WO2021110927A1 - Procédé de prédiction de la réponse au traitement du cancer par immunothérapie anti-pd1 - Google Patents

Procédé de prédiction de la réponse au traitement du cancer par immunothérapie anti-pd1 Download PDF

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WO2021110927A1
WO2021110927A1 PCT/EP2020/084657 EP2020084657W WO2021110927A1 WO 2021110927 A1 WO2021110927 A1 WO 2021110927A1 EP 2020084657 W EP2020084657 W EP 2020084657W WO 2021110927 A1 WO2021110927 A1 WO 2021110927A1
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genes
response
cancer
immunotherapy
immune checkpoint
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María Isabel BARRAGÁN MALLOFRET
Martina ÁLVAREZ PÉREZ
Miguel Ángel BERCIANO GUERRERO
Manuel COBO DOLS
Alicia GARRIDO ARANDA
Alfonso SÁNCHEZ MUÑOZ
Francisco Javier OLIVER MARTOS
Pedro JIMENEZ GALLEGO
Emilio ALBA CONEJO
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Servicio Andaluz De Salud
Universidad De Málaga
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Priority to EP20825148.8A priority Critical patent/EP4069869A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates to the field of medicine, and more specifically to the field of precision medicine. It refers to a series of functional biomarkers for response to anti-PD1 treatment, which can be implemented to make therapeutic decisions, in a method to predict the response to said treatment.
  • Immune checkpoint blockade has been clinically applied in multiple cancers and revolutionized the field of cancer treatment. It has demonstrated acceptable toxicity and durable response in responders.
  • Programmed death 1 (PD1) blockade is a common ICB treatment in advanced melanoma patients, as second or third line treatment. PD1 is expressed on T lymphocytes and is the dominant inhibitory immune checkpoint for maintaining the self-tolerance of T cells. Activated PD1 signalling restrains the T cell cytotoxicity towards cancer cells (Ribas, 2012). It negatively affects chemokine and cytokine production, as well as the proliferation of CD4+ and CD8+ T cells. Treatment with Nivolumab, a PD1 antibody, yields 20% higher objective response rate than chemotherapy in advanced melanoma patients with better tolerability (Falchook, 2015).
  • immune checkpoint blockade demonstrated durable responses and acceptable toxicity, resulting in the regulatory approval of 8 checkpoint inhibitors to date for 15 cancer indications.
  • ICB immune checkpoint blockade
  • FIG. 1 Association of gene expression levels with overall survival in cutaneous melanoma. Kaplan-Meier analysis of overall survival (B) in 16 patients with metastatic cutaneous melanoma treated with Nivolumab. The most significant associations of gene expression levels and overall survival are shown.
  • FIG. 1 Tumour infiltrating cell component analysis by CiberSortX. Out of all cellular types present in the tumor stroma of the 16 metastatic melanoma patients, good and bad responders presented different distributions. Good responders were significantly enriched in plasmablasten, while bad responders were significantly enriched in CD8+ exhausted T cells, and BC1 subtype of B cells.
  • FIG 3 Heatmap that represent the expression of the different genes differentially expressed according to the study sample.
  • the p value adjusted is less than 0.05
  • there is a large number of DE genes and a certain grouping can be seen in the expression of some genes in some non-responders and responders, but it is not described clearly.
  • Figure 3B in which the adjusted p-value has been limited to less than 0.01, an expression pattern is clearly seen in some subsets of genes (orange boxes) in a group of non-responders (green box) and responders (purple box).
  • Figure 4 The total number of receptor clonotypes is associated to a good response to Nivolumab.
  • Circle plot with the heatmap of the DE expressed genes in good versus bad responders (external circle), the total BCR clonotypes sum (yellow squares), and the heatmap depicting the amount of HLA loci in each patient (internal circle).
  • Figure 8 Validation of 35 out of the 140 DE genes that constitute our firm in another cohort of melanoma patients treated with anti-PD1. In yellow, those genes that also form part of the prognostic signature. In red, a gene that is among the most common BCR clonotypes in good responders.
  • FIG. 9 Multispectral fluorescence validation of the B cell CD19 biomarkers as a response marker to anti-PD1.
  • A) CD 19 is significantly associated with response in a cohort subgroup of 12 patients, specifically plasmablasts (CD19+, CD20-, CD138-) show a trends towards significant association with responders.
