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WO2024239087A1 - Iintg-hpv method for direct determination of the physical patterns of the hpv genome in relation to the genome of infected cells - Google Patents

Iintg-hpv method for direct determination of the physical patterns of the hpv genome in relation to the genome of infected cells Download PDF

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WO2024239087A1
WO2024239087A1 PCT/BR2024/050210 BR2024050210W WO2024239087A1 WO 2024239087 A1 WO2024239087 A1 WO 2024239087A1 BR 2024050210 W BR2024050210 W BR 2024050210W WO 2024239087 A1 WO2024239087 A1 WO 2024239087A1
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hpv
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Eleonidas MOURA LIMA
Marla Simone LOPES RODRIGUES
Rodrigo VILAR MARQUES
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Definitions

  • the present invention relates to the areas of molecular biology and in vitro molecular diagnostics in relation to the direct determination of the patterns of the episomal, mixed and integrated physical status of the genome of the Human Papilloma Virus - HPV and other viruses, which present similar behavior, in relation to the genome of infected cells, applied to the diagnosis and prognosis of cancers, which have as their main cause the infection followed by the integration of the viral genome into the genome of infected cells, such as Cervical Cancer - CC.
  • the main cause in the etiology of CC is infection by the Human Papilloma Virus – HPV followed by the integration of its genome into the genome of infected cells (Zur Hausen, 2002).
  • HPV belonging to the Papillomaviridae family, Papillomavirus genus are non-enveloped viruses with icosahedral symmetry and a circular, double-stranded DNA genome with approximately 8,000 base pairs (Zheng & Baker, 2006).
  • the HPV genome has two main regions, a coding region and a non-coding region. This second region controls the transcription and duplication of the viral genome and is called the LCR ( long control region).
  • the coding region consists of six genes that are expressed early and two genes that are expressed late, called E ( Early ) and L ( Late ), respectively, totaling eight open reading frames (ORFs).
  • E Early
  • L Late
  • ORFs open reading frames
  • the early region or E encodes the non-structural proteins: E1, E2, E4, E5, E5 and E7, while the late region or L is responsible for the synthesis of the L1 and L2 proteins, which will form the viral capsid (Villa et al. 2002).
  • the inactivation of the E2 gene is key in the genesis of CC and other cancers, which are caused by infection with high-risk HPV subtypes followed by the integration of its genome into the genome of the infected cell, since its gene product acts directly in the regulation of the transcriptions of the oncogenic genes E6 and E7, silencing them.
  • the E2 gene is inactivated, because the recombination sites between the viral genome and the genome of the infected cell usually occur in the sequence of the E2 gene (Dall et al., 2008).
  • the molecular mechanisms related to the malignant phenotype include the action of the E7 protein in the inactivation of the tumor suppressor protein pRb , as well as the E6 protein being involved in the inactivation of the p53 protein, which is a tumor suppressor, accelerating its proteolytic degradation.
  • the association of viral proteins with the products of tumor suppressor genes presents variability according to the oncogenic potential of the HPV subtype (Ghittoni et al., 2010).
  • HPV subtypes detected in CC samples are HPV subtypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73 and 82, all of which belong to the alpha-papillomavirus genus (Castellsagué et al., 2016).
  • HPV subtypes 16 and 18 are the most prevalent, representing 70% of cases and having the greatest geographical distribution in the world (de Sanjose et al., 2010).
  • the rates of viral integration found in carcinomas resulting from infection by HPV subtypes 16 and 18 by indirect methods are approximately between 80% and 100%, respectively (Pett & Coleman, 2007).
  • the evolution of CC considering the patterns of the physical status of the genome of high-risk HPV subtypes in relation to the genome of infected cells, comprises three stages: the first or initial stage, which is characterized by viral infection with maintenance of the integrity of the viral genome in the circular form or episomal pattern, at this stage there is a possibility of regression of the infection and the lesion; the second or intermediate stage is characterized by the coexistence of the episomal and integrated patterns, called a mixed pattern; and the third or late stage is characterized only by the presence of the integrated pattern.
  • the stages that present the integrated pattern are responsible for the carcinogenic process, with no possibility of regression in 99% of cases (Vinokurova et al., 2008).
  • the main PCR-based protocols that suggest indirect analysis of the physical status of the HPV genome compared to the genome of infected cells are: a) amplification of HPV oncogenic transcripts (Klaes et al., 1999), b) amplification and relative analysis between the E2 and E6 genes (Yoshinouchi et al., 1999), c) amplification of the integrated HPV sequence by franking with cellular sequences (DIPS) (Luft et al., 2001) and d) amplification of the fusion region using specific primers and restriction enzymes (Thorland et al., 2000).
  • the IntG-HPV molecular method is characterized as a disruptive innovation in the areas of molecular biology for diagnosis, prognosis, screening and prevention of Cervical Cancer, among other neoplasms, which are caused by high-risk oncological HPV subtypes in in natura samples, since it directly determines the patterns of the episomal, mixed and integrated physical status of the viral genome in relation to the genome of infected cells, at low cost, quickly and with national technology.
  • the IntG-HPV molecular method directly determines the patterns of the episomal, mixed and integrated physical status of the viral genome in relation to the genome of infected cells, at low cost, quickly and using in natura samples.
  • the IntG-HPV molecular method is characterized as a disruptive innovation in the areas of molecular biology for diagnosis, prognosis, screening and prevention of Cervical Cancer, among other neoplasms, which are caused by high oncological risk HPV subtypes.
  • FIG. 1 Presents the results of the amplification of the PCR-Multiplex reaction of the IntG-HPV method in samples previously genotyped for HPV-16 in cervical desquamation cells with low-grade lesions and in a DNA sample from the CCU cell line SiHa , evidencing the episomal and integrating patterns of HPV-16, respectively.
  • FIG. 2 Presents the results of the amplification of the PCR-Multiplex reaction of the IntG-HPV method in samples previously genotyped for HPV-16 in cervical desquamation cells with low-grade lesions and in a DNA sample from the CC cell line Ca Ski , evidencing the episomal and mixed patterns of HPV-16, respectively.
  • FIG. 3 Presents the results of the amplification of the PCR-Multiplex reaction of the IntG-HPV method in samples previously genotyped for HPV-16 in cervical desquamation cells with low-grade lesions and in DNA sample from the CC cell line Ca Ski and DNA sample from the CC cell line SiHa , evidencing the episomal, mixed and integrated patterns of HPV-16, respectively.
  • the IntG-HPV METHOD FOR DIRECT DETERMINATION OF PATTERNS OF THE PHYSICAL STATUS OF THE HPV GENOME IN RELATION TO THE GENOME OF INFECTED CELLS targets HPV regions that directly determine the patterns of the Episomal, Mixed and Integrated physical status of the HPV genome in relation to the genome of the infected cell, but is not limited to the diagnosis and prognosis of HPV infection in the screening and prevention of CC.
  • the IntG-HPV method innovates in the direct determination of the integration of the viral genome into the genome of the infected cell and can be applied to other viruses, which have a circular DNA genome and present molecular behavior similar to HPV, such as human polyomavirus, which causes Merkel cell skin cancer (Feng et al. 2018).
  • the innovation of the IntG-HPV method consists of the direct determination of the patterns of the Episomal, Mixed and Integrated physical status of the genome of high-risk oncological HPV subtypes in in natura samples of desquamation cells and/or biopsies of the genital tract, among others, amplified preferentially by Multiplex PCR in recombinant and non-recombinant regions of the HPV genome in relation to the genome of infected cells.
  • the IntG-HPV METHOD FOR DIRECT DETERMINATION OF PATTERNS OF THE PHYSICAL STATUS OF THE HPV GENOME IN RELATION TO THE GENOME OF INFECTED CELLS comprises two (2) steps, namely:
