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  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
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  • G01N2333/205—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Campylobacter (G)

Definitions

• the present invention relates to a biomarker or a combination of biomarkers in salivary samples that, on the one hand, help to the diagnosis of Epidermoid Cancer of the Head and Neck and, on the other hand, predict the response to treatment of a subject with this disease.
• Epidermoid Cancer of the Head and Neck is a malignant neoplastic entity originated in the epithelium that covers the mucosa of the aerodigestive tract. Its annual incidence of 700,000 patients places it in sixth positition of the world ranking in terms of more frequent cancers. Of the 53,640 new cases of head and neck tumours (oral cavity, pharynx and larynx) that were detected in the United States in 2013, 95% of them corresponded to epidermoid cancer of the head and neck.
• Spain is one of the countries with the highest incidence of epidermoid cancer of the head and neck in the entire European continent, proof of this are the 11,513 cases of patients with epidermoid cancer of the head and neck that were diagnosed in 2002.
• saliva as a diagnostic tool or evolutionary follow-up of certain pathologies or diseases, has acquired great relevance in the field of biomedicine.
• the main advantage of the use of saliva as a diagnostic or follow-up tool is that consists on a non- invasive method, easy to collect, inexpensive, difficult to alter and stable under non refrigerated conditions.
• the human oral microbiota of which the salivary microbiota is also part comprises the set of microorganisms and pathogens that are components of the oral cavity. Its function is to protect the oral cavity and prevent the development of diseases.
• massive sequencing techniques has allowed to study changes in the composition of the oral microbiota of healthy and sick patients. Some of the studies carried out so far, show that sites with oncological character have a lower diversity of species.
• salivary species such as Capnocytophaga gingivalis, Prevotelle melaninogenica, and Streptococcus Mitis correlate with epidermoid cancer of the head and neck.
• Dysbiosis between epidermoid head and neck tumour sites and healthy tissues shows a decrease in Actinobacteria and Firmicutes phylo when compared with healthy tissues.
• analysis of the results at genus or species level does not show the same trend.
• Epidermoid cancer of the head and neck is frequently diagnosed in advanced stages of the disease, conferring it a grim prognosis. At present, it seems paradoxical that, with the available means, there are no effective programs for the early detection of this type of tumours.
• Rothia is the only one that has been used as an oral biomarker with clinical indications for carcinoma detection in the oral and oropharyngeal cavity until now [Lim Y, Fukuma N, Totsika M, Kenny L, Morrison M, Punyadeera C: The Performance of an Oral Microbiome Biomarker Panel in Predicting Oral Cavity and Oropharyngeal Cancers. Front Cell Infect My 2018, 8],
• salivary microbiota shows high potential as a prognostic and diagnostic marker for the development of screening programs helping to the early diagnosis of epidermoid cancer of the head and neck in high-risk patients, and on the other hand it has potential to diagnose and predict both evolution and follow-up of patients with epidermoid cancer of the head and neck, being able to complement the known classic diagnostic / prognostic factors.
• Alloprevotella genus and, especially, its Alloprevotella rava and Alloprevotella tannerae species are at lower levels in salivary biological samples of patients with epidermoid cancer of the head and neck than in general population.
• sequences SEQ I D NO 1 to SEQ I D NO 3 are representative sequences of this genus and, therefore, useful for identification of bacteria belonging to this genus in such samples through a PCR procedure or by means of any sequencing technique, being of special interest the molecular diagnostic techniques by microarray.
• SEQ ID NO 2 >be884fc0a4b0abbl3e06f9dal978b834
• GGGTATCAAAC GGGTATCAAAC
• Other representative sequences of this genus will be known by the person skilled in the art and they will serve to identify those bacteria belonging to this genus in a saliva or breath sample.
• Prevotella genus is at lower levels in salivary biological samples of patients with epidermoid cancer of the head and neck than in general population. Furthermore, it is noted that the following sequences SEQ I D NO 4 to SEQ I D NO 6, are representative sequences of this genus and, therefore, useful for the identification of bacteria belonging to this genus in such samples through PCR or any other sequencing technique, including the molecular diagnostic techniques used by DNA microarray.
• the Campylobacter genus is at lower levels in salivary biological samples of patients with epidermoid cancer of the head and neck than in general population. Furthermore, it is noted that the following sequences SEQ I D NO 7 to SEQ I D NO 9, are representative sequences of this genus and, therefore, useful for the identification of bacteria belonging to this genus in such samples through PCR or any other sequencing technique.