  • B) Lymphoid structures containing B cells, CD8 T cells, macrophages and myeloid cells are identified in the tumors of some good responders (representative image).
  • Figure 10 Technical validation of the RNA-seq by using RQ-PCR.
  • A Pearson correlation between TaqMan target genes.
  • B Scatterplot with correlations between RQ-PCR and TPM (RNA-seq) results. Samples with low expression in RQ-PCR have in most cases resulted in low expression in TPM as well.
  • the present invention shows the potential biomarkers for patient selection and therapy stratification in melanoma patients.
  • inventors used coding and non-coding transcriptome analysis in bulk tumour samples from melanoma patients treated with the anti-PD1 agent Nivolumab to identify a signature of 140 genes associated with response of which 58 were also prognostic, that unmasked a pattern of high B lymphocyte activity under response.
  • the expression of the genes related to the responding phenotype was associated with the overall survival (OS).
  • This study addresses the lack of definite biomarkers of response to ICB in metastatic melanoma patients, by studying the differential gene expression of the responders versus the non-responders to Nivolumab using a transcriptomic approach that includes coding and non coding transcripts.
  • TILs tumor infiltrating lymphocytes
  • the immune and particular B cell specific transcriptomic signature associated with the responder phenotype allows to envisage a novel mechanism of resistance where regulatory T lymphocytes and B lymphocytes, among several other immune populations that infiltrate the tumor, are disbalanced in a context of resistance to treatment with Nivolumab.
  • various genes that conform the transcriptomic signature of the response are able to predict overall survival and constitute good candidates as biomarkers.
  • the present invention represent a step-forward in cancer precision immunotherapy, and a base for understanding the complexity of the immune system-tumor interactions that trigger a resistance phenotype in melanoma patients treated with ICB.
  • the initial cohort is 21 patients, in which there are 3 of the uveal subtype, 2 of the mucous subtype and 16 patients of the cutaneous subtype.
  • Responding or non-responding criteria include:
  • Non-respondent progression in less than 3 months from the beginning of immunotherapy.
  • Non-severe those non-responders who previously had a poor prognosis due to immunotherapy treatment due to present some adverse symptoms such as brain metastasis or an "animal-like" tumor, so the non-response to immunotherapy may not be related to the treatment but to the tumor profile of the patient.
  • the term "responder” refers to those patients who have a complete or partial response to the drug and with the term “nonresponder” to those who do not respond to it so that the tumor continues to progress in the short term after the beginning of immunotherapeutic treatment.
  • one aspect of the invention refers to the use of the 140 genes described in Table 5 or any combinations thereof for prognosticating or predicting the response to anti-PD1 treatment of a subject suffering from a cancer disease.
  • the invention relates to the simultaneous use of the 140 genes described in Table 5 for predicting the response to anti-PD1 treatment of a human subject suffering from a cancer disease.
  • Out of the 140 genes, 58 also serve for prognosticating the overall survival of the patients
  • the use of the 140 genes described in Table 5, or the use of the 58 genes described in Table 6, can be independent or in any combination thereof, or can be used simultaneously.
  • inventions refers to an in vitro method of predicting or prognosticating the response of a human subject to anti-PD1 and/or anti PD-L1 immune checkpoint inhibition immunotherapy, hereinafter first method of the invention, wherein the subject is suffering from a cancer disease, and wherein the method comprises using, as an indicator a) expression levels of the genes of Table 5 to predict the response, b) expression levels of the genes of Table 6 (selected from Table 5) to prognosticate the response, and wherein the result is indicative of a positive response if the expression levels of genes highlighted in grey (shadowed) in Tables 5 are over-expressed while the genes in white in Tables 5 are infra-expressed.
  • the /firstmethod of the invention also comprising determine the protein CD19, and wherein the result is indicative of a positive response if the expression levels of protein CD19 is over-expressed.
  • Another aspect of the invention relates to a method for obtaining useful data, hereinafter the first method of the invention, for prognosticating or predicting the response to anti-PD1 treatment of a subject suffering from a cancer disease, wherein said method comprises using, as an indicator, expression levels of the 140 genes described in Table 5.