  • the first step involves the application of the IntG-HPV Method, which is characterized by the production of specific amplicons from the primer sets of the recombinant and non-recombinant regions of the HPV genome in relation to the genome of infected cells, preferably by PCR-Multiplex ;
  • the second stage is characterized by the amplicon analysis processes, which determine the Episomal, Mixed and Integrated molecular patterns;
  • the IntG-HPV method consists of producing specific amplicons from primer sets of the recombinant and non-recombinant regions of the HPV genome in relation to the genome of infected cells, preferably by Multiplex PCR , where the results can be analyzed either by Real Time PCR system or by polyacrylamide gel - PAG or agarose.
  • the integration of the HPV genome into the genome of infected cells is a determining factor for the establishment and progression of the neoplastic process in infected tissues, such as in cervical cells, which, when infected and followed by integration of the HPV genome, evolve into CC.
  • the IntG-HPV method presents an innovative approach for determining the physical status of the HPV genome, especially of high-risk HPV subtypes, such as HPV-16.
  • This method determines the change in the HPV genome from the episomal form to the integrated form directly in infected cells, and is a direct method for determining the integration of the genome of high-risk HPV subtypes into the genome of infected cells.
  • the IntG-HPV method analyzes the regions of the E1, E2, E4, E5, E6 and E7 genes of the HPV subtype of interest. In the episomal pattern, specific amplicons are generated for the HPV subtype of interest. In the integrated pattern, depending on which recombination site participates in the integration mechanism, there will be an absence of specific amplicons .
  • the amplification of the IntG-HPV method was performed preferably by PCR-Multiplex using DNA samples from the CCU cell lines SiHa, Ca Ski and in 35 DNA samples from cervical desquamation cells with low-grade lesions and positive for HPV-16, preferably in the final volume of 50 ⁇ L, being 4 ⁇ L of the primer mix at a concentration of 100pMol for each recombination region and 2 ⁇ L of samples (maximum 50ng of DNA per reaction), 19 ⁇ L of ultrapure water free of DNAse and RNAse and 25 ⁇ L of MasterMix 2X (with Hot start DNA polymerase).
  • the set of primers used in the Multiplex PCR of the IntG-HPV method for HPV-16 preferably correspond to the sequences: SEQ ID NO:1; SEQ ID NO:2; SEQ ID NO:3; SEQ ID NO:4; SEQ ID NO:5; SEQ ID NO:6 and SEQ ID NO:7 .
  • the conditions of the PCR-Multiplex reaction of the IntG-HPV method were preferably, 1 cycle of 95°C for 3 minutes and 40 cycles, being 95°C for 30 seconds, 62°C for 1 minute and 30 seconds and 72°C for 2 minutes, and 1 final extension cycle of 72°C for 5 minutes.
  • the 88bp, 313bp, 450bp and 657bp amplicons are preferentially generated.
  • the absence of the 657bp amplicon is associated with the integration site of the HPV-16 genome, involving the regions adjacent to the E7 gene in a clockwise direction.
  • the absence of the 313bp amplicon is associated with the integration site of the HPV-16 genome, involving the regions adjacent to the L2 gene in a counterclockwise direction.
  • the 450bp amplicon is produced from the non-recombinant regions.
  • its amplification is constant and serves as a reference for determining the mixed pattern.
  • RR Relative Ratio
  • the Relative Ratio is calculated by dividing the constant amplicon intensity or amplification value (IA k ) by the non-constant amplicon intensity or amplification value (IA n ) for each amplicon.
  • the percentage of integration of the HPV subtypes of interest is calculated by multiplying the Relative Ratio by 100 and dividing the product by the intensity or amplification value of the constant amplicon (IA k ).
  • Example 1 Analysis of the physical status of the HPV-16 genome in a DNA sample from the SiHa CC cell line by the IntG-HPV method.
  • the IntG-HPV method was validated in a DNA sample extracted from the SiHa CC cell line, which is a suitable in vitro model for this purpose.
  • This cell line was immortalized by the integration of the HPV-16 genome and established in 1970 from a CC sample of a 55-year-old patient (Friedl F, et al. 1970).
  • the set of primers of the IntG-HPV method for HPV-16 preferably correspond to the sequences: SEQ ID NO:1; SEQ ID NO:2; SEQ ID NO:3; SEQ ID NO:4; SEQ ID NO:5; SEQ ID NO:6 and SEQ ID NO:7 .
  • the preferred conditions of the PCR-Multiplex reaction of the IntG-HPV method were 1 cycle of 95°C for 3 minutes and 40 cycles, being 95°C for 30 seconds, 62°C for 1 minute and 30 seconds and 72°C for 2 minutes, and 1 final extension cycle of 72°C for 5 minutes.
  • the results of the IntG-HPV method suggest the association of the Episomal pattern of the HPV-16 genome with the genome of the uterine cervical desquamation cells with low-grade lesions and the association of the Integrated pattern of the HPV-16 genome with the genome of the SiHa CC cell line through the absences of the 88bp and 657bp amplicons , which are associated with the recombination site of the HPV-16 genome, involving the regions adjacent to the E7 gene in a clockwise direction, as shown in Figure 1.
  • Example 2 Analysis of the physical status of the HPV-16 genome in a DNA sample from the Ca Ski CC cell line by the IntG-HPV Method.
  • the IntG-HPV method was validated on a DNA sample extracted from the Ca Ski CC cell line.
  • This cell line can be used in testing and calibration in ISO 17025 accredited laboratories to challenge assay performance, validate or compare test methods, and establish sensitivity, linearity, and specificity during validation or implementation of assays related to in vitro molecular diagnostics (ATCC, 2022).
  • the Ca Ski CC cell line was immortalized by the integration of a few copies of the HPV-16 genome and established in 1977 from cervical carcinoma epidermoid cells of a 40-year-old patient (Pattillo et al., 1977).
  • the set of primers of the IntG-HPV method for HPV-16 preferably correspond to the sequences: SEQ ID NO:1; SEQ ID NO:2; SEQ ID NO:3; SEQ ID NO:4; SEQ ID NO:5; SEQ ID NO:6 and SEQ ID NO:7 .
  • the preferred conditions of the Multiplex PCR reaction of the IntG-HPV method were 1 cycle of 95°C for 3 minutes and 40 cycles, being 95°C for 30 seconds, 62°C for 1 minute and 30 seconds and 72°C for 2 minutes, and 1 final extension cycle of 72°C for 5 minutes.
  • the results of the IntG-HPV method suggest the association of the Mixed pattern of the HPV-16 genome with the genome of the Ca Ski CC cell line and the association of the Episomal pattern of the HPV-16 genome with the genome of the uterine cervical desquamation cells with low-grade lesions.
  • the Ca Ski cell line presents a reduction in the intensity of the 657bp amplicon , which is associated with the recombination site of the HPV-16 genome, involving the regions adjacent to the E7 gene in a clockwise direction, as shown in Figure 2.
  • Example 3 Analysis of the physical status of the HPV-16 genome in DNA from cervical desquamation cells with low-grade lesions and positive for HPV-16 by the IntG-HPV method.
  • the amplifications of the PCR-Multiplex reaction of the IntG-HPV method were performed in duplicate using as positive control for the Integrated and Mixed standards the DNA of the CCU SiHa and Ca Ski cell lines, respectively, being preferably used in the final volume of 50 ⁇ L, being 4 ⁇ L of the primer mix at a concentration of 50 pMol for each recombination region and 2 ⁇ L of samples (maximum 50ng of DNA per reaction), 19 ⁇ L of DNAse and RNAse-free ultrapure water and 25 ⁇ L of MasterMix 2X with Hot start DNA polymerase and 2% DMSO.
  • the set of primers of the IntG-HPV method for HPV-16 preferably correspond to the sequences: SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 6 and SEQ ID NO: 7 .
  • the preferred conditions of the PCR-Multiplex reaction of the IntG-HPV method were 1 cycle of 95°C for 3 minutes and 40 cycles, being 95°C for 30 seconds, 62°C for 1 minute and 30 seconds and 72°C for 2 minutes, and 1 final extension cycle of 72°C for 5 minutes.
  • the IntG-HPV METHOD FOR DIRECT DETERMINATION OF PATTERNS OF THE PHYSICAL STATUS OF THE HPV GENOME IN RELATION TO THE GENOME OF INFECTED CELLS supports the industrial production of in vitro diagnostic kits for diagnosis, prognosis, screening and prevention of Cervical Cancer, among other neoplasms, which are caused by high-oncological risk HPV subtypes.
  • sequence listing is as follows: SEQ ID NO:1; SEQ ID NO:2; SEQ ID NO:3; SEQ ID NO:4; SEQ ID NO:5; SEQ ID NO:6 and SEQ ID NO:7 .
  • NPL 1. Publication entitled Analysis by multiplex PCR of the physical status of human papillomavirus type 16 DNA in cervical cancers. J Clin Microbiol. 1999 Nov;37(11):3514-7, by Yoshinouchi et al.
  • NPL 3. Publication entitled Type-dependent integration frequency of human papillomavirus genomes in cervical lesions. Cancer Res. 2008 Jan 1;68(1):307-13, by Vinokurova et al.

