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    • 专利摘要:
    The invention presents methods of using Fourier transform infrared (FTIR) microspectroscopy for common and specific characterization of carcinogenesis in human skin tumors. The invention provides the user with the method to analyse intra- and inter-molecular interactions for nucleic acids and proteins expressed in infrared (IR) spectra of human skin epidermal cancers towards understanding the molecular, cellular and tissue changes that occur during skin carcinogenesis. More particularly, presented analytical method has the advantage to simultaneously observe DNA-RNA, DNA-DNA, DNA-protein and protein-protein interactions by means of their expressed interacting activity levels within one type of tumour and between different types of tumours, both commonly and specifically, with further indication of the grade of activity in benign, premalignant and malignant skin tissue cells.
    公开号:AU2013224476A1
    申请号:U2013224476
    申请日:2013-02-21
    公开日:2014-10-09
    发明作者:Natalja EIKJE
    申请人:MC PROFESSIONAL OU；SKREBOVA IRINA；
    IPC主号:G01N21-35
    专利说明:
    WO 2013/123950 PCT/EE2013/000001 ANALYTICAL METHOD FOR COMMON AND SPECIFIC CHARACTERIZATION OF SKIN CARCINOGENESIS BY FTIR MICROSPECTROSCOPY BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention is directed towards providing an optical method for non-destructive characterization of carcinogenesis in skin cancerous tissues based on Fourier transform infrared (FTIR) microspectroscopy. More specifically, the invention provides with an analytical method for simultaneous observation and farther characterization of intra- and inter molecular interactions for nucleic acids and proteins, commonly and 1 WO 2013/123950 PCT/EE2013/000001 specifically expressed in IR spectra of each type of pathology, that also finds use for indication of the grade of neoplastic activity in cells in human skin cancerous tissues. 2. Prior Art Publications and other reference materials referred to herein, including reference cited therein, are incorporated herein by reference in their entirety and are numerically referenced in the following text and respectively grouped in the appended Bibliography, which immediately preceds the claims. In many countries skin cancer affects more people than all other cancers combined [1], but there is not yet enough understanding about the molecular, cellular and tissue changes that occur during skin carcinogenesis, that will have impact on clinical diagnosis, monitoring and treatment of skin cancer. For many years the mouse skin model provided a possibility to study multistage carcinogenesis mechanisms [2], but without direct relevance of this model system to human skin cancers. Up-to-date literature describes skin cancer development is, in part, under genetic control, and transcription factors (TFs), which are sequence-specific DNA-binding proteins, that control transcriptional activation or regression and thus control cell growth, being important in this process [3]. However, existing methods do not permit studying mechanisms of cancerogenesis directly on human beings and do not answer to all existing questions at present. For better understanding of molecular and cellular mechanisms involved in skin cancer development, there is a need for a method to directly characterize carcinogenesis from hyperplasia to invasive cancer, from one type of cancer to another type of cancer, from one patient to the group of patients with the same pathology. Skin tissue IR microspectroscopy technique is a tool for spectroscopic evaluation of unstained tissue of skin biopsies. It is an objective method for the analysis of skin tissue section that uses the chemical composition of the tissue as an indicator for healthy or pathological state of the cells in the tissue. IR spectral infonnation objectively and quantitatively determines 2 WO 2013/123950 PCT/EE2013/000001 variations in the chemical composition of skin cells and tissue, that can be further "converted" to medical knowledge. It is a sensitive tool for studying isolated biomolecules alone and in interactions. Feasibility of application of IR microspectroscopy for spectral characterization of the most common skin precancers and cancers has been already introduced in the 900-1700 cm-1 region, revealing the most visible changes related to protein confonnation and nucleic acid bases, that in general showed modifications and enhancement with progression to malignancy in that region [4]. Detailed alignment of the peaks for nucleic acids and proteins with spectral variations in IR spectra from skin epidermal cancers described appearance of DNA peak at about 965 cm-1; the multiplet (DNA/RNA) at about 1055 cm-1; DNA/RNA triad peaks at about 1071, 1084/1085, 1095 cm-1; at about 1245 cm-1 as a combination for DNA and amide III; a number of non-specific (non-descriptive) proteins at about 1310, 1390 and 1450 cm-1; the amide II vibration at about 1540 cm-1; and the amide I vibration at about 1650 cm-i [5]. Nevertheless, the problem has not been yet addressed towards common and specific characterization of carcinogenesis in human skin tumours by FTIR microspectroscopy. Therefore, it is another purpose of this invention to provide an optical analytical method for objective evaluation of intra- and inter-molecular interactions for nucleic acids and proteins expressed in infrared (IR) spectra of epidennal skin cancers towards understanding the molecular, cellular and tissue changes that occur during skin carcinogenesis. SUMMARY OF THE INVENTION In a first aspect, the invention provides a method of using FTIR microspectroscopy for characterization of common and specific carcinogenesis in human biopsied cancerous tissue samples. The sample can be comprised of normal, benign, precancerous or cancerous cells on a tissue sample. The invented method is based on simultaneous characterization of intra- and inter-molecular interactions for nucleic acids and proteins by means of their expressed interacting activity levels in infrared (IR) spectra 3 WO 2013/123950 PCT/EE2013/000001 of benign, premalignant and