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WO2008132167A2 - Indicateurs de diagnostic, de pronostic et/ou prédictifs du cancer du sein - Google Patents

Indicateurs de diagnostic, de pronostic et/ou prédictifs du cancer du sein Download PDF

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WO2008132167A2
WO2008132167A2 PCT/EP2008/055077 EP2008055077W WO2008132167A2 WO 2008132167 A2 WO2008132167 A2 WO 2008132167A2 EP 2008055077 W EP2008055077 W EP 2008055077W WO 2008132167 A2 WO2008132167 A2 WO 2008132167A2
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est
protein
assay
expression
breast cancer
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PCT/EP2008/055077
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WO2008132167A3 (fr
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Lorraine O'driscoll
Padraig Doolan
Martin Clynes
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Dublin City University
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57415—Specifically defined cancers of breast
    • 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
    • C12Q2600/118—Prognosis of disease development

Definitions

  • the invention relates to a novel panel of mRNAs that have potential as diagnostic, prognostic and/or predictive indicators of breast cancer. Members of this panel also have potential as novel therapeutic targets.
  • Biomarkers are defined as molecules which are associated with the presence of cancer, the identification and measurement of which may be useful in patients' diagnosis/prognosis, predicting response to therapy, screening populations that may be at risk of developing breast cancer, and may be potential targets for future "targeted" therapies.
  • microarrays have opened the possibility of unbiased searches for more useful markers and, perhaps, even more importantly, simultaneous analysis of multiple markers and identification of key pathways which may provide a better understanding of development and progression of cancer and ultimately lead to better cancer therapies.
  • the relevance of microarray analysis of breast cancer has been indicated by a number of research groups. In what is considered to be the landmark study in this area, Van't Veer et al. [8] performed expression microarray analysis of 78 sporadic lymph node-negative tumours ( ⁇ 5 cm in diam.) arising in women under 55 yrs. old. Approx.
  • SNAP25-interacting protein i.e. SNIP
  • SNIP is a hydrophilic 145 kDa protein that comprises two coiled-coil domains, two highly charged regions, and two proline-rich domains.
  • SNIP was originally described as selectively expressed in the brain, co- distributing with SNAP-25 in most brain regions.
  • SNIP is tightly associated with the brain cytoskeleton and may serve as a linker protein connecting SNAP-25 to the sub-membranous cytoskeleton, where it is involved in regulating neurosecretion (11).
  • PRAME Preferentially expressed antigen in melanoma
  • PRAME encodes a 509 amino acid protein and, although its function has not yet been defined, PRAME is reported to be a dominant repressor of retinoic acid receptor (RAR) signalling, inhibiting RA-induced differentiation, growth arrest and apoptosis (13).
  • RAR retinoic acid receptor
  • Lipocalin 2 is an iron-binding secreted protein that converts embryonic kidney mesenchyme to epithelia and it may be a useful biomarker for early detection of various renal injuries. Lipocalin 2 is abundantly expressed in adipose tissue and liver. Lipocalin 2 has previously been studied in some cancer types, but its relevance is unclear.
  • pancreatic adenocarcinoma 15
  • pancreatic adenocarcinoma 15
  • studies of a colon cancer cell line suggest lipocalin 2 to be a metastatic suppressor (14).
  • Studies of cells in culture suggest lipocalin 2 to be a candidate paracrine factor that may mediate estrogen-induced proliferation in normal breast tissue (17).
  • 17b-estradiol + medroxyprogesterone acetate treatment has been reported to repress lipocalin 2 expression (18), while N-(4-Hydroxyphenyl)retinamide induces its expression (19).
  • Neuromedin U [Affy. ID: 206023_at; NM 006681; synonyms: none listed]: a structurally highly conserved neuropeptide, is ubiquitously distributed - with highest levels found in the gastrointestinal tract and pituitary. Neuromedin U is involved in alternation of ion transport in the gut and it induces stimulation of smooth muscle. Alevizos et al. (20) reported down-regulation of neuromedin U mRNA levels in oral cancer.
  • neuromedin U Recombinant expression of neuromedin U in esophageal squamous cell carcinoma cell lines resulted in inhibited growth in colony- forming assays, suggest its role as a tumour suppressor gene (21), while analysis of K562 cells suggested neuromedin U to act as an autocrine growth factor for human myeloid leukemia cells and to stimulate growth of primary AML cells (22).
  • the secreted polypeptide neuromedin U has been identified as one of a number of potential markers for ovarian cancer (23), and is abundantly expressed in non-small cell lung cancer (NSCLC), with a significant association reported between neuromedin U expression and poor prognosis (24).