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Abstract

The invention relates to the areas of molecular biology and in-vitro molecular diagnostics, consisting of the direct determination of the physical status of the HPV genome in relation to the genome of infected cells, that can develop neoplastic processes, such as Cervical Cancer - CC. The IntG-HPV method was validated on DNA samples from the SiHa and CaSki UCC cell lines and from cervical desquamation cells with low-grade lesions and positive for HPV-16. The comparative analysis using the Fisher's exact test, showed a statistically significant difference-p-value > 0.05. Thusthe results suggest associations of the Episomal, Mixed and Integrated patterns of the HPV-16 genome with the aforementioned shedding cells, the CaSki cell line and the cell line SiHa, respectively. Therefore, the IntG-HPV method is novel, and it is a molecular diagnostic and prognostic test for CC screening and prevention.

Description

MÉTODO IntG-HPV PARA DETERMINAÇÃO DIRETA DOS PADRÕES DOS STATUS FÍSICO DO GENOMA DO HPV EM RELAÇÃO AO GENOMA DAS CÉLULAS INFECTADASIntG-HPV METHOD FOR DIRECT DETERMINATION OF PATTERNS OF PHYSICAL STATUS OF THE HPV GENOME IN RELATION TO THE GENOME OF INFECTED CELLS

A presente invenção se refere às áreas de biologia molecular e diagnóstico molecular in vitro em relação à determinação direta dos padrões dos status físico epissomal, misto e integrado do genoma do Papiloma Vírus Humano - HPV e outros vírus, que apresentam comportamento semelhante, frente ao genoma das células infectadas, aplicado ao diagnóstico e prognóstico dos cânceres, que apresentam como causa principal a infecção seguida pela integração do genoma viral ao genoma das células infectadas, a exemplo do Câncer do Colo do Útero - CCU. Em relação ao produto dos reagentes e kits para o teste de diagnóstico molecular dos padrões dos status físico do genoma do HPV em células infectadas, a exemplo de DNA de linhagens celulares do CCU e DNA de amostras de células de descamação do colo do útero com lesões de baixo grau e positivas para o HPV-16, sendo determinados diretamente pelo método IntG-HPV e amplificados preferencialmente por PCR-Multiplex. The present invention relates to the areas of molecular biology and in vitro molecular diagnostics in relation to the direct determination of the patterns of the episomal, mixed and integrated physical status of the genome of the Human Papilloma Virus - HPV and other viruses, which present similar behavior, in relation to the genome of infected cells, applied to the diagnosis and prognosis of cancers, which have as their main cause the infection followed by the integration of the viral genome into the genome of infected cells, such as Cervical Cancer - CC. In relation to the product of the reagents and kits for the molecular diagnostic test of the patterns of the physical status of the HPV genome in infected cells, such as DNA from CC cell lines and DNA from samples of cervical desquamation cells with low-grade lesions and positive for HPV-16, being determined directly by the IntG-HPV method and amplified preferably by Multiplex PCR.

A principal causa na etiologia do CCU é infecção pelo Papiloma Vírus Humano – HPV seguida pela integração do seu genoma ao genoma das células infectadas (Zur Hausen, 2002).The main cause in the etiology of CC is infection by the Human Papilloma Virus – HPV followed by the integration of its genome into the genome of infected cells (Zur Hausen, 2002).

O HPV pertencente à família Papillomaviridae, gênero Papillomavirus são vírus não envelopados, de simetria icosaédrica e possuindo um genoma de DNA de fita dupla e circular com aproximadamente 8.000 pares de bases (Zheng & Baker, 2006).HPV belonging to the Papillomaviridae family, Papillomavirus genus, are non-enveloped viruses with icosahedral symmetry and a circular, double-stranded DNA genome with approximately 8,000 base pairs (Zheng & Baker, 2006).

O genoma do HPV possui duas regiões principais, uma região codificante e outra região não codificante. Essa segunda região controla a transcrição e duplicação do genoma viral, sendo denominada LCR (long control region). A região codificante é constituída por seis genes, que se expressam precocemente e dois genes que se expressam tardiamente, sendo denominados respectivamente de E (Early) e L (Late), totalizando oito open reading frames (ORF). A região precoce ou E codifica as proteínas não estruturais: E1, E2, E4, E5, E5 e E7, já a região tardia ou L é responsável pela síntese das proteínas L1 e L2, que irão formar o capsídeo viral (Villa et al. 2002).The HPV genome has two main regions, a coding region and a non-coding region. This second region controls the transcription and duplication of the viral genome and is called the LCR ( long control region). The coding region consists of six genes that are expressed early and two genes that are expressed late, called E ( Early ) and L ( Late ), respectively, totaling eight open reading frames (ORFs). The early region or E encodes the non-structural proteins: E1, E2, E4, E5, E5 and E7, while the late region or L is responsible for the synthesis of the L1 and L2 proteins, which will form the viral capsid (Villa et al. 2002).

A inativação do gene E2 é a chave no processo da gênese do CCU e outros cânceres, que tem como causa infecção dos subtipos de HPV de alto risco oncológico seguida da integração do seu genoma ao genoma da célula infectada, uma vez que, o seu produto gênico atua diretamente na regulação das transcrições dos genes oncogênicos E6 e E7, silenciando-os, quando ocorre a integração do genoma viral, o gene E2 é inativado, porque os sítios de recombinação entre o genoma viral e o genoma da célula infectada ocorre de regra na sequência do gene E2 (Dall et al., 2008).The inactivation of the E2 gene is key in the genesis of CC and other cancers, which are caused by infection with high-risk HPV subtypes followed by the integration of its genome into the genome of the infected cell, since its gene product acts directly in the regulation of the transcriptions of the oncogenic genes E6 and E7, silencing them. When the integration of the viral genome occurs, the E2 gene is inactivated, because the recombination sites between the viral genome and the genome of the infected cell usually occur in the sequence of the E2 gene (Dall et al., 2008).

Os mecanismos moleculares relacionados ao fenótipo maligno compreendem a atuação da proteína E7 na inativação da proteína supressora de tumor pRb, bem como, a proteína E6 está envolvida na inativação da proteína p53, que é supressora de tumor, acelerando sua degradação proteolítica. A associação das proteínas virais aos produtos dos genes supressores de tumores apresenta variabilidade de acordo com o potencial oncogênico do subtipo de HPV (Ghittoni et al., 2010).The molecular mechanisms related to the malignant phenotype include the action of the E7 protein in the inactivation of the tumor suppressor protein pRb , as well as the E6 protein being involved in the inactivation of the p53 protein, which is a tumor suppressor, accelerating its proteolytic degradation. The association of viral proteins with the products of tumor suppressor genes presents variability according to the oncogenic potential of the HPV subtype (Ghittoni et al., 2010).

Os principais subtipos de HPV de alto risco oncológico detectados em amostras de CCU são os subtipos HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73 e 82, todos pertencem ao gênero alpha-papillomavirus (Castellsagué et al., 2016).The main high-risk oncological HPV subtypes detected in CC samples are HPV subtypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73 and 82, all of which belong to the alpha-papillomavirus genus (Castellsagué et al., 2016).

Os subtipos HPV 16 e 18 são os mais prevalentes, representam 70% dos casos e apresentam maior distribuição geográfica no mundo (de Sanjose et al., 2010).HPV subtypes 16 and 18 are the most prevalent, representing 70% of cases and having the greatest geographical distribution in the world (de Sanjose et al., 2010).

As taxas de integração viral encontrada em carcinomas em decorrência da infecção pelos subtipos de HPV 16 e 18 pelos métodos indiretos são aproximadamente entre 80% e 100%, respectivamente (Pett & Coleman, 2007).The rates of viral integration found in carcinomas resulting from infection by HPV subtypes 16 and 18 by indirect methods are approximately between 80% and 100%, respectively (Pett & Coleman, 2007).

A evolução do CCU, considerando os padrões dos status físico do genoma dos subtipos de HPV de alto risco oncológico em relação ao genoma das células infectadas, compreendem três estágios: o primeiro estágio ou inicial, o qual é caracterizado pela infecção viral com a manutenção da integridade do genoma viral na forma circular ou padrão epissomal, nesse estágio há possibilidade de regressão da infecção e da lesão; o segundo estágio ou intermediário é caracterizado pela coexistência dos padrões epissomal e integrado, denominado padrão misto e o terceiro estágio ou tardio é caracterizado apenas pela presença do padrão integrado. Os estágios que apresentam o padrão integrado são responsáveis pelo processo carcinogênico, sem possibilidade de regressão em 99% dos casos (Vinokurova et al., 2008).The evolution of CC, considering the patterns of the physical status of the genome of high-risk HPV subtypes in relation to the genome of infected cells, comprises three stages: the first or initial stage, which is characterized by viral infection with maintenance of the integrity of the viral genome in the circular form or episomal pattern, at this stage there is a possibility of regression of the infection and the lesion; the second or intermediate stage is characterized by the coexistence of the episomal and integrated patterns, called a mixed pattern; and the third or late stage is characterized only by the presence of the integrated pattern. The stages that present the integrated pattern are responsible for the carcinogenic process, with no possibility of regression in 99% of cases (Vinokurova et al., 2008).

Os principais protocolos com base na PCR, que sugerem a análise indireta do status físico do genoma do HPV frente ao genoma das células infectadas são: a) amplificação dos transcritos oncogênicos do HPV (Klaes et al., 1999), b) amplificação e anlálise relativa entre o gene E2 e E6 (Yoshinouchi et al., 1999), c) amplificação da sequência integrada do HPV franqueando com sequencias celulares (DIPS) (Luft et al., 2001) e d) amplificação da região de fusão utilizando primers específicos e enzimas de restrição (Thorland et al., 2000).The main PCR-based protocols that suggest indirect analysis of the physical status of the HPV genome compared to the genome of infected cells are: a) amplification of HPV oncogenic transcripts (Klaes et al., 1999), b) amplification and relative analysis between the E2 and E6 genes (Yoshinouchi et al., 1999), c) amplification of the integrated HPV sequence by franking with cellular sequences (DIPS) (Luft et al., 2001) and d) amplification of the fusion region using specific primers and restriction enzymes (Thorland et al., 2000).

Em material de patente que trata sobre o status físico do genoma do HPV, em 2002, os invetores Lorincz & Lazar relacionado a anlálise relativa entre as expressões dos gene E6 e E7 a depender do coeficiente obtido pode ser inferido a condição integrada do HPV em células infectadas (US6355424).In patent material dealing with the physical status of the HPV genome, in 2002, inventors Lorincz & Lazar related the relative analysis between the expressions of the E6 and E7 genes, depending on the coefficient obtained, the integrated condition of HPV in infected cells can be inferred (US6355424).