• SEQ ID NO 12 >40a5d7d7947a9b9f428d2a4de54030d2
• Catonella genus is present at lower levels in salivary biological samples of patients with epidermoid cancer of the head and neck compared with general population.
• SEQ I D NO 13 to SEQ I D NO 15 are representative sequences of this genus and, therefore, useful for identification of bacteria belonging to this genus in such samples through PCR or any other sequencing technique, including the molecular diagnostic techniques used by DNA microarray.
• SEQ ID NO 14 >60000a298e962dd45ea8f64f83dl3296
• SEQ ID NO 15 >8a4f390e5a28fa4b319a417165699560
• the Porphyromona genus is present at lower levels in salivary biological samples of patients with epidermoid cancer of the head and neck compared with general population.
• the following sequences SEQ I D NO 16 to SEQ I D NO 18, are representative sequences of this genus and, therefore, useful for identification of bacteria belonging to this genus in such samples through PCR or any other sequencing technique, including the molecular diagnostic techniques used by DNA microarray.
• SEQ ID NO 18 >b79c9d865f361911a29c5ccda8af5804
• the Fretibacterium genus is at lower levels in salivary biological samples of patients with epidermoid cancer of the head and neck than in general population. Furthermore, it is noted that the following sequences SEQ I D NO 19 to SEQ I D NO 21, are representative sequences of this genus and, therefore, useful for identification of bacteria belonging to this genus in such samples through PCR or any other sequencing technique, including the molecular diagnostic techniques used by DNA microarray.
• a first aspect of the present invention relates to the in vitro use of the level or concentration of bacteria in a salivary or breath sample, belonging to the genus of Alloprevotella, Prevotella, Campylobacter, Rothia, Catonella, Porphyromona, Fretibacterium, or any combination thereof, for the diagnosis of head and neck cancer, esophageal cancer, lung cancer, stomach cancer or carcinomas or epidermoid cancers selected from the list consisting of epidermoid cancer of the esophagus, epidermoid cancer of the lung, epidermoid carcinoma of the stomach, or epidermoid cancer of the head and neck, in a patient, or to obtain useful data that enable such diagnosis.
• bacteria belonging to the Alloprevotella genus are Alloprevotella rava and / or Alloprevotella tannerae species.
• An alternative embodiment of the first aspect of the invention refers to a method for in vitro diagnosis or collection of useful data to support in such diagnosis, of a subject suspected of resembling head and neck cancer, esophageal cancer, lung cancer, stomach cancer or epidermoid cancers selected from the list consisting of epidermoid cancer of the esophagus, epidermoid cancer of the lung, epidermoid carcinoma of the stomach, or epidermoid cancer of the head and neck, which comprises the use as an indicator in a salivary or breath sample obtained from such subject, of the bacteria level or concentration in such sample belonging to the genus of Alloprevotella, Prevotella, Campylobacter, Rothia, Catonella, Porphyromona, Fretibacterium, or any combination thereof, where if the level or concentration in such salivary sample of
• the method comprises the use as an indicator in a salivary or breath sample obtained from such subject, of at least the level or concentration in such sample of bacteria belonging to the genus of Alloprevotella, where if the level or concentration in the salivary sample of bacteria belonging to such genus is decreased or significantly lower than in a salivary sample obtained from a healthy subject or compared with a reference value, this is indicative of that such subject shows head and neck cancer, esophageal cancer, lung cancer, stomach cancer or carcinomas or epidermoid cancers selected from the list that consists of epidermoid cancer of the esophagus, epidermoid cancer of the lung, epidermoid cancer of the stomach, or epidermoid cancer of the head and neck.
• the method comprises the use as an indicator in a salivary or breath sample obtained from such subject, of at least the level or concentration in such sample of bacteria belonging to the genus of Prevotella, where if the level or concentration in such salivary sample of bacteria belonging to such genus, is decreased or significantly lower than in a salivary or breath sample obtained from a healthy subject or compared with a reference value, this is indicative of that such subject shows head and neck cancer, esophageal cancer, lung cancer, stomach cancer or carcinomas or epidermoid cancers selected from the list that consists of epidermoid cancer of the esophagus, epidermoid cancer of the lung, epidermoid cancer of the stomach, or epidermoid cancer of the head and neck.
• the method comprises the use as an indicator in a salivary or breath sample obtained from such subject, of at least the level or concentration in such sample of bacteria belonging to the genus of Campylobacter, where if the level or concentration in such salivary or breath sample of bacteria belonging to such genus, is decreased or significantly lower than in a salivary or breath sample obtained from a healthy subject or compared with a reference value, this is indicative of that such subject shows head and neck cancer, esophageal cancer, lung cancer, stomach cancer or carcinomas or epidermoid cancers selected from the list consisting of epidermoid cancer of the esophagus, epidermoid cancer of the lung, epidermoid cancer of the stomach, or epidermoid cancer of the head and neck .
• the method comprises the use as an indicator in a salivary or breath sample obtained from such subject, of at least the level or concentration in such sample of bacteria belonging to the genus of Catonella, where if the level or concentration in such salivary or breath sample of bacteria belonging to such genus, is decreased or significantly lower than in a salivary or breath sample obtained from a healthy subject or compared with a reference value, this is indicative of that such subject shows head and neck cancer, esophageal cancer, lung cancer, stomach cancer or carcinomas or epidermoid cancers selected from the list consisting of epidermoid cancer of the esophagus, epidermoid cancer of the lung, epidermoid cancer of the stomach, or epidermoid cancer of the head and neck.
• the method comprises the use as an indicator in a salivary or breath sample obtained from such subject, of at least the level or concentration in such sample of bacteria belonging to the genus of Porphyromona, where if the level or concentration in such salivary or breath sample of bacteria belonging to such genus, is decreased or significantly lower than in a salivary or breath sample obtained from a healthy subject or compared with a reference value, this is indicative of that such subject shows head and neck cancer, esophageal cancer, lung cancer, stomach cancer or carcinomas or epidermoid cancers selected from the list consisting of epidermoid cancer of the esophagus, epidermoid cancer of the lung, epidermoid cancer of the stomach, or epidermoid cancer of the head and neck.
• the method comprises the use as an indicator in a salivary or breath sample obtained from such subject, of at least the level or concentration in such sample of bacteria belonging to the genus of Fretibacterium, where if the level or concentration in such salivary or breath sample of bacteria belonging to such genus, is decreased or significantly lower than in a salivary or breath sample obtained from a healthy subject or compared with a reference value, this is indicative of that such subject shows head and neck cancer, esophageal cancer, lung cancer, stomach cancer or carcinomas or epidermoid cancers selected from the list consisting of epidermoid cancer of the esophagus, epidermoid cancer of the lung, epidermoid cancer of the stomach, or epidermoid cancer of the head and neck.
• the method comprises the use as an indicator in a salivary or breath sample obtained from such subject, of at least the level or concentration in such sample of bacteria belonging to the genus of Rothia, where if the level or concentration in such salivary or breath sample of bacteria belonging to such genus, is decreased or significantly lower than in a salivary or breath sample obtained from a healthy subject or compared with a reference value, this is indicative of that such subject shows head and neck cancer, esophageal cancer, lung cancer, or stomach cancer or carcinomas or epidermoid cancers selected from the list consisting of epidermoid cancer of the esophagus, epidermoid cancer of the lung, epidermoid cancer of the stomach, or epidermoid cancer of the head and neck.
• the epidermoid cancer of the head and neck is considered as the set of malignant tumours produced in the mucosa epithelium of the aerodigestive tracts.
• This type of cancer represents the 90% of the head and neck cancers and according to their characteristics are classified into three groups:
• pharynx and larynx group including epidermoid carcinomas mainly related to tobacco and alcohol and with a greater or lesser degree of differentiation.
• Nostrils-nasosinusal cavities group including epidermoid cancers produced in the lining epithelium of the nasal and paranasal sinuses.
• - Nasopharynx group including epidermoid cancers mainly related to the Epstein-Barr virus (EBV).
• EBV Epstein-Barr virus
• carcinoma or epidermoid cancer are not only related to epidermoid cancer of the head and neck, but also to epidermoid cancer of the esophagus, epidermoid cancer of the lung and / or epidermoid cancer of the stomach.
• Epidermoid esophageal carcinoma develops in the inner tissue of the esophagus. It usually occurs in the upper and middle part of the esophagus. It is the most common esophageal cancer in the world.
• Epidermoid cancer of the lung occurs in the lung tissues, usually in the cells covering the respiratory tract (bronchi) that connect the trachea to the lung. This type of cancer represents between 25 and 30 % of lung cancers.