  • Another aspect of the invention refers to a method of prognosticating or predicting the response to anti-PD1 treatment of a subject suffering from a cancer disease, wherein said method comprises using, as an indicator, expression levels of the 140 genes described in
  • the cancer disease is selected from the list consistin on melanoma, lung cancer, renal cell carcinoma, Hodgkin lymphoma, head and neck cancer, colon cancer, liver cancer, or combinations thereof. More preferably the cancer disease is melanoma, and more preferably metastatic cutaneous melanoma.
  • the anti-PD1 treatment is an anti-PD1 antibody, more preferably the anti-PD1 antibody is selected from Pembrolizumab and/or Nivolumab, and most preferably is Nivolumab.
  • subject refers to animals, preferably mammals, and more preferably, humans. Then, subject is preferred a human subject, and is not intended to be limiting in any respect; it may be of any age, sex of physical condition.
  • the methods of the present invention may be applied with samples from individuals of either sex, i.e. men or women, and at any age.
  • the profile determined by the present invention is predictive and prognostic.
  • “Response” refers to the clinical outcome of the subject, “Response” may be expressed as overall survival or progression-free survival. Survival of cancer patients is generally suitably expressed by Kaplan-Meier curves, named after Edward L. Kaplan and Paul Meier who first described it (Kaplan, Meier: Amer. Statist. Assn. 53:457-481).
  • the Kaplan-Meier estimator is also known as the product limit estimator. It serves for estimating the survival function from life-time data.
  • a plot of the Kaplan-Meier estimate of the survival function is a series of horizontal steps of declining magnitude which, when a large enough sample is taken, approaches the true survival function for that population.
  • the Kaplan- Meier estimator may be used to measure the fraction of patients living for a certain amount of time after beginning of chemotherapy and/or radiotherapy.
  • the clinical outcome predicted may be the (overall/progression-free) survival in months/years from the time point of taking the sample. It may be survival for a certain period from taking the sample, such as of six months or more, one year or more, two years or more, three years or more, four years or more, five years or more, six years or more.
  • “survival” may refer to “overall survival” or “progression-free-survival”.
  • the response is clinical outcome, which is “overall survival” (OS).
  • OS overall survival
  • “Overall survival” denotes the chances of a patient of staying alive for a group of individuals suffering from a cancer.
  • the decisive question is whether the individual is dead or alive at a given time point.
  • the techniques are selected from the list consisting on: a. a gene profiling method, such as a microarray, or a Next Generation Sequencing panel and/or b. a method comprising PCR, such as real time PCR; and/or c. northern Blot and/or d. an immunohistochemistry method; and/or e. an elisa-based method
  • RQ-PCR real time quantitative PCR
  • the AACt-method will involve a control sample and a treatment sample. For each sample, a target gene and an endogenous control (as described below) gene are included for PCR amplification from (typically serially diluted) aliquots. Typically several replicates are used for each diluted concentration to derive amplification efficiency. PCR amplification efficiency can be defined as percentage amplification (from 0 to 1).
  • a software typically measures for each sample the cycle number at which the fluorescence (indicator of PCR amplification) crosses an arbitrary line, the threshold. This crossing point is the Ct value. More dilute samples will cross at later Ct values.
  • Ct for an RNA or DNA from the mRNA gene of interest is divided by Ct of nucleic acid from the endogenous control, such as non-tumoral tissue, to normalize for variation in the amount and quality of RNA between different samples. This normalization procedure is commonly called the AACt-method (Schefe et al., 2006, J. Mol. Med. 84: 901-10).
  • the response is determined by using multispectral immunofluorescence.
  • the used of this technique entails the utilisation of the random trees algorithm classifier.
  • the myeloid and lymphoid cell panel targeted the myeloid marker CD11b, the phagocytic cell marker CD68 of macrophages, CD3+ and CD8+ T cells, CD20+ B lymphocytes, and the melanoma marker MELAN-A.
  • the melanoma-associated B cells panel included the CD19+ and CD20+ B cells, CD138+ plasma cells.
  • IF multiplex immunofluorescence development and validation workflow and protocols have been implemented as previously described (PMID: 32591586; PMID: 30742120;). Briefly, 4- micron sections of formalin-fixed paraffin-embedded (FFPE) tissue from 16 melanomas were deparaffinised and antigen retrieval was performed using DAKO PT-Link heat induced antigen retrieval with low pH (pH6) or high pH (pH 9) target retrieval solution (DAKO).