O método molecular IntG-HPV, caracteriza-se com uma inovação disruptiva nas áreas de biologia molecular para diagnóstico, prognóstico, rastreamento e prevenção do Câncer do Colo do Útero, entre outras neoplasias, que são causadas por subtipos de HPV de alto risco oncológico em amostras in natura, uma vez que, determina diretamente os padrões do status físico epissomal, misto e integrado do genoma viral em relação ao genoma das células infectadas, à baixo custo, com celeridade e com tecnologia nacional.The IntG-HPV molecular method is characterized as a disruptive innovation in the areas of molecular biology for diagnosis, prognosis, screening and prevention of Cervical Cancer, among other neoplasms, which are caused by high-risk oncological HPV subtypes in in natura samples, since it directly determines the patterns of the episomal, mixed and integrated physical status of the viral genome in relation to the genome of infected cells, at low cost, quickly and with national technology.

Os métodos supracitados fornecem análises indiretas, apresentam custo elevado e informação limitada. Como resultado, sugerem apenas os padrões epissomal e integrado, sendo o padrão misto alcançado por meio inferência precária (Woodman et al., 2007).The aforementioned methods provide indirect analyses, are expensive and provide limited information. As a result, they suggest only episomal and integrated patterns, with the mixed pattern being achieved through poor inference (Woodman et al., 2007).

O método molecular IntG-HPV determina diretamente os padrões do status físico epissomal, misto e integrado do genoma viral em relação ao genoma das células infectadas, à baixo custo, com celeridade e amostras in natura.The IntG-HPV molecular method directly determines the patterns of the episomal, mixed and integrated physical status of the viral genome in relation to the genome of infected cells, at low cost, quickly and using in natura samples.

O método molecular IntG-HPV, caracteriza-se com uma inovação disruptiva nas áreas de biologia molecular para diagnóstico, prognóstico, rastreamento e prevenção do Câncer do Colo do Útero, entre outras neoplasias, que são causadas por subtipos de HPV de alto risco oncológico.The IntG-HPV molecular method is characterized as a disruptive innovation in the areas of molecular biology for diagnosis, prognosis, screening and prevention of Cervical Cancer, among other neoplasms, which are caused by high oncological risk HPV subtypes.

Fig.1Fig.1

[Fig. 1] Apresenta os resultados da amplificação da reação PCR-Multiplex do método IntG-HPV em amostras previamente genotipadas para o HPV-16 em células de descamação do colo do útero com lesões de baixo grau e em amostra de DNA da linhagem celular de CCU SiHa, evidenciando os padrões epissomal e integrando do HPV-16, respectivamente. [Fig. 1] Presents the results of the amplification of the PCR-Multiplex reaction of the IntG-HPV method in samples previously genotyped for HPV-16 in cervical desquamation cells with low-grade lesions and in a DNA sample from the CCU cell line SiHa , evidencing the episomal and integrating patterns of HPV-16, respectively.

Fig.2Fig.2

[Fig. 2] Apresenta os resultados da amplificação da reação PCR-Multiplex do método IntG-HPV em amostras previamente genotipadas para o HPV-16 em células de descamação do colo do útero com lesões de baixo grau e em amostra de DNA da linhagem celular de CCU Ca Ski, evidenciando os padrões epissomal e misto do HPV-16, respectivamente. [Fig. 2] Presents the results of the amplification of the PCR-Multiplex reaction of the IntG-HPV method in samples previously genotyped for HPV-16 in cervical desquamation cells with low-grade lesions and in a DNA sample from the CC cell line Ca Ski , evidencing the episomal and mixed patterns of HPV-16, respectively.

Fig.3Fig.3

[Fig. 3] Apresenta os resultados da amplificação da reação PCR-Multiplex do método IntG-HPV em amostras previamente genotipadas para o HPV-16 em células de descamação do colo do útero com lesões de baixo grau e em amostra de DNA da linhagem celular de CCU Ca Ski e amostra de DNA da linhagem celular de CCU SiHa, evidenciando os padrões epissomal, misto e integrado do HPV-16, respectivamente. [Fig. 3] Presents the results of the amplification of the PCR-Multiplex reaction of the IntG-HPV method in samples previously genotyped for HPV-16 in cervical desquamation cells with low-grade lesions and in DNA sample from the CC cell line Ca Ski and DNA sample from the CC cell line SiHa , evidencing the episomal, mixed and integrated patterns of HPV-16, respectively.

O MÉTODO IntG-HPV PARA DETERMINAÇÃO DIRETA DOS PADRÕES DOS STATUS FÍSICO DO GENOMA DO HPV EM RELAÇÃO AO GENOMA DAS CÉLULAS INFECTADAS, tem como alvo regiões do HPV, que determinam diretamente os padrões dos status físico Epissomal, Misto e Integrado do genoma do HPV em relação ao genoma da célula infectada, porém não se limita ao diagnóstico e prognóstico da infecção por HPV no rastreamento e na prevenção do CCU.The IntG-HPV METHOD FOR DIRECT DETERMINATION OF PATTERNS OF THE PHYSICAL STATUS OF THE HPV GENOME IN RELATION TO THE GENOME OF INFECTED CELLS targets HPV regions that directly determine the patterns of the Episomal, Mixed and Integrated physical status of the HPV genome in relation to the genome of the infected cell, but is not limited to the diagnosis and prognosis of HPV infection in the screening and prevention of CC.

O método IntG-HPV inova na determinação direta da integração do genoma viral ao genoma da célula infectada e pode ser aplicada a outros vírus, que possuam genoma de DNA circular e apresentam comportamento molecular semelhante ao HPV, a exemplo dos poliomavírus humano, que causa o câncer de pele das células de Merkel (Feng et al. 2018).The IntG-HPV method innovates in the direct determination of the integration of the viral genome into the genome of the infected cell and can be applied to other viruses, which have a circular DNA genome and present molecular behavior similar to HPV, such as human polyomavirus, which causes Merkel cell skin cancer (Feng et al. 2018).

A inovação do método IntG-HPV consiste na determinação direta dos padrões dos status físico Epissomal, Misto e Integrado do genoma dos subtipos de HPV de alto risco oncológico em amostras in natura de células de descamação e/ou biopsias do trato genital, entre outros, amplificados preferencialmente por PCR-Multiplex em regiões recombinantes e não recombinantes do genoma do HPV em relação ao genoma das células infectadas. The innovation of the IntG-HPV method consists of the direct determination of the patterns of the Episomal, Mixed and Integrated physical status of the genome of high-risk oncological HPV subtypes in in natura samples of desquamation cells and/or biopsies of the genital tract, among others, amplified preferentially by Multiplex PCR in recombinant and non-recombinant regions of the HPV genome in relation to the genome of infected cells.

O MÉTODO IntG-HPV PARA DETERMINAÇÃO DIRETA DOS PADRÕES DOS STATUS FÍSICO DO GENOMA DO HPV EM RELAÇÃO AO GENOMA DAS CÉLULAS INFECTADAS, compreende duas (2) etapas, a saber: The IntG-HPV METHOD FOR DIRECT DETERMINATION OF PATTERNS OF THE PHYSICAL STATUS OF THE HPV GENOME IN RELATION TO THE GENOME OF INFECTED CELLS, comprises two (2) steps, namely:

A partir da determinação prévia do subtipo de HPV de alto risco oncológico, a primeira etapa, trata-se da aplicação do Método IntG-HPV, que é caracterizado pela produção de amplicons específicos a partir dos conjuntos primers das regiões recombinantes e não recombinantes do genoma do HPV em relação ao genoma das células infectadas, preferencialmente por PCR-Multiplex;From the prior determination of the high-risk oncological HPV subtype, the first step involves the application of the IntG-HPV Method, which is characterized by the production of specific amplicons from the primer sets of the recombinant and non-recombinant regions of the HPV genome in relation to the genome of infected cells, preferably by PCR-Multiplex ;

A segunda etapa é caracterizada pelos processos de análise dos amplicons, os quais determinam os padrões moleculares Epissomal, Misto e Integrado;The second stage is characterized by the amplicon analysis processes, which determine the Episomal, Mixed and Integrated molecular patterns;

O detalhamento da invenção encontra-se nos parágrafos seguintes.The details of the invention are found in the following paragraphs.

O método IntG-HPV consiste na produção de amplicons específicos a partir dos conjuntos primers das regiões recombinantes e não recombinantes do genoma do HPV em relação ao genoma das células infectadas preferencialmente por PCR-Multiplex, onde os resultados podem ser analisados tanto por sistema de PCR Real Time quanto por gel de poliacrilamida – PAG ou agarose. A integração do genoma do HPV ao genoma das células infectadas é fator determinante para o estabelecimento e progressão do processo neoplásico nos tecidos infectados, a exemplo nas células do colo do útero, as quais quando sofrem infecção seguida de integrado do genoma do HPV evoluem para o CCU.The IntG-HPV method consists of producing specific amplicons from primer sets of the recombinant and non-recombinant regions of the HPV genome in relation to the genome of infected cells, preferably by Multiplex PCR , where the results can be analyzed either by Real Time PCR system or by polyacrylamide gel - PAG or agarose. The integration of the HPV genome into the genome of infected cells is a determining factor for the establishment and progression of the neoplastic process in infected tissues, such as in cervical cells, which, when infected and followed by integration of the HPV genome, evolve into CC.

