Volatile organic compounds as markers for pancreatic cancer diagnosis and prognosis

Abstract

The present invention relates to the use of volatile organic compounds (VOCs) present in an individual's blood serum as diagnostic and / or prognostic biomarkers of pancreatic cancer. The invention also relates to a method for identifying VOCs in blood samples as diagnostic and prognostic biomarkers of pancreatic cancer. Lastly, the invention further relates to pancreatic cancer diagnosis and prognosis methods based on the detection of the markers in blood serum samples, and to kits and devices for carrying out the methods.

Description

[0001] DESCRIPTION

[0002] Volatile organic compounds as markers for the diagnosis and prognosis of pancreatic cancer

[0003] Field of the invention

[0004] The present invention belongs to the technical field of medicine, in particular it belongs to the technical field of oncology and, more particularly, it relates to volatile organic compounds suitable for the early detection and monitoring of pancreatic tumors.

[0005] Background of the invention

[0006] Pancreatic adenocarcinoma is one of the most lethal cancers today, with very high incidence rates and very worrying survival rates. The latest clinical data show that this cancer ranks 12th in terms of global incidence, but 8th in terms of mortality (World Health Organization. Global cancer incidence and mortality, https: / / www.iarc.who.int / 2023.4-4-2023). Furthermore, it is estimated that there will be a worrying increase in incidence (+77.7%) and mortality (+79.9%) from 2018 to 2040 (Rawla P. et al. (2019) Epidemiology of Pancreatic Cancer: Global Trends, Etiology and Risk Factors. World J Oncol 10:10-27). There are clear risk factors associated with this tumor, some of which are modifiable (tobacco, alcohol, diet, obesity, etc.) while others are non-modifiable (age, sex, blood type, or family history, among others) (McGuigan A.et al (2018) Pancreatic cancer: A review of clinical diagnosis, epidemiology, treatment and outcomes. World J Gastroenterol 24:4846-4861). The location of the pancreas and the lack of symptoms in the early stages of the cancerous process mean that this type of tumor is generally discovered in late stages, preventing surgical treatment and contributing to the low 5-year survival rate, which is around 10% (American Cancer Society. Survival in pancreatic cancer. https / / www.cancer.org / . 2023. 4-4-2023). Currently, biopsy is the only method for the unequivocal diagnosis of this cancer in conjunction with imaging techniques (CT, PET), but there is no early diagnostic method that can be applied to perform population screening similar to those performed for colon or breast cancer (Mottram R. et al (2021) Factors associated with attendance at screening for breast cancer: a systematic review and meta-analysis. BMJ Open 11:e046660).Several biomarkers for pancreatic cancer have been described, such as blood levels of the antigen 19-9 (CA 19-9) or carcinoembryonic antigen (CEA). However, these markers lack sufficient specificity and sensitivity to be useful as a diagnostic marker and are generally used for patient follow-up once diagnosed (Zhao, B.; et al., Diagnostic value of serum carbohydrate antigen 19-9 in pancreatic cancer: a systematic review and meta-analysis. European Journal of Gastroenterology & Hepatology 2022, 34). Therefore, there is a great need to identify suitable biomarkers for the early detection of pancreatic cancer and their application in screening campaigns.

