CES: a chemoendocrine index based on PAM50 for breast cancer with positive hormone receptors with an

专利摘要:
CES: a chemoendocrine index based on PAM50 for breast cancer with positive hormone receptors with an intermediate risk of recurrence. This new method refers to the development of a chemoendocrine index (CES), based on the well-known PAM50 analysis to predict whether a patient with breast cancer responds to chemotherapy or endocrine therapy, specifically in a patient with breast cancer HR +/HER2- above the risk of recurrence (ROR) and the intrinsic subtypes of PAM50. Specifically, the clinical utility of this CES predictor is based on the intermediate ROR group of PAM50, where the proportion of each CES group (sensitive to endocrine therapy, intermediate and sensitive to chemotherapy) is greater than 25%. (Machine-translation by Google Translate, not legally binding)
公开号:ES2674327A1
申请号:ES201601012
申请日:2016-11-28
公开日:2018-06-28
发明作者:Aleix PRAT；Charles M. Perou；Alba EMILIO
申请人:Geicam - Grupo Espanol De Investigacion En Cancer De Mama；Geicam Grupo Espanol De Investig En Cancer De Mama；
IPC主号:

专利说明:

CES: a chemoendocrine index based on PAM50 for breast cancer with positive hormonal receptors with an intermediate risk of recurrence
DESCRIPTION
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The new method is related to the field of prediction of the efficacy of treatment in patients with breast cancer. Specifically, this new method is related to the prediction of the efficacy of chemotherapy or endocrine therapy in patients with breast cancer with positive hormonal receptors and negative HER2 (HR + / HER2-), regardless of intrinsic subtype and risk of recurrence ( ROR), preferably in the case where the patient is classified as a patient with intermediate risk of recurrence.
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STATE OF THE TECHNIQUE
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Approximately 70% of invasive breast cancers at diagnosis are HR + / HER2- (TCGA: Comprehensive molecular portraits of human breast tumors. Nature 2012; 490: 61-70; Prat A, et al. Journal of Clinical Oncology . 2013, 31: 203-209). However, HR + / HER2- disease is clinically and biologically heterogeneous and other subclassifications are necessary to better adapt current and future treatments (Ades F., et al. Journal of Clinical Oncology. 2014, 32: 2794-2803; Prat A, et al. Mol Oncol. 2011, 5: 5-23, Prat A, et al. Nat Rev Clin Oncol 2012, 9: 48-57).

During the last decade, molecular characterization studies have identified and thoroughly investigated the two main molecular subtypes in HR + / HER2- disease (i.e., Luminal A and B) (TCGA: Comprehensive molecular portraits of human breast tumors). 2012, 490: 61-70; Prat A, et al. Journal of Clinical Oncology. 2013. 31: 203-209; Perou CM, et al. Nature. 2000, 406: 747-752). Tumors of the Luminal A subtype have a better prognosis at 5 and 10 years of 30 follow-up compared to tumors of the Luminal B subtype regardless of the classic clinical-pathological variables (e.g., tumor size and nodal status ) and (neo) adjuvant treatment (i.e., endocrine therapy and chemotherapy) (TCGA: Comprehensive molecular portraits of human breast tumors. Nature 2012; 490: 61-70; Prat A, et al. Journal of Clinical Oncology. 35 2013 , 31: 203-209, Martín M, et al. Breast Cancer Research and Treatment. 2013, 138: 457-466, Prat A, et al. Journal of the National Cancer Institute. 2014, 106). In
In terms of sensitivity to treatment, Luminal A subtype tumors achieve significantly lower pathological response rates (RpC) than those of Luminal B subtype tumors after neoadjuvant chemotherapy with several drugs (Usary J, et al. Clinical Cancer Research. 2013, 19: 4889-4899; von Minckwitz G, et al. Journal of Clinical Oncology. 2012, 30: 1796-1804; Prat A, et al. 5 Breast Cancer Research and Treatment. 2012, 135: 301-306; Prat A, et al. BMC Medicine. 2015, 13: 1-11). However, the difference in sensitivity to endocrine therapy between the two luminal subtypes is less clear (Ellis MJ, et al. Journal of Clinical Oncology. 2011, 29: 2342-2349; Dunbier AK, et al. Steroids. 2011,76 : 736-740).
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Adjuvant endocrine therapy is currently recommended for 5-10 years in all patients with early breast cancer HR + / HER2-, while chemotherapy is recommended in patients with intermediate and high risk tumors (Goldhirsch A, et al. Personalizing the treatment of women with early breast cancer: highlights of the St Gallen International Expert Consensus on the Primary Therapy of 15 Early Breast Cancer 2013. Annals of Oncology). However, the relationship between therapy and risk justifies the continuation of the investigation taking into account that the risk is associated with the two factors related to tumor biology and clinical-pathological characteristics such as tumor size and nodal status, while The ability to respond to treatment is usually considered independent of the clinical-pathological factors.

