Screening Volatile Biomarkers of Lung Cancer in Breath

Abstract

Using IMS to screen for volatile biomarkers of lung cancer in breath.

Description

TITLE: Screening Volatile Biomarkers of Lung Cancer in Breath Field of the Invention The invention is directed to the field of chemical screening. Background of the Invention Death caused by cancer diseases is a serious problem in modern society. The most common type of cancer in the world is lung cancer with millions of new cases diagnosed every year. Early detection is a desirable goal as it allows treatment to increase survival rate. Screening tests are based on x-ray, ultrasonography, computed tomography (CT) and MRI scan. These techniques have limitations in reliably discriminating between cancer patients and healthy subjects. Existing non-invasive screening methods include using proteomic and genetic technologies detecting molecular biomarkers. The possibility of using breath analysis to improve the management of cancer has led to the identification of volatile organic compounds (VOCs) linked to cancer. The concept has been established. However, despite the vast amount of work on this subject, the study of breath has not made it into clinical practice yet. Obstacles include identifying the best sampling method and origin of these VOCs, and instrumentation and data analysis standardization. References 1. Felix Schmidt et al. Lung Cancer 175 (2023) 131-140 2. M.Westhoff et al . Thorax 64 (2009), 744-748 3. Michael Phillips et al. CHEST 123 (2003) June 6. 4. Thriumani et al. BMC Cancer (2018) 18 :362. 5. Cristian Azorin et al. Analytical Chimica Acta 1271 (2023), 341. 6. Dorota M. Ruszkiewicz et al. Breath Analysis (2022) pp 45-71. 7. Meggie Hakim et al. Chemical Reviews (2012) April 26. Dx.doi.org / 10.1021 / cr300174a 8. Raquel Cumeras, Xavier Correig. Volatile Organic Compound Analysis in Biomedical Diagnosis Applications. Chapter 3, ppg. 53-78. Apple Academic Press Inc. 2021. Summary of the Invention The advantages of breath testing are, amongst others: It is non-invasive, the patient breath into a chemical treated filter to capture VOCs in his / her breath, followed by vapor analysis of the sample.Breath samples closely reflect the blood concentrations of some biological substances. Breath is much less complicated mixture than serum or urine and offers fast sample analysis. Once patients have been diagnosed and treated, they need to be followed-up, and breath tests can be carried out regularly to confirm that the disease is not coming back, these tests could be invasive, costly and give radiation. Therefore, VOCs analysis could also have a role in patients follow up. The advantage here when compared to using this methodology to diagnose cancer is that a VOCs profile of the patient with cancer is already available. Another important usefulness of the breath test is the screening of a population at high risk of developing lung cancer. The speed of analysis, low consumable of this approach offers screening capability for large number of people with low cost to the health system. When cancer develops into the body, it produces oxidative stress at the cellular level, which causes the emission of specific volatile biomarkers into the blood. From there, through exchange via the lungs, these biomarkers arrive at the exhaled air and are released through the breath. The breath analysis studies performed so far suggested that each type of cancer ( or disease in general) has its own breath print. Exhaled breath is a valuable medium to study how metabolites change with physiology. Previous studies have shown that the breath biomarkers panel will change with disease or its physiology; moreover, different biomarkers can be detected in different disease or physiology in breath. Many studies have used GC-MS to characterize exhaled breath condensate from subjects identified with lung cancer. From extensive literature search, 12 VOCs reported in breath samples were associated with lung cancer. Most of the compounds were aldehydes, ketones and aromatic hydrocarbons. These studies listed in the reference section demonstrated there is a difference in chemical profiles between lung cancer individuals and control subjects. These findings suggest that breath VOCs analysis may provide a tool that can be used to discriminate an individual’s history of lung cancer. Ion mobility breathalyzer exhibits low detection limits for volatile organic compounds pertaining to reported VOCs in people breath suffering from lung cancer. The instrument has excellent sensitivity and specificity using both ion polarity and chemical ionization reagents. Pattern recognition algorithm is built into the instrument to assist in the identification of target VOCs. The method can be modified to include additional marker chemicals for lung cancer and other respiratory diseases.