[0007] Volatile organic compounds (VOCs) are the end products of metabolism and can be detected in blood, exhaled air, urine, and other body fluids by gas chromatography (Makos P. et al. (2019) Methods of assaying volatile oxygenated organic compounds in effluent samples by gas chromatography-A review. J Chromatogr A 1592:143-160). The untargeted analysis of these VOCs is known as volatolomics and is a technique that allows the identification of fluctuations in these low molecular weight compounds due to various diseases and other internal or external changes (Rondanelli M. et al. (2019) Volatile Organic Compounds as Biomarkers of Gastrointestinal Diseases and Nutritional Status. J Anal Methods Chem 2019:7247802). It has been proven that many diseases such as cystic fibrosis (Kramer R. et al (2015) A rapid method for breath analysis in cystic fibrosis patients.Eur J Clin Microbiol Infect Dis 34:745- 751), el cáncer (Schmidt K, Podmore I (2015) Solid phase microextraction (SPME) method development in analysis of volatile organic compounds (VOCS) as potential biomarkers of cancer. J Mol Biomark Diagn 6:6), la diabetes (Das S. et al (2016) Significance of Exhaled Breath Test in Clinical Diagnosis: A Special Focus on the Detection of Diabetes Mellitus. J Med Biol Eng 36:605-624), la tuberculosis (Zetola NM. et al (2017) Diagnosis of pulmonary tuberculosis and assessment of treatment response through analyses of volatile compound patterns in exhaled breath samples. J Infect 74:367-376), el síndrome de colon irritable (Ahmed I. et al (2013) An investigation of fecal volatile organic metabolites in irritable bowel syndrome. PLoS One 8:e58204), o las enfermedades infecciosas (Sethi S. et al (2013) Clinical application of volatile organic compound analysis for detecting infectious diseases.Clin Microbiol Rev 26:462-475) can be monitored through changes in VOC levels in patient fluids. Previous studies have attempted to identify diagnostic compounds in pancreatic cancer. For example, Kobayashi et al. found several metabolites that changed significantly in pancreatic cancer patients (Kobayashi T, et al (2013) A novel serum metabolomics-based diagnostic approach to pancreatic cancer Cancer Epidemiol Biomarkers Prev 22:571-579); Urayama et al. described a number of amino acids, bile acids and polar lipids whose levels were altered in the plasma of pancreatic cancer patients (Urayama S. et al (2010) Comprehensive mass spectrometry based metabolic profiling of blood plasma reveals potent discriminatory classifiers of pancreatic cancer Rapid Commun Mass Spectrom 24:613-620) Markar et al.found 10 VOCs in exhaled air that were modified in these patients (Markar SR et al (2018) Profile of exhaled-breath volatile organic compounds to diagnose pancreatic cancer Br J Surg 105:1493-1500)-, Princivalle et al. found another 10 VOCs associated with pancreatic cancer in expired air (Princivalle A, et al (2018) Pancreatic ductal adenocarcinoma can be detected by analysis of volatile organic compounds (VOCs) in alveolar air. BMC Cancer 18:529). The volatolome of the urine of these patients and their controls has also been studied. For example, Nissinen et al. They managed to distinguish patients from their controls with a sensitivity of 79% and a specificity of 79% (Nissinen SI et al (2019) Detection of Pancreatic Cancer by Urine Volatile Organic Compound Analysis.Anticancer Res 39:73-79); Arasaradnam et al used a similar technique and obtained a sensitivity of 91% and a specificity of 83% (Arasaradnam RP, et al (2018) Noninvasive Diagnosis of Pancreatic Cancer Through Detection of Volatile Organic Compounds in Urine. Gastroenterology 154:485-487); finally, Daulton et al. achieved a sensitivity of 72% and a specificity of 96% in urine (Daulton E et al (2021) Volatile organic compounds (VOCs) for the non-invasive detection of pancreatic cancer from urine. Taianta 221:121604).

[0008] However, volatolome has not been investigated in the blood serum of patients with pancreatic cancer so far.

[0009] Blood serum has the advantage of being easily obtained from routine blood draws and is easy to preserve. Furthermore, numerous specimen banks hold large collections of serum, greatly facilitating the development and monitoring of potential biomarkers identified with this methodology.

[0010] Following significant research work, the authors of the present invention have demonstrated for the first time that some of the VOCs present in the blood serum of patients with pancreatic cancer have diagnostic and prognostic biomarker characteristics with a high degree of sensitivity and specificity (AUC > 0.98).

[0011] Furthermore, the exact chemical composition of some of these VOCs has been determined, thus facilitating their industrial development as the basis for widely used diagnostic and prognostic devices. Early detection of pancreatic cancer, especially among the at-risk population, can significantly increase the survival rate of these patients.