malignant skin cancers, in comparison to IR spectra from healthy, i.e. normal/unchanged, skin tissue. More particularly, provided method allows to describe common and specific features of skin carcinogenesis for each type of cancer and between different types of cancers, to assess the activity of neoplastic cells in each patient and between the patients by FTIR microspectroscopy. Skin epidermal carcinogenesis is a multi-stage process, that involves at least 3 mechanistically distinct steps - initiation, promotion and progression. Skin cancer generally develops in the epidermis. Most common malignant skin cancers are basal cell carcinoma (BCC), squamous cell carcinoma (SCC) and malignant melanoma (MM), that have been used as examples for the user of invention. To further provide the user with the invention steps, Bowen's disease, has been chosen as an example of precancerous skin lesion, well-known as intraepidernal carcinoma in situ. Benign compound nevus has been chosen as a common example of benign skin tumour in the presented invention. The method can be carried out manually and comprises the following steps: i. obtaining FTIR spectra from a multitude of pathological sites on a sample to obtain a multitude of measurements for each sample ii. detennining the specified wavenunber region between 900 and 1300 cm 1 for nucleic acids and the specified wavenumber region between 1300 and 1700 cm-1 for proteins in each measured IR spectrum in. nonnalizing each of IR spectrum to aide I peak iv. averaging epidermal measurements to obtain an average spectrum for the sample In the above method, the biopsied tissue samples are prepared for measurements by using FTIR microspectroscopy by the following steps: i. strictly sequential 2 sample cuts having a thickness of 6 micrometers ii. staining 1 sample cut with hematoxilin and eosin for histopathological evaluation iii. air-drying 1 sample cut on CaF2 slide glass for collection of FTIR spectra 4 WO 2013/123950 PCT/EE2013/000001 Although skin tissue is used throughout this description as the representative tissue, it should be understood that the invention method is not limited to measuring epidennal skin tissue and can be exploited with other tissues, as will be apparent to the skilled person. All the above and other characteristics and advantages of the invention will be further described. DETAILED DESCRIPTION OF THE INVENTION The invention will now be further explained through description of preferred embodiments. All the FTIR microscopy measurements were performed using a JEOL Co. (Tokyo, Japan) FT-IR spectrometer, a model IR-MAU200. Before each measurement, a calibration was performed using a sample provided with the instrument by the manufacturer and proper operating conditions of FTIR microspectrometer was confirmed. The data used in the examples below is derived from tissue specimen from the biopsy archive of the Department of Dermatology, Tokushima University School of Medicine, Tokushima, Japan, and a courtesy of Dr. T. Ikehara from the Department of Orthopedics, Tokushima University School of Medicine, Tokushima, Japan. The tissue samples used for FT-IR microspectroscopy were in the dry state, while the corresponding slides observed by light microscope were stained with hematoxilin and eosin for the identification of the tissue structure. The aperture size used in the measurements was 25 over 25 micrometers. Initially, the background spectrum was collected. After the measurement site was chosen using the visible light, the microscope was changed to IR mode. The number of co-added scans were increased to 127 to achieve high signal to noise ratio. The measured spectra covered the wavenumber range between 800 and 4000 cm-1, at a resolution of 4 cm-1. A database of having totally measured 198 spectra in the epidermis, both 5 WO 2013/123950 PCT/EE2013/000001 pathological and non-pathological, was created from 13 patients with malignant tumours: basal cell carcinoma (BCC) (6 patients), squamous cell carcinoma (SCC) (4 patients), malignant melnoma (MM) (3 patients); from 4 patients with benign tumours: benign compound nevus (4 patients); from 3 patients with precancers: Bowen's disease (3 patients); and from 4 healthy subjects. The invention method included the following steps for determination of the activity levels for nucleic acids and proteins in the IR spectra of BCC, SCC, MM, Bowen's disease and benign compound nevus, in comparison to IR spectra from healthy subjects: " calculation of mean values of each determined nucleic acids peaks " calculation of mean values of each detennined protein (specific and non-specific) peak " a comparison of the intensities of all determined nucleic acids peaks in the 900-1300 cm-i region with the intensity of the peak at about 1245 cm-i by calculating the following intensity peaks ratios: 1(965)/I (1245), I(1055)/I(1245), I(max peak among DNA/RNA triad peaks)/I(1245). " a comparison of the intensities of all determined peaks for specific and nonspecific proteins in the 1300-1700 cm-i region with the intensity of amide I peak at about 1650 cm-i by calculating the following peaks ratios: I(1310)/I(1650), I(1390)/I(1650), I(1450)/I(1650), 1(1540)/I (1650). " a calculation of the level of activity of DNA-RNA vs. DNA-protein interactions by the following ratio: I(sum mean DNA/RNA triad peaks)/I(1245). . a comparison of the level of activity of multiplet (DNA/RNA) with the level(s) of activity of the peaks in DNA/RNA triad. Below is a summary of the data employed from the patients with BCC, SCC, MM, Bowen's disease, benign compound nevus and healthy subjects, provided in examples. INTRA- AND INTER-MOLECULAR INTERACTIONS FOR 6 WO 2013/123950 PCT/EE2013/000001 NUCLEIC ACIDS EXAMPLES EXAMPLE 1 BCC Spectral data epidermally measured from 6 patients with BCC on nucleic acids peaks mean values (965 cm-i, 1055 cm-i, the most prominent peak in DNA/RNA triad peaks), the intensity ratios and proportional ratios (sum mean DNA/RNA triad peaks vs. 1245 cm-i). 