• Epidermoid cancer of the stomach is a gastric epidermoid cancer that originates in the stomach. It is a very rare tumour, but it should be considered as a possibility of a gastric neoplasm. It is usually diagnosed in advanced stages, which gives it a poor prognosis.
• the present invention sufficiently describes the representative nucleotide sequences (i.e SEQ ID No. 1 to SEQ No. 21) that allow identifying the presence as well as the concentration of each of the genera mentioned herein in salivary or breath samples.
• a second aspect of the present invention relates to an in vitro method for monitoring the evolution of a subject with head and neck cancer, esophageal cancer, lung cancer, gastric cancer or epidermoid cancers selected from the list consisting of epidermoid cancer of the esophagus, epidermoid cancer of the lung, epidermoid cancer of the stomach , or epidermoid cancer of the head and neck , which comprises the following steps:
• a third aspect of the present invention relates to an in vitro method for monitoring the evolution of a subject with head and neck cancer, esophageal cancer, lung cancer, stomach cancer or epidermoid cancers selected from the list consisting of epidermoid cancer of the esophagus, epidermoid cancer of the lung, epidermoid cancer of the stomach, or epidermoid cancer of the head and neck, which comprises the following steps:
• a fourth aspect of the invention refers to an in vitro method for predicting the therapeutic response of a patient diagnosed with head and neck cancer, esophageal cancer, lung cancer, stomach cancer or epidermoid cancers selected from the list consisting of epidermoid cancer of the esophagus, epidermoid cancer of the lung, epidermoid cancer of the stomach, or epidermoid cancer of the head and neck, where such method comprises the following steps:
• a fifth aspect of the invention refers to an in vitro method for predicting the therapeutic response of a patient diagnosed with head and neck cancer, esophageal cancer, lung cancer, stomach cancer or epidermoid cancers selected from the list consisting of epidermoid cancer of the esophagus, epidermoid cancer of the lung, epidermoid cancer of the stomach, or epidermoid cancer of the head and neck, where such method comprises the following steps:
• Molecular therapies monoclonal antibodies.
• Molecular therapies with cetuximab a monoclonal antibody that binds to the EGFR antigen (Epidermal Growth Factor Receptor) in epidermoid cancer of the head and neck; and
• the reference value is preferably understood as the result of the data of a mathematical algorithm that uses the concentration and / or the total amount of each bacterium belonging to one or more of the genera proposed in the present invention for general population or a healthy subject.
• the best value for sensitivity and specificity will be proposed automatically using the algorithm.
• This algorithm will provide values together with the proposed one, as well as changing sensitivity and specificity values providing more information to physicians and allowing them to decide the best test and cut-off value for each patient or specific situation.
• a bacterium belonging to any proposed genera of the salivary or breath sample from such subject is possible to determine a bacterium belonging to any proposed genera of the salivary or breath sample from such subject, but not limiting to it, through massive sequencing of the saliva or breath genoma obtaining by this way the total number of bacterial sequences in the saliva together with the total number of other bacteria in that saliva sample.
• determination is performed by PCR or real-time PCR method.
• such bacteria levels or concentration refer to the total amount of bacteria belonging to genus category out of the total bacteria in such sample.
• the levels or concentration of bacteria belonging to the genus of Alloprevotella, Prevotella, Campylobacter, Catonella, Porphyromona, Fretibacterium, or any combination thereof are determined via an amplification reaction of a nucleic acid preparation derived from such sample using a pair of primers able to amplify one or more representative regions of such genera of bacteria.
• the amplification reaction is carried out by a real-time polymerase chain reaction.
• the detection of the amplification product is accomplished through a fluorescent intercalating agent.
• detection of the amplification product /s is performed by means of a labelled probe, wherein preferably the probe comprises at its 5 'end a reporter pigment and at its 3' end a "quencher” pigment or silencer or buffer.
• the method for the first to fifth aspect of the invention or any of its embodiments further comprises the storage of the method results on a data carrier, preferably wherein such data carrier is readable by computer.
• the method of the first to fifth aspect of the invention or any of its embodiments include at least the implementation of the comparative step and optionally the provision of a result as a consequence of such comparison by a computer program.
• An eighth aspect of the invention refers to a kit including one or more primer pairs able to amplify the bacteria belonging to the genus of Alloprevotella, Prevotella, Campylobacter, Catonella, Porphyromona, Fretibacterium, or any combination thereof.