  • SlOO calcium binding protein (SlOOP) [Affy. ID: 204351_at; NM 005980; synonym: MIG9] is a 95 amino acid member of the SlOO protein family of calcium-binding proteins, was initially purified from placenta. SlOOP expression has been detected in a number of cancer types, including pancreatic, breast, colon, prostate and lung. SlOOP expression increases with advancement of pancreatic lesions to invasive adenocarcinomas (26) and it has been proposed as an early developmental marker of pancreatic carcinogenesis (27). In breast cancer, SlOOP over-expression in immortalised human breast epithelial cells suggests its involvement is an early event in breast cancer cell immortalisation (28, 29).
  • SlOOP has been identified as one of the genes whose expression is differentially regulated in luminal epithelium (30). A role in cell cycle imbalance occurring during breast carcinogenesis has been suggested (31) and its expression is regulated by seven progestins analysed (32). SlOOP is one of the genes whose expression is up-regulated in cancer (33). Schor et al. (34) described a positive association between SlOOP protein over-expression and high-risk breast cancer lesions, with SlOOP expression strongly associated with ER status. Approx. 53% of breast tumours have been shown to express SlOOP protein, with expression associated with unfavourable survival outcome for patients (35). MAGE-D4 [Affy.
  • MAGE-El is a family member of genes encoding an antigen expressed on melanoma cells that are recognised by T lymphocytes. MAGE-D4 is expressed predominantly in the brain and ovaries. MAGE-D4 is expressed in renal cell carcinomas glioblastomas (36). In NSCLC there are significantly higher levels of MAGE-D4 in tumour compared to normal lung specimens, with higher levels in squamous cell carcinomas compared to adenocarcinomas and in poorly differentiated tumours compared to well differentiated tumours. MAGE-D4 expression directly associated with proliferative activity of tumour cells, but not pathological stage of tumour (37).
  • NCS-I neuronal calcium sensor- 1
  • VILIPs visinin-like proteins
  • SOXIl (Sex-determining region Y)-box 11/SRY Box 11 (SOXIl) [Affy. ID: 204914 s at; NM_003108; synonyms: none listed] is a SOX gene family of transcription factor development regulators. Epression of SOXl 1 is required for neuron survival and neurite growth and is over-expressed in fetal, compared to adult brain, and in malignant gliomas (39). Schmidt et al. (40) have suggested that a SOXl 1 -derived peptide as a suitable candidate for a glioma T cell-based immunotherapy. SOXl 1 is expressed strongly in classical medulloblastomas, but only weakly in desmoplastic medulloblastomas (41).
  • EST_ AI014551 [Affy. ID: 230945_at; NM 005735; ACTRlB; synonyms: ARPlB, CTRN2] and now described as ARPl actin-related protein 1 homolog B, centractin B (yeast) (ACTRlB).
  • ARPl actin-related protein 1 homolog B centractin B (yeast) (ACTRlB).
  • This gene encodes a 42.3 kD subunit of dynactin, a macromolecular complex consisting of 10 subunits. Dynactin binds to both microtubules and cytoplasmic dynein.
  • This subunit is an actin-related protein. These two proteins are of equal length and share 90% amino acid identity. They are present in a constant ratio of approximately 1 : 15 in the dynactin complex.
  • MGC9712 [Affy. ID: 1558281_a_at; NM 152689; synonym: FLJ24011] is a hypothetical protein. While the coding sequence is predicted, the mRNA record is supported by experimental evidence.
  • EST_AI733801 [Affy. ID: 237923_at; NM 009586 & NM 005069; synonyms: SIM, MGC119447] is now described as Single-minded homolog 2, a member of the bHLH-PAS family of transcription factors (SIM transcription factor) (mSIM).SIMl and SIM2 genes are Drosophila single-minded (sim) homologs.
  • the Drosophila sim gene encodes a transcription factor that is a master regulator of fruit fly neurogenesis.
  • SIM2 maps within the so-called Down syndrome chromosomal region.
  • SIM2s SIM2 short
  • RKO colon cancer cell line
  • P4502B7P1 (formerly described as P4502B6) [Affy. ID: 210272_at & 206754_s_at (NR_001278); synonyms: CYP2B, CYP2B7, CYP2B7P]: Analysis of P4502B7 mRNA in normal and tumour lung tissue indicated reduced mRNA levels in tumour compared to normal tissue, with no correlation found between levels of expression in tumour and histology diagnosis or smoking history (46, 47). P4502B7 mRNA expression was not detected in non-neoplastic oesophageal mucosa (48).
  • P4504B1 [Affy. ID: 210096_at; NM 000779; synonym: P-450HP] metabolises certain pro- toxic xenobiotics (incl. valproic acid, 3-methylindole, and aromatic amines), leading to tissue- specific toxicities in several experimental animals.
  • Bladder-tumour patients have a significantly higher expression level of P4504B1 mRNA than non-bladder tumour patients (49).
  • P4504B1 mRNA was detected by RT-PCR in the majority of cases but not in controls (50).
  • Two studies of CYP4B1 (also known as P4504B1) in breast cancer have been published. Iscan et al.
  • Protein tyrosine phosphatase, receptor type N, 2 [Affy. ID: 203029_s_at; NM 002847 & NM 130842 & NM 130843; synonyms: IAR, ICAAR, PTPRP, IA-2beta] encodes a protein which is a member of the protein tyrosine phosphatase (PTP) family.
  • PTPs are molecules that regulate a variety of cellular processes, including cell growth, differentiation, mitotic cycle, and oncogenic transformation (for Review see 53).
  • PTP and PTPRN are both found to be major autoantigens associated with insulin-dependent diabetes mellitus.
  • PTPNP2 is an alternative isoform to IA-2beta, arising due to alternative splicing of the same gene, and PTPNP2 mRNA expression to be highly restricted in human fetal brain and adult brain and pancreas, transcribing a catalytically inactive receptor protein tyrosine phosphatase, and to be involved in synaptic bouton endocytosis.
  • Insulinoma-associated 1 [Affy. ID: 206502 s at; NM 002196; synonyms: IAl, IA-I] gene is intronless and encodes a protein containing both a zinc finger DNA-binding domain and a putative prohormone domain. This gene is a sensitive marker for neuroendocrine differentiation of human lung tumours. The gene has been described as expressed exclusively during early embryonal development, but re-expressed at high levels in neuroendocrine tumours, suggesting the regulatory region of this gene to be a potential novel tool for transcriptionally-targeted cancer gene therapy against neuroendocrine tumours, including small cell lung cancers (54, 55).
  • Transmembrane protein 25 [Affy. ID: 226647_at; NM 032780; synonym: FLJ14399] was identified and characterized as a novel member of the immunoglobulin superfamily.
  • TMEM25 isoform 1 (BC042896) encodes a 366 amino acid transmembrane protein and TMEM25 isoform 2 (AY358919) encodes a 322 amino acid secreted protein.
  • TMEM25 mRNA was detected in brain (cerebellar cortex and hippocampus), as well as in neuroblastoma, brain tumors, and gastric cancer.
  • TMEM25 is proposed as a target of pharmacogenomics in the field of oncology and regenerative medicine (56 ). NO Syn.
  • NOSTRIN Nitric oxide synthase trafficker
  • NOSTRTN Nitric oxide synthase trafficker
  • FLJ40165 fis/RALBPl associated Eps domain containing 2 REPS2 [Affy.
  • REPS2 RALBPl associated Eps domain containing 2
  • the product of this gene is part of a protein complex that regulates the endocytosis of growth factor receptors.
  • the encoded protein directly interacts with a GTPase activating protein that functions downstream of the small G protein RaI. Its expression can negatively affect receptor internalisation and inhibit growth factor signaling.
  • MRG2, DKFZp547H236 is a member of the TALE family of homeodomain proteins, is reported as widely expressed at low levels in both hematopoietic and non-hematopoietic tissues including liver, pancreas and brain, with higher levels found in appendix.
  • the murine homolog of Meis 1 is known to be activated in myeloid leukaemia by retroviral insertion.
  • Meis 1 and Hoxa9 in leukemia specimens and leukemia cell lines indicated frequent co-expression of these genes in acute myelogenous leukaemia (AML) of all sub-types except promyelocytic leukaemia, suggesting that co- activation of Meis 1 and Hoxa9 is a common event in AML.
  • Meis 1 knock-down in neuroblastoma cell line resulted in reduced proliferation rates (58).
  • over-expression of Meis 1 cDNA in human and murine myeloid, lymphoid, and fibroblast cell lines strongly induces apoptosis through a caspase-dependent mechanism (59).
  • Breast carcinoma amplified sequence 1 (BCASl) [Affy. ID: 204378_at; NM 003657; synonyms: AIBCl, NABCl] is also known as novel amplified in breast cancer (NABCl) is amplified in a variety of tumors and associated with more aggressive tumor phenotypes.
  • BCASl /NABCl was found to be highly expressed in three amplified breast cancer cell lines and in one breast tumour although not in the breast cancer cell line MCF7. Although not consistently expressed, BCAS1/NABC1 is reported to be a candidate oncogene and amplified in adenocarcinomas of the gastroesophageal junction (60) and pancreas (61). Studies involving over-expression of BCASl /NABCl in NIH3T3 cells have suggested, however, that BCASl is not a prototypical oncogene (62).
  • Microarray analysis of a tamoxifen-sensitive (MaCa3366) and tamoxifen-resistant (MaCa3366/TAM) cell line indicated BCAS1/NABC1 to be differentially expressed (63).
  • a study of breast tumour and colorectum tumour by Correa et al. (64) reported BCASl to be over-expressed in breast tumours and down-regulated in colorectal tumours.
  • IGFRl is believed to play a role in maintaining the malignant phenotype in cancer, including cancer cell proliferation and metastasis and disruption of IGFRl activation has been shown to inhibit growth and motility of a broad range of cancer cells (65, 66).