[0012] Description of the figures

[0013] Figure 1 shows an example of a chromatogram of a human serum sample, showing distinct peaks corresponding to VOCs along with the two internal markers (IS1 and IS2). Some of the VOCs are identified by their codes.

[0014] Figure 2 shows a principal components diagram (PCA) that perfectly separates healthy controls (triangles) from cancer patients (circles).

[0015] Figure 3 shows the ROO curves and expression diagrams for two of the VOCs with the greatest discriminatory capacity: V197 (A) and V328 (B).

[0016] Figure 4 shows the coincidences between the mass spectra obtained with the commercial reagents (upper graphs) and the VOCs identified in the study (lower graphs) for four of the identified compounds: butoxymethylbenzene (V328, A); toluene (V40, B); 2-ethyl-1-hexanol (V172, C) and pentylbenzene (V274, D). Description of the invention

[0017] In order to resolve needs and inconveniences existing in the state of the art in relation to the detection of pancreatic cancer and, specifically, in the absence of studies on the volatolome of blood serum in these patients, the present invention shows for the first time the diagnostic and prognostic capacity of VOCs present in blood serum for use as biomarkers for the early detection and monitoring of this disease.

[0018] Thus, one object of the present invention is to be able to detect pancreatic cancer in blood serum samples from patients using VOCs, present in said samples, as diagnostic and prognostic biomarkers.

[0019] To achieve this objective, the present inventors conducted a case / control study in which the VOC pattern was measured in serum. Specifically, a total of 433 VOCs were identified in the human serum samples. Of these, 125 VOCs showed significant variations (p<0.05) when comparing their levels in cases and controls (diagnostic biomarkers). Bonferroni statistical correction determined that there were 40 VOCs whose p value remained less than 0.05 after correction. A principal components analysis (PCA) showed a high discriminatory power between cases and controls. A partial least squares regression model (PLS-DA) was performed to select the best VOCs. For the 40 selected VOCs, the area under the curve (AUC) was between 0.98 and 0.85.

[0020] When the validation cohort (5 patients, 5 controls) was used, the discriminatory utility of 11 previously identified VOCs could be confirmed.

[0021] When analyzing the predictive value (discriminating between patients who survived more than one year from diagnosis and those who did not), 16 VOCs were found to have discriminatory capacity (p < 0.05). In ROC analyses, three of these VOCs had an AUC > 0.87 (AUC = 0.93 for V109).

[0022] The exact chemical nature of four of the biomarker candidates was identified.

[0023] These were toluene, 2-ethyl-1-hexanol, pentylbenzene, and butoxymethylbenzene. All of these compounds are capable of classifying patients with an AUC > 0.90. Notably, butoxymethylbenzene has an AUC = 0.98.

[0024] Based on the above, a first aspect of the present invention relates to the use of VOCs present in the blood serum of an individual as diagnostic and / or prognostic biomarkers of pancreatic cancer.

[0025] For the purposes of this invention, "diagnostic biomarkers" are defined as VOCs capable of distinguishing individuals with pancreatic cancer from those without; and "prognostic biomarkers" are defined as VOCs capable of distinguishing individuals who will survive more than one year after diagnosis.

[0026] In a particular embodiment, the VOCs are selected from butoxymethylbenzene, toluene, 2-ethyl-1-hexanol, pentylbenzene and their combinations as diagnostic biomarkers for pancreatic cancer.

[0027] In a second aspect of the present invention, a method is contemplated for identifying a set of VOCs indicative of pancreatic cancer (hereinafter, method for identifying VOCs), comprising the steps of: collecting a blood serum sample from a patient with pancreatic cancer; determining the levels of VOCs in said sample; comparing the levels of VOCs in the blood serum sample from the patient with pancreatic cancer with the levels of VOCs in a control sample; and identifying those VOCs that have significantly different levels in the blood serum sample from the cancer patient compared to the control sample, thereby determining a set of VOCs indicative of pancreatic cancer.

[0028] In a particular embodiment, the set of VOCs indicative of pancreatic cancer comprises at least one of butoxymethylbenzene, toluene, 2-ethyl-1-hexanol and pentylbenzene.

[0029] In a third aspect of the invention, a method of diagnosing and / or prognosing pancreatic cancer in an individual is contemplated, comprising: i) collecting a blood serum sample from an individual;

[0030] (i) determining the levels of at least one VOC from a set of VOCs indicative of pancreatic cancer in the sample from the individual, wherein the set of VOCs indicative of pancreatic cancer are identified according to the method for identifying VOCs described above; and (iii) comparing the levels of the at least one VOC in the sample from the individual with the levels of said at least one VOC in a control sample, where a statistically significant difference (p<0.05) in the levels of said at least one VOC in the sample from the individual, compared to the levels of said compound in the control sample, is indicative of pancreatic cancer.

[0031] For the purposes of the present invention, the individual is defined as a subject who is at risk of developing pancreatic cancer, a subject who is suspected of having pancreatic cancer, or a subject who is suffering from pancreatic cancer.

[0032] Step i) for determining the levels of volatile organic compounds in a blood serum sample can be carried out using techniques available in the state of the art such as gas chromatography, high-performance liquid chromatography (HPLC), ultra-performance liquid chromatography (UPLC), mass spectrometry or infrared spectrometry.

[0033] In a particular embodiment, the VOC levels in the blood serum sample are measured in (i) by gas chromatography. The VOC levels measured by gas chromatography are determined by measuring the area delimited by each peak of the chromatogram and the baseline.

[0034] In a preferred embodiment, step ¡i) comprises determining in the sample obtained in i) the levels of at least one VOC selected from butoxymethylbenzene, toluene, 2-ethyl-1-hexanol, pentylbenzene and combinations thereof, where a statistically significant decrease (p<0.05) in the levels of at least one of these VOCs in the sample of the individual, compared to the levels of said at least one VOC in a control sample, allows diagnosing pancreatic cancer.

[0035] In particular, chromatographic peak area values ​​lower than 80.9 in the case of toluene, 3.205 in the case of 2-ethyl-1-hexanol, 0.0228 in the case of pentylbenzene and 0.00351 in the case of butoxymethylbenzene are indicative of pancreatic cancer.

[0036] In a fourth aspect, a panel of biomarkers for the diagnosis of pancreatic cancer is contemplated that comprises at least one VOC selected from butoxymethylbenzene, toluene, 2-ethyl-1-hexanol, and pentylbenzene present in blood serum.

[0037] Finally, in another main aspect of the invention, a kit for the in vitro diagnosis and / or prognosis of pancreatic cancer is contemplated, comprising the means necessary to quantify in a blood serum sample the levels of at least one VOC from the set of VOCs identified according to the method for identifying VOCs described above, adapted to implement the diagnostic and / or prognostic method of the invention.

[0038] In particular, the kit comprises the means necessary to quantify in a blood serum sample of an individual the levels of at least one VOC selected from butoxymethylbenzene, toluene, 2-ethyl-1-hexanol, pentylbenzene and their combinations, adapted to implement the diagnostic method described.

[0039] In a particular embodiment, the kit comprises a fixed or portable measuring device for the levels of at least one VOC selected from butoxymethylbenzene, toluene, 2-ethyl-1-hexanol, pentylbenzene, and combinations thereof, for use in hospitals, medical offices, pharmacies, or at the patient's home. The device may contain a sensor (including solid-state sensors) capable of detecting and quantifying the described biomarkers.

[0040] EXAMPLES

[0041] Below are some specific examples of embodiments of the invention that serve to illustrate the invention.

[0042] 1. Study design and sample collection

[0043] The study was designed as a case-to-case comparison (pancreatic cancer patients / healthy controls). Approval was initially obtained from the La Rioja Drug Research Ethics Committee (CEImLar), and all subjects signed an informed consent form. Twenty patients diagnosed with pancreatic cancer (65% men, age = 65.2 ± 9.0 years) and 19 healthy volunteers with the same sex and age distribution (63% men, age = 63.4 ± 7.7 years) were initially recruited (Table 1). A second group of patients (n = 5) and healthy volunteers (n = 5) was subsequently recruited as a validation cohort. All blood samples were taken at San Pedro Hospital in Logroño. Blood was collected in red-capped tubes (for serum separation) and, after centrifugation at 3,000 xg for 10 min at 4°C, the resulting serum was aliquoted into 1 mL samples that were stored at -80°C until use.Ultrapure water extracted into the same tubes and stored in the same manner was used as a negative control to eliminate volatile compounds caused by the storage system.

[0044] Table 1. Sex and age distribution of the study subjects. Statistical tests used: Chi square (*) or Student's t test (t).

[0045] 2. Volatolomic analysis

[0046] Serum samples were analyzed using a Vahan CP-3800 gas chromatograph coupled to a Saturn 2200 ion trap mass spectrometer, equipped with a CombiPal autosampler CTC loading system (Agilent). Zebron ZB-35 GC columns (30 m, 0.25 mm ID, Phenomenex) were used with helium as the carrier gas at a flow rate of 1.0 mL / min.

[0047] Each serum sample was thawed at 4°C and homogenized. 250 μl were separated into a 20 mL vial, to which 25 μl (1 μg / mL) of D10-ethylenebenzene and D4-1,4-dichlorobenzene (Merck) were added as internal standards. Each sample was incubated for 1 min at 50°C, and volatile elements were transferred to a CAR / PDMS / DVB fiber (Supelco) for 20 min at 50°C. Desorption was carried out for 30 s at 250°C directly in the injection port. After each use, the fibers were regenerated by treatment at 270°C for 15 min. The VOC separation program began with exposure to 50°C for 15 min, then the temperature was ramped 5°C every min to 100°C, held at 100°C for 1 min, and then ramped to 250°C at 25°C / min for another 2 min. The total analysis time per sample was 36 min. VOC signals were analyzed in full-scan mode between 40 and 300 m / z.Three technical replicates were made of each sample.

[0048] 3. Data collection and statistical analysis

[0049] MassHunter software (Agilent) was used to process the chromatograms. A total of 433 components (V1–V433) were identified in the human serum samples (Figure 1). For each VOC, the most abundant signal was selected as the quantitative ion, and the area under the curve was integrated into the chromatogram. A table was constructed with all the data, including the mean of the three replicates for each VOC. The area of ​​each peak was normalized with the internal standards in each sample. For the univariate study, the normality (Shapiro-Wilk test) and homoscedasticity (F test) of the case and control groups were checked for each VOC. If the samples followed a normal distribution, a two-tailed Student t test (or a Mann-Whitney U test in the case of non-normal samples) was used to study the difference between areas.The initial p values ​​were corrected with the Bonferroni multiple comparisons method, obtaining 40 VOCs that maintained their statistical significance less than 0.05 (Table 2).

[0050] Table 2. Chromatographic and validation parameters for the 40 VOCs selected for their discriminatory capacity. Signals were normalized by the relative peak area of ​​D4-1,4-dichlorobenzene as an internal control. Repeatability and intermediate precision values ​​were calculated by ANOVA from three replicates of a control group, performed on three consecutive days.

[0051] To identify predictive biomarkers, VOCs were selected that allowed us to separate patients who survived more than one year after diagnosis from those who did not. Sixteen VOCs were found to have statistically significant predictive capacity (Table 3). When analyzing the ROC curves for these metabolites, three of them had AUC > 0.86 (Table 4).

[0052] Table 3. List of VOCs that showed different behavior when comparing patients who survived more than one year with those who did not. Normalized mean values, fold change (FC), and p values ​​after performing a Student t test are shown.

