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WO 2012/116979 Al 7 September 2012 (07.09.2012) P O P C T

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WO 2012/116979 Al 7 September 2012 (07.09.2012) P O P C T (12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date WO 2012/116979 Al 7 September 2012 (07.09.2012) P O P C T (51) International Patent Classification: (81) Designated States (unless otherwise indicated, for every G01N33/574 (2006.01) kind of national protection available): AE, AG, AL, AM, AO, AT, AU, AZ, BA, BB, BG, BH, BR, BW, BY, BZ, (21) International Application Number: CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, DO, PCT/EP2012/053349 DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, (22) International Filing Date: HR, HU, ID, IL, IN, IS, JP, KE, KG, KM, KN, KP, KR, 28 February 2012 (28.02.2012) KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD, ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, (25) Filing Language: English OM, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SC, SD, (26) Publication Language: English SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (30) Priority Data: 11156242.7 28 February 201 1 (28.02.201 1) (84) Designated States (unless otherwise indicated, for every kind of regional protection available): ARIPO (BW, GH, (71) Applicant (for all designated States except US): RHEIN- GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, SZ, TZ, ISCHE FRIEDRICH-WILHELMS-UNIVERSITAT UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, BONN [DE/DE]; Regina-Pacis-Weg 3, 53 113 Bonn (DE). TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, (72) Inventors; and LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, (75) Inventors/Applicants (for US only): DEBALD, Manuel SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, [DE/DE]; Haager Weg 93, 53 127 Bonn (DE). WALGEN- GW, ML, MR, NE, SN, TD, TG). BACH-BRUNAGEL, Gisela [DE/DE]; Waldauweg 15, 53 127 Bonn (DE). Published: (74) Agent: VIERING, JENTSCHURA & PARTNER; with international search report (Art. 21(3)) Kennedydamm 55/RoBstr., 40476 Dusseldorf (DE). [Continued on next page] (54) Title: METHOD OF DIAGNOSING BREAST CARCINOMA (57) Abstract: The present invention relates to methods of p-value < 0.05 diagnosing a breast carcinoma, determining the prognosis of a patient diagnosed with breast carcinoma and determining 25- the efficacy of a treatment regimen of breast carcinoma in a patient, using Calponin-h2 and /or CALML 5 as markers. 20 Furthermore, the invention relates to a kit and a marker pan el for use in these methods. 151 1 · me an--4. 0 Fig. 10 o o w o 2012/116979 Al II 11 II I 1 Illlll I I II III II llll III II I II before the expiration of the time limit for amending the — with sequence listing part of description (Rule 5.2(a)) claims and to be republished in the event of receipt of amendments (Rule 48.2(h)) Method of diagnosing breast carcinoma Field of thej nve t n. The present invention relates to methods diagnosing a breast carcinoma, determining the prognosis of a patient diagnosed with breast carcinoma and determining the efficacy of a treatment regimen of breast carcinoma in a patient, using Calponin-h2 and /or CALML 5 as markers. Furthermore, the invention relates to a kit and a marker panel for use in these methods. Background Breast cancer is the most common cancer in women. Every year more than 1.3 million women worldwide are diagnosed with breast cancer, whereby nearly 500.000 patients die due to the fatal course of this disease. The early diagnosis of breast cancer in a potentially curable stage improves the prognosis and consecutively reduces mortality of breast cancer diagnosed patients. Currently, clinical breast examination, imaging by mammography as well as tumor biopsy are the only methods recommended for breast cancer screening in the non-high risk population. The established screening by breast examination and mammography is able to detect breast cancer in early stages and has been shown to reduce mortality of patients diagnosed for breast cancer. Nevertheless, screening by mammography, especially in patients younger than 50 years, remains controversial. This is in particular due to significant rates of false negative as well as false positive results leading to overdiagnosis and overtherapy. Importantly, young women (<50 years) with a high density of the breast show low sensitivities in mammography. The sensitivity of mammography in women aged 50 years and older ranges from 68% to 90%, in women aged 40-49 the sensitivity is lower, with an estimate of between 62% and 76%. Likewise, a meta¬ analysis of randomized controlled trials showed a decreased mortality reduction of 15% in young women (39 - 49 years) undergoing regular screening by mammography, compared to 30% in women aged 49-59. Therefore, established mammography screening programs are mainly