  • EST_BF109381 now termed Sideroflexin 4 [Affy. ID: 230637_at; NM 178867, NM_213649 & NM_213650] has been mapped to chromosome 10q25-26 and spans more than 24.7kb of the genomic DNA. It is 1428 base pair in length and the putative protein contains 305 amino acids with a conserved predicted five-transmembrane-domains structure.
  • the present invention is the first whole genome microarray study analysing a novel large bank of breast tumour and normal specimens, where there is extensive follow-up information available on the clinicopatho logical characteristics of the patients and their outcome, in terms of both relapse-free survival (RFS) and overall survival (OS).
  • RFS relapse-free survival
  • OS overall survival
  • a further object is to meet the increasing demand for accurate diagnosis of breast cancers and to facilitate personalised medical treatment.
  • a further object is to enable the determination of the success rate of a particular treatment on a patient.
  • a still further object is to provide an assay for the determination of susceptibility to breast cancer, for the diagnosis of breast cancer and the prognosis for cancer patients.
  • a panel of biomarkers could come at least in part, help to overcome the problem of patients receiving chemotherapy or radiotherapy from which they derive no benefit, and also meet the increasing demand for accurate diagnosis and/or prognosis. With increasing numbers of personalised drugs entering the market, is has become important to determine the success rate of a particular treatment on a patient, which in turn stresses the need for IVDs. Summary of the Invention
  • the invention provides a panel of biomarkers useful in the diagnosis and treatment of breast cancer.
  • Some markers are diagnostic markers for breast cancer, as they are identified in breast tumours, but are not present in normal breast tissue while some are prognostic markers, expression of which are associated with spread to lymph nodes, and outcome in terms of relapse-free survival and overall survival time from diagnosis.
  • Other markers are predictive markers in that they are associated with response to chemotherapy.
  • the markers may be therapeutic targets in the development of alternative or more effective breast cancer treatments.
  • an assay for breast cancer comprising mRNA selected from at least one of the mRNAs encoding Lipocalin 2; SNIP (SNAP25- interacting protein); Neuromedin U; SlOO Ca-binding protein P; Melanoma antigen fam.D, 4; Preferentially exp. antigen in melanoma (PRAME); Frequenin Homolog 21; SRY Boxl l; EST AI014551; MGC9712; EST AI870951; EST AI733801; PoIy(A) binding protein;
  • IGFRl Insulin-like GFl receptor
  • the assay may be a diagnostic assay for breast cancer comprising mRNA selected from at least one of the mRNAs encoding Melanoma antigen fam.D,4; Preferentially exp. antigen in melanoma (PRAME); Frequenin Homolog 21; EST AI014551; MGC9712; EST AI870951; EST AI733801; Insulinoma-assoc.
  • EST.BF224050 FLJ13236; FLJ45983; EST BF109381; BM992214; EST BG281679; EST AWOl 5140 , a polypeptide encoded by any one of these mRNAs, a protein comprising a polypeptide encoded by any one of the mRNAs or a antibody raised against such a polypeptide or protein.
  • the assay may also be a prognostic assay for breast cancer comprising mRNA selected from at least one of the mRNAs encoding Lipocalin 2; Neuromedin U; SNIP (SNAP25-interacting protein); S 100 Ca-binding protein P; Melanoma antigen fam.D, 4; Preferentially exp.
  • the assay may also be a predictive assay for breast cancer following chemotherapy treatment comprising mRNA selected from at least one of the mRNAs encoding SNIP (SNAP25- interacting protein); SlOO Ca-binding protein P; Melanoma antigen fam.D, 4; Preferentially exp. antigen in melanoma (PRAME); SRY Boxl l; MGC9712; ESTAI733801; P4502B7P1; EST (241368); EST/Disintegrin-like & metalloprotease Type 1, 15; NO Syn. Trafficker; EST/similar to pleckstrin & Sec7 domain-containing 3; TMEM25 (Transmembrane protein 25); FLJ40165 fis; Hypo.
  • mRNA selected from at least one of the mRNAs encoding SNIP (SNAP25- interacting protein); SlOO Ca-binding protein P; Melanoma antigen fam.D, 4; Preferentially exp. antigen in
  • the assay may be a real time PCR assay, a customised micro array assay or an immuno- histochemical assay. All such assays are well known to those of skill in the art. Where the assay is an immuno-histochemical assay the antibody maybe labelled with a suitable label.
  • Suitable labels include coloured labels, fluorescent labels or radioactive labels.
  • the invention also relates to use of any one of the mRNAs, DNAs, cDNAs, polypeptides, proteins or antibodies defined above in a method of identifying therapeutic targets for the treatment of breast cancer or in identifying suitable methods of treatment for breast cancer patients.
  • the invention provides a solid support onto which one or more of the mRNAs, DNAs, cDNAs, polypeptides, proteins or antibodies defined above have been fixed.