[0053] Table 4. ROC analysis for those VOCs that showed discriminatory ability as prognostic biomarkers (survivors vs. non-survivors at one year). The AUC, cutoff, sensitivity, and specificity for each are shown.

[0054] Before conducting the multivariate analysis, the data were log-transformed, centered around the mean, and divided by the standard deviation of each variable for scaling. Unsupervised principal component analysis (PCA) was performed to identify potential outliers and to assess the discriminatory power of the VOC profile.

[0055] To identify the most significant VOCs, a partial least squares regression model (PLS-DA, Figure 2) was run, receiver operating characteristic (ROC) plots were constructed, and their area under the curve (AUC) was calculated (Figure 3). The predictive accuracy of potential biomarkers was assessed using permutation tests, including 1000 permutations per analysis.

[0056] When the validation cohort was studied, the predictive value was confirmed for 23 VOCs, among which are the compounds with the highest discriminant value, V197 and V328 (Table 5).

[0057] Table 5. Results of the validation group. The number of false positives and negatives in the new cohort is indicated. The p value is also included.

[0058] 4) VOC Identification For those VOCs with potential as biomarkers, a tentative identification was initially made with the MassHunter program trying to relate the exact mass spectrum with the NIST17 library. When commercially available compounds existed for the candidates, these were purchased and their retention time on the column and their mass spectrum were compared with those obtained from human samples (Figure 4).

Claims

MODIFIED CLAIMS received by the International Bureau on 20 December 2024 (20.12.2024) 1. A method for diagnosing pancreatic cancer in an individual comprising: i) determining in vitro, in a blood serum sample from an individual, the levels of at least one VOC selected from butoxymethylbenzene, 2-ethyl-1-hexanol, pentylbenzene and combinations thereof; and (i) comparing the levels determined in (i) of the at least one VOC in the sample from the individual with the levels of said at least one VOC in a control sample, where a statistically significant decrease in the levels of said at least one VOC in the sample from the individual compared to the levels of said compound in the control sample is indicative of pancreatic cancer.

2. The method according to claim 1 wherein the levels of VOCs in the blood serum sample are measured by gas chromatography.

3. The method according to claim 2, wherein the VOC levels measured by chromatography are determined by measuring the area under the peak in the chromatogram.

4. The method according to claim 3 wherein chromatographic peak area values ​​less than 3.205, in the case of 2-ethyl-1-hexanol, 0.0228, in the case of pentylbenzene and 0.00351, in the case of butoxymethylbenzene, are indicative of pancreatic cancer.

5. Panel of biomarkers for the diagnosis of pancreatic cancer comprising at least one VOC selected from butoxymethylbenzene, pentylbenzene and 2-ethyl-1-hexanol, present in blood serum MODIFIED SHEET (ARTICLE 19) In relation to the examiner's comments on the possible lack of inventive step based on the documents located in the International Search Report, we consider that none of such documents describe the measurement of VOCs in the blood serum of patients with pancreatic cancer for diagnostic purposes. Document D1 is limited to describing the tests performed in air and urine, but not in serum. The present invention shows for the first time that some of the VOCs present in the blood serum of patients with pancreatic cancer have diagnostic and prognostic biomarker characteristics with a high degree of sensitivity and specificity (AUG > 0.98). In particular, the following compounds: butoxymethylbenzene (V328, A); (V40, B); 2-ethyl-1 -hexanol ( V172, C) and pentylbenzene (V274, D). The fact that these VOCs present in blood serum have been shown to be effective in the diagnosis and prognosis of pancreatic cancer is not only novel but is also non-obvious in the light of the current state of the art. The teachings contained in the documents representing the closest prior art according to the ISR, either considered individually or as a whole, do not render it obvious to a person skilled in the art that certain VOCs present in blood serum such as butoxymethylbenzene , 2-ethyl-1 -hexanol and pentylbenzene, can be used as biomarkers for pancreatic cancer given their high diagnostic and prognostic efficacy for this particular type of cancer. DECLARACION SEGUN EL ARTICULO 19 (1 )

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