  • FFPE formalin-fixed paraffin-embedded
  • a random trees algorithm classifier was trained separately for each cell marker by an experienced pathologist (CEA) annotating the tumour regions.
  • CCA experienced pathologist
  • Interactive feedback on cell classification performance is provided during training in the form of markup image, improving significantly the accuracy of machine learning-based phenotyping.
  • PMID: 29203879, PMID: 32591586 All phenotyping and subsequent quantifications were performed blinded to the sample identity. Cells close to the border of the images were removed to reduce the risk of artifacts.
  • CD11b+, CD68+, CD3+, CD8+, and CD20+ were further subclassified as CD11b+, CD68+, CD3+, CD8+, and CD20+.
  • CD4+ T cells were defined as CD3+ CD8-.
  • MELAN-A was used to visualize the melanoma cells.
  • melanoma-associated B cells panel subpopulations were then classified as: i) total B cells (CD19+ CD20-, CD19+ CD20+ and CD19- CD20+ cells), ii) plasmablasts (CD19+ CD20- CD138- cells), iii) plasma cell-like (CD19+ CD20- CD138+ cells), and iv) mature plasma cell (CD19- or + CD20- CD138+ cells), as previously described (PMID: 31519915). Cells negative for these markers were defined as ‘other cell types’.
  • a biological sample include different types of samples from tissues, as well as from biological fluids, such as blood, serum, plasma, cerebrospinal fluid, peritoneal fluid, faeces.
  • said samples are samples from tissues and most preferably, said samples of tissues originate from tumour tissue of the individual the response of which is to be predicted, and may originate from biopsies.
  • the cancer could be any immunogenic type of cancer that is susceptible to clinical benefit from immunotherapy.
  • the cancer disease as defined in any of the methods of the invention is melanoma, non-small cell lung cancer, head and neck cancer or combinations thereof..
  • Prognosis depends on the stage of the cancer and, in this sense, it is important to find good prognosis markers for survival after treatment for this specific disease and thereby the usefulness of the biomarkers of the present invention in the prognosis of this disease.
  • Another aspect of the present invention refers to anyone of the methods of the invention, wherein the method is a drug response predictive method which is performed in vitro using a biological sample originating from the human subject, and wherein at the time point of taking the sample from the human subject, the human subject has not been treated yet with anti- PD1 and/or anti PD-L1 immune checkpoint inhibition immunotherapy.
  • the outcome for evaluation of the response is the clinical response using the RECIST criteria at a specific time after the beginning of the treatment.
  • Another aspect of the present invention refers to anyone of the methods of the invention, wherein the method is a prognostic method which is performed in vitro using a biological sample originating from the human subject, and wherein at the time point of taking the sample from the human subject, the human subject has not been treated yet with anti-PD1 immune and/or anti PD-L1 checkpoint inhibition immunotherapy.
  • the outcome for evaluation of the prognosis is the overall survival.
  • Another aspect of the invention refers to a pharmaceutical composition comprising with anti- PD1 and/or anti PD-L1 immune checkpoint inhibition immunotherapy for treating a human subject of group 1 as identifiable by any of the methods of the invention.
  • the anti-PD1 immune checkpoint inhibition immunotherapy is selected from the list consisting on: Pembrolizumab (Keytruda), Nivolumab (Opdivo), Cemiplimab (Libtayo) or combinations thereof.
  • the anti PD-L1 immune checkpoint inhibition immunotherapy is selected from the list consisting on: Atezolizumab (Tecentriq), Avelumab (Bavencio), Durvalumab (Imfinzi) or combinations thereof.
  • the present invention also provides a kit or device suitable to put into practice the method of the invention.
  • the kit comprises at least one oligonucleotide(s) capable of hybridizing with the mRNAs of any of the 140 genes of the drug response predictor signature of Table 5, and/or 58 genes of the prognosis predictor signature of Table 6.
  • the kit or device is based on the predictive power of the method of the present invention.
  • the reference value indicative for non-response (and/or a reference value indicative for response) may be provided with the kit.
  • the expression of each target gene can be calculated, i.e. relative to, such as the endogenous control samples exemplified above.
  • the endogenous control can thus also be comprised within the kit.