Consequently, there is a need for tools to identify heterogeneous subgroups with HR + / HER2 breast cancer - with different prognosis and different treatment sensitivity in order to select the type and degree of treatment most appropriate for the patient.
DESCRIPTION OF THE INVENTION

Breast cancer with positive hormone receptors (HR +) is clinical and biologically heterogeneous and it is necessary to identify subgroups with different prognosis and sensitivity to treatment. Here we present the development and clinical validation in multiple studies of a predictor based on gene expression, which uses the well-known PAM50 analysis, which is associated with the response to chemotherapy and endocrine therapy in early breast cancer beyond the risk of recurrence ( ROR) and of the 35 intrinsic subtypes of PAM50. The clinical utility of this PAM50-based chemoendocrine predictive index (CES) is found in the group with ROR
intermediate of PAM50, where the proportion of each CES group (sensitive to endocrine therapy, intermediate and sensitive to chemotherapy) is greater than 25%.

Our results are the first to confirm, in randomized cases, an inverse relationship between sensitivity to endocrine therapy and chemotherapy in 5-estrogen-positive breast cancer (ER +) cancer. Previous tests have shown an inverse relationship between proliferation and biological processes related to RE in regards to sensitivity to endocrine therapy and chemotherapy in ER + breast cancer. For example, two independent studies demonstrated an inverse correlation between an index of 200 ER-related genes, or between the expression of 10 TAU, an ER-related gene, and sensitivity to endocrine therapy and chemotherapy (Symmans WF, et al. Journal of Clinical Oncology. 2010, 28: 4111-4119; Andre F, et al. Clinical Cancer Research. 2007, 13: 2061-2067). Likewise, the high recurrence rate measured by Oncotype DX (Genomic Health, Inc., Redwood, CA) predicted little or no benefit from adjuvant tamoxifen treatment in trial 15 NSABP-B14, but at the same time also predicted a considerable benefit of adjuvant CMF chemotherapy in the NSABP-B20 trial (Paik S, et al. New England Journal of Medicine. 2004, 351: 2817-2826; Paik S, et al. Journal of Clinical Oncology. 2006, 24: 3726-3734 ). These results coincide with our results, which show that virtually all patients with high ROR disease are identified as CES-C. However, this new method also highlights that, in the disease with high ROR / CES-C, not all ER + / HER2 samples are luminal (i.e., Luminal A or B) since non-luminal disease can also be identified (i.e., Basal-like and HER2 enriched). According to our results (Fig. 2), the benefit of chemotherapy in tumors with high ROR / not 25 luminals in HR + / HER2- disease is probably even greater than in tumors with high ROR / B luminals.

The results of this new method also indicate that the main driver of sensitivity to endocrine therapy and chemotherapy in RE + / HER2- disease is 30 Basal-like intrinsic biology versus Luminal A. To reflect both biological states in each sample, the correlation coefficients of each of the samples at both PAM50 centroids (ie, Luminal A and Basal-like) have been calculated and then both coefficients are subtracted (the correlation coefficient of luminal A (CC Luminal A) versus the Basal-like correlation coefficient (CC Basal-Like). Therefore, instead of choosing a genetic profile (e.g., a proliferation-based profile) of the numerous profiles that can be distinguished between both subtypes
One way or another, it was decided for each tumor to incorporate in an index the intrinsic Basal-like versus luminal A status of each tumor identified by the PAM50 subtype predictor. It should be noted that the PAM50 genes were originally selected for their ability to reflect the intrinsic biology shown by 1900 genes (the so-called intrinsic gene list). In fact, in the TCGA, the intrinsic subtype 5 defined by PAM50 reflected the vast majority of the biological diversity shown by the set of molecular data analyzed (TCGA: Comprehensive molecular portraits of human breast tumors. Nature. 2012, 490: 61-70 ).

From a clinical perspective, the data of this new method corroborates the current guidelines for the systemic treatment of early breast cancer HR + / HER2-. On the one hand, patients with a low ROR index and a low tumor burden (i.e. <10% risk of recurrence distant at 10 years) are recommended than endocrine therapy alone (Harris LN, et al. Journal of Clinical Oncology . 2016). Indeed, the results shown with this new method indicate that these patients have 15 tumors with a high sensitivity to endocrine therapy and low sensitivity to chemotherapy. On the other hand, patients with high-risk HR + / HER2 disease are recommended to receive treatment with endocrine therapy and chemotherapy. According to the data of this new method, this group is the only one with a high benefit of chemotherapy and low endocrine therapy. With respect to endocrine therapy in this group, the main problem is that we do not have survival data indicating that CES-C tumors do not benefit at all from endocrine therapy. Therefore, the withdrawal of a potentially effective treatment strategