SALIVARY BIOMARKERS FOR THE DETECTION OF EPIDERMOID CANCER OF THE HEAD AND NECK

Abstract

Present invention relates to the in vitro use of the level or concentration in a salivary or breath sample of bacteria belonging to the Alloprevotella, Prevotella, Campylobacter, Rothia, Catonella, Porphyromona, Fretibacterium genus, or any combination thereof, for the diagnosis of carcinomas or epidermoid cancers, especially epidermoid cancer of the head and neck, in a patient, or to obtain useful data that allow such a diagnosis.

Claims

1. Method for the in vitro diagnosis of a subject suspected of having epidermoid cancer of the oral cavity, pharynx and larynx, that comprises the use, as an indicator, in a salivary or breath sample obtained from said subject, of the concentration in such sample of bacteria belonging to the Alloprevotella genus, where if the concentration in such salivary or breath sample of bacteria belonging to Alloprevotella is significantly lower than in a salivary sample obtained from a healthy subject or with respect to a reference value, this is indicative that such subject has epidermoid cancer of the oral cavity, pharynx and larynx.

2. The method according to claim 1, wherein the bacteria belonging to the Alloprevotella genus is selected from the group consisting of Alloprevotella rava and/or Alloprevotella tannerae species.

3. The method according to any of claim 1 or 2, wherein a further indicator is used and said indicator is the concentration in said sample of bacteria belonging to the Prevotella genus, where if the concentration in such salivary or breath sample of bacteria belonging to the Prevotella genus is significantly lower than in a salivary or breath sample obtained from a healthy subject or with respect to a reference value, this is indicative that such subject has epidermoid cancer of the oral cavity, pharynx and larynx.

4. The method according to any of claims 1 to 3, wherein a further indicator is used and said indicator is the level or concentration in said sample of bacteria belonging to the Campylobacter genus, where if the level or concentration in such salivary or breath sample of bacteria belonging to Campylobacter is significantly lower than in a salivary or breath sample obtained from a healthy subject or with respect to a reference value, this is indicative that such subject has epidermoid cancer of the oral cavity, pharynx and larynx.

5. The method according to any of claims 1 to 4, wherein a further indicator is used and said indicator is the level or concentration in said sample of bacteria belonging to the Catonella genus, where if the level or concentration in such salivary or breath sample of bacteria belonging to Catonella is significantly lower than in a salivary or breath sample obtained from a healthy subject or with respect to a reference value, this is indicative that such subject has epidermoid cancer of the oral cavity, pharynx and larynx.

6. The method according to any of claims 1 to 5, wherein a further indicator is used and said indicator is the concentration in said sample of bacteria belonging to the Porphyromona genus, where if the level or concentration in such salivary or breath sample of bacteria belonging to Porphyromona is significantly lower than in a salivary or breath sample obtained from a healthy subject or with respect to a reference value, this is indicative that such subject shows epidermoid cancer of the oral cavity, pharynx and larynx.

7. The method according to any of claims 1 to 6, wherein a further indicator is used and said indicator is the concentration in such sample of bacteria belonging to the Fretibacterium genus, where if the level or concentration in such salivary or breath sample of bacteria belonging to Fretibacterium is significantly lower than in a salivary sample obtained from a healthy subject or with respect to a reference value, this is indicative that such subject has epidermoid cancer of the oral cavity, pharynx and larynx.

8. In vitro use of a kit comprising one or more pairs of primers able to amplify bacteria belonging to the Alloprevotella genus, and optionally Prevotella, Campylobacter, Catonella, Porphyromona, or Fretibacterium, for the implementation of the diagnostic method of any of claims 1 to 7.

9. Kit as defined in claim 8, to implement the methodology according to any of claims 1 to 7.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0083] FIG. 1. Rarefaction curves in cases and controls.

[0084] FIG. 2. Analysis of major components in cases and controls.

[0085] FIG. 3. Clusterization between different microorganisms at the genus level.

[0086] FIG. 4. Beta diversity: genera.

[0087] FIG. 5. Alpha diversity: genus.

[0088] FIG. 6. Differences between groups of genera described in table 2.

[0089] FIG. 7. ROC curve (the area under the curve is shown in table 2) for the Alloprevotella genus.

[0090] FIG. 8. ROC curve (the area under the curve is shown in table 2) for the Prevotella genus.

[0091] FIG. 9. ROC curve (the area under the curve is shown in table 2) for the Campylobacter genus.

[0092] FIG. 10. ROC curve (the area under the curve is shown in table 2) for the Rothia genus.

[0093] FIG. 11. ROC curve (the area under the curve is shown in table 2) for the Catonella genus.

