytology tests and urine molecular test methods are not adequate alone. Even when cancer cells are detected, a cancer diagnosis must be confirmed by other diagnostic tests, such as urethrocystoscopy and a biopsy. Therefore, other methods which will give better sensitivity and specificity together with other advantages are deeply needed. Fourier transform infrared (FT-IR) spectroscopy is one of those techniques which have been previously applied to bladder cancer issues and tissue cultures [Matysiak et al., 2006]. However, in none of the studies, bladder wash or urine samples have been used.

Fourier transform infrared (FT-IR) spectroscopy is a valuable analytical technique which studies molecular changes rapidly and simultaneously, in various biological samples. It detects the molecular changes from vibration changes in the functional groups of many organic and inorganic compounds. The shifts in peak positions, changes in the bandwidth and area values of the bands give valuable structural and functional information, which may have diagnostic value.

The advantages of FT-IR spectroscopy can be summarized as follows:

■ It can be applied to the analysis of any kind of material. Samples may be solutions, viscous liquids, suspensions, inhomogeneous solids or powders [Haris and Severcan, 1999].

^■ A very small sample size is adequate for the spectral investigations.

■ It is a very precise measurement method, which does not require external calibration. In addition, it is rapid, sensitive, and easy to perform [Manoharan et al., 1993, Ci et al., 1999].

■ It is a non-destructive technique [Severcan et al., 1999; Cakmak et al., 2003].

■ The instrument components are easy to use and data processing is simple with the computer software. In addition, system permits permanent data storage, manipulation of data and quantitative calculations [Yono et al., 1996, Ci et al., 1999]. ■ Digital subtraction (that is, point-by-point subtraction of the separate spectra by a computer) can be used to produce good difference spectra. This method has great advantages in obtaining infrared spectra in aqueous solutions [Campbell, 1984].

■ Small sample quantities are sufficient to analyze and in vivo studies are possible [Mendelsohn et al., 1986].

■ Since a computer is already used to obtain the Fourier transform, it is easy to perform many scans to improve the signal-to-noise ratio (noise adds up as the square root of the number of scans, whereas signal adds linearly). The signal to noise ratio has been dramatically improved by the averaging of numbers of scans per sample [Beaten et al. _f 1998].

■ Kinetic and time-resolved studies are possible [Mendelson et al., 1986; Severcan et al. _f 1999].

■ Frequency and bandwidth values can be determined routinely with uncertainties of better than ±0.05 cm ^"1 .

■ The price of the FT-IR spectrometer is considerably reasonable.

- FT-IR spectroscopy has been widely used in the molecular investigation and diagnosis of many diseases ranging from diabetes and malignancy to infectious diseases (Rigas et al., 1990, Fung et al., 1996; Boyar et al., 2003; Severcan et al., 2005; Toyran et al., 2006; Dogan et al., 2007; ]. In different cancer types, different tissue types have been used and the changes between the healthy and cancerous tissues have been investigated at molecular level [Andrus et al., 1998; Sahu et al., 2004; Li et al., 2005].

Especially in cancer, early diagnosis is very important. Rapid detection of the tumors significantly reduces mortality and the cost associated with the treatment. Most commonly used diagnosis method in hospitals is based on the investigation of the tissue samples (biopsies) by a pathologist. Spectroscopic techniques not only can detect [Li et al., 2005] the tumors but also classify [Andrus et al., 1998] tumors rapidly, with the use of a very small sample size. FT-IR can detect changes towards cancer at the molecular level before it can be seen In pathology. This technique has been previously used for early diagnosis of diabetes [Toyran et al. 2006].

Although FT-IR spectroscopy has several applications in cancer diagnosis, its application to bladder cancer is very limited. In the previous FT-IR studies of bladder cancer, bladder tissues and tissue cultures were investigated [Matysiak et al., 2006]. However, in none of the studies, bladder wash or urine samples have been used. In prior art, CA 2 616 277 Al and WO 2006 012522 Al numbered documents concern the BTM and UBTM proteins, which are urine markers. These documents do not include any methods which uses bladder wash sample or urine sediment.

