Method for the quantification of pd-l1 expression

Abstract

A highly sensitive for determining the expression of PD-L1 that is based on a RT-qPCR in a RNA sample of, for example, Circulating Tumor Cells (CTC) or fresh frozen primary tumor tissues. In particular, to a method for the detection of PD-L1 mRNA positive CTCs or primary tumor tissues (fresh frozen) based on the quantitative determination of the molecular marker (PD-L1) in biological samples of patients suffering from cancer. By using the method, detection can take place before, during or after the immune therapy or any other treatment in order to provide significant information concerning guiding or monitoring of the anti-PD-L1 agents effectiveness. This RT-qPCR assay could comprise a promising companion diagnostic test in order to evaluate the PD-L1 expressional status on CTC or tumor tissue, providing clinical applications, which could have an important impact on therapeutic interventions since the expression of PD-L1 is associated with response to immunotherapy.

1 - 47 . (canceled) 
     
     
         48 . A method for either (a) assessing the outcome of treatment of malignant neoplastic disease in a subject during and after immunotherapy with checkpoint inhibitors where an indication of effective treatment is a relative change in decreasing amount of PD-L1 mRNA in a sample relative to a previous sample analyzed in the steps of repeating the method herein, or (b) assessing recurrence of malignant neoplastic disease in a subject during and after immunotherapy with checkpoint inhibitors where an indication of the recurrence is an over time increase in the amount of PD-L1 mRNA in a sample relative to a previous sample analysed in the steps of repeating the method herein, comprising the quantification of the Programmed Death Ligand 1 (PD-L1) in a liquid biopsy sample of Circulating Tumor Cells (CTC), and said method comprising:
 i. subjecting the liquid biopsy sample to reverse transcription using RNA present in the sample as a template in order to synthesize a corresponding cDNA sequence,   ii. forming a reaction mixture comprising the sample, nucleic acid amplification reagents, a target primer pair, a target hydrolysis probe, said target primer pair and target hydrolysis probe being capable of hybridizing to PD-L1 mRNA, and said target primer pair comprising a forward target primer and a reverse target primer wherein at least one of said forward or reverse target primers is capable of hybridizing to an intron spanning site of the target sequence of PD-L1 mRNA,   iii. subjecting the reaction mixture to amplification conditions optimized to generate at least one copy of a nucleic acid sequence complementary to a target sequence, and said target sequence being a mRNA transcript of the PD-L1 mRNA sequence (SEQ ID NO: 1),   iv. determining the amount of PD-L1 mRNA in the liquid biopsy sample,   v. normalizing the expression of PD-L1 with respect to an expression of a reference gene whose expression levels remain constant between different cell types, and   vi. comparing the amount of PD-L1 mRNA expressed in the said sample to a positive control which is a sample comprising PD-L1 mRNA and a negative control which is a sample without comprising PD-L1 mRNA in order to estimate an overexpression of the PD-L1 mRNA sequence.   
     
     
         49 . The method according to  claim 48 , further comprising designing the forward target primer of PD-L1 to hybridize between exon 4 and 5 of the PD-L1 mRNA sequence. 
     
     
         50 . The method according to  claim 49 , further comprising using at least a sequence 5′-GTCATCCCAGAA-3′ (SEQ ID NO: 3) as the forward PD-L1 target primer comprises or using a sequence 5′-GCTGAATTGGTCATCCCAGAA-3′ (SEQ ID NO: 4) as the forward PD-L1 target primer. 
     
     
         51 . The method according to  claim 50 , further comprising designing the reverse target primer of PD-L1 to hybridize between exon 5 and 6 of the PD-L1 mRNA sequence. 
     
     
         52 . The method according to  claim 51 , further comprising using at least a sequence 5′-CATTCTCCCTT-3′ (SEQ ID NO: 5) as the reverse PD-L1 target primer or using a sequence 5′-TTTCACATCCATCATTCTCCCTT-3′ (SEQ ID NO: 6) as the reverse PD-L1 target primer comprises. 
     