[0094] FIG. 12. ROC curve (the area under the curve is shown in table 2) for the Porphyromonas genus.

[0095] FIG. 13. ROC curve (the area under the curve is shown in table 2) for the Fretibacterium genus.

EXAMPLE

Materials and Methods

Study Population

[0096] All patients older than 18 years with an anatomopathological diagnosis of epidermoid carcinoma of pharyngolaryngeal localization were studied.

Samples

[0097] Salivary and tissue samples were collected by the Otorhinolaryngology and Molecular Biology service, before signature of the informed consent by all patients who underwent treatment, of benign and malignant head and neck tumours during the period between January 2008 and December 2012, both inclusive. Thereby obtaining an initial total sample of 179 patients.

[0098] Subsequently, between these samples a total of 20 patients were chosen and, together with salivary samples from 20 healthy volunteers, an analysis of the hypervariable region of the r516 gene was performed in saliva samples. The results of the sequencing were analyzed in the biomedical research center of Bioithas, located in the scientific park of Alicante. Patients in both groups were selected in order to maintain the homogeneity in their sociodemographic characteristics.

[0099] Population included in the final analysis of the data was defined according to the following criteria:

Inclusion Criteria:

[0100] 18-90 age group. [0101] Location of the original primary tumour in pharynx, larynx or cervical ganglion. [0102] Anatomopathological diagnosis of epidermoid carcinoma. [0103] Curative or palliative surgical treatment. [0104] Collection of biological samples in surgery room: blood, saliva, tumour tissue and healthy tissue.

Exclusion Criteria:

[0105] Age less than 18 years or over 90 years. [0106] Primary tumour of localization other than larynx, pharynx and/or lymph nodes. [0107] Anatomopathological diagnosis of benign and malignant tumours of non-epidermoid variants: adenocarcinoma, papillary carcinoma, mucoepidermoid carcinoma, anaplastic carcinoma, melanoma, lymphoepithelioma and paraganglioma. [0108] Treatment with non-surgical organ preservation. [0109] Extranodal metastatic disease concomitant with the primary tumour. [0110] Insufficient tumour samples, with inability for anatomopathological study. [0111] Low number of salivary samples.

Preparation and Collection of Biological Samples

[0112] Biological samples were collected in the Otorhinolaryngology operating room at the time prior to the surgical intervention and in a fasting state of patients.

[0113] Saliva samples were collected using lentins placed in the sublingual region for a period of 10 minutes and prepared by the researcher. No method was used for the stimulation of salivary production.

[0114] All samples were identified and sent to the Research Unit of the Clinical Analysis Service (Laboratory of Molecular Therapy and Cancer Biomarkers), where they were processed. Saliva was centrifuged at 2500 rpm, 5 minutes, the supernatant was aliquotted and frozen at −80° C. until next use.

Statistic Analysis

[0115] Contingency tables and “Chi square (X.sup.2)” statistical test were used. Comparison of means with a dichotomous variable was evaluated using “Student's t” statistical test, as long as one variable was quantitative and the other categorical. In cases which the qualitative variable had more than 2 categories, the one-way variance analysis (ANOVA) was used.

[0116] Different analysis curves were elaborated: ROC curves (acronym of Receiver Operating Characteristic), for the graphic representation of Sensitivity versus 1—Specificity. Optimal cut-off point was determined for each salivary microbiome pattern by searching for the Yoiden index (IY) or maximum sensitivity and specificity point. Area under the curve (AUC) was calculated by the numerical integration of the ROC curves. Concentration with the highest AUC was identified as the strongest predictor for detection of epidermoid cancer of the head and neck. Data were analyzed using SPSS© (version 15.0) software.

Results

[0117] During the study period, a total of 20 patients with tumour pathology and 20 healthy patients corresponding to the control group were analyzed. Clinical-pathological characteristics of these patients are summarized in Table 1 below.

TABLE-US-00008 TABLE 1 Clinico-pathological characteristics of the patients under study Feature No. of cases (%) Age <55 yo  8 (10,5) 50-59 yo 26 (34,2) 60-69 yo 25 (32,9) 70-79 yo 12 (15,8) >80 yo  5 (6,6) Sex Male 75 (98,7) Female  1 (1,3) Tobacco Yes 69 (92,0) No  6 (8,0) Alcohol Yes 55 (72,4) No 21 (27,6) Comorbidities Diabetes 12 (15,8) EPOC 27 (35,5) Heart disease 19 (25,0) Liver disease  6 (7,9) Location Larynx 69 (90,8) Supreglottis 26 (37,1) Glottis 42 (60,0) Subglottis  2 (2,9) Pharynx  7 (9,2) Clasification pT T1 13 (17,8) T2 20 (27,4) T3 28 (38,4) T4 12 (16,4) Clasification pN N0 53 (69,7) N1  7 (9,2) N2 10 (13,2) N3  6 (7,9) Stages Stage I 13 (17,1) Stage II 20 (26,3) Stage III 22 (28,9) Stage IV 21 (27,6) Stage type Initial 33 (43,4) Advanced 43 (56,6)

[0118] Patients range from 38 to 86 years old with an average of 61.9 years old and a predominance of males (98.7%). 92% of the patients had a smoking habit and 72.4% had an enolic one. The majority had tumour of laryngeal location (90.8%), being the most frequent group the one corresponding to glottic tumours (60.0%).