2 BCC patterns in 6 patients, based on expressed mean values vs. proportional ratios: " 965mean < 1055mean < DNA/RNA triad max peak mean e 965mean < 1055mean > DNA/RNA triad max peak mean 1) 0.35 < 0.44 < 0.45 vs. [1:1] 2) 0.31 < 0.43 < 0.48 vs. [1:1] 3) 0.35 < 0.42 > 0.40 vs. [1:1] 4) 0.11 <0.19>0.18 vs. [0.8:1] 5) 0.08 <0.14 <0.16 vs. [0.8:1] 6) 0.09 - 0.09 < 0.23 vs. [0.8:1] 1 BCC pattern in 6 patients, based on calculated intensity ratios vs. proportional ratios: * I (965)/I(1245) < I(1055)/I(1245) < I (DNA/RNA triad max peak)/I (1245) (independent on the grade of activity) 1) 0.75 < 0.94 < 0.96 vs. [1:1] 2) 0.71 < 0.98 < 1.09 vs. [1:1] 3) 0.88 < 1.05 < 1.00 vs. [1:1] 7 WO 2013/123950 PCT/EE2013/000001 4) 0.50 < 0.87 < 0.82 vs. [0.8:1] 5) 0.38 < 0.67 < 0.76 vs. [0.8:1] 6) 0.45 < 0.45 < 1.15 vs. [0.8:1] EXAMPLE 2 SCC Spectral data epidennally measured from 4 patients with SCC on nucleic acids peaks mean values (965 cm-i, 1055 em-i, the most prominent peak in DNA/RNA triad peaks), the intensity ratios and proportional ratios (sum mean DNA/RNA triad peaks vs. 1245 cm-i). 3 SCC patterns in 4 patients, based on expressed mean values vs. proportional ratios: 965mean <1055mean > DNA/RNA triad max peak mean vs. [1:1] 965mean <1055mean < DNA/RNA triad max peak mean vs. [0.9: 1] 965mean > 1055mean < DNA/RNA triad max peak mean vs. [0.7:1] 1) 0.60 < 0.66 > 0.65 vs. [1:1] 2) 0.26 < 0.30 < 0.35 vs. [0.9:1] 3) 0.19 > 0.13 < 0.16 vs. [0.7:1] 4) 0.07 > (-) < 0.12 vs. [0.7:1] 2 SCC patterns in 4 patients, based on calculated intensity ratios vs. proportional ratios: * I (965)i(1245) < 1(1055)/1(1245) > I (DNA/RNA triad max peak)/J (1245) (for high activity levels of the peak at about 1055 cm-1) * 1 (965)/1(1245) > I(1055)/I(1245) < I (DNA/RNA triad max peak)/I (1245) (for low activity levels of the peak at about 1055 cm-1) 1) 0.91 < 1.00 > 0.99 vs. [1:1] 8 WO 2013/123950 PCT/EE2013/000001 2) 0.70 < 0.81 > 0.78 vs. [0.9:1] 3) 0.41 < 0.77 > 0.71 vs. [0.7:1] 4) 0.91 > (-) <0.76 vs. [0.7:1] ,EXAMPLE 3 MM Spectral data epidermally measured from 3 patients with Mv on nucleic acids peaks mean values (965 cm-i, 1055 cm-i, the most prominent peak in DNA/RNA triad peaks), the intensity ratios and proportional ratios (sum mean DNA/RNA triad peaks vs. 1245 cm-i). 2 MM patterns in 3 patients, based on expressed mean values vs. proportional ratios: e 965mean > 1055mean < DNA/RNA triad max peak mean e 965mean < 1055mean < DNA/RNA. triad max peak mean 1) 0.19 > 0.14 < 0.27 vs. [0.8:1] 2) 0.25 > (-) < 0.35 vs. [0.7:1] 3) 0.23 < 0.26 < 0.30 vs. [0.8:1] 2 MM patterns in 3 patients, based on calculated intensity ratios vs. proportional ratios: * I (965)/I(1245) < 1(1055)1(1245) < I (DNA/RAA triad max peak)/I (1245) * I (965)1(1245) > 1(1055)1(1245) < I (DNA/RNA triad max peak)/I (1245) 1) 0.61 < 0.69 < 0.79 vs. [0.8:1] 2) 0.63 > 0.47 < 0.90 vs. [0.8:1] 3) 0.52 > (-) < 0.73 vs. [0.7:1] 9 WO 2013/123950 PCT/EE2013/000001 EXAMPLE 4 BOWEN'S DISEASE (BD) Spectral data epidermally measured from 4 patients with BCN on nucleic acids peaks mean values (965 cm-i, 1055 cm-i, the most prominent peak in DNA/RNA triad peaks), the intensity ratios and proportional ratios (sum mean DNA/RNA triad peaks vs. 1245 cm-i). 1 BD pattern in 3 patients, based on mean values: a 965mean < 1055mean < DNAIRNA triad max peak mean (independent on the grade of activity) 1) (-) (-) 0.07 vs. [0.7:1] 2) 0.09 < 0.15 < 0.18 vs. [0.8:1] 3) 0.23 < 0.26 < 0.29 vs. [0.9:1] 1 BD pattern in 3 patients, based on the intensity ratios: 1 (965)1(1245) < I (1055)/1(1245) < I (DNA/RNA triad max peak)1(1245) (independent on the grade of activity) 1) (-) (-) 0.64 vs. [0.7:1] 2) 0.41 < 0.68 < 0.82 vs. [0.8:1] 3) 0.70 < 0.79 < 0.88 vs. [0.9:1] EXAMPLE 5 BENIGN COMPOUND NEVUS (BCN) Spectral data epidermally measured from 4 patients with BCN on nucleic acids peaks mean values (965 cm-i, 1055 cm-i, the most prominent peak in DNA/RNA triad peaks), the intensity ratios and proportional ratios (sum mean DNA/RNA triad peaks vs. 1245 cm-i). 10 WO 2013/123950 PCT/EE2013/000001 1 BCN pattern in 4 patients, based on expressed mean values vs. proportional ratios: # 965mean < 1055mean < DNAI/RNA triad max peak mean 1) 0.15 < 0.22 < 0.23 vs. [0.8:1] 2) 0.15 < 0.19 < 0.22 vs. [0.7:1] 3) 0.18 < 0.24 < 0.27 vs. [0.9:1] 4) 0.12 < 0.17 < 0.19 vs. [0.7:1] 1 BCN pattern in 4 patients, based calculated intensity ratios vs. proportional ratios: I (965)/1(1245) < I(1055)/1(1245) < I(DNA/RNA triad max peak)/I (1245) (independent on the grade of activity) 1) 0.48 < 0.71 < 0.78 vs. [0.8:1] 2) 0.47 < 0.59 < 0.72 vs. [0.7:1] 3) 0.56 < 0.75 < 0.84 vs. [0.9:1] 4) 0.48 < 0.68 < 0.76 vs. [0.7:1] EXAMPLE 6 HEALTHY SKIN (HS) HS pattern, based on expressed mean values vs. proportional ratios: * 965mean > (no peak expression) < DNA/RNA triad max peak mean 1) 0.15 > (-)< 0.27 vs. [0.5:1] 2) 0.24 > (-)< 0.47 vs. [0.5:1] 3) 0.17 > (-) < 0.22 vs. [0.7:1] 4) 0.22 > (-)< 0.30 vs. [0.7:1] HS pattern, based on calculated intensity ratios vs. proportional ratios: e I (965)/I (1245) > (no peak expression) < I (DNA/RNA triad max peak)/I (1245) 11 WO 2013/123950 PCT/EE2013/000001 1) 0.41 > (-)< 0.75 vs. [0.5:1] 2) 0.51 > (-)< 1.00 vs. [0.5:1] 3) 0.38 > (-) < 0.49 vs. [0.7:1] 4) 0.51 > (-)< 0.70 vs. [0.7:1] RESULTS Based on the presented Examples 1-5, the activity level of the inultiplet at about 1055 cm-1 strongly correlated with the activity level of the most prominent peak expressed in DNA/RNA triad (1071, 1084, 1095 cm-1) in 5 out of 6 patients with BCC, in 3 out of 4 patients with SCC and only in 1 out of 3 patients with MM, as well in all patients with BCN and BD. However, calculated proportional ratios differed for mean values and the intensity ratios between malignant and benign/premalignant, being higher in malignant skin tumours. Pattern recognition on DNA-RNA and DNA-DNA interactions as 1965 < 11055 < I max level DNA/RNA triadpeak were the most clearly observed in all 6 patients with BCC, independent on expressed activity levels of all peaks of nucleic acids. The same pattern recognition on DNA-RNA and DNA-DNA interactions were seen in all patients with Bowen's disease and benign compound nevus, again independently on the activity levels expressed by all peaks of nucleic acids. Pattern recognition on