  • Fig. Ub Summary of qRT-PCR Validation Analysis Work-Flow for each "Test" mRNA Detailed Description of the Drawings
  • Fig. 1 A number of clinical and pathologic parameters were abstracted from patients' charts including age, post-operative treatment and follow-up, tumour stage, and hormonal analysis. Pathologic material was examined on each case by SK. Tumours were typed 69) and graded (70) as previously described. Staging was performed according to the TNM system of the UICC (71). Nineteen non-cancerous breast biopsies were also included in these studies to represent normal breast tissue. A summary of the analysis of in the study is illustrated in Fig. 1.
  • RNA Extraction For RNA analyses, dissected tumours that had been snap-frozen in liquid nitrogen and then stored at -70/-80 0 C until required were homogenised, on ice, in 2 ml TriReagent (Sigma; Poole, England) and total RNA was subsequently isolated according to the manufacturer's instructions. RNA quantity and purity were assessed at 260 nm and 280 nm using a Nanodrop (ND- 1000; Labtech. International); the A 2 60/A 2 80 ratio of pure RNA is approximately 2. RNA qualitatively was evaluated using an Agilent bioanalyser (Agilent 2100; Agilent Technologies). For Microarray Analysis RNA Amplification & Labelling
  • Microarray hybridization Hybridisation solution (1 mol/1 NaCl, 20 mmol/1 EDTA, 100 mmol/1 2-(N-morpholino) ethanesulfonic acid, and 0.01% Tween 20) was used to pre-hybridise Affymetrix; U133 Plus 2.0 oligonucleotide microarrays for 10 minutes at 45°C and 60 rpm.
  • the pre-hybridisation solution was removed and replaced with 200 ⁇ l hybridisation solution containing 0.05 ⁇ g/ ⁇ l fragmented cRNA.
  • the arrays were hybridised for 16 hours at 45°C and 60 rpm.
  • Arrays were subsequently washed (Affymetrix Fluidics Station 400) and stained with streptavidin-phycoerythrin (Stain Buffer, 2 mg/ml acetylated BSA and 10 ⁇ g/ml streptavidin R-phycoerythrin; Molecular Probes, Inc., Eugene, OR), and were scanned on an Affymetrix GCS GeneChip GeneArray scanner. Resulting data was analysed using GCOS, dCHIP, and GeneSpring (Agilent Technologies). Normalisation and Filtering CeI files obtained from the GCOS server were processed and normalized by dCHIP
  • a filter was designed to include a fold change of at least 1.2 fold between normal and BCC specimens, a difference of at least 100 Affymetrix arbitrary units between normal and BCC average values, and a t-test between normal and BCC (with a p-value cut-off ⁇ 0.05).
  • cDNA Formation on mRNA Template Following priming with oligo (dT) at 65°C for 5 minutes, followed by 1 minute incubation on ice, cDNA was synthesised from 100 ng total RNA, using Superscript III RNase H- (with increased thermal stability; Invitrogen), RNase OUT Ribonuclease (active against RNase A, B and C; Invitrogen) and a cocktail of dNTPs, by incubating at 50 0 C for 1 hour, followed by 70 0 C for 15 minutes, in a 40 ⁇ l reaction volume.
  • RNase H- with increased thermal stability; Invitrogen
  • RNase OUT Ribonuclease active against RNase A, B and C; Invitrogen
  • cocktail of dNTPs by incubating at 50 0 C for 1 hour, followed by 70 0 C for 15 minutes, in a 40 ⁇ l reaction volume.
  • Primer Design Primers and TaqMan probes were designed using Primer Express Software 2.0 ensuring that, for optimum amplification efficiency, PCR primers produce an amplicon of 50- 150bp; flanking the probe, without overlap; with no more than two Gs and/or Cs of the 3' end; and with a T m of 58-6O 0 C.
  • the qPCR temperature profile of all reactions was 50 0 C for 2 minutes 95°C for 10 minutes, 40 cycles of 95 0 C and 6O 0 C for 1 minute.
  • Individual specimens were analysed in triplicate. Analysis including all components except cDNA (replaced with H20) showed no contamination of reaction components. Additionally, controls including RNA, but lacking reverse transcriptase enzyme and lacking oligo(dT), respectively, verified no DNA/pseudogene contamination of starting material. Threshold cycle (C T ) results were subsequently normalised to 2 suitable endogenous controls - ⁇ -actin and GAPDH - and calibrated against a pooled cDNA from breast specimens, using the comparative C ⁇ method, 2 " ⁇ CT (72). Statistical analysis
  • tumours Eighty- one tumours were invasive ductal carcinoma, seventeen were invasive lobular and five were tumours of special type (two tubular and three mucinous). Eleven tumours were grade 1; thirty- nine were grade 2; and fifty-three were grade 3.