  • the kit may further include, with no type of limitation, buffers, agents to prevent contamination, protein degradation inhibitors, etc. Therefore, the kit may include all the supports and receptacles necessary for its implementation and optimization. Preferably, the kit further comprises the instructions for carrying out any of the methods of the invention.
  • the kit is selected from (a) a kit suitable for PCR, (b) a kit suitable for Northern Blot, (c) a kit suitable for microarray analysis, (d) a kit suitable for Next Generation Sequencing, and a kit suitable for immunohistochemistry (CD19). Any two or more of these embodiments may also be combined, so that the kit may comprise, for example both (a) and (c).
  • this PCR is typically real-time quantitative PCR (RQ- PCR), a sensitive and reproducible gene expression quantification technique.
  • RQ- PCR real-time quantitative PCR
  • a Northern Blot involves the use of electrophoresis to separate RNA samples by size and subsequent detection with an oligonucleotide(s) (hybridization probe) complementary to (part of) the target sequence of the RNA of interest.
  • oligonucleotide(s) are immobilized in spots on a (preferably solid) surface.
  • the kit comprises a microarray.
  • An RNA microarray is an array on a solid substrate (usually a glass slide or silicon thin-film cell) that assays large amounts of different RNAs which are detectable via specific probes immobilized on spots on the solid substrate.
  • Each spot contains a specific nucleic acid sequence, typically a DNA sequence, known as probes (or reporters). While the number of spots is not as such limited, there is a preferred embodiment in which the microarray is customized to the methods of the invention. In one embodiment, such a customized microarray comprises fifty spots or less, such as thirty spots or less, including twenty spots or less.
  • the kit comprises a number of capture probes for specific genes that are hybridised in suspension and subsequently amplified by PCR and sequenced in a low performance sequencer since the total number of reads needed for the targeted sequencing is low; therefore, it can be implemented in clinical routine laboratories.
  • a further embodiment of the invention refers to a kit suitable for detecting the level of expression of the genes of Table 5 and/or Table 6, or the protein CD19 which comprises a media having affixed thereto a capture antibody capable of complexing with any of biomarker proteins encoding by the genes of Table 5 and/or Table 6, or the protein CD19, or a fragment thereof and an assay for the detection of a complex of the biomarker and the capture antibody.
  • the kit may be used and the use is not particularly limited, although use in the method of the invention in any of its embodiments is preferred.
  • the kit may also be automated, or can be incorporated in devices capable of carrying out the methods of the invention automatically.
  • COMPUTER IMPLEMENTED INVENTION Another aspect of the invention relates to computer-readable storage means comprising program instructions capable of making a computer perform the steps of any of the methods of the invention
  • the invention also extends to computer programs adapted so that any processing means can carry out the methods of the invention.
  • Such programs may take the form of source code, object code, an intermediate source of code, and object code, for example, as in partially compiled form, or in any other form suitable for use in implementing the processes according to the invention.
  • Computer programs also encompass cloud applications based on this procedure.
  • the invention encompasses computer programs arranged on or within a carrier.
  • the carrier can be any entity or device capable of supporting the program.
  • the carrier may be made up of said cable or another device or medium.
  • the carrier could be an integrated circuit in which the program is included, the integrated circuit being adapted to execute, or to be used in the execution of, the corresponding processes.
  • the programs could be embedded in a storage medium, such as a ROM, a CD ROM or a semiconductor ROM, a USB memory, or a magnetic recording medium, for example, a floppy disk or a disk. Lasted.
  • the programs could be supported on a transmittable carrier signal.
  • it could be an electrical or optical signal that could be transported through an electrical or optical cable, by radio or by any other means.
  • the invention also extends to computer programs adapted so that any processing means can carry out the methods of the invention.
  • Such programs may take the form of source code, object code, an intermediate source of code, and object code, for example, as in partially compiled form, or in any other form suitable for use in implementing the processes according to the invention.
  • Computer programs also encompass cloud applications based on this procedure.
  • Another aspect of the invention relates to a computer-readable storage medium comprising program instructions capable of causing a computer to carry out the steps of any of the methods of the invention.
  • a transmittable signal comprising program instructions capable of causing a computer to carry out the steps of any of the methods of the invention.
  • the terms “subject”, “patient” or “individual”' are used herein interchangeably to refer to all the animals classified as mammals and includes but is not limited to domestic and farm animals, primates and humans, for example, human beings, non-human primates, cows, horses, pigs, sheep, goats, dogs, cats, or rodents.