• such kit is used to implement a method for the detection of bacteria from a sample of saliva or breath isolated from a human subject, that comprises the following steps: i) Contacting the sample to be analyzed with a reaction mixture, containing specific primers able to amplify bacteria belonging to the genus of Alloprevotella, Prevotella, Campylobacter, Catonella, Porphyromona, Fretibacterium, or any combination thereof, preferably for the production of Multiplex PCR. ii) Amplification by means of polymerase chain reaction.
• Identification of the products formed in the prior step being such information indicative of the levels or concentration of bacteria belonging to one or more of the genera of Alloprevotella, Prevotella, Campylobacter, Catonella, Porphyromona, Fretibacterium, or any combination thereof.
• the present invention protects the methodology per se abovementioned, as well as its use for the implementation of any of the methodologies described in the first to seventh aspects of the invention. Also, the present invention protects the use of the kit of the eighth aspect of the invention for implementation of the methodology according to any of the first to fifth aspects of the present invention.
• this eighth aspect of the invention preferably it provides a method for detecting bacteria belonging to Alloprevotella rava and / or Alloprevotella tannerae.
• fragments of DNA included or comprised in sequences 1 to 21 are amplified.
• amplification products which allow to identify the different species and bacterial groups are detected using probes.
• these probes have between 15 to 25 nucleotides in length.
• the primers can be designed by multiple alignment with programs such as CLUSTAL X, which allow the identification of highly conserved regions that serve as a template.
• PCR inhibitors such as humic and fulvic acids, heavy metals, heparin, etc. that result in false negatives, and although there are methods that reduce the concentration of these types of molecules, it is recommendable (see J. Hoorfar et al., "Making Internal Amprolization Control Mandatory for Diagnostic PCR” J. of Clinical Microbiology, Dec 2003, pp.5835) PCR tests containing an Internal Amplification Control (IAC).
• IAC Internal Amplification Control
• This IAC consist of no more than a fragment of DNA, which is amplified simultaneously together with the target sample, so its absence at the end of the tests is indicative of the presence of factors, that have caused an undesired development of the PCR.
• the term "specific” implies that the primers comprise a nucleotide sequence totally complementary to the genes or gene fragments employed by the present invention.
• the knowledge gained on the composition of the salivary microbiota in patients undergoing treatment along with the development of the diagnostic or prognostic KIT based on this salivary microbiota is of crucial importance, since it would allow the oncology specialist who treats these patients to have data on the probability of a patient showing or not epidermoid cancer of the head and neck in his first visits, in addition to the probability of response to treatment, as well as patient evolution.
• Beta diversity genera.
• ROC curve (the area under the curve is shown in table 2) for the Campylobacter genus.
• ROC curve (the area under the curve is shown in table 2) for the Porphyromonas genus.
• ROC curve (the area under the curve is shown in table 2) for the Fretibacterium genus.
• 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.
• Anatomopathological diagnosis of benign and malignant tumours of non-epidermoid variants adenocarcinoma, papillary carcinoma, mucoepidermoid carcinoma, anaplastic carcinoma, melanoma, lymphoepithelioma and paraganglioma.
• 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.
• Contingency tables and "Chi square (X 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.
• 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.
• 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 3. Characteristics of salivary microbiota results obtained from ROC curves.
• figure 6 and figure 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.
• figure 6 and figure 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.
• figure 6 and figure 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.
• figure 6 and figure 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.
• 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 Rsl6 could be useful for the diagnosis, preferably for the early diagnosis of disease and relapse of epidermoid cancer of the mouth and pharynx.
• 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.

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• 2019
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• 2020
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  • 2020-04-06 US US17/601,382 patent/US12584178B2/en active Active
  • 2020-04-06 ES ES20719135T patent/ES3053185T3/es active Active
  • 2020-04-06 JP JP2021560447A patent/JP7557789B2/ja active Active
  • 2020-04-06 CN CN202080041403.4A patent/CN114008458A/zh active Pending
  • 2020-04-06 AU AU2020252417A patent/AU2020252417B2/en active Active
  • 2020-04-06 WO PCT/EP2020/059761 patent/WO2020201575A1/en not_active Ceased
  • 2020-04-06 KR KR1020217036142A patent/KR20210149144A/ko active Pending
  • 2020-04-06 EP EP20719135.4A patent/EP3948285B1/en active Active
  • 2020-04-06 PL PL20719135.4T patent/PL3948285T3/pl unknown

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