DNA-RNA and DNA-DNA interactions as 1965 < Ioss > I max level DNA/RNA triad peak was clearly seen in 3 SCC patients with the highest activity levels of the multiplet at about 1055 cm-1. In 2 patients with MM pattern recognition on DNA-RNA and DNA-DNA interactions as 1965 > 110ss < I max level DNA/RNA triad peak was unique and not seen in any other pathologies, with the lowest activity level of the multiplet. In 4 healthy subjects pattern recognition on DNA-RNA and DNA-DNA interactions as 1965 > (no peak expression) < I max level DNA/RNA triad peak was different from all measured above pathologies, with no expression of the 12 WO 2013/123950 PCT/EE2013/000001 multiplet at about 1055 cm-1 in the epidenmis. INTRA- AND INTER-MOLECULAR INTERACTIONS FOR PROTEINS EXAMPLE 1 BCC 1),BCC: 0.5 > 0.4 = 0.4 = 0.4 < 0.7 < 1.0 vs. [1:1] 2) BCC: 0.5 > 0.4 = 0.4 = 0.4 < 0.7 < 1.0 vs. [1:1] 3) BCC: 0.4 = 0.4 < 0.5 = 0.5 < 0.8 < 1.0 vs. [1:1] 4) BCC: 0.2 = 0.2 < 0.3 = 0.3 < 0.7 < 1.0 vs. [0.8:1] 5) BCC: 0.2 = 0.2 < 0.3 = 0.3 < 0.8 < 1.0 vs. [0.8:1] 6) BCC: 0.2 = 0.2 < 0.3 < 0.3 < 0.7 < 1.0 vs. [0.8:1] EXAMPLE 2 SCC 1) SCC: 0.7 > 0.6 < 0.7 = 0.7 < 0.9 < 1.0 vs. [1:1] 2) SCC: 0.4 = 0.4 = 0.4 = 0.4 < 0.7 < 1.0 vs. [0.9:1] 3) SCC: 0.2 = 0.2 < 0.3 = 0.3 < 0.6 < 1.0 vs. [0.7:1] 4) SCC: 0.2 = 0.2 = 0.2 = 0.2 < 0.6 < 1.0 vs. [0.7:1] EXAMPLE 3 MM 1) MM: 0.4 = 0.4 < 0.5 = 0.5 < 0.8 < 1.0 vs. [0.8:1] 13 WO 2013/123950 PCT/EE2013/000001 2) MM: 0.3 = 0.3 < 0.4 = 0.4 < 0.8 < 1.0 vs. [0.8:1] 3) MM: 0.5 > 0.4 < 0.5 = 0.5 < 0.8 < 1.0 vs. [0.7:1] EXAMPLE 4 SCC vs. BOWEN's DISEASE (BD) 1) SCC: 0.7 > 0.6 < 0.7 = 0.7 < 0.9 < 1.0 vs. [1:1] 2) SCC: 0.4 = 0.4 = 0.4 = 0.4 < 0.7 < 1.0 vs. [0.9:1] 3) BD: 0.3 < 0.3 < 0.4 = 0.4 < 0.8 < 1.0 vs. [0.9:1] 4) BD: 0.2 = 0.2 < 0.3 = 0.3 < 0.7 < 1.0 vs. [0.8:1] 5) SCC: 0.2 = 0.2 < 0.3 = 0.3 < 0.6 < 1.0 vs. [0.7:1] 6) SCC: 0.2 = 0.2 = 0.2 = 0.2 < 0.6 < 1.0 vs. [0.7:1] 7)BD: 0.1=0.1<0.2=0.2<0.6<1.0 vs. [0.7:1] EXAMPLE 5 MM vs. BENIGN COMPOUND NEVUS (BCN) 1) BCN: 0.3 > 0.2 < 0.3 < 0.4 < 0.7 < 1.0 vs. [0.9:1] 2) BCN: 0.3 > 0.2 < 0.3 < 0.4 < 0.7 < 1.0 vs. [0.9:1] 3) BCN: 0.3 > 0.2 < 0.3 = 0.3 < 0.7 < 1.0 vs. [0.8:1] 4) BCN: 0.3 > 0.2 < 0.3 = 0.3 < 0.7 < 1.0 vs. [0.7:1] 5) BCN: 0.3 = 0.3 < 0.4 = 0.4 < 0.8 < 1.0 vs. [0.7:1] 1) MM: 0.4 = 0.4 < 0.5 = 0.5 < 0.8 < 1.0 vs. [0.8:1] 2) MM: 0.3 = 0.3 < 0.4 = 0.4 < 0.8 < 1.0 vs. [0.8:1] 3) MM: 0.5 > 0.4 < 0.5 < 0.5 < 0.8 < 1.0 vs. [0.7:1] EXAMPLE 6 14 WO 2013/123950 PCT/EE2013/000001 ALL MEASURED PATHOLOGIES 1) BCC: 0.4 = 0.4 = 0.4 < 0.7 < 1.0 vs. [1:1] 2) BCC: 0.4 = 0.4 = 0.4 < 0.7 < 1.0 vs. [1:1] 3) BCC: 0.4 < 0.5 = 0.5 < 0.8 < 1.0 vs. [1:1] 4) SCC: 0.6 < 0.7 = 0.7 < 0.9 < 1.0 vs. [1:1] 5) SCC: 0.4 = 0.4 = 0.4 < 0.7 < 1.0 vs. [0.9:1] 6) MM: 0.4 < 0.5 = 0.5 < 0.8 < 1.0 vs. [0.8:1] 7) MM: 0.3 < 0.4 = 0.4 < 0.8 < 1.0 vs. [0.8:1] 8) BCC: 0.2 < 0.3 = 0.3 < 0.7 < 1.0 vs. [0.8:1] 9) BCC: 0.2 < 0.3 = 0.3 < 0.7 < 1.0 vs. [0.8:1] 10) BCC: 0.2 < 0.3 = 0.3 <0.7 < 1.0 vs. [0.8:1] 11) SCC: 0.2 < 0.3 = 0.3 < 0.6 < 1.0 vs. [0.7:1] 12) SCC: 0.2 = 0.2 = 0.2 < 0.6 < 1.0 vs. [0.7:1] 13) MM: 0.4 < 0.5 < 0.5 < 0.8 < 1.0 vs. [0.7:1] RESULTS Presented data on DNA-protein interactions were mostly expressed between intensities of DNA/RNA triad peaks and non-descriptive proteins at 1310, 1390 and 1450 cm-1, that were in agreement between BCC and SCC, but not in MM. Protein-protein interactions were similar between the patients, indicating the grade of activity in cells in tissues. Among the patients with BCC and SCC the levels of non-descriptive proteins generally differed between 0.2 and 0.7. In 3 patients with BCC I (sum mean DNA/RNA triad peaks)1(1245) was [1 : 1] and their levels of non-descriptive proteins were stable at the level of 0.4-0.5. Similar levels were observed in 3 patients with MM, but when their J(sum mean DNA/RNA triad peaks)/I(1245) were [0.7 : 1] and [0.8 : 1]. In all patients with SCC the levels of non-descriptive proteins were the most sensitive to clearly indicate the activity of I(sum mean DNA/RNA triad peaks)/I(1245). Protein-protein interactions were observed by the correlation between I (amide II)/I(amide ) and mean values-of the peaks at about 1310, 1390 and 15 WO 2013/123950 PCT/EE2013/000001 1450 cm-1. Protein-protein interactions were similar between skin cancers, generally indicating low, medium and high activity levels in cells in tissues. BIBLIOGRAPHY 1. Federman, D.G., Concato, J., Kirsner, R.S., "Screening for skin cancer: absence of evidence", Arch. Dermatol., 145, (2009), 926. 2. DiGiovanni, J., "Multistage carcinogenesis in mouse skin", Pharmacol. Ther., 54 (1), (1992), 63-128. 3. Chimento, S.M., Kirsner, R.S., "Understanding the role of c-Jun and Jun B transcription factors in skin cancer development", J. Invest. Dennatol., 131, (2011), 1002. 4. Eikje, N.S., Aizawa, K., Ozaki, Y., "Vibrational spectroscopy for molecular characterization and diagnosis of benign, premalignant and malignant skin tumours", Biotechnology Annual Review, Vol. 11, (2005), 191-226. 5. Eikje, N.S., "Numerical modeling and analytical treatment of IR spectra in the diagnosis of skin cancers", In: SFM 2010: Optical Technologies in Biophysics and Medicine XII (Eds. Tuchin, V.V., Genina, E.A.), Proc SPIE, 7999, (2011), 799909. CLAIMS 1. A method of using Fourier transfonn infrared microspectroscopy for common and specific characterization of carcinogenesis in human skin tumors, comprising of: (i) comparative assessment of expressed mean values for nucleic acids and proteins, specific and non-specific, in FTIR spectra from epidermis of benign, premalignant and malignant skin lesions (ii) comparative calculation of the intensity ratios for all assigned peaks for nucleic acids vs. the peak at about 1245 cm-1 in FTIR spectra of benign, premalignant and malignant skin lesions (iii) comparative calculation of the intensity ratios for all assigned peaks for proteins, specific and non-specific, vs. the amide I peak at about 1650 cm-1 in FTIR spectra from epidernis of benign, premalignant and malignant skin 16 WO 2013/123950 PCT/EE2013/000001 lesions (iv) determination of a pattern, both common and/or specific, based on comparative assessment