  • the subject is a male or female human being of any age or race.
  • Patient biopsies were selected to exclude lymph node metastases and to include only samples with availability of clinical data and information on progression after treatment.
  • the cohort is distributed in responders (11) and non-responders (10), where the distinction criteria is described as follows:
  • Non-responders progression in less than 3 months from the start of immunotherapy. Of them, a subgroup of “severe” non-responders are define as those who progressed in less than 60 days.
  • Responders patients with maintained partial or complete response for a year or in treatment during at least one year.
  • Nivolumab was assessed according to the “Response Evaluation Criteria in Solid Tumors” criteria (RECIST v1.1 guide). The study follows the Declaration of Helsinki and has been vetted and aproved by the Ethical Committee of Malaga. All patients signed an Informed Consent to participate in the study.
  • tumour-specific area in FFPE melanoma samples was predefined by a pathologist. Two to four 10 pm slides were dissected for nucleic acid extraction, using the microtome HM 340E (Thermo Scientific). RNA was extracted with the RNeasy FFPE kit following the manufacturer instructions (Qiagen; Ref. 73504).
  • RNA-Seq libraries were prepared using TruSeq Stranded Total RNA Gold (lllumina; Ref.20020598) and indexed by IDT for lllumina - TruSeq RNA UD Indexes (lllumina; Ref. 20020591). These libraries include coding and non-coding RNA by ribosomal RNA depletion. In order to obtain a better exclusion of ribosomal RNA, the manufacturer protocol was modified including a double-depletion. Libraries concentration (0.1-1 micrograms) was determined by Qubit dsDNA BR kit, and the size distribution was examined by Agilent Tapestation 2200. Each libraries contained 0.1 -Paired-end reads (75bp c 2) were acquired from the lllumina NextSeq 550 platform according to the corresponding protocol.
  • RNA-seq data was perfomed using real-time quantitative PCR (RT-qPCR) of the following selected genes based on the gene expression patterns and the representation of tumor and immune system candidate biomarkers: TNFRSF11B, IGLV6- 57, IGHA1, and GRIA1.
  • RNA from selected samples of Discovery cohort presenting with high and low expression for the genes of study was retrotranscribed into cDNA and subjected to RT-qPCR.
  • the housekeeping control gene ACTB was used for normalization.
  • each tissue section was subjected to three or six successive rounds of antibody staining, each round consisting of protein blocking with 20% normal goat serum (Dako) in phosphate-buffered saline (PBS), incubation with primary antibody, biotinylated anti-mouse/rabbit secondary antibodies and Streptavidin-HRP (Dako), followed by TSA visualization with Opal fluorophores (Akoya Biosciences) diluted in 1X Plus Amplification Diluent (Akoya Biosciences).
  • PBS normal goat serum
  • PBS phosphate-buffered saline
  • the myeloid and lymphoid cell panel included: CD11b (Rabbit monoclonal, clone EPR1344, 1:1000, Abeam, product number ab133357), CD68 (Mouse monoclonal, clone PG-M1, ready-to-use, Agilent, product number IR613), CD3 (Rabbit polyclonal, IgG, ready-to-use, Agilent, product number IR503), CD8 (Mouse monoclonal, clone C8/144B, ready-to-use, Agilent, product number GA62361-2), CD20 (Mouse monoclonal, lgG2a, clone L26, ready- to-use, Agilent, product number GA604), and MELAN-A (Mouse monoclonal, clone A103, ready-to-use, Agilent, product number IR63361).
  • CD11b Rabbit monoclonal, clone EPR1344,
  • the melanoma-associated B cells panel included: CD19 (Mouse monoclonal, clone LE- CD19, ready-to-use, Agilent, product number GA656), CD20 (Mouse monoclonal, lgG2a, clone L26, ready-to-use, Agilent, product number GA604), CD138 (Mouse monoclonal, lgG1, clone M 115, 1:100, Agilent, product number M7228).
  • nuclei were counterstained with spectral DAPI (Akoya Biosciences) and sections mounted with Faramount Aqueous Mounting Medium (Dako).
  • Fastq quality control was performed with FastQC.