• Specific biomarkers indicating of lung cancer disease • non-radiation source based on combined corona and insulator producing a cross high field ionization of the VOCs sample. • Method applicable to other respiratory diseases, like TB, COVID-19 and others • Chemically treated filter to capture the VOCs and enrich the sample to allow detection at low ppb-pptv levels. • Formulated coating of filter consisting of fullerene and diphenyl oxide. • Filters of cotton lintel, fiberglass, Teflon or metallic substrate • breathalyzer has internal gas cleaning system to provide low moisture and hydrocarbon into the drift region of the instrument and during desorption cycle. • Fast breath analysis in span of 10-20 seconds followed by extensive data processing and reporting. • Deconvoluted overlapping peaks and back subtraction to deal with complex spectrum • Matching detection algorithm with programmed lung cancer profile. Brief Description of the Drawings Figure 1 is a schematic of measurement by IMS; Figure 2 is graph showing amplitude versus mass. Detailed Description of the Preferred Embodiments Methodology Using IMS Detection System The breathalyzer includes a fast ion mobility spectrometer (IMS) with front internal separator unit and thermal vaporization of the breath sample collected on a chemically treated filter such as cotton lintel, fiberglass, Teflon or metallic substrate. The filter is inserted into the heated desorbed module to vaporize the collected sample into the ionization source of the IMS. Each ionized molecule is detected in the fast-switching polarity instrument and ion spectra profile of the VOCs are compared with programmed substances for positive detection. Analysis time is 10-20 seconds from time of insertion of the sample filter. There is no rapid method for detecting cases of lung cancer and the need for new screening test that is non-invasive, fast, and accurate is addressed in this invention. We tested the hypothesis that volatile organic compounds (VOCs) in the breath might provide new biomarkers of active lung cancer cases. The hypothesis is based on limited literature studies that were reported over the past 20 years covering a broad range of identifiedbiomarkers from patients with respiratory conditions. Aldehydes, alkyl alcohols, aromatic hydrocarbons, aromatic hydrocarbons, and others identified VOCs by gas chromatograph mass spectrometry (GC-MS). The analytical approach of this invention is to develop methodology to characterize biomarkers profiles on people breath, which identify their respiratory disease in both ion polarity. The steps to followed in the development of invention for lung cancer’s VOCs are as follows: - Raw data acquisition using GC-IMS or IMS, other sensor like surface ionization detection, mass spectrometry with front chemical ionization source. - Data pre-processing, which involves, baseline correction, noise filtering, deconvolution, peak alignment and normalization and scaling. - Data analysis using pattern recognition algorithm software. - VOCs identification, based on front separation and specific reduced mobility parameters. Data pre-processing improves signal quality by removing bias and variance. Methods such as deconvolution, alignment, noise filtering, baseline correction, and normalization can reduce instrument error. Furthermore, analytical signals may contain overlapped biomarkers in one single signal. Deconvolution procedures are implemented in the breathalyzer to separate constituent compounds from one broad signal. The pre-processed data is easily be treated by VOCs identification tools. Multi-peaks analysis is used in both positive and negative ion polarity of the IMS to improve identification of the programmed VOCs and reduce false positives and negative results. Breath Collection and Assay The method of breath collection involves the subject to breathe into the chemically treat disposable filter20 consecutive breathes to allow alveolar breath to be captured onto the adsorber film, which acts as an enrichment of the sample. The filter is inserted into the heated desorber inlet of the breathalyzer for rapid screening of programmed VOCs. Screening time is 10-20 seconds per sample with results immediately shown on the touch screen of the unit. Table 1 lists the key VOCs in lung cancer patients.TABLE 1 Channel Designation TYPE LC1 C6H12O aldehyde LC2 C7H14O aldehyde LC3 C4H8O ketone LC4 C9H18O Aldehyde LC5 C8H16O Aldehyde LC6 C5H8O Aldehyde LC7 C5H10O Ketone LC8 C4H8O2 Hydroxyketone LC9 C7H7O Aromatic aldehyde LC10 C3H6O Aldehyde LC11 C8H10 Alkyl benzene LC12 C9H12 Alkyl benzene Principles and Operating Conditions Ion mobility spectrometry (IMS) is an analytical technique based on the gas phase separation of ionized analytes under a weak electric field at atmospheric pressure. Ion velocities (vd) are directly related to the intensity of the applied electric field E and the mobility constant (K) of target species through the following equation: vd=KE Mobility constant (K) is characteristic of each analyte and is related to the mass, charge, size, and shape of the ionized compounds. K is expressed in units of cm2 / V.sec, and is affected by properties of the drift gas, temperature, and pressure. Usually, K values are normalized to allow comparison between data obtained by using different instruments and compensate for variations in drift tube temperature, pressure, and physical characteristics of the drift tube assembly. Ko= K (273 / T)(P / 760) Where T is temperature (degree Kelvin) and P is pressure(mmHg) inside the drift tube. Normalized reduced mobility constant Ko is programmed to identify each VOCs in both positive and negative ion modes using an internal calibrant substance as the reference peak in the mobility spectrum. Thermal desorber set at 200oC and inlet temperature 170oC.Drift tube temperature at 140-160oC and drift gas at 300cc / min Internal gas generator of low moisture 2ppm and hydrocarbon 1ppm. Pressure at ambient atmospheric pressure Field strength ̃470V / cm and ion injection gate width 100-160µsec. Scan average 12, scan duration 10msec, scan delay 1.38msec Date point per scan 200 and sampling rate 25-50µsec Non-radioactive ionization source consisting of combined corona-discharge with insulating barrier to induced a high filed plasma where ionization occurs. All VOCs of a sample react with the reactant ions. In a scenario in which the sample is composed of a single volatile analyte, M, the reaction between this generic compound and the reactant ions occurs according to the reaction: M + NH4+ ( H2O ) n ↔ MH++ NH3 + nH2O by proton transfer to M Clustering reaction can also occur to form MH+(NH3)(H2O) ion. If the concentration of the analyte is sufficiently high, the monomer can continue to react with the remaining M molecules, giving rise to the formation of protonated dimers. There is even the possibility of larger clusters being formed, as follows: MH+ + M = M2H+ and possible clustering M2H+(NH3)(H2O)n. IMS is able of working in two different modes, positive and negative. The previous reactions exemplify the positive mode procedure. For the negative mode, the reactant ions are formed due to the reaction of the primary ions with O2-(H2O)n ions. The reaction in the negative ion mode can proceed by clustering with internal chloride ion dopant to form specific negative ions of the form (M+Cl-(H2O)n) . Once created, the product ions are exposed to a homogeneous and weak electric field (470V / cm) that accelerates the ions through the IMS tube. Figure 1 represents the action of this field in the interior of the IMS tube during a complete analysis. Fig 1. Is a schematic of a complete measurement by IMS. The sample is ionized in the ionization chamber, and the formed ions are exposed to an electric field that will enable the detection of each VOC at their specific drift and reduced mobility constants. A front GC separation will also provide another ID dimension tool for complex mixture of VOCs.Results And Discussion Reduced mobilities (Ko) were calculated for peaks found in mobility spectra of the test compounds and product ions ranged from 0.96 to 1.74 cm2 / V.sec. The spectra were obtained in positive and negative polarity modes. Limits of detection for each compound are listed in terms of amount injected onto the collector filter. Calculated average concentration was determined based on breath rate of 2 liters / min for ten consecutive breaths, assuming 100% collection efficiency of the adsorbent coating on the sample card. TABLE 2 Limits of Detection Channel Designation LOD (ng) Concentration ng / cc LC4 0.1 0.3 LC7 0.5 1.5 LC3 1 3 LC2 1 3 LOD: amount deposited on the sample card in nanogram for signal / noise of 3. Concentration of VOC on breath for 2L / min breathing rate and ten breathes in ng / cm3. VOCs Quantification Through IMS Moving from the sampling procedures and VOCs identification to their quantification, the relation between the VOCs concentration and the signal measured at the Faraday plate is a rather complex process, which involves kinetics and thermodynamics. At this point, it should be considered that, as mentioned, one of the main limitations for the quantification through IMS is the ion clustering present in the drift tube, which can affect the IMS sensitivity. Another issue that should be considered is that IMS presents some limitations regarding the analysis of high-concentration compounds due to the saturation of the ionization source and the depletion of reactant ions. IMS devices that use radioactive ionization sources, like nickel source, present a linear behavior for concentration versus intensity but solely for very low concentration levels, usually in low nanogram or parts per billion concentrations. However, when the concentration is seen to increase, the calibration curve exhibits a plateau, limiting the concentration range of analysis. In this way, a typical calibration curve for this type of IMS has a logarithmic behavior. Since most of VOCs present in breath samples are low concentration, quantitative analysis is feasible to permits to setup an alarm threshold for positive indication of a person suffering from HAPE. One of the frequently report VOCs for lung cancer is alkyl aldehyde, which is programmed in the instrument and is detected in the positive ion mode. Method of ionization through ammonia chemical ionization reagent used in the ionization source is by proton transferfrom the ammonia-water cluster reagent ion. The ion-molecule reaction proceeds in the following manner. C8H16O + NH4(H2O)+ -------> C8H16OH+ + NH3 + H2O The sensitivity is in the low nanogram level collected on the sample filter. Calibration of aldehyde is shown in Figure 2 in terms of picogram deposited on the filter versus peak amplitude in digital counts. The concentration on breath would be in the low parts per billion level for this lung cancer chemical marker. Generally, the concentration on people breath would be in low ppb to high ppt (parts per billion and parts per trillion volume / volume), which falls in the linear range of the detector. Specificity of the method relies on the chemical ionization of individual volatile compounds in both positive and negative ion modes, ion separation and the detection algorithm method. Multi-peak detection algorithm with pattern recognition identifies the VOCs for specific disease.

Claims

CLAIMS:

1. A method of screening for lung cancer, comprising the step of screening for volatile biomarkers of lung cancer in breath.