  • Sixty-six tumours were oestrogen receptor positive and thirty- four were oestrogen receptor negative (oestrogen receptor status was determined by Enzyme Immuno-Assay (EIA); a positive result was defined as more than 200 frnol/g protein). Oestrogen receptor status was not available for 3 patients. Forty-five tumours had no axillary metastases and fifty-eight tumours had metastasised to axillary lymph nodes.
  • Results are shown in Table 2.
  • SNIP/P140Cap mRNA was detected in 36.9% (38/103) see table 2 of the breast tumour specimens analysed and was found not to be expressed in any of the normal breast specimens.
  • PRAME mRNA was detected in 53.4% (55/103) of the breast tumour specimens analysed (and in 36.8% (7/19) of the normal breast specimens).
  • Neuromedin U was expressed in almost half (-48%) of breast cancer cases, but only -6% of normal breast tissues.
  • SlOOP was expressed in the majority of breast tumours, but in ⁇ 25% of normal breast tissue specimens.
  • MAGE-D4 was expressed in >40% of tumours, but undetectable in normal breast tissue and is a potential diagnostic biomarker for breast cancer.
  • Frequenin homolog 21 was absent from normal breast tissue, but present in approx. 12% breast tumours. SRY Box 11 was expressed in more than half (-55%) of breast cancer cases, but only -6% of normal breast tissues.
  • EST AI014551/ACTR1B was undetected in normal breast tissue, but was present in approx. 52% of breast tumours.
  • MGC9712 was undetected in normal breast tissue, but was present in 40% of breast tumours.
  • EST AI870951 was undetected in normal breast tissue, but was found to be expressed by approx. 41% of breast tumours.
  • EST AI733801 was expressed in approx. 35% of breast tumours, but absent from normal breast tissue. Insulinoma-associated 1 (INSMl) was expressed in approx.
  • EST/Sushi-domain containing 3 was expressed in approx. 70% of breast tumours, but ⁇ 24% of normal breast specimens.
  • EST BF224050 was expressed in approx. 57% of breast tumours, but in no normal breast specimens.
  • FLJ13236 was expressed in approx. 51% of breast tumours, but in no normal breast specimens.
  • FLJ45983 was expressed in approx. 43% of breast tumours, but in no normal breast specimens.
  • EST BF109381/Sideroflexin 4 was expressed in approx. 60% of breast tumours, but in no normal breast specimens.
  • BM992214 was expressed in approx. 35% of breast tumours, but in no normal breast specimens.
  • EST BG281679 was expressed in approx.
  • Kaplan-Meier analysis indicated no correlation between RFS and expression of SNIP/PI 40Cap mRNA.
  • tumour grade p ⁇ 0.0005
  • Table 2 also shows results for other markers. Lipocalin 2 has not previously been studied in a large bank of breast tissue specimens. In this study its expression in tumours was associated with ER-negativity and with poorer outcome in terms of both disease- free survival and overall survival.
  • Neuromedin U expression tended to be found in higher grade tumours and to be significantly associated with poorer outcome for patients, in terms of both disease- free survival and overall survival.
  • SlOOP expression is significantly associated with spread of tumour to the lymph nodes and with poor prognosis, in terms of both RFS and OS.
  • MAGE-D4 tended to associate with spread of cancer to lymph nodes and its presence was significantly associated with ER-negativity, higher grade tumours, and with shortened RFS and OS (potential poor prognosis factor).
  • Frequenin homolog 21 expression is apparently an unfavourable indicator - it tended to associate with larger tumours and was significantly associated with ER-negativity and with poor outcome in terms of RFS and OS.
  • SRY Box 11 expression was associated with ER-negativity and higher grade tumours, being indicative (prognostic) of poor outcome for patients in terms of both RFS and OS and apparently also predictive of shortened OS time from diagnosis.
  • EST AI014551/ACTR1B was associated with shortened RFS and OS from time of diagnosis, and similarly MGC9712 was associated with shortened OS from time of diagnosis.
  • EST AI870951 was associated with ER-negativity, higher grade tumours, and reduced RFS and OS times from diagnosis.
  • EST AI733801 expression is associated with higher grade tumours, spread to lymph nodes, and shortened RFS and OS times.
  • PoIy(A) binding protein nuclear 1/EST transcript was significantly associated with favourable outcome from time of diagnosis, in terms of both RFS and OS.
  • Expression of P4502B7P1 was significantly associated with ER-positivity, lower grade tumours, and favourable outcome from time of diagnosis, in terms of both RFS and OS (indicting prognostic relevance).
  • P4504B1 expression to be significantly associated with favourable outcome for breast cancer patients, in terms of both RFS and OS.
  • PTPRN2 expression WAS significantly associated with favourable outcome for breast cancer patients, in terms of both RFS and OS.