Portanto, o método IntG-HPV apresenta uma abordagem inovadora para a determinação dos status físico do genoma do HPV, em especial, dos subtipos de HPV de alto risco oncológico, a exemplo do HPV-16. O referido método, determina a mudança do genoma do HPV da forma epissomal para forma integrada diretamente nas células infectadas, sendo um método direto para determinação da integração do genoma dos subtipos de HPV de alto risco oncológico ao genoma das células infectadas.Therefore, the IntG-HPV method presents an innovative approach for determining the physical status of the HPV genome, especially of high-risk HPV subtypes, such as HPV-16. This method determines the change in the HPV genome from the episomal form to the integrated form directly in infected cells, and is a direct method for determining the integration of the genome of high-risk HPV subtypes into the genome of infected cells.

O método IntG-HPV analisa as regiões dos genes E1, E2, E4, E5, E6 e E7 do subtipo de HPV de interesse. No padrão epissomal são gerados obrigatoriamente amplicons específicos para o subtipo HPV de interesse, no padrão integrada, a depender de qual sítio de recombinação participa do mecanismo de integração, haverá ausência de amplicon específico.The IntG-HPV method analyzes the regions of the E1, E2, E4, E5, E6 and E7 genes of the HPV subtype of interest. In the episomal pattern, specific amplicons are generated for the HPV subtype of interest. In the integrated pattern, depending on which recombination site participates in the integration mechanism, there will be an absence of specific amplicons .

A amplificação do método IntG-HPV foi realizada preferencialmente por PCR-Multiplex utilizado amostras de DNA das linhagens celulares de CCU SiHa, Ca Ski e em 35 amostras de DNA de células de descamação do colo uterino com lesões de baixo grau e positivas para o HPV-16, preferencialmente no volume final 50μL, sendo 4µL do mix dos primers na concentração de 100pMol para cada região de recombinação e 2µL de amostras (no máximo 50ng de DNA por reação), 19µL água ultrapura livre de DNAse e RNAse e 25µL de MasterMix 2X (com DNA polimerase Hot start).The amplification of the IntG-HPV method was performed preferably by PCR-Multiplex using DNA samples from the CCU cell lines SiHa, Ca Ski and in 35 DNA samples from cervical desquamation cells with low-grade lesions and positive for HPV-16, preferably in the final volume of 50 μL, being 4 µL of the primer mix at a concentration of 100pMol for each recombination region and 2 µL of samples (maximum 50ng of DNA per reaction), 19 µL of ultrapure water free of DNAse and RNAse and 25 µL of MasterMix 2X (with Hot start DNA polymerase).

O conjunto de primers utilizado na PCR-Multiplex do método IntG-HPV para o HPV-16 correspondem preferencialmente as sequências: SEQ ID NO:1; SEQ ID NO:2; SEQ ID NO:3; SEQ ID NO:4; SEQ ID NO:5; SEQ ID NO:6 e SEQ ID NO:7. The set of primers used in the Multiplex PCR of the IntG-HPV method for HPV-16 preferably correspond to the sequences: SEQ ID NO:1; SEQ ID NO:2; SEQ ID NO:3; SEQ ID NO:4; SEQ ID NO:5; SEQ ID NO:6 and SEQ ID NO:7 .

As condições da reação PCR-Multiplex do método IntG-HPV foram preferencialmente, 1 ciclo de 95°C por 3 minutos e 40 ciclos, sendo 95°C por 30 segundos, 62 ºC por 1minuto e 30 segundos e 72°C por 2 minuto, e 1 ciclo de extensão final de 72°C por 5 minutos.The conditions of the PCR-Multiplex reaction of the IntG-HPV method were preferably, 1 cycle of 95°C for 3 minutes and 40 cycles, being 95°C for 30 seconds, 62°C for 1 minute and 30 seconds and 72°C for 2 minutes, and 1 final extension cycle of 72°C for 5 minutes.

A exemplo do status físico Epissomal do genoma do HPV-16, quando amplificado por PCR-Multiplex do método IntG-HPV são gerados preferencialmente os amplicons 88pb, 313pb, 450pb e 657pb.As an example of the Episomal physical status of the HPV-16 genome, when amplified by PCR-Multiplex of the IntG-HPV method, the 88bp, 313bp, 450bp and 657bp amplicons are preferentially generated.

A ausência do amplicon 657pb está associada ao sítio de integração do genoma do HPV-16, envolvendo as regiões adjacentes ao gene E7 no sentido horário.The absence of the 657bp amplicon is associated with the integration site of the HPV-16 genome, involving the regions adjacent to the E7 gene in a clockwise direction.

A ausência do amplicon 313pb está associada ao sítio de integração do genoma do HPV-16, envolvendo as regiões adjacentes ao gene L2 no sentido anti-horário.The absence of the 313bp amplicon is associated with the integration site of the HPV-16 genome, involving the regions adjacent to the L2 gene in a counterclockwise direction.

A ausência dos amplicons 88pb e 657pb está associada ao sítio de integração do genoma do HPV-16, envolvendo as regiões adjacentes ao gene E7 no sentido horário.The absence of the 88bp and 657bp amplicons is associated with the integration site of the HPV-16 genome, involving the regions adjacent to the E7 gene in a clockwise direction.

Uma vez, o genoma do HPV-16 integrado ao genoma das células infectadas, o amplicon 450pb é produzido a partir das regiões não recombinantes. Assim, sua amplificação é constante e serve como referência para determinação do padrão misto.Once the HPV-16 genome is integrated into the genome of infected cells, the 450bp amplicon is produced from the non-recombinant regions. Thus, its amplification is constant and serves as a reference for determining the mixed pattern.

Para diferenciar o padrão misto do padrão epissomal há necessidade de estabelecer a Razão Relativa (RR) entre a intensidade ou amplificação do amplicon constante pela a intensidade ou amplificação dos amplicons “não constante”, separadamente, sendo a RR menor ou igual 1 para cada amplicon analisado, o genoma do HPV se encontra no padrão epissomal ou não integrado. Quando a RR maior que 1 para quaisquer amplicons analisados, o genoma do HPV se encontra no padrão integrado ao genoma das células infectadas.To differentiate the mixed pattern from the episomal pattern, it is necessary to establish the Relative Ratio (RR) between the intensity or amplification of the constant amplicon and the intensity or amplification of the “non-constant” amplicons , separately. If the RR is less than or equal to 1 for each amplicon analyzed, the HPV genome is in the episomal or non-integrated pattern. When the RR is greater than 1 for any amplicon analyzed, the HPV genome is in the pattern integrated into the genome of the infected cells.

A Razão Relativa é calculada dividindo o valor intensidade ou amplificação do amplicon constate (IAk) pelo valor intensidade ou amplificação do amplicon não constate (IAn) para cada amplicon. The Relative Ratio is calculated by dividing the constant amplicon intensity or amplification value (IA k ) by the non-constant amplicon intensity or amplification value (IA n ) for each amplicon.

O percentual de integração dos subtipos de HPV de interesse é calculado multiplicando Razão Relativa por 100 e dividindo o produto pelo valor intensidade ou amplificação do amplicon constate (IAk).The percentage of integration of the HPV subtypes of interest is calculated by multiplying the Relative Ratio by 100 and dividing the product by the intensity or amplification value of the constant amplicon (IA k ).

Modificações de alguns detalhes no que se refere à execução do método poderão ser introduzidas. Essas modificações não implicarão na alteração dos princípios fundamentais que estão substanciados no quadro reivindicatório. Os exemplos descritos a seguir ilustram, porém não limitam a natureza da invenção.Modifications to some details regarding the execution of the method may be introduced. These modifications will not imply any change in the fundamental principles that are substantiated in the claims. The examples described below illustrate, but do not limit, the nature of the invention.