  • Fastq files were trimmed using tool HISAT2(v 2.1.0) with a customized index built using combined rRNA data from HGNC, ENA, SILVA, and additional manually curated sequences from NCBI. Trimmed fastq files were mapped against reference (genome build GRCh38) using STAR (v 2.5.1b) and read quantification was done with the same tool. % of uniquely mapped reads and M mapped reads were computed with Qualimap.
  • Pathway analysis was done with R in-house scripts using two different approaches: gene set enrichment analysis (GSEA and DAVID) and network based pathway analysis.
  • Packages used were STRINGdb, clusterprofiler, pathfindR.
  • MPC Counter was used to infer the abundance of different immune cells populations of the tumor infiltrate using the normalized counts of RNA-seq.
  • MIXCR and Seq2HLA for HLA, TCR and BCR profiling, and GATK for calculation of the Tumour Mutational Burden (TMB) from our bulk RNA-seq dataset.
  • TMB Tumour Mutational Burden
  • 30 COSMIC mutational signatures we evaluated in our cohort. Survival analysis was performed using the Kaplan-Meier method.
  • the coding and non coding transcriptome of 21 melanoma samples of different subtypes (16 cutaneous, 3 uveal and 2 mucosal melanomas respectively;) was generated using RNA sequencing with ribosomal depletion with RNA-Seq. Given the known molecular differences of the different subtypes of melanoma probably due to diverse carcinogenic events (Hayward et al., 2017), we explored common and subtype-specific response expression signatures in our cohort. In all melanoma subtypes, 22 genes conformed a common signature that features the response to anti-PD1 (Table 1).
  • the expression signature of the responding patients with cutaneous melanoma was more extensive, with 140 differentially expressed genes, of which 11 genes overlapped the general signature (Table 2).
  • 108 genes were down-regulated while 32 genes were up-regulated compared to responders.
  • the BP enrichment analysis demonstrated the dramatic difference of B lymphocytes- related biological processes including phagocytosis, B cell/complement activation, and immunoglobulin production among PD1 blockade responders and non responders. Similarly, immunoglobulin receptor binding and monomeric IgA immunoglobulin complex, were discovered in MF and CC enrichment analysis.
  • the T cells that were significantly associated to the treatment were the exhausted CD8 T cells, while circumscribing our analysis to the B cell lineage, plasmablasts and the subtype of B cells BC1 were significantly enriched in good and bad responders, respectively, refining the characterisation of the B cells-related subtypes involved in the efficacy of Nivolumab.
  • CD19 B cell marker as a protein biomarker of response to anti-PD-1 in melanoma, identifying a trend towards statistical association of plasmablasts (Figure 9).
  • Fc-gamma receptor signaling pathway involved in phagocytosis 40 1.64E-52 phagocytosis, engulfment 39 1.25E-51 positive regulation of B cell activation 39 1.05E-47
  • Table 5 List of the 140 differentially expressed genes of the 16 samples of cutaneous metastatic melanoma that are significant by restricting a set value of less than 0.05, an absolute value of FoldChange> 1.5 and the base value Mean> 10. In grey (shaded), the upregulated genes.
  • Table 7 Ranking of counts of the 26 clonotypes that are enriched in good responders to Nivolumab.
  • Genome Analysis Toolkit A MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res. 20, 1297-1303.

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Abstract

L'invention concerne des biomarqueurs, des procédés in vitro et un kit ou un dispositif permettant la prédiction ou le pronostic de la réponse d'un sujet humain à une immunothérapie par inhibition de point de contrôle immunitaire anti-PD1 et/ou anti-PD-L1.
PCT/EP2020/084657 2019-12-04 2020-12-04 Procédé de prédiction de la réponse au traitement du cancer par immunothérapie anti-pd1 WO2021110927A1 (fr)

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WO2023227818A1 (fr) * 2022-05-26 2023-11-30 Servicio Andaluz De Salud Profil d'arncirc pour prédire la réponse à l'immunothérapie chez des patients cancéreux

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023280973A1 (fr) * 2021-07-07 2023-01-12 Evaxion Biotech A/S Procédé de prédiction de réponse à une immunothérapie anticancéreuse
WO2023227818A1 (fr) * 2022-05-26 2023-11-30 Servicio Andaluz De Salud Profil d'arncirc pour prédire la réponse à l'immunothérapie chez des patients cancéreux

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