  • EST (241368) expression WAS significantly associated with ER-positivity, lower grade tumours, and overall favourable outcome for breast cancer patients, in terms of both RFS and
  • EST/Disintegrin-like & metalloprotease Type 1 15 expression tended to associate with lower grade tumours, and was significantly associated with favourable outcome for breast cancer patients, in terms of both RFS and OS.
  • insulinoma-associated 1 (INSMl) transcript tended to be associated with smaller tumours and was significantly associated favourably with longer times to relapse and death from breast cancer.
  • NO Syn. trafficker (NOSTRIN) expression was associated with lower grade tumours, and extended time to relapse and death from breast cancer.
  • EST/similar to pleckstrin and Sec7 domain containing 3 expression was associated with smaller tumours, and extended time to relapse and death from breast cancer.
  • Transmembrane protein 25 (TMEM25) expression was associated with favourable RFS and OS times in analysis of all cases. Expression of REPS2 mRNA and FLJ32685 mRNA were both associated with lower grade tumours and favourable RFS and OS times.
  • Meisl was associated with ER-positivity, smaller, lower grade tumours and favourable RFS and OS times.
  • EST_BF109381/Sideroflexin 4 expression was associated with lower grade, smaller tumours that did not spread to the lymph nodes, with favourable outcome in terms of both RFS and OS times from diagnosis.
  • BM992214 was associated with ER-positivity, lower grade tumours, and favourable outcome in terms of both RFS and OS times from diagnosis.
  • EST BG281679 was associated with smaller, lower grades tumours, and extended survival time from diagnosis.
  • EST AWOl 5140 was associated with lower grades tumours and extended survival time from diagnosis.
  • marker mRNA is predictive of response to adjuvant chemotherapy
  • SlOOP is predictive of shortened overall survival time from diagnosis.
  • MAGE-D4 was predictive of poor response, in terms of both RFS and OS to adjuvant chemotherapy.
  • MGC9712 was apparently predictive of shortened RFS times.
  • EST AI733801 expression had predictive potential, in terms of both RFS and OS for adjuvant chemotherapy.
  • EST/Disintegrin-like & metalloprotease Type 1 15 transcript associated with extended time from diagnosis to relapse.
  • NOSTRTN mRNA was predictive of favourable OS times.
  • Expression of EST/similar to pleckstrin and Sec7 domain containing 3 mRNA was predictive of favourable OS times.
  • Transmembrane protein 25 (TMEM25) expression has predictive (associated with favourable
  • REPS2 expression was associated with extended survival time from diagnosis.
  • FLJ32685 expression was associated with extended RFS and OS times from diagnosis.
  • prognostic factors currently relied upon are those determined by clinical or standard pathological approaches namely lymph node status, tumour size, histological grade, nuclear grade and tumour histology.
  • Predictive factors are limited to ER, progesterone receptor, and herl/neu, which are used to predict response to hormonal treatment and herceptin, respectively; no reliable predictive markers for response to chemotherapy have been identified.
  • SNIP/PI 40Cap mRNA was detected in approximately 37% of the breast tumours analysed and its expression was significantly associated with increased tumour size. Although an association was not found between expression of SNIP/PI 40Cap mRNA and RFS, a significant association was evident between expression of this transcript and disease outcome, in terms of overall survival; with the presence of SNIP/PI 40Cap mRNA associated with shorter survival from diagnosis. Indeed, multivariate analysis indicates that, similarly to advanced tumour grades and spread of cancer to lymph nodes, SNIP/PI 40Cap mRNA expression is an independent unfavourable prognostic factor for OS. The results from this study were further analysed to investigate a potential predictive relevance for SNIP/P140Cap.
  • PRAME expression Although the incidence of PRAME expression has been assessed in many cancer types by other researchers, the clinicopatho logical relevance of its expression has, in general, been limited to neuroblastoma, leukaemia and multiple myeloma. As described previously, analysis of blood disorders has indicated that PRAME is transcribed in leukaemia cells, but not in normal bone marrow or peripheral blood mononuclear cells (73, 74). However, reports on the clinical relevance of PRAME expression in blood diseases have been conflicting.
  • PRAME has been suggested as a specific marker for acute megakaryoblastic leukaemia, with no expression of this mRNA detected in transient myeloproliferative disorder (75) and recent studies have concluded that PRAME quantification by qPCR appears to be suitable for monitoring minimal residual disease in PRAME-positive leukaemia (76, 77).
  • PRAME mRNA found in approximately 60% of cases
  • PRAME mRNA found in approximately 60% of cases
  • PRAME protein expression at diagnosis did not correlate with established clinical or pathologic characteristics namely, ER status of patients, lymph node status, histological sub-type, histological grade, tumour size or subsequent treatment with adjuvant chemotherapy.