JP 1 016 80 98 A numbered document includes an investigation of cancer and degree of invasive from the DNA of TCC cancer cell by investigating a new protein or peptide.

In prior art, US 5 541 076 A and WO 9 503 547 Al numbered documents concern the detection of bladder tumor from the bladder wash sample by investigating the basement membrane components and their polypeptide components.

The document US 6 350 571 Bl, relates to a novel methods for detecting and evaluating bladder cancer by detecting hyaluronic acid (HA) and hyluronidase (HAase) in bladder sample.

In prior art, US 6 521 409 Bl, US 6 630 301 Bl, US 6 939 675 B2, US 7 288 380 Bl, US 7 387 874 B2, US 7 399 592 B2 and WO 9 734 015 Al numbered documents include the investigation of extracellular tumor nucleic acids in plasma and serum fractions. These documents do not include any detection method in bladder samples.

Summary of the Invention

Briefly stated, the present invention provides a novel method for diagnosis of bladder cancer, at low and high grade, from the bladder wash with higher sensitivity and specificity together with other advantages that the other methods provide. To achieve this, Fourier transform infrared (FT-IR) spectroscopy together with cluster analysis based on spectral variations were employed for the first time, for the rapid and sensitive diagnosis of bladder cancer.

Furthermore, although the information revealed is not necessary for the routine application of FT-IR spectroscopy into the bladder cancer diagnosis, the results of the detail FT-IR analysis of the control and diseased samples and the significant values were presented to give better idea about the disease-induced variations.

Aims of the Invention

In developing of the method for the detection of bladder tumor from the bladder wash sample (bladder instillation) by using FT-IR spectroscopy, it is aimed to; • Develop a method^ which is able to detect tumors with a higher sensitivity and specifity, even in low grade tumors,

• Shorten time for diagnosis,

• Early detection, as a result early therapy of the tumors,

• Automation in the analysis of sample and diagnose,

• Bringing objectivity and standardization on the grading of the bladder tumor,

• Prevention of the invasive tools.

Description of the Drawings

Figure - 1: Normalized Average FT-IR Spectra Belonging to the Control, Papilloma, Neoplasy and Carcinoma in 3795-2996 cm ^"1 Region. (The spectra were normalized with respect to the amide A band located at 3282 cm ^"1 ).

Figure - 2: Normalized Average FT-IR Spectra Belonging to the Control, Papilloma, Neoplasy and Carcinoma in 2999-2800 cm ¹ Region. (The spectra were normalized with respect to the CH ₂ asymmetric band located at 2936 cm ^"1 ).

Figure - 3: Normalized Average FT-IR Spectra Belonging to the Control, Papilloma, Neoplasy and Carcinoma in 1800-400 cm ^"1 Region. (The spectra were normalized with respect to the PO ^"2 symmetric stretching band located at 1086 cm ^"1 ).

Figure - 4: Hierarchical cluster analysis performed on the FT-IR spectra of control (n=34) and diseased samples containing all tumor types (n=37), showing the differentiation of all tumor types from the control in the spectral region of 2954- 2979 cm ^"1 .

Figure - 5: Hierarchical cluster analysis performed on the FT-IR spectra of control (n=34) and diseased samples containing all tumor types (n=37), showing the differentiation of all tumor types from the control in the spectral region of 2907- 2923 cm ^"1 .

Figure - 6: Hierarchical cluster analysis performed on the FT-IR spectra of control (n=34) and diseased samples containing all tumor types (n=37) showing the differentiation of all tumor types from the control in the spectral region of 1444- 1457 cm ^"1 . Figure - 7: Hierarchical cluster analysis performed on the FT-IR spectra of control (n=34) and diseased samples containing all tumor types (n=37) showing the differentiation of all tumor types from the control in the spectral region of 1015- 1033 cm ^"1 .

Figure - 8: Hierarchical cluster analysis performed on the FT-IR spectra of control (n =34) and diseased samples containing all tumor types (n=37) showing the differentiation of all tumor types from the control in the spectral region of 637- 649 cm ¹ .

Figure - 9: Hierarchical cluster analysis performed on the FT-IR spectra of control (n=34) and diseased samples containing all tumor types (n=37) showing the differentiation of all tumor types from the control in the spectral region of 625- 637 cm ^"1 .

Detailed Description of the Invention

In present invention, Totally 71 individuals who were diagnosed with bladder tumor are recruited to this study, of whom 4 were women (5.6%) and 67 (94.4%) were men. In some of the patients, the tumor has relapsed and caused some cellular changes in different levels, and in some patients no tumor relapsation has occurred. Based on these relapsations which are diagnosed by pathologic observations, patients were divided to four groups as:

• Control group (n=34) which has normal bladder epithelial tissue;

• Papilloma group (n=9) which has uretelial papilloma ;

• Neoplasy group (n=6) which has uretelial neoplasm with lower malignancy potential;

• Carcinoma group (n=22) which has low and high grade papillary uretelial carcinoma.

The groups were formed according to the histopathology results.

In present invention, the bladder wash sample is used in the diagnosis of the bladder tumor by using FT-IR spectroscopy.

In bladder wash, or in other words bladder instillation procedure, as it is known in the prior art, the bladder is filled with a sterile solution containing one of a number of ingredients that work directly on the bladder wall. The solution is put into the bladder through a catheter (hollow tube) that is placed through the urethra. When the bladder of the person is emptied, the sample which is taken from the person is called as bladder wash sample. This sample is very close to the urine of that person. In the evaluation of hematuria the diagnostic method for bladder cancer in the present technique is invasive cystoscopy. After the diagnosis and treatment the follow up schedule is cystoscopy because of high recurrence rate.

During cystoscopy in present invention, bladder tissue is observed with the camera; tissue biopsy is taken from the suspected area and sent to the pathologist. Also the bladder wash sample of 5 - 15 ml is collected for cytology. This sample is centrifuged for 30 minutes at 10,000 rpm. The supernatant is removed and the pellet is used for urine cytology examination. The pellet left is stored at -80 °C until FT-IR analysis.

The biopsy and urine cytology results are obtained from the pathologist and compared with FT-IR results.

For FT-IR studies in present invention, the well-known routine pellet preparation method is applied. The frozen samples stored at -80° C, are dried in the lyophilizer. The samples are then ground in an agate mortar in order to obtain bladder wash powder. 0.4 mg of powder is mixed with 100 mg potassium bromide KBr. In present invention, the sample to KBr ratio is 1:250. However in sample preparation procedure other ratio values can also be used as long as the absorbance is kept as not very high. The preferred absorbance value is below 1. The mixture is then subjected to a pressure of 100 kg/cm ² (1200 psi) for 6 minutes in an evacuated die to produce a KBr pellet to be used in FT-IR spectrometer. The spectra are collected in mid-infrared region. The cluster analysis method that is based on the spectral variations is applied to the spectra in different regions, in order to discriminate control and diseased groups.

In present invention, Cluster analysis classifies objects, via a tree diagram calculated using the Ward's algorithm. Constructed with the OPUS 5.5 software, the dendrogram graphically represents the cluster analysis groups. For cluster analysis, second derivatives of the spectra are calculated and subsequently vector normalized over the investigated frequency range. Cluster analysis is applied to distinguish between the spectra of control and diseased groups, such as Carcinoma, Neoplasy and Papilloma.

As seen from Figures 1 - 3, the control and tumorous samples show different FT-IR spectra. Based on these spectral variations, hierarchical cluster analyses are performed in order to control and bladder tumors (even in low grade tumors like neoplasy and papilloma). Hierarchial clustering of control and diseased samples using second derivative spectra in the spectral ranges of 2954-2979 cm ^"1 / 2907-2923 cm ^"1 , 1444-1457 cm ^"1 , 1015-1033 cm ^"1 , 637- 649 cm ^"1 and 625-637 cm ^"1 give best sensitivity values. As a result, an overall sensitivity (including all individuals with tumor) of 91.8% in spectral range of 1444-1457 cm ^"1 are reached, while an overall sensitivity of 83.8% in spectral range of 2954-2979 cm ^"1 and 2907- 2923 cm ^"1 , 75.7% in spectral ranges of 637-639 cm ^"1 and 625-637 cm ^"1 , and 72.9% in spectral region of 1015-1033 cm ^"1 are obtained.

The clustering of control and neoplasy samples or control and papilloma samples are not performed separately, since the sample size in neoplasy and papilloma groups are insufficient. Therefore, all these disease groups are considered as one disease group, and sensitivity and specificity values are calculated accordingly. However, even with the small sample size in low grade tumors, especially in the spectral ranges of 1444-1457 cm ^"1 , 2954- 2979 cm ^'1 and 2907-2923 cm ^"1 , the sensitivities for papilloma group are obtained as 77.8%, 66.7% and 66.7% respectively, which clearly indicates the superiority of FTIR spectroscopy in bladder cancer diagnosis.

In the study, urine cytology has a sensitivity of only 11.1%. The sensitivity for neoplasy group is 83.3% in these three spectral ranges while the urine cytology has a sensitivity of only 16.7

"Sensitivity and specificity values of the results of cluster analysis in the spectral ranges of interest" and "Sensitivity values of the results of cytology for the samples used in the present study" are given in tables below:

Table 1A: Sensitivities and specifities of the spectral ranges used for clustering Control Papilloma Neoplasy Carcinoma

Sensitivity (%) 100 11.1 16.7 45.5

Table IB: Sensitivity values of the results of cytology for the samples used present invention

In summary as can be seen from Table 1A, with the help of the method discussed in the present invention, higher sensitivity is achieved, even for low grade tumors. Especially the band located at 1444-1457 cm ^"1 , gave the best differentiation. Using this band, the sensitivity is obtained as 100% for carcinoma, 83.3% for Neoplasy and 77.8 % for Papilloma with overall sensitivity as 91.8%.

With the help of the present invention, the bladder tumor is diagnosed with high sensitivity and specificity and prevention of invasive tools is achieved on the diagnosis and follow up bladder tumors. Higher sensitivity and specificity is one of the advantages of the present invention provides higher detection success for bladder tumor. In addition, early detection of the low grade tumors may lead to early therapy of the tumor so higher success rate.

Other than the sensitivity advantage of our method over cytology, the present invention gives the results in much shorter time and it is much less cost-effective compared to cytology. In addition, one of the important advantages of the technique is that, the results are not dependent on the subjective observation of a pathologist, but to computational analysis of the spectra. In other words it is operator independent. Furthermore, the spectral changes may give information about the tumoral changes long before they become visible. So this information may be very important in early diagnosis.

Supplementary Tables and Figures:

Although it is not necessary for routine application of FT-IR spectroscopy in medical diagnosis, in order to prove the spectral changes obtained from the FT-IR analysis are significant, the general band assignments of bladder wash samples, the numerical summary of the detailed differences in the band frequencies of control and carcinoma groups spectra, the numerical summary of the detailed differences in the band areas of control and carcinoma groups spectra, the numerical summary of the significant differences in the band frequencies and areas of control and neoplasy groups spectra and the numerical summary of the significant differences in the band frequencies and areas of control and papilloma groups spectra are given in tables and figures in this section as supplementary materials.

Table 2: General band assignments of bladder wash samples

Table 3: Numerical summary of the detailed differences in the band frequencies of control and carcinoma groups spectra. The values are the mean ± standard deviation for each sample

C-N antisymmetric p<0.001**

1462.04±1.62 1462.77±0.51

stretching *

6

CH ₂ bending 1452.54±0.48 1453.04±0.62 p<0.01**

P0 ₂ ^" symmetric

7 1086.71± 1.30 1087.69±0.87 p<0.05* stretching

p<0.001**

8 C-0 stretching 1052.06±1.85 1053.62±0.33

p<0.001**

9 C-0 bending 1022.62±1.46 1024.64±0.99 *

p<0.05*

10 C-C stretching 967.22±0.33 967.40±0.31

(at limit) p<0.001**

11 C-O-C ring 930.92±1.32 932.30±0.39 *

Table 4: Numerical summary of the detailed differences in the band areas of control and carcinoma groups spectra. The values are the mean ± standard deviation for each sample

Area

Control Carcinoma

Band p values

(n=34) (n=22)

1 N-H and O-H stretching 238.12±33.66 263.81±45.60 p<0.05*

CH ₃ antisymmetric

2 2.77±0.38 3.06±0.48 p<0.05* stretching

CH ₂ antisymmetric

3 5.74±1.03 6.86±1.22 p<0.01** stretching

4 CH ₃ symmetric stretching 7.04±1.35 8.27±1.22 p<0.01**

5 Amide I (C=0 stretching) 10.10±6.38 5.35±2.75 p<0.05*

C-N antisymmetric

6 stretching and CH ₂ 18.14±1.84 20.04±2.18 p<0.01** bending

P0 ₂ ^" symmetric

7 19.09±2.68 20.93±3.06 p<0.05* stretching p<0.001**

8 C-0 stretching 2.08±0.30 2.52±0.42 9 C-0 bending 12.51±1.90 14.18±2.22 p<0.05*

10 C-C stretching 0.44±0.26 0.67±0.16 p<0.01**

11 C-O-C ring 3.55±0.77 4.28±0.72 p<0.01**

Table 5: Numerical summary of the significant differences in the band frequencies and areas of control and neoplasy groups spectra. The values are the mean ± standard deviation for each sample

Table 6: Numerical summary of the significant differences in the band frequencies and areas of control and papilloma groups spectra. The values are the mean ± standart deviation for each sample CH ₃ symmetric ^{^}

4 2877.38±6.22 2872.96±6.03 p<0.05* stretching

5 C=0 stretching 1664.85±1.01 1663.09±2.50 p<0.05*

6 CH ₂ bending : 1452.54±0.48 1453.13±0.51 p<0.05*

8 C-0 stretching 1052.06±1.85 1052.95±1.77 p<0.05*

9 C-0 bending 1022.62±1.46 1024.02±1.30 p<0.05*

11 C-O-C ring 930.92±1.32 931.82±1.12 p<0.05*

Area

Band Control(n=34) Papilloma (n=9) p values

N-H and O-H p<0.05*

1 238.12±33.66 272.58±45.87

stretching (at limit)

CH ₃ antisymmetric

2 2.77±0.38 3.21±0.58 p<0.05* stretching

C-N antisymmetric

6 stretching and CH ₂ 18.14±1.84 20.67±2.64 p<0.05* bending

P0 ₂ ^" symmetric

7 19.09±2.68 22.22±3.28 p<0.05* stretching

8 C-0 stretching 2.08±0.30 2.61±0.64 p<0.05*

9 C-0 bending 12.51±1.90 14.80±2.38 . . p<0.05*

The bands were identified and assigned for each group according to the literature. The results were expressed as mean±standard deviation. For the statistical comparison of control vs carcinoma group, neoplasy group and papilloma group, Mann-Whitney U test is used. A p value of less than 0.05 was considered significant (p<0.05*, p<0.01**, and p<0.001***).

Figure - 10: The representative infrared spectra of bladder wash sample of the control group in 4000-400 cm ^"1 region.

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