ExemplosExamples

Exemplo 1: Análise do status físico do genoma do HPV-16 em amostra de DNA da linhagem celular de CCU SiHa pelo método IntG-HPV. O método IntG-HPV foi validado em amostra de DNA extraído da linhagem celular de CCU SiHa, que é um modelo in vitro adequado para essa finalidade. A referida linhagem celular foi imortalizada pela integração do genoma do HPV-16 e estabelecida em 1970 a partir de amostra de CCU de uma paciente de 55 anos (Friedl F, et al. 1970). As amplificações por reação PCR-Multiplex do método IntG-HPV foram realizadas em duplicata, bem como, em amostras de células de descamação do colo uterino (N = 35 amostras) com lesões de baixo grau e positivas para o HPV-16, sendo usado preferencialmente no volume final 50μL, sendo 4µL do mix dos primers na concentração de 50pMol para cada região de recombinação e 2µL de amostras (no máximo 50ng de DNA por reação), 19µL água ultrapura livre de DNAse e RNAse e 25µL de MasterMix 2X com DNA polimerase Hot start e 2% de DMSO. O conjunto dos primers do método IntG-HPV para o HPV-16 correspondem preferencialmente as sequências: SEQ ID NO:1; SEQ ID NO:2; SEQ ID NO:3; SEQ ID NO:4; SEQ ID NO:5; SEQ ID NO:6 e SEQ ID NO:7. As condições preferenciais da reação PCR-Multiplex do método IntG-HPV foram, 1 ciclo de 95°C por 3 minutos e 40 ciclos, sendo 95°C por 30 segundos, 62 ºC por 1minuto e 30 segundos e 72°C por 2 minuto, e 1 ciclo de extensão final de 72°C por 5 minutos. Os resultados do método IntG-HPV em DNA da linhagem celular de CCU SiHa quando comparado às amostras de células de descamação do colo uterino com lesões de baixo grau e positivas para o HPV-16 pelo Teste Exato de Fisher, apresentaram diferenças estatisticamente significativa valor-p > 0,05. Assim, os resultados do método IntG-HPV sugerem a associação do padrão Epissomal do genoma do HPV-16 ao genoma das células de descamação do colo uterino com lesões de baixo grau e a associação do padrão Integrado do genoma do HPV-16 ao genoma da linhagem celular de CCU SiHa por meio das ausências dos amplicons 88pb e 657pb, que estão associadas ao sítio de recombinação do genoma do HPV-16, envolvendo as regiões adjacentes ao gene E7 no sentido horário, como apresentado na Figura 1.Example 1: Analysis of the physical status of the HPV-16 genome in a DNA sample from the SiHa CC cell line by the IntG-HPV method. The IntG-HPV method was validated in a DNA sample extracted from the SiHa CC cell line, which is a suitable in vitro model for this purpose. This cell line was immortalized by the integration of the HPV-16 genome and established in 1970 from a CC sample of a 55-year-old patient (Friedl F, et al. 1970). The amplifications by PCR-Multiplex reaction of the IntG-HPV method were performed in duplicate, as well as in samples of cervical desquamation cells (N = 35 samples) with low-grade lesions and positive for HPV-16, being preferably used in the final volume of 50 μL, being 4 µL of the primer mix at a concentration of 50pMol for each recombination region and 2 µL of samples (maximum 50ng of DNA per reaction), 19 µL of ultrapure water free of DNAse and RNAse and 25 µL of MasterMix 2X with Hot start DNA polymerase and 2% DMSO. The set of primers of the IntG-HPV method for HPV-16 preferably correspond to the sequences: SEQ ID NO:1; SEQ ID NO:2; SEQ ID NO:3; SEQ ID NO:4; SEQ ID NO:5; SEQ ID NO:6 and SEQ ID NO:7 . The preferred conditions of the PCR-Multiplex reaction of the IntG-HPV method were 1 cycle of 95°C for 3 minutes and 40 cycles, being 95°C for 30 seconds, 62°C for 1 minute and 30 seconds and 72°C for 2 minutes, and 1 final extension cycle of 72°C for 5 minutes. The results of the IntG-HPV method in DNA from the CCU cell line SiHa when compared to samples of cervical desquamation cells with low-grade lesions and positive for HPV-16 by Fisher 's Exact Test, showed statistically significant differences ( p-value > 0.05). Thus, the results of the IntG-HPV method suggest the association of the Episomal pattern of the HPV-16 genome with the genome of the uterine cervical desquamation cells with low-grade lesions and the association of the Integrated pattern of the HPV-16 genome with the genome of the SiHa CC cell line through the absences of the 88bp and 657bp amplicons , which are associated with the recombination site of the HPV-16 genome, involving the regions adjacent to the E7 gene in a clockwise direction, as shown in Figure 1.

Exemplo 2: Análise do status físico do genoma do HPV-16 em amostra de DNA da linhagem celular de CCU Ca Ski pelo Método IntG-HPV. O método IntG-HPV foi validado em amostra de DNA extraído da linhagem celular de CCU Ca Ski, a referida linhagem celular pode ser usada em testes e calibração em laboratórios credenciados pela ISO 17025, para contestar o desempenho de ensaios, validar ou comparar métodos de teste e estabelecer sensibilidade, linearidade e especificidade durante a validação ou implementação dos ensaios relacionados ao diagnóstico molecular in vitro (ATCC, 2022), a linhagem celular de CCU Ca Ski foi imortalizada pela integração de algumas cópias do genoma do HPV-16 e estabelecida em 1977 a partir de células epidermóides do carcinoma cervical de uma paciente de 40 anos (Pattillo et al., 1977). As amplificações da reação PCR-Multiplex do método IntG-HPV foram realizadas em duplicata, bem como, em amostras de células de descamação do colo uterino (N = 35 amostras) com lesões de baixo grau e positivas para HPV-16, sendo usado preferencialmente no volume final 50μL, sendo 4µL do mix dos primers na concentração de 50pMol para cada região de recombinação e 2µL de amostras (no máximo 50ng de DNA por reação), 19µL água ultrapura livre de DNAse e RNAse e 25µL de MasterMix 2X com DNA polimerase Hot start e 2% de DMSO. O conjunto dos primers do método IntG-HPV para o HPV-16 correspondem preferencialmente as sequências: SEQ ID NO:1; SEQ ID NO:2; SEQ ID NO:3; SEQ ID NO:4; SEQ ID NO:5; SEQ ID NO:6 e SEQ ID NO:7. As condições preferenciais da reação de PCR-Multiplex do método IntG-HPV foram, 1 ciclo de 95°C por 3 minutos e 40 ciclos, sendo 95°C por 30 segundos, 62 ºC por 1minuto e 30 segundos e 72°C por 2 minuto, e 1 ciclo de extensão final de 72°C por 5 minutos. Os resultados do método IntG-HPV em DNA de linhagem celular de CCU Ca Ski quando comparado às amostras de células de descamação do colo uterino com lesões de baixo grau e positivas para o HPV-16 pelo Teste Exato de Fisher apresentaram diferença estatisticamente significativa valor-p > 0,05. Assim, os resultados do método IntG-HPV sugerem a associação do padrão Misto do genoma do HPV-16 ao genoma da linhagem celular de CCU Ca Ski e a associação do padrão Epissomal do genoma do HPV-16 ao genoma das células de descamação do colo uterino com lesões de baixo grau. Portanto, a linhagem celular Ca Ski apresenta a redução da intensidade do amplicon 657pb, a qual está associada ao sítio de recombinação do genoma do HPV-16, envolvendo as regiões adjacentes ao gene E7 no sentido horário, como apresentado na Figura 2.Example 2: Analysis of the physical status of the HPV-16 genome in a DNA sample from the Ca Ski CC cell line by the IntG-HPV Method. The IntG-HPV method was validated on a DNA sample extracted from the Ca Ski CC cell line. This cell line can be used in testing and calibration in ISO 17025 accredited laboratories to challenge assay performance, validate or compare test methods, and establish sensitivity, linearity, and specificity during validation or implementation of assays related to in vitro molecular diagnostics (ATCC, 2022). The Ca Ski CC cell line was immortalized by the integration of a few copies of the HPV-16 genome and established in 1977 from cervical carcinoma epidermoid cells of a 40-year-old patient (Pattillo et al., 1977). The amplifications of the PCR-Multiplex reaction of the IntG-HPV method were performed in duplicate, as well as in samples of cervical desquamation cells (N = 35 samples) with low-grade lesions and positive for HPV-16, being preferably used in the final volume of 50 μL, being 4 μL of the primer mix at a concentration of 50 pMol for each recombination region and 2 μL of samples (maximum 50ng of DNA per reaction), 19 μL of ultrapure water free of DNAse and RNAse and 25 μL of MasterMix 2X with Hot start DNA polymerase and 2% DMSO. The set of primers of the IntG-HPV method for HPV-16 preferably correspond to the sequences: SEQ ID NO:1; SEQ ID NO:2; SEQ ID NO:3; SEQ ID NO:4; SEQ ID NO:5; SEQ ID NO:6 and SEQ ID NO:7 . The preferred conditions of the Multiplex PCR reaction of the IntG-HPV method were 1 cycle of 95°C for 3 minutes and 40 cycles, being 95°C for 30 seconds, 62°C for 1 minute and 30 seconds and 72°C for 2 minutes, and 1 final extension cycle of 72°C for 5 minutes. The results of the IntG-HPV method in DNA from the CCU Ca Ski cell line when compared to samples of cervical desquamation cells with low-grade lesions and positive for HPV-16 by Fisher 's Exact Test showed a statistically significant difference ( p-value > 0.05). Thus, The results of the IntG-HPV method suggest the association of the Mixed pattern of the HPV-16 genome with the genome of the Ca Ski CC cell line and the association of the Episomal pattern of the HPV-16 genome with the genome of the uterine cervical desquamation cells with low-grade lesions. Therefore, the Ca Ski cell line presents a reduction in the intensity of the 657bp amplicon , which is associated with the recombination site of the HPV-16 genome, involving the regions adjacent to the E7 gene in a clockwise direction, as shown in Figure 2.

Exemplo 3: Análise do status físicos do genoma do HPV-16 em DNA de células de descamação do colo uterino com lesões de baixo grau e positivas para o HPV-16 pelo método IntG-HPV. O método IntG-HPV foi validado em amostras de DNA de células de descamação do colo uterino (N = 35 amostras) com lesões de baixo grau e positivas para o HPV-16. As amplificações da reação PCR-Multiplex do método IntG-HPV foram realizadas em duplicata tendo como controle positivo para os padrões Integrado e Misto os DNA das linhagens celulares de CCU SiHa e Ca Ski, respectivamente, sendo usado preferencialmente no volume final 50μL, sendo 4µL do mix dos primers na concentração de 50pMol para cada região de recombinação e 2µL de amostras (no máximo 50ng de DNA por reação), 19µL água ultrapura livre de DNAse e RNAse e 25µL de MasterMix 2X com DNA polimerase Hot start e 2% de DMSO. O conjunto dos primers do método IntG-HPV para o HPV-16 correspondem preferencialmente as sequências: SEQ ID NO:1; SEQ ID NO:2; SEQ ID NO:3; SEQ ID NO:4; SEQ ID NO:5; SEQ ID NO:6 e SEQ ID NO:7. As condições preferenciais da reação PCR-Multiplex do método IntG-HPV foram, 1 ciclo de 95°C por 3 minutos e 40 ciclos, sendo 95°C por 30 segundos, 62 ºC por 1minuto e 30 segundos e 72°C por 2 minuto, e 1 ciclo de extensão final de 72°C por 5 minutos. Os resultados do método IntG-HPV em DNA das linhagens celulares de CCU SiHa e Ca Ski quando comparado às amostras de células de descamação do colo uterino com lesões de baixo grau e positivas para o HPV-16 pelo Teste Exato de Fisher apresentaram diferença estatisticamente significativa valor-p > 0,05. Assim, os resultados obtidos pelo método IntG-HPV sugerem a associação do padrão Epissomal às amostras de células de descamação do colo uterino com lesões de baixo grau, a associação do padrão Misto do genoma do HPV-16 ao genoma da linhagem celular de CCU Ca Ski e a associação do padrão Integrado ao genoma da linhagem celular de CCU SiHa, como apresentado na Figura 3.Example 3: Analysis of the physical status of the HPV-16 genome in DNA from cervical desquamation cells with low-grade lesions and positive for HPV-16 by the IntG-HPV method. The IntG-HPV method was validated in DNA samples from cervical desquamation cells (N = 35 samples) with low-grade lesions and positive for HPV-16. The amplifications of the PCR-Multiplex reaction of the IntG-HPV method were performed in duplicate using as positive control for the Integrated and Mixed standards the DNA of the CCU SiHa and Ca Ski cell lines, respectively, being preferably used in the final volume of 50 μL, being 4 μL of the primer mix at a concentration of 50 pMol for each recombination region and 2 μL of samples (maximum 50ng of DNA per reaction), 19 μL of DNAse and RNAse-free ultrapure water and 25 μL of MasterMix 2X with Hot start DNA polymerase and 2% DMSO. The set of primers of the IntG-HPV method for HPV-16 preferably correspond to the sequences: SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 6 and SEQ ID NO: 7 . The preferred conditions of the PCR-Multiplex reaction of the IntG-HPV method were 1 cycle of 95°C for 3 minutes and 40 cycles, being 95°C for 30 seconds, 62°C for 1 minute and 30 seconds and 72°C for 2 minutes, and 1 final extension cycle of 72°C for 5 minutes. The results of the IntG-HPV method in DNA of the CCU SiHa and Ca Ski cell lines when compared to samples of cervical desquamation cells with low-grade lesions and positive for HPV-16 by Fisher 's exact test showed a statistically significant difference ( p-value > 0.05). Thus, the results obtained by the IntG-HPV method suggest the association of the Episomal pattern with samples of uterine cervical desquamation cells with low-grade lesions, the association of the Mixed pattern of the HPV-16 genome with the genome of the CC cell line Ca Ski and the association of the Integrated pattern with the genome of the CC cell line SiHa, as shown in Figure 3.

O MÉTODO IntG-HPV PARA DETERMINAÇÃO DIRETA DOS PADRÕES DOS STATUS FÍSICO DO GENOMA DO HPV EM RELAÇÃO AO GENOMA DAS CÉLULAS INFECTADAS subsidia a produção industrial de Kits de diagnóstico in vitro para diagnóstico, prognóstico, rastreamento e prevenção do Câncer do Colo do Útero, entre outras neoplasias, que são causadas por subtipos de HPV de alto risco oncológico.The IntG-HPV METHOD FOR DIRECT DETERMINATION OF PATTERNS OF THE PHYSICAL STATUS OF THE HPV GENOME IN RELATION TO THE GENOME OF INFECTED CELLS supports the industrial production of in vitro diagnostic kits for diagnosis, prognosis, screening and prevention of Cervical Cancer, among other neoplasms, which are caused by high-oncological risk HPV subtypes.

zur Hausen (2002) relaciona a infecção por HPV com a carcinogêne do câncer do colo do útero.zur Hausen (2002) relates HPV infection to the carcinogenesis of cervical cancer.

Dall et al. (2008) relaciona a inativação do gene E2 como a perda do controle gênico dos genes E6 e E7.Dall et al. (2008) relates the inactivation of the E2 gene to the loss of genetic control of the E6 and E7 genes.

Pett & Coleman (2007) obsevaram que no câncer do colo do útero os subtipos de HPV de alto risco oncológico 16 e 18 estão intergados ao genoma da células cancerosas, sendo o subtipo HPV-16 em 90% dos casos e o subtipo HPV-18 em 100% dos casos analisados.Pett & Coleman (2007) observed that in cervical cancer, high-risk HPV subtypes 16 and 18 are integrated into the genome of cancer cells, with the HPV-16 subtype in 90% of cases and the HPV-18 subtype in 100% of cases analyzed.

Yoshinouchi et al. (1999) sugerem a análise indireta do status físico HPV anlálise relativa entre o gene E2 e E6.Yoshinouchi et al. (1999) suggest indirect analysis of HPV physical status by relative analysis between the E2 and E6 gene.

Klaes et al. (1999) sugerem a análise indireta do status físico HPV pela análise dos transcritos oncogênicos do HPV.Klaes et al. (1999) suggest indirect analysis of HPV physical status by analysis of HPV oncogenic transcripts.

Thorland et al. (2000) sugerem a análise indireta do status físico HPV por meio da amplificação da região de fusão utilizando primers específicos e enzimas de restrição.Thorland et al. (2000) suggest indirect analysis of HPV physical status by amplifying the fusion region using specific primers and restriction enzymes.

Luft et al. (2001) sugerem a análise indireta do status físico HPV pela amplificação da sequência integrada do HPV franqueando com sequencias celulares.Luft et al. (2001) suggest indirect analysis of HPV physical status by amplification of the integrated HPV sequence cross-linked with cellular sequences.

Não se aplica a referência a depósito de material biológico Reference to deposit of biological material does not apply

A listagem de sequencia é a seguinte: SEQ ID NO:1; SEQ ID NO:2; SEQ ID NO:3; SEQ ID NO:4; SEQ ID NO:5; SEQ ID NO:6 e SEQ ID NO:7. The sequence listing is as follows: SEQ ID NO:1; SEQ ID NO:2; SEQ ID NO:3; SEQ ID NO:4; SEQ ID NO:5; SEQ ID NO:6 and SEQ ID NO:7 .

As lista de citações é a seguinte:The list of citations is as follows:

zur Hausen H. Nat Rev Cancer. 2002 May;2(5):342-50.zur Hausen H. Nat Rev Cancer. 2002 May;2(5):342-50.

Zheng ZM, Baker CC. Front Biosci. 2006 Sep 1;11:2286-302.Zheng ZM, Baker CC. Front Biosci. 2006 Sep 1;11:2286-302.

Villa LL, Bernard HU, Kast M, et al. Virus Res. 2002 Nov;89(2):163-73.Villa LL, Bernard HU, Kast M, et al. Virus Res. 2002 Nov;89(2):163-73.

Dall KL, Scarpini CG, Roberts I, et al. Cancer Res. 2008 Oct 15;68(20):8249-59. Dall KL, Scarpini CG, Roberts I, et al. Cancer Res. 2008 Oct 15;68(20):8249-59.

Ghittoni R, Accardi R, Hasan U, et al. Virus Genes. 2010 Feb;40(1):1-13.Ghittoni R, Accardi R, Hasan U, et al. Virus Genes. 2010 Feb;40(1):1-13.

Castellsagué X, Ault KA, Bosch FX, et al. Papillomavirus Res. 2016 Dec;2:61-69.Castellsagué X, Ault KA, Bosch FX, et al. Papillomavirus Res. 2016 Dec;2:61-69.

de Sanjose S, Quint WG, Alemany L, et al. Lancet Oncol. 2010 Nov;11(11):1048-56.de Sanjose S, Quint WG, Alemany L, et al. Lancet Oncol. 2010 Nov;11(11):1048-56.

Pett M, Coleman N. J Pathol. 2007 Aug;212(4):356-67.Pett M, Coleman NJ Pathol. 2007 Aug;212(4):356-67.

Vinokurova S, Wentzensen N, Kraus I, et al. Cancer Res. 2008 Jan 1;68(1):307-13.Vinokurova S, Wentzensen N, Kraus I, et al. Cancer Res. 2008 Jan 1;68(1):307-13.

Klaes R, Woerner SM, Ridder R, et al. Cancer Res. 1999 Dec 15;59(24):6132-6.Klaes R, Woerner SM, Ridder R, et al. Cancer Res. 1999 Dec 15;59(24):6132-6.