of expressed mean values, for nucleic acids in FTIR spectra from epidermis of benign, premalignant and malignant skin lesions, in comparison to a determined pattern of healthy epidermis (v) determination of a pattern, common for benign, premalignant and malignant skin tumours, based on comparative calculation of the intensity ratios, for nucleic acids and proteins in FTIR spectra of benign, premalignant and malignant lesions, in comparison to determined pattern of healthy epidennis (vi) detennination of a pattern, specific for each type of pathology, based on comparative calculation of the intensity ratios, for nucleic acids and proteins in FTIR spectra from epidermis of benign, premalignant and malignant skin lesions, in comparison to determined pattern of healthy epidermis 2. A comparative analytical method, comprised of steps in Claim 1, for an individual patient and between the patients with any type of benign, premalignant and malignant skin lesions 3. A comparative analytical method, comprised of steps in Claim 1, for one type of pathology, related to any analyzed benign, premalignant and malignant skin lesions 4. A comparative analytical method, comprised of steps in Claim 1, for different types of epidennal skin cancers 5. A method according to Claims 1-4, for indication of the grade of activity in neoplastic cells of measured skin tumors, comprising the following steps: * calculation of the level of activity of DNA-RNA vs. DNA-protein interactions by the following proportional ratio: I(sum mean DNA/RNA triad peaks)/I(1245) " calculation of mean values of each assigned nucleic acids peak, in comparison to a proportional ratio, i.e. sum mean DNA/RNA triad peaks vs. 1245 cm-i " calculation of mean values of each assigned protein (specific and nonspecific) peak, in comparison to a proportional ratio, i.e. sum mean DNA/RNA triad peaks vs. 1245 cm-i * a comparison of the intensities of all determined nucleic acids peaks in the 900-1300 cm-i region with the intensity of the peak at about 1245 cm-i by calculating the following intensity peaks ratios: 1(965)/I 17 WO 2013/123950 PCT/EE2013/000001 (1245), I(1055)/I(1245), I(max peak among DNA/RNA triad peaks)/I(1245), in comparison to a proportional ratio, i.e. smn mean DNA/RNA triad peaks vs. 1245 cm-1 * a comparison of the intensities of all detennined peaks for specific and non-specific proteins in the 1300-1700 cm-i region with the intensity of amide I peak at about 1650 cm-i by calculating the following peaks ratios: I(1310I)/(1650), I(1390)/I(1650), I(1450)/I(1650), 1(1540)/I (1650), in comparison to a proportional ratio, i.e. sum mean DNA/RNA triad peaks vs. 1245 cm-i . a comparison of the level of activity of multiplet (DNA/RNA at about 1055 cm-1) with the level(s) of activity of the peaks in DNA/RNA triad (at about 1071, 1084, 1095 cm-1) 6. A comparative method of analysing a systematic progression of the grade of activity between benign, premalignant and malignant skin tissue cells, comprising of steps in Claim 5 7. A method of analysing a systematic progression of the grade of activity in malignant skin tissue cells in different types of pathologies, comprising of steps in Claim 5 8. A method of analysing a systematic progression of the grade of activity in malignant skin tissue cells within one type of pathology, comprising of steps in Claim 5 18
    权利要求:
    Claims (9)
    [1] 1. A method for common and specific characterization of skin epidermal carcinogenesis, to analyse intra- 'and inter-molecular interactions for nucleic acids and proteins expressed in DNA- and protein-specific sequences, alone and in combination, determined in the FTIR spectrum of a tissue sample over a range of frequency bands for nucleic acids and proteins, using pattern recognition technique to commonly and specifically feature benign, premalignant and malignant cancerous tissues against healthy (i.e. histopathologically unchanged) in the epidermis, comprising of' (i) DNA sequence detection by means of determination of DNA-specific frequency bands over a range of known bands at about 965 cm~ 1 , 1055 cm- 1 , and in the triad at about 1071 cm 1 , 1084/1085 cm- 1 , 1095 cm', to find the following patterns, as: (a) "DNA(965), DNA/RNA triad (1071, 1084/1085, 1095)", specific for healthy tissue; (b) "DNA(965), DNA/RNA(1055), DNA/RNA triad (1071, 1084/1085, 1095)", specific for benign, premalignant and malignant cancerous tissue (ii) mean value calculation of each frequency band, detected in DNA sequence as "DNA (965), DNA/RNA(1055), DNAIRNA triad (1071, 1084/1085, 1095)", to find further patterns, as: (a) "DNA(965mean) < DNA/RNA(1055mean) < DNA/RNAtriad (1071, 1084/1085/1095max peak mean)", characteristic for skin benign tumours; (b) "DNA(965mean) < DNA/RNA(1055mean) < DNA/RNAtriad (1071, 10 8 4 /1085/1095max peak mean)", characteristic for skin premalignant tumours; (c) "DNA(965mean) < DNA/RNA(1055mean) < DNA/RNAtriad (1071, 1084/1085/1095max peak mean)", as well "DNA(965mean) < DNAIRNA(1055mean) > DNA/RNAtriad (1071, 1084/1085/1095max peak mean)", as well "DNA(965mean) > DNA/RNA(1055mean) < DNA/RNAtriad (1071, 1084/1085/1095max peak mean)", 22 WO 2013/123950 PCT/EE2013/000001 characteristic for skin malignant tumours. (iii) mean intensity calculation of each frequency band detected in DNA sequence as "DNA(965), DNA/RNA(1055), DNA/RNA triad (1071, 1084/1085, 1095)", in a ratio, calculated in comparison with mean intensity of the peak at about 1245 cm-1, i.e. DNA/amide III, to find further patterns, as: (a) "DNA(I(6 5 sean)/I(12 45 mean)) < DNA/RNA(I(i 0 5 5 mean)/I(1 24 mean)) < DNA/RNAtriad (I(DNA/RNA triad max peak mean)/I(1245mean))", characteristic for skin benign tumours; (b) "DNA(I( 9 65 mean)/I(12 4 5 mean)) < DNA/RNA(I(o 5 5mean)/I(1 24 5mean)) < DNA/RNAtriad (I(DNA/RNA triad max peak mean)/I(1245mean))", characteristic for skin premalignant tumours; (c) "DNA(I( 9 6 5 mean)/I(12 4 5 mean)) < DNARNA(I(io 5 5 mean)I(1 2 4 5 mean) < DNAIRNAtriad (I(DNA/RNA triad max peak mean)/I(1245mean))", as well "DNA(I(965mean)/I(1245mean)) < DNA/RNA(I(1o55mean)/I(1245mean)) > (I(DNA/RNA triad max peak mean)/I (1245mean))", as