[0119] From the obtained salivary samples, those genera with significant differences between groups were analyzed. These results are shown below in table 2.

TABLE-US-00009 TABLE 2 Test Statistics.sup.a g__Rothia g__Porphyromonas g__Alloprevotella g__Prevoteila 7 g__Campylobacter g__Catonella g__Fretibacterium Marin- 72,000 70,000 13,500 66,000 31,000 74,500 53,000 Whitney U Wilcoxon W 282,000 280,000 223,500 266,000 241,000 284,500 263,000 Z −3,462 −4,228 −5,085 −4,214 −4,573 −3,422 −4,255 Asym Sig. 0.001 0.000 0.000 0.000 0.000 0.001 0.000 (2-tailed) .sup.aGrouping Variable: caso 1

[0120] Additionally, and on the basis of table 2 and FIGS. 6 to 13, table 3 shows the different areas under the curve for each of the genera showing statistically significant differences between groups in the present study:

TABLE-US-00010 TABLE 3 Characteristics of salivary microbiota results obtained from ROC curves. ROC Area Under the Curve Typical Asymptotic Cut-off Contrast variables Area error significance values Sensibility Specificity Prevotella 0.86 8 0.65 0.95 Alloprevotella genus 0.966 19 0.95 0.85 Alloprevotella rava 0.966 19 −0.95 −0.85 Alloprevotella Tannarae 0.966 19 −0.95 −0.85 Campylobacter genus 0.923 61.5 1 0.80 Rothia genus 0.820 9563 0.75 0.85 Catonella genus 0.814 54 0.85 0.75 Porphyromona genus 0.825 1.5 65 1 Fretibacterium genus 0.868 7.5 0.80 0.85

[0121] As shown in table 3, FIG. 6 and FIG. 7, both in absolute values and in the presence percentage of Alloprevotella genus in salivary samples and, especially, of Alloprevotella rava and Alloprevotella tannera species, there is a negative correlation between subjects with epidermoid cancer of mouth and pharynx and healthy subjects.

[0122] As shown in table 3, FIG. 6 and FIG. 8, both in absolute values and in the presence percentage of Prevotella genus in salivary samples, there is a negative correlation between subjects with epidermoid cancer of the mouth and pharynx and healthy subjects.

[0123] As shown in table 3, FIG. 6 and FIG. 9, both in absolute values and in the presence percentage of Campylobacter genus in salivary samples, there is a negative correlation between subjects with epidermoid cancer of mouth and pharynx and healthy subjects.

[0124] As shown in table 3, FIG. 6 and FIG. 10, both in absolute values and in the presence percentage of Rothia genus in salivary samples, there is a positive correlation between subjects with epidermoid cancer of mouth and pharynx and healthy subjects.

[0125] As shown in table 3, FIG. 6 and FIG. 11, both in absolute values and in the presence percentage of Catonella genus in salivary samples, there is a negative correlation between subjects with epidermoid cancer of the mouth and pharynx and healthy subjects.

[0126] As shown in table 3, FIG. 6 and FIG. 12, both in absolute values and in the presence percentage of Porphyromona genus in salivary samples, there is a negative correlation between subjects with epidermoid cancer of the mouth and pharynx and healthy subjects.

[0127] As shown in table 3, FIG. 6 and FIG. 13, both in absolute values and in the presence percentage of Fretibacterium genus in salivary samples, there is a negative correlation between subjects with epidermoid cancer of the mouth and pharynx and healthy subjects.

[0128] Design of a RNA quantification test (qPCR) by any of the known methods including metagenomics, massive sequencing, qPCR, DNA microarray, and the correlation of these values with the values found by sequencing the hypervariable region Rs16 could be useful for the diagnosis, preferably for the early diagnosis of disease and relapse of epidermoid cancer of the mouth and pharynx.

[0129] Additionally, the use of probiotics that modulate the salivary microbiota of these patients could be a preventive or intervention treatment for patients with epidermoid cancer of the larynx and pharynx mouth.