  • PRAME expression is associated with unfavourable outcome for breast cancer patients - with a significant correlation between expression of this transcript in breast tumours and shortened RFS and OS from diagnosis. Indeed, multivariate analysis indicates that, similarly to tumour grades, tumour sizes and ER status, PRAME mRNA expression in breast tumours is an independent unfavourable prognostic factor for both RFS and OS.
  • prognostic markers are associated with the spread of the cancer to the lymph notes and poor outcome in terms of relapse- free survival and overall survival from the time of diagnosis.
  • Predictive markers are associated with the response to chemotherapy with CMF +/- adriamycin.
  • prognostic markers associated with good prognosis for breast cancer: PoIy(A) binding protein AI492388 P4502B7P1 P4504B1 Protein tyrosine phosphatide receptor type N,2
  • TMEM25 Transmembrane protein 25
  • Insulin-like GFl receptor IGFRl
  • Table 2 36 Panel Association with Clinical & Pathological Information Available on the 104 Breast Tumours Analysed
  • mRNA gene transcript
  • Ts the % of tumours in which this mRNA was detected (e.g. 48% of the 104 tumours);
  • Ns the % of normal breast specimens in which this mRNA was detected
  • G/B whether the presence of this mRNA is a good/favourable indicator or a bad/unfavourable indicator for the patient;
  • RFS P values showing significant association with RFS (these P values were derived from the KM curves in the 1 st doc. attached); G/B: whether the presence of this mRNA is a good/favourable indicator or a bad/unfavourable indicator for the patient;
  • ER whether or not the presence of the given mRNA is significantly associated with estrogen receptor status i.e. ER presence or absence.
  • ER + i.e. ER presence
  • LN Lymph node status; whether or not the presence of the given mRNA is significantly associated with the spread of cancer to the lymph nodes.
  • tumours that have not spread to the lymph nodes are "better” and “more contained” than those that have spread.
  • Tumours that spread to the nodes, if not caught in time, may be on their way to metastasising to form secondaries in other organs.
  • Spread to lymph nodes is an important consideration for breast cancer and its follow-on therapy;
  • tumour Size Bigger tumours would be considered worse to have than smaller tumours.
  • Pedersen N Pedersen MW. Lan MS. Breslin MB. Poulsen HS. (2006). Cancer Gene Ther.

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Abstract

L'invention porte sur un nouveau panneau de marqueurs d'ARNm qui ont un potentiel en tant qu'indicateurs de diagnostic, de pronostic et/ou prédictifs du cancer du sein. Des éléments de ce panneau ont également un potentiel en tant que nouvelles cibles thérapeutiques.
PCT/EP2008/055077 2007-04-26 2008-04-25 Indicateurs de diagnostic, de pronostic et/ou prédictifs du cancer du sein WO2008132167A2 (fr)

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GB2496150A (en) * 2011-11-02 2013-05-08 Provost Fellows & Scholars College Of The Holy Undivided Trinity Of Queen Elizabeth Near Dublin Biomarker and target for responsiveness and resistance to cancer targeting agents
WO2013057323A3 (fr) * 2011-10-21 2013-06-27 The Provost, Fellows, Foundation Scholars, And The Other Members Of The Board, Of The College Of The Holy And Undivided Trinity Of Queen Elizabeth, Near Dublin Marqueur et cible pour la sensibilité et la résistance à des agents anticancéreux
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WO2009138850A1 (fr) * 2008-05-13 2009-11-19 Istituto Nazionale Di Genetica Molecolare - Ingm Cellules hematopoietiques exprimant la proteine susd3 et ligands associes
CN104080924A (zh) * 2011-08-16 2014-10-01 昂科赛特公司 用于治疗和诊断乳腺癌的方法和组合物
WO2013057323A3 (fr) * 2011-10-21 2013-06-27 The Provost, Fellows, Foundation Scholars, And The Other Members Of The Board, Of The College Of The Holy And Undivided Trinity Of Queen Elizabeth, Near Dublin Marqueur et cible pour la sensibilité et la résistance à des agents anticancéreux
GB2496150A (en) * 2011-11-02 2013-05-08 Provost Fellows & Scholars College Of The Holy Undivided Trinity Of Queen Elizabeth Near Dublin Biomarker and target for responsiveness and resistance to cancer targeting agents
WO2016144267A1 (fr) * 2015-03-12 2016-09-15 Agency For Science, Technology And Research Dosage multigène
CN108026585A (zh) * 2015-03-12 2018-05-11 路胜基因（新加坡）私人有限公司 多基因检测
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US11242565B2 (en) 2015-03-12 2022-02-08 Lucence Life Sciences Pte Ltd. Multigene assay
CN112449684A (zh) * 2018-09-12 2021-03-05 菲亚诺斯蒂克斯有限责任公司 用于诊断肝病的方法
CN115838802A (zh) * 2022-08-29 2023-03-24 山东第一医科大学第一附属医院(山东省千佛山医院) lncRNA PTPRN2-AS1作为肾透明细胞癌预后标志物的应用

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