addressed to older women (50-69 years). However, breast cancer a especially important issue for the non-screened women belonging to the younger subgroup, as among this population one fifth of new breast cancers cases occurs, which are in this subgroup often aggressive and in a fast growing form. Moreover, breast cancer is the most common cause of cancer death among young females aged 20-59 years. For this reason, there i a special need for a test which allows reliable diagnosis of breast cancer in young women. In addition, mammography usually requires the patient to make an appointment with a mammography center for analysis. These centers are rare and thus the patient is usually required to travel a significant distance to the center. For this reason about half of patients leave out an institutional invitation to a screening. Accordingly, there is a demand for a diagnostic method which can be employed by any physician and that avoids inconvenience for the patients. In addition, the slots for the breast cancer diagnostic test are rare in screening centers and thus appointments are seldom given to the patient. A frequent control of the patient is particularly important if the treatment of a breast cancer patient is to be monitored. Hence, there is need for a breast cancer diagnostic test which allows frequent analysis of patients. Also for economic reasons, as mammography is a high priced technology, there is a demand for a more cost effective breast cancer test. During the last two decades, genomic and proteomic technologies have significantly increased the number of potential DNA, RNA and protein biomarkers in breast cancer. But so far, there is no reliable non-invasive test available for the clinical routine. Regarding proteomic approaches, a major challenge lies in the complexity of the human proteome an its dynamic state. Even current Mass Spectrometry (MS) based technologies are still failing to achieve large access to low abundant proteins in complex biological samples. However, low abundant proteins represent numerous potential tumor specific biomarkers. Nuclear matrix proteins (NMPs) represent only 1% of the total cell proteome. In 974, the nuclear matrix has been first described as the structural framework scaffolding of the nucleus, consisting of the peripheral lamins, protein complexes, an internal ribonucleic protein network and residual nucleoli. Most of the nuclear matrix proteins (NMPs) are common to all cell types, but numerous NMPs are tissue and cell type specific. The main characteristics of cancer cells are alterations in the size and shape of the nucleus that reflect the analogous alteration of the nuclear matrix. Recently, alterations of several NMP have been shown to be cancer specific biomarkers (Leman et al., J Cell Biochem, 104(6): 1988-1993, 2008). These findings have been successfully developed into non-invasive, blood- and urine-based test with high sensitivity and specificity for prostate, bladder as well as colon cancer, which have now to be validated in correctly designed large studies to demonstrate their clinical utility (Van Le et al.. Urology, 66(6): 1256-1260, 2005; Leman et al., Cancer Res, 67(12): 5600-5605, 2007; Leman et al, Urology, 69(4): 714-720; 2007; Walgenbach-Brunagel et al., .1 Cell Biochem, 104(1): 286-294, 2008). However, for none of the above detailed tests suitability as a breast cancer diagnostic test has been demonstrated. Accordingly, there is still need in the field for a reliable, preferably non¬ invasive, rapid diagnostic test which allows diagnosis of breast cancer. Summary of the Invention The present invention relates to a method of diagnosing breast carcinoma n a patient, wherein the method comprises determining the level of Calponin-li2 and/or CALML5 in a sample obtained from the patient, wherein if the level of Calponin-h2 and/or CALML5 is increased said patient is diagnosed with breast carcinoma. In a further aspect, the present invention relates to a method of determining the prognosis of a patient diagnosed with breast carcinoma, wherein the method comprises determining the level of Calponin-h2 and/or CALML5 in a sample obtained from the patient, wherein, if the level of Calponin-h2 and/or CALML5 is increased, said patient has an increased likelihood of an adverse outcome. The present invention is further directed to a method of determining the efficacy of a treatment regimen of breast carcinoma in a patient, wherein the method comprises determining the level of Calponin-h2 and/or CALML5 in a first sample obtained from the patient before said treatment regimen has commenced and a second sample obtained from the patient during or after said treatment regimen, wherein a decrease of the level of Calponin-h2 and/or CALML5 in the second sample relative to the first sample indicates that the treatment is effective.
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