well "DNA(I(965mean)/I(1245mean)) > DNA/RNA((O55mean)/I(1245mean)) < (I(DNA/RNA triad max peak mean)/I (1245mean))", characteristic for skin malignant tumours. (iv) calculation of proportional ratio, i.e. sum mean DNAIRNA triad peaks at about 1071, 1084/1085, 1095 cm- 1 vs. DNA/amide III peak at about 1245 cm- 1 , to characterize epidermal tissue towards skin cancer development within the following ranges, as: (a) [0.5:11 - [0.7:1], specific for healthy epidermis; (b) [0.7:1] - [0.9:11, specific for skin benign tumours; (c) [0.8:1] - [0.9:11, specific for skin premalignant tumours; (d) [0.7:11-[1:11, specific for skin malignant tumours, BUT dependent on the type of tumour. (v) mean value calculation of each protein-specific frequency band in detected protein sequence, for descriptive and non-descriptive proteins at about 1310 cm-1, 1390 cm- 1 , 23 WO 2013/123950 PCT/EE2013/000001 1450 cm', 1540 cm' and 1645 cm 1 , wherein (a) expressed mean values of non-descriptive, i.e. non-specific, protein at about 1310 cm 1 determine the starting level, i.e. activity level, in detected protein sequences from healthy, benign, premalignant and malignant cancerous tissues (b) mean values of non-descriptive, i.e. non-specific, protein at about 1310 cm' in protein sequences are ranged in strong correlation with gradation in proportional ratios (c) mean values of non-descriptive, i.e. non-specific protein at about 1310 cm 1 in protein sequences are always at similar values expressed by activity levels of DNA/amide III peak at about 1245 cm4 (d) mean values of non-desciptive, i.e. non-specific, proteins at about 1390 cm 1 and 1450 cm 1 are expressed equally in protein sequences of premalignant and malignant cancerous tissues, but not of benign cancerous tissue vs. [0.9:1] (e) mean values of specific proteins at about 1540 cm 1 are expressed in strong correlation with gradation in proportional ratios, but differ for benign, premalignant and malignant cancerous tissues (vi) a comparative analysis of the activity of expressed mean values in protein sequences related to benign, premalignant and malignant cancerous tissues, as: (a) specific for benign skin tumours, with reference to benign compound nevus, characterized by: * 2 patterns of non-specific protein sequences, as "(1310) < (1390) = (1450)" and "(1310) < (1390) < (1450)" * the pattern, "(1310) < (1390) (1450)", is for ranged mean values that is correlated with [0.7:1] and [0.8:11 proportional ratios * the pattern, "(1310) < (1390) < (1450)" is for ranged mean values that is in strong correlation with [0.9:11 proportional ratio * mean values for non-specific proteins, ranged within 0.2-0.4, and mean values for specific protein at about 1540 cm 1 , ranged within 0.7-0.8, do not show correlation with any of the gradation in proportional ratios between [0.7:11-[0.9:11 (b) specific for premalignant cancerous tissue, with reference to Bowen's disease, characterized by: * one-patterned non-specific protein sequences, as "(1310) < (1390) = (1450)" 24 WO 2013/123950 PCT/EE2013/000001 * this pattern is strongly correlated with gradation in proportional ratios " mean values for non-specific proteins, ranged within 0.1-0.4, and mean values for specific protein at about 1540 cm 1 , ranged within 0.6-0.8, increase with gradation increment in proportional ratios from [0.7:1] to [1:11 (c) specific for malignant cancerous tissue, with reference to BCC, characterized by: * 2 patterns of non-specific protein sequences, as "(1310) = (1390) = (1450)" and "(1310) < (1390) = (1450)" 0 the pattern "(1310) = (1390) = (1450)" is only correlated with the gradation in [1:11 proportional ratios * mean values for non-specific proteins, ranged within 0.2-0.5 in the pattern as of "(1310) < (1390) = (1450)", and mean values for specific proteins at about 1540 cm', ranged within 0.7-0.8, are expressed in strong correlation with gradation increment in proportion ratios from [0.8:11 to [1:11 with reference to SCC, characterized by: * 2 patterns of non-specific protein sequences, as "(1310) = (1390) = (1450)" and "(1310) < (1390) = (1450)" * these patterns are not correlated with the gradation in proportional ratios e high-ranged mean values for non-specific proteins within 0.2-0.7, and mean values for specific proteins at about 1540 cm' ranged within 0.6-0.9, are expressed in strong correlation with gradation increment in proportional ratios from [0.7:1] to [1:1] with reference to MM, characterized by: * one-patterned non-specific protein sequences, as "(1310) < (1390) = (1450)" * low-ranged mean values for non-specific proteins within 0.3-0.5 are expressed with mean values of 0.8 for specific proteins at about 1540 cm 1 , that are higher than for benign tumour
    [2] 2. A comparative analytical method, comprised of steps in Claim 1, to further indicate common and specific features in examined tissue towards skin cancer development by means of comparing expressed mean values and mean intensities of each frequency 25 WO 2013/123950 PCT/EE2013/000001 band, detected in DNA- and protein-specific sequences, with proportional ratios, to indicate the grade of activity in cells of benign, premalignant and malignant cancerous tissues, as well to analyse a systemic progression of the grade of activity within one type of tumour and between different types of tumour, comprising of' (i) detection of the lowest and the highest mean values and intensities in all patterned DNA sequences specific to each kind of cancerous tissue to commonly characterize cancer progression, as: (a) in benign cancerous tissue with reference to benign compound nevus with the lowest mean values, as "DNA(0. 12) < DNA/RNA(0.17) < DNA/RNA triad(0.19) vs. [0.7:11 and the lowest intensities, as "DNA(0.48) <DNA/RNA(0.68) < DNA/RNA triad(0.76) vs. [0.7:1]; with the highest mean values, as "DNA(0.18) < DNA/RNA(0.24) < DNA/RNA triad(0.27) vs. [0.9:1], and with the highest intensities, as "DNA(0.56) <DNA/RNA(0.75) < DNA/RNA triad(0.84) vs. [0.9:1]. (b) in precancerous tissue with reference to Bowen's disease with the lowest mean values, as "DNA(0.09) < DNA/RNA(0.15) < DNA/RNA triad(0. 