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(12) Patent Application Publication (10) Pub. No.: US 2016/0068916 A1 Nekarda Et Al

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(12) Patent Application Publication (10) Pub. No.: US 2016/0068916 A1 Nekarda Et Al US 2016.0068916A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2016/0068916 A1 Nekarda et al. (43) Pub. Date: Mar. 10, 2016 (54) TEST KITS (30) Foreign Application Priority Data (71) Applicant: Pacific Edge Limited, Dunedin (NZ) Dec. 23, 2005 (NZ) ........................................ 544.432 (72) Inventors: Hjalmar Nekarda, Taufkirchen (DE); Publication Classification Jan Friederichs, Munich (DE); Bernhard Holzmann, Munich (DE); (51) Int. Cl. Robert Rosenberg, Munich (DE); CI2O I/68 (2006.01) Anthony Edmund Reeve, Dunedin (52) U.S. Cl. (NZ); Michael Alan Black, Dunedin CPC ........ CI2O I/6886 (2013.01); C12O 2600/1 18 (NZ); John Lindsay McCall, Auckland (2013.01); C12O 2600/158 (2013.01); C12O (NZ); Yu-Hsin Lin, Dunedin (NZ): 2600/16 (2013.01) Robert Craig Pollock, Dunedin (NZ) (57) ABSTRACT This invention relates to prognostic signatures, and compo (73) Assignee: Pacific Edge Limited, Dunedin (NZ) sitions and methods for determining the prognosis of cancer in a patient, particularly for colorectal cancer. Specifically, (21) Appl. No.: 14/860,024 this invention relates to the use of genetic markers for the prediction of the prognosis of cancer, such as colorectal can (22) Filed: Sep. 21, 2015 cer, based on signatures of genetic markers. In various aspects, the invention relates to a method of predicting the likelihood of long-term Survival of a cancer patient, a method Related U.S. Application Data of determining a treatment regime for a cancer patient, a (60) Division of application No. 12/214,782, filed on Jun. method of preparing a treatment modality for a cancer patient, 20, 2008, which is a continuation of application No. among other methods as well as kits and devices for carrying PCT/NZ2006/000343, filed on Dec. 22, 2006. out these methods. Patent Application Publication Mar. 10, 2016 Sheet 1 of 9 US 2016/006891.6 A1 FIG. New Zealand CRC data set German CRC data Set Oligo-spotted arrays, Affymetrix arrays (Sample n is 149, 47 relapsed, (Sample, n=55, 26 relapsed, 102 non-relapsed; genes, nr 038) 29 non-reiapsed; genes, nr 5260) Class predicition Class predicition identify a 19 gene signature, called German signature Germar CRC data set with NZ NZ CRC data set with Geria signature genes signature genes (Sample, n=55, genes, n-18) (Sample, rs149; genes, n-4) Patent Application Publication Mar. 10, 2016 Sheet 2 of 9 US 2016/006891.6 A1 FG, 2. ----- | st w w v . “rs----- , r wr 23 . g : '....... ; go y ... s. 5. sos w wg M, v. is P-elis 50,000 ... SE---- Predicted recurrentsecret S -P-value Predictax <0.000 cr-ferrent e - - - - Predicted recurrent c T. w ve------e.truerrrrrrrrrroom gec or soverryw - 0 0 20 30 40 50 60 1 20 30 4, 5, 80 Risease free survival months} Disease free surviva (onths) C D fro- wned g o is - a assaakassassississ r. was as 8 3 wer 3 - 3 x Y a in . 2 *A : is . P-waite 46.49 P-waite 4039 - Predicted non-recurrent - Fredicted non-recurrent - - - - Precicted fectifier - - - - Pfeiced feetiret d s Morrowmirrowroo. s ---r-r-sur 0 0 20 3 43 50 60 2 30 ( 50 80 Disease free survival months} isease free survival months Patent Application Publication Mar. 10, 2016 Sheet 3 of 9 US 2016/006891.6 A1 FG, 3 do a W ** - & a dtra - - - - - a a A- a v w w y a A P. s r. v. v. ap w Aw s war Stage if (predictedio-recurent * * * Stage it (predicted rectivent} * * * * * Stage if predicted non-recurret ' ' ' ' Stage 3 predicted recurrent) 10 2. 30 40 53 6) Disease free survival months) g & s s 3 s . &ss. a w w w w aw w ruit; p una' r t Malea - rew --- - -------- s Stage pedicted for-exuifei - - - Stage is predicted ?ectifret) Stage i3 predicted ro-recurrent 3 * * * * Stage (predicted recipient) i O 2. 3) 4. Sc 6. Disease free survival months) Patent Application Publication Mar. 10, 2016 Sheet 4 of 9 US 2016/006891.6 A1 •,ç•k?$ ***??{}--*J{};1r--;******************************•4s??»),«ºgaer. *· so so go go go 8:SS 83 geod goisse Acago S.A digitoxic So Kyoid Patent Application Publication Mar. 10, 2016 Sheet 5 of 9 US 2016/006891.6 A1 ***********,………… {}{}, ******® *** {}{}} (panu?uoo)?ºr? 99' Sg 8.3avo pegsses Áf383.33 Rosiodoxic Patent Application Publication Mar. 10, 2016 Sheet 6 of 9 US 2016/006891.6 A1 { ),*{?3}********###***********•** *:**;·.x xx O E. O 90 gC. ic 33ssage 33&so es; SSaga: Agoauco s, so took S.A. c. logicatio, Patent Application Publication Mar. 10, 2016 Sheet 7 of 9 US 2016/006891.6 A1 s s OS" is City eaac gess said goes to a taogi Odo Patent Application Publication Mar. 10, 2016 Sheet 8 of 9 US 2016/006891.6 A1 F.G. 6 Number of Appearances in “Top 108' List SO 2:3 wer o i e d s: Patent Application Publication Mar. 10, 2016 Sheet 9 of 9 US 2016/006891.6 A1 FIG 7 Number of Appearances in “Top 100” List. Wilcoxon s i i nod-test : C s 3 US 2016/006891.6 A1 Mar. 10, 2016 TEST KITS accuracy of prediction can be enhanced by combining the markers together into a prognostic signature for, providing for RELATED APPLICATIONS much more effective individual tests than single-gene assays. 