Yoshinouchi M, Hongo A, Nakamura K, et al. J Clin Microbiol. 1999 Nov;37(11):3514-7.Yoshinouchi M, Hongo A, Nakamura K, et al. J Clin Microbiol. 1999 Nov;37(11):3514-7.

Luft F, Klaes R, Nees M, Dürst M, et al. Int J Cancer. 2001 Apr 1;92(1):9-17.Luft F, Klaes R, Nees M, Dürst M, et al. Int J Cancer. 2001 Apr 1;92(1):9-17.

Thorland EC, Myers SL, Persing DH, et al. Cancer Res. 2000 Nov 1;60(21):5916-21.Thorland EC, Myers SL, Persing DH, et al. Cancer Res. 2000 Nov 1;60(21):5916-21.

PTL:1. US6355424PTL:1. US6355424

NPL:1. Publicação intitulada Analysis by multiplex PCR of the physical status of human papillomavirus type 16 DNA in cervical cancers. J Clin Microbiol. 1999 Nov;37(11):3514-7, por Yoshinouchi et al.NPL:1. Publication entitled Analysis by multiplex PCR of the physical status of human papillomavirus type 16 DNA in cervical cancers. J Clin Microbiol. 1999 Nov;37(11):3514-7, by Yoshinouchi et al.

NPL:2. Publicação intitulada Detection of high-risk cervical intraepithelial neoplasia and cervical cancer by amplification of transcripts derived from integrated papillomavirus oncogenes. Cancer Res. 1999 Dec 15;59(24):6132-6, por Klaes et al.NPL:2. Publication entitled Detection of high-risk cervical intraepithelial neoplasia and cervical cancer by amplification of transcripts derived from integrated papillomavirus oncogenes. Cancer Res. 1999 Dec 15;59(24):6132-6, by Klaes et al.

NPL:3. Publicação intitulada Type-dependent integration frequency of human papillomavirus genomes in cervical lesions. Cancer Res. 2008 Jan 1;68(1):307-13, por Vinokurova et al.NPL:3. Publication entitled Type-dependent integration frequency of human papillomavirus genomes in cervical lesions. Cancer Res. 2008 Jan 1;68(1):307-13, by Vinokurova et al.

Claims (5)

O MÉTODO IntG-HPV PARA DETERMINAÇÃO DIRETA DOS PADRÕES DOS STATUS FÍSICO DO GENOMA DO HPV EM RELAÇÃO AO GENOMA DAS CÉLULAS INFECTADAS, caracterizado por se utilizar da ação dos seguintes componentes: a) conjunto de primers específicos para o método IntG-HPV, que apresentam sequências específicas para amplificação direta de amplicons das regiões recombinantes e não recombinantes do genoma de HPV preferencialmente por PCR-Multiplex; b) processo de análise dos amplicons do método IntG-HPV para determinação direta do status físico do HPV, que avaliam a presença ou ausência de amplicons para determinação direta dos padrões moleculares Epissomal e Integrado do genoma do HPV em relação ao genoma da célula infectada; c) processo de análise dos amplicons do método IntG-HPV para determinação indireta do status físico do HPV, que avaliam a diferença de intensidade entre os amplicons não constantes e o amplicon constante para determinação do padrão molecular Misto do genoma do HPV em relação ao genoma da célula infectada.THE IntG-HPV METHOD FOR DIRECT DETERMINATION OF PATTERNS OF THE PHYSICAL STATUS OF THE HPV GENOME IN RELATION TO THE GENOME OF INFECTED CELLS, characterized by using the action of the following components: a) set of primers specific for the IntG-HPV method, which present specific sequences for direct amplification of amplicons of the recombinant and non-recombinant regions of the HPV genome preferably by PCR-Multiplex ; b) process of analysis of amplicons of the IntG-HPV method for direct determination of the physical status of HPV, which evaluate the presence or absence of amplicons for direct determination of the Episomal and Integrated molecular patterns of the HPV genome in relation to the genome of the infected cell; c) process of analyzing amplicons from the IntG-HPV method for indirect determination of the physical status of HPV, which evaluates the difference in intensity between non-constant amplicons and the constant amplicon for determining the Mixed molecular pattern of the HPV genome in relation to the genome of the infected cell. MÉTODO IintG-HPV, conforme a reivindicação 1, caracterizado por ser utilizado em teste diagnóstico e prognóstico molecular para cânceres causados pela infecção seguida de integração do genoma viral ao genoma das células infectadas por determinar diretamente dos padrões moleculares epissomal e integrado do genoma do HPV e de outros vírus, sendo aplicado preferencialmente ao rastreamento e a prevenção do Câncer do Colo do Útero - CCU. IintG-HPV METHOD, according to claim 1, characterized by being used in molecular diagnostic and prognostic testing for cancers caused by infection followed by integration of the viral genome into the genome of infected cells by directly determining the episomal and integrated molecular patterns of the HPV genome and other viruses, being preferably applied to the screening and prevention of Cervical Cancer - CCU. MÉTODO IntG-HPV, conforme as reivindicações 1 e 2, caracterizado por utilizar amostras in natura de descamação de células e/ou biopsias do trato genital e outros tratos, sendo aplicado preferencialmente ao rastreamento e a prevenção do Câncer do Colo do Útero - CCU. IntG-HPV METHOD, according to claims 1 and 2, characterized by using in natura samples of cell desquamation and/or biopsies of the genital tract and other tracts, being preferably applied to the screening and prevention of Cervical Cancer - CCU. KIT PARA DETECÇÃO DIRETA IN VITRO DOS PADRÕES EPISSOMAL, MISTO E INTEGRADO DO GENOMA DO HPV AO GENOMA DA CÉLULA INFECTADA EM AMOSTRAS IN NATURA DE DESCAMAÇÃO DE CÉLULAS E/OU BIOPSIAS DO TRATO GENITAL E OUTRAS TRATOS, caracterizado por conter pelo menos: mastermix para realização da reação PCR-Multiplex do método IntG-HPV em único passo, de acordo com as reivindicações 1, 2 e 3, caracterizada por conter preferencialmente os reagentes: o conjunto de primers referente às sequências SEQ ID NO:1; SEQ ID NO:2; SEQ ID NO:3; SEQ ID NO:4; SEQ ID NO:5; SEQ ID NO:6 e SEQ ID NO:7 no mínimo 50 ρMol cada, dNTPs no mínimo 10nM, Tris-HCl pH 8.8 no mínio 20 mM, DMSO no mínimo 2%, DNA polímerase hot start no mínimo 1UI e MgCl2no mínimo 2mM.KIT FOR DIRECT IN VITRO DETECTION OF EPISOMAL, MIXED AND INTEGRATED PATTERNS OF THE HPV GENOME WITH THE GENOME OF THE INFECTED CELL IN NATURAL SAMPLES OF CELL DESCALATION AND/OR BIOPSIES OF THE GENITAL TRACT AND OTHER TREATS, characterized by containing at least: mastermix for performing the PCR-Multiplex reaction of the IntG-HPV method in a single step, according to claims 1, 2 and 3, characterized by preferably containing the reagents: the set of primers referring to the sequences SEQ ID NO:1; SEQ ID NO:2; SEQ ID NO:3; SEQ ID NO:4; SEQ ID NO:5; SEQ ID NO:6 and SEQ ID NO:7 at least 50 ρMol each, dNTPs at least 10nM, Tris-HCl pH 8.8 at least 20 mM, DMSO at least 2%, hot start DNA polymerase at least 1UI and MgCl 2 at least 2mM. KIT PARA DETECÇÃO DIRETA IN VITRO DOS PADRÕES EPISSOMAL, MISTO E INTEGRADO DO GENOMA DO HPV AO GENOMA DAS CÉLULAS INFECTADAS EM AMOSTRAS IN NATURA DE DESCAMAÇÃO DE CÉLULAS E/OU BIOPSIAS DO TRATO GENITAL E OUTRAS TRATOS, conforme as reivindicações 1, 2, 3 e 4, caracterizado por ser utilizado no diagnóstico in vitro para determinação direta dos padrões moleculares epissomal, misto e integrado do genoma do HPV em amostras in natura de descamação de células e/ou biopsias do trato genital e outros tratos, sendo aplicado preferencialmente ao rastreamento e a prevenção do Câncer do Colo do Útero - CCU.KIT FOR DIRECT IN VITRO DETECTION OF EPISOMAL, MIXED AND INTEGRATED PATTERNS OF THE HPV GENOME IN THE GENOME OF INFECTED CELLS IN NATURA SAMPLES OF CELL DESCALATION AND/OR BIOPSIES OF THE GENITAL TRACT AND OTHER TREATS, according to claims 1, 2, 3 and 4, characterized by being used in in vitro diagnosis for direct determination of episomal, mixed and integrated molecular patterns of the HPV genome in natural samples of cell desquamation and/or biopsies of the genital tract and other tracts, being preferably applied to the screening and prevention of Cervical Cancer - CC.
PCT/BR2024/050210 2023-05-23 2024-05-23 Iintg-hpv method for direct determination of the physical patterns of the hpv genome in relation to the genome of infected cells Pending WO2024239087A1 (en)

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