18) vs. [0.8:1], and the lowest intensities, as "DNA(0.41) <DNAIRNA(0.68) < DNA/RNA triad(0.82) vs. [0.8:11; with the highest mean values, as "DNA(0.23) < DNAIRNA(0.26) < DNA/RNA triad(0.29) vs. [0.9:11, and with the highest intensities, as "DNA(0.70) <DNA/RNA(0.79) < DNAIRNA triad(0.88) vs. [0.9:1]. (c) in malignant cancerous tissue, 26 WO 2013/123950 PCT/EE2013/000001 with reference to SCC, with the lowest mean values, as "DNA(0.19) > DNA/RNA(0.13) < DNA/RNA triad(0.16) vs. [0.7:1], and with the lowest intensities, as "DNA(0.41) < DNA/RNA(O.77) > DNAIRNA triad(0.71) vs. [0.7:1]. with the highest mean values, as "DNA(0.60) < DNA/RNA(0.66) > DNA/RNA triad(0.65) vs. [1:1], and with the highest intensities, as "DNA(0.91) < DNA/RNA(1.00) > DNA/RNAtriad(0.99) vs. [1:11; with reference to BCC, with the lowest mean values, as "DNA(0.08) < DNAIRNA(0.14) < DNA/RNA triad(0.16) vs. [0.8:1], and with the lowest intensities, as "DNA(0.38) < DNA/RNA(0.67) < DNA/RNA triad(0.76) vs. [0.8:1]; with the highest mean values, as "DNA(0.35) < DNA/RNA(0.44) < DNA/RNA triad(O.45) vs. [1:1], and with the highest intensities, as "DNA(0.75) < DNA/RNA(0.94) < DNA/RNA triad(O.96) vs. [1:1]. with reference to MM, with the lowest/highest mean values, as "DNA(0.23) < DNA/RNA(0.26) < DNA/RNA triad(0.30) vs. [0.8:11, as "DNA(0. 19) > DNA/RNA(0.14) < DNA/RNA triad(0.27) vs. [0.8:1]; with the lowest/highest intensities, as "DNA(0.61) < DNA/RNA(0.69) < DNA/RNA triad(0.79) vs. [0.8:1], as "DNA(0.63) > DNA/RNA(0.47) < DNA/RNA triad(0.90) vs. [0.8:11. 27 WO 2013/123950 PCT/EE2013/000001 (ii) correlating the lowest/highest expressed mean values and calculated intensities in detected DNA sequences with proportional ratios, to specifically characterize cancer progression within one type of tumour, as: (a) in benign skin tumours, with reference to benign compound nevus, characterized by: -one pattern of DNA sequence as "DNA(965) < DNA/RNA(1055) < DNA/RNA triad(1071, 1084/1085, 1095)" -this pattern is independent on the grade of activity by proportional ratios -this pattern is characterized in low-ranged mean values and calculated intensities when compared with premalignant and malignant skin tumours, that are well correlated with gradation in proportional ratios (b) in premalignant skin tumours, with reference to Bowen's disease, characterized by: -one pattern of DNA sequence as "DNA(965) < DNA/RNA(1055) < DNA/RNA triad(1071, 1084/1085, 1095)" -this pattern is independent on the grade of activity by proportional ratios -this pattern is characterized by well-correlated with gradation in proprotional ratios intermediate ranges of mean values and calculated intensities, that are higher than for benign skin tumours, but lower than for malignant skin tumours (c) in malignant skin tumours, with reference to BCC, characterized by: -2 patterns of DNA sequences: 1) "DNA(965) < DNA/RNA(1055) < DNA/RNA triad(1071, 1084/1085, 1095)"; 2) "DNA(965) < DNAIRNA(1055) > DNA/RNA triad(1071, 1084/1085, 1095)" -DNA sequence pattern "DNA(965) < DNA/RNA(1055) < DNAIRNA triad(1071, 1084/1085, 1095)" is dominant, independent whether based on the expressed mean values or calculated intensities -these patterns are independent on the grade of activity by proportional ratios -these patterns are characterized in high-ranged mean values and calculated intensities that are well-correlated with gradation in proportional ratios, when compared with benign and premalignant skin tumours 28 WO 2013/123950 PCT/EE2013/000001 with reference to SCC, characterized by: -3 patterns of DNA sequences as "DNA(965) < DNA(1055) > DNA/RNA triad", "DNA (965) < DNA(1055) < DNA/RNA triad" and "DNA(965) > DNA(1055) < DNA/RNA triad", based on expressed mean values, that are dependent on the grade of activity by proportional ratios and the acitivity of DNA/RNA peak at about 1055 cm 1 , -one-patterned DNA sequence "DNA(965) < DNA(1055) > DNA/RNA triad", based on calculated intensities, that are dependent on the grade of activity by proportional ratios and the acitivity of DNA/RNA peak at about 1055 cm 1 -all patterns, independent whether based on expressed mean values or calculated intensities, are characterized by high ranges, that are higher in comparison to other malignant skin tumours, and are well-correlated with gradation in proportional ratios with reference to MM, characterized by: -2 patterns of DNA sequences as "DNA(965) < DNA(1055) < DNA/RNA triad" and "DNA (965) > DNA(1055) < DNA/RNA triad", independent whether based on the expressed mean values or calculated intensities -these patterns are independent on the grade of activity by proportional ratios -these patterns are characterized by the highest levels of expressed mean values and calculated intensities vs. lower gradation in proportional ratios, when compared with the other types of malignant skin tumours (iii) comparative protein sequence analysis for determination of systematic progression of the grade of activity between benign, premalignant and malignant skin tissue cells, as: (a) specific to indicate a progression from premalignant to malignant cancerous tissue against healthy epidermal tissue, characterized in protein sequences by expressed mean values in relation to gradation in proportional ratios, as: with reference to healthy epidermal skin, i.e. HS, Bowen's disease (in situ cancer), i.e. BD, vs. SCC - in relation to [0.7:11 29 WO 2013/123950 PCT/EE2013/000001 BD: 0.1 < 0.2 = 0.2 < 0.6 < 1.0 SCC: 0.2 = 0.2 = 0.2 < 0.6 < 1.0 SCC: 0.2 < 0.3 = 0.3 < 0.6 < 1.0 HS: 0.4 = 0.4 = 0.4 = 0.4 < 0.8 < 1.0 HS: 0.5 < 0.6 > 0.5 =0.5 < 0.8 < 1.0 -in relation to [0.9:11 BD: 0.3 <0.4 = 0.4 < 0.8 < 1.0 SCC: 0.4 =0.4 = 0.4 <0.7 < 1.0 (b) specific to indicate a progression from benign to malignant cancerous tissue in known pathologies to transform from benign to malignant skin tumours against healthy epidermal tissue, characterized in protein sequences by expressed mean values in relation to gradation in proportional ratios, as: with reference to healthy epidermal skin, i.e. HS, benign compound nevus, i.e. BCN vs. malignant melanoma (MM) - in relation to [0.7:1]: BCN:0.3<0.4=0.4<0.8< 1.0 BCN: 0.2 < 0.3 = 0.3 < 0.7 < 1.0 MM: 0.2 < 0.3 = 0.3 < 0.6 < 1.0 HS: 0.4 = 0.4 = 0.4 = 0.4 <0.8 < 1.0 HS: 0.5 < 0.6 > 0.5 =0.5 < 0.8 < 1.0 - in relation to [0.8:1] BCN:0.2<0.3=0.3<0.7< 1.0 MM: 0.4 <0.5 = 0.5 < 0.8 < 1.0 MM: 0.3 < 0.4 =0.4 < 0.8 < 1.0 (c) specific to indicate a progression from benign to premalignant, from premalignant to malignant skin cancerous tissue, against healthy epidermal tissue, characterized in protein sequences by expressed mean values in relation to gradation in proportional ratios, as: 30 WO 2013/123950 PCT/EE2013/000001 -in relation to [0.7:1] BCN: 0.2 < 0.3 = 0.3 < 0.7 < 1.0 BCN: 0.3 < 0.4 = 0.4 < 0.8 < 1.0 BD:0.1<0.2=0.2<0.6< 1.0 SCC: 0.2 = 0.2 = 0.2 < 0.6 < 1.0 SCC: 0.2 < 0.3 = 0.3 < 0.6 < 1.0 MM: 0.4 < 0.5 = 0.5 < 0.8 < 1.0 HS: 0.4 = 0.4 = 0.4 = 0.4 < 0.8 < 1.0 HS: 0.5 < 0.6 > 0.5 =0.5 < 0.8 < 1.0 in relation to [0.8:11 BCN: 0.2 < 0.3 = 0.3 < 0.7 < 1.0 BD: 0.2 < 0.3 = 0.3 < 0.7 < 1.0 BCC: 0.2 < 0.3 =0.3 < 0.7 < 1.0 MM: 0.3 < 0.4 = 0.4 < 0.8 < 1.0 MM: 0.4<0.5=0.5<0.8< 1.0 in relation to [0.9:11 BCN: 0.2 < 0.3 < 0.4 < 0.7 < 1.0 BD: 0.3 < 0.4 = 0.4 < 0.8 < 1.0 SCC: 0.4 = 0.4 = 0.4 < 0.7 < 1.0 in relation to [1:11 BCC: 0.4 = 0.4 = 0.4 < 0.7 < 1.0 BCC: 0.4 = 0.4 = 0.4 <0.8 < 1.0 BCC: 0.4 <0.5 = 0.5 <0.8 < 1.0 SCC: 0.6 < 0.7 = 0.7 < 0.9 < 1.0
    [3] 3. The method as claim 1, wherein the detecting step is performed by infrared spectrometer and/or infrared microspectrometer.
    [4] 4. The method as claim 1, wherein said infrared spectra are determined by FT-IR spectroscopy.
    [5] 5. The method as claim 1 (i), wherein said DNAIRNA triad peak (1071, 1084/1085, 1095) is defined by the maximum peak in a triad. 31 WO 2013/123950 PCT/EE2013/000001
    [6] 6. The method as claim 1 (iii), wherein said finding patterns in DNA- specific sequences, based on the following intensity ratios by calculating mean intensity of each DNA specific frequency band in relation to mean intensity of a combined peak of DNA and amide III at about 1245 cm- 1 as 1(965)/1(1245), I(1055)/I(1245),I(DNA/RNA triad max peak)/I(1245); and in protein-specific sequences, based by calculating mean intensity of each protein-specific frequency band in relation to mean intensity of normalized amide I peak at about 1650 cm 1 of "1.00" as I(11)/1(1650), I(1390)/I(165o), I(1450)/I(1650), 1(1540)/I(1650), that makes them numerically equal to mean values of protein-specific bands
    [7] 7. The method as claimed in claims 1-2, wherein said epidermal healthy, benign, premalignant and malignant skin cancerous tissue is human tissue, that is also applicable to animal tissue.
    [8] 8. The method as claimed in claims 1-2, wherein said known skin epidermal benign, premalignant and malignant skin cancerous tissue is selected from the group, consisting of benign nevi, Bowen's disease, malignant melanoma, squamous cell carcinoma, basal cell carcinoma, that is applicable to other benign, premalignant and malignant skin tumours.
    [9] 9. The method as claimed in claims 1-2, wherein said epidermal benign, premalignant and malignant cancerous tissue is skin tissue, that is also applicable to tissue from other organs. 32 WO 2013/123950 PCT/EE2013/000001 STATEMENT UNDER ARTICLE 19 (1) Amended claims for PCT/EE2013/000001 precisely indicate features of the invention necessary to define the claimed subject matter, and recite each and every step of a method in the claims but, which, in combination, are part of the prior art. As a result, there have been provided a number of dependent claims, claiming specific forms of the invention, claimed in the independent claims, even though these features of dependent claims should be considered as constituting in themselves an invention. The amendments of the claims do not go beyond the content of this international application as originally filed. Originally filed broadly worded claims 1 to 8 are very general and contain no specific features to distinguish them from those in the prior art. Therefore, amendments to the claims are proposed, as original Claim 1 amended; original Claims 2 to 4 cancelled; original Claims 5 to 8 are replaced by Claim 2; new Claims 3-9 are added, in order to: * clearly formulate idea of inventive step in a method claim to protect the invention of a particular use for known things * clearly formulate idea of inventive step how the comparisons and calculations that are referred to in the claims would actually be used, in order to objectively evaluate intra and inter-molecular interactions expressed in the patterns of DNA- and protein-specific sequences for common and specific characterization of carcinogenesis, to indicate the grade of activity and a systematic progression in cancerous tissues, and the method as it is written requires each calculation and comparison step to be made e introduce inventive aspects of the invention in dependent claims as grouped together to the extent and in the most practical way possible to state concisely all features of the invention it is desired to protect by the description of the original application 33
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    同族专利:
    公开号 | 公开日
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    JP2019207235A|2019-12-05|
    SG10201912241WA|2020-02-27|
    KR20140130191A|2014-11-07|
    JP6991442B2|2022-01-12|
    US20190317015A1|2019-10-17|
    KR102282013B1|2021-07-27|
    WO2013123950A4|2013-10-24|
    US10809187B2|2020-10-20|
    EP2825866A1|2015-01-21|
    SG11201405048TA|2014-09-26|
    MY172569A|2019-12-03|
    WO2013123950A1|2013-08-29|
    JP2021192034A|2021-12-16|
    AU2013224476B2|2017-02-02|
    JP2015508166A|2015-03-16|
    CA2901954C|2021-05-04|
    IL234048D0|2014-09-30|
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    法律状态:
    2017-06-01| FGA| Letters patent sealed or granted (standard patent)|
    优先权:
    申请号 | 申请日 | 专利标题
    US201261595050P| true| 2012-02-21|2012-02-21||
    US61/595,050||2012-02-21||
    PCT/EE2013/000001|WO2013123950A1|2012-02-21|2013-02-21|Analytical method for common and specific characterization of skin carcinogenesis by ftir microspectroscopy|
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