0001. This application is a Divisional application filed Also provided for is the application of techniques, such as under 35 U.S.C. S 120 and 37 C.F.R. 1.53(b), which claims statistics, machine learning, artificial intelligence, and data priority to U.S. patent application Ser. No. 13/214,782, which mining to the prognostics signatures to generate prediction models. In another embodiment, expression levels of the is a Continuation under 35 U.S.C. S.1.1.11(a) of PCT/NZ2006/ markers of a particular prognostic signature in the tumour of 000343, International Filing Date 22 Dec. 2006, which claims a patient can then be applied to the prediction model to deter the benefit of New Zealand Provisional Patent Application mine the prognosis. No. 544432 filed Dec. 23, 2005, each of which is incorporated 0008. In certain embodiments, the expression level of the by reference herein in its entirety. markers can be established using microarray methods, quan FIELD OF THE INVENTION titative polymerase chain reaction (qPCR), or immunoassays. 0002. This invention relates to test kits, methods and com BRIEF DESCRIPTION OF THE FIGURES positions for determining the prognosis of cancer, particu larly colorectal cancer, in a patient. Specifically, this inven 0009. This invention is described with reference to spe tion relates to the use of genetic markers for determining the cific embodiments thereofand with reference to the figures, in prognosis of cancer, such as colorectal cancer, based on prog which: nostic signatures. 0010 FIG. 1 depicts a flow chart showing the methodol ogy for producing the prognostic signatures from 149 New BACKGROUND OF THE INVENTION Zealand (NZ) and 55 German (DE) colorectal cancer (CRC) samples. New Zealand RNA samples were hybridized to oli 0003 Colorectal cancer (CRC) is one of the most common gonucleotide spotted arrays, with a 22-gene signature pro cancers in the developed world, and its incidence is continu duced via leave one out cross validation (LOOCV), and then ing to increase. Although the progression of colorectal cancer independently validated by LOOCV using the 55 sample DE from benign polyp to adenoma to carcinoma is well Studied data set. German RNA samples were hybridized to Affyme (1), the molecular events influencing the transition and estab trix arrays, with a 19-gene signature produced via LOOCV. lishment of metastasis are less well understood. The progno and then independently validated by LOOCV using the NZ sis and treatment of CRC currently depends on the clinico data set. pathological stage of disease at the time of diagnosis, and 0011 FIG. 2 depicts a Kaplan-Meier analysis of disease primary Surgical treatment. Unfortunately disease stage alone free survival time with patients predicted as high versus low does not allow accurate prediction of outcome for individual risk of tumour recurrence: FIG. 2a, using NZ 22-gene signa patients. If patient outcomes could be predicted more accu ture on 149 tumours from NZ patients: FIG. 2b, using DE rately treatments could be tailored to avoid under-treating 19-genesignature on 55 tumours from DE patients: FIG.2c, patients destined to relapse, or over-treating patients who NZ prognostic signature validated on 55 tumours from DE would be helped by surgery alone. patients: FIG. 2d. DE prognostic signature validated on 149 0004. Many attempts have been made to identify markers tumours from NZ patients. P-values were calculated using the that predict clinical outcome in CRC. Until recently most log-rank test. studies focused on single proteins or gene mutations with 0012 FIG. 3 depicts a Kaplan-Meier analysis of disease limited Success in terms of prognostic information (2). free survival time with patients predicted as high versus low Microarray technology enables the identification of sets of risk of tumour recurrence: FIG. 3a, using the 22-gene NZ genes, called classifiers or signatures that correlate with can signature on NZ patients with Stage II and Stage III disease; cer outcome. This approach has been applied to a variety of FIG. 3b, using the 19-gene DE signature on NZ patients with cancers, including CRC (3-5), but methodological problems Stage II and Stage III disease. and a lack of independent validation has cast doubt over the 0013 FIG. 4 shows the predictive value of signatures of findings (6.7). Furthermore, doubts about the ability of clas varying lengths for prognosis of colorectal cancer.
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