     
         53 . The method according to  claim 48 , further comprising using at least a sequence 5′-6FAM-GCACATCCTCCA-BBQ-3′ (SEQ ID NO: 7) as the target PD-L1 hydrolysis probe. 
     
     
         54 . The method according to  claim 53 , further comprising using a sequence 5′-6FAM-ACCTCTGGCACATCCTCCAAATGAAAG-BBQ-3′ (SEQ ID NO: 8) and two fluorescent particles as the target PD-L1 hydrolysis probe. 
     
     
         55 . The method according to  claim 53 , further comprising selecting the reference gene from the group consisting of hypoxanthine phosphoribosyl transferase (HPRT), β2-microglobulin (B2M), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and β-actin (ACTB), 18S ribosomal RNA (18S rRNA), 28S ribosomal RNA (28S rRNA), α-tubulin (TUBA), albumin (ALB), ribosomal protein L32 (RPL32), TATA sequence binding protein (TBP), cyclophilin C (CYCC), Eelongation factor 1α (EF1A), RNA polymerase II (RPII). 
     
     
         56 . The method according to  claim 55 , further comprising using β2-microglobulin (B2M) mRNA sequence (SEQ ID NO: 2) as the reference gene, using at least a sequence of 5′-GCCGTGTGAAC-3′ (SEQ ID NO: 9) as the forward B2M reference primer or using a sequence 5′-GCCTGCCGTGTGAACCATGT-3′ (SEQ ID NO: 10) as the forward B2M reference primer, and using at least the sequence of 5′-CTTCAAACCTC-3′ (SEQ ID NO: 11) the reverse B2M as the reference primer or a sequence 5′-AAATGCGGCATCTTCAAACCTC-3′ (SEQ ID NO: 12) as the reverse B2M reference primer. 
     
     
         57 . The method according to  56 , further comprising using fluorescent particles as the target or reference hydrolysis probe. 
     
     
         58 . The method according to  claim 57 , further comprising using a fluorescent reporter covalently attached to a 5′ end of the target or a reference hydrolysis probe and a fluorescent quencher dye attached to a 3′ end as the fluorescent particles of the target or reference hydrolysis probe. 
     
     
         59 . The method according to  claim 58 , further comprising using 6-fluorescein (FAM) as the fluorescent reporter dye and BlackBerry Quencher (BBQ) as the fluorescent quencher dye. 
     
     
         60 . The method according to  claim 56 , further comprising using at least a sequence of 5′-6FAM-CTCGATCCCAC-BBQ-3′ (SEQ ID NO: 13) as the reference B2M hydrolysis probe. 
     
     
         61 . The method according to  claim 60 , further comprising using a sequence 5′-6FAM-CATGATGCTGCTTACATGTCTCGATCCCAC-BBQ-3′ (SEQ ID NO: 14) and two fluorescent particles as the reference B2M hydrolysis probe. 
     
     
         62 . The method according to  claim 48 , further comprising selecting the malignant neoplastic disease from the group consisting of breast, urothelial, colorectal, esophageal, gastric, hepatocellular carcinoma, lung, melanoma, nasopharyngeal, multiple myeloma, renal cell carcinoma, lymphomas, oropharyngeal squamous cell carcinoma, cervical, glioblastoma, malignant mesotheliomas, ovarian and pancreatic cancer. 
     
     
         63 . A kit for amplifying a PD-L1 target sequence and determining the expression of PD-L1 mRNA in a CTC sample from a human, for use in the method according to  claim 48 , comprising;
 a) nucleic acid amplification reagents,   b) a target primer pair being capable of hybridizing to PD-L1 mRNA,   c) a reference primer pair being capable of hybridizing to mRNA of a reference gene,   d) a target hydrolysis probe being capable of hybridizing to PD-L1 mRNA   e) a reference hydrolysis probe being capable of hybridizing to mRNA of the reference gene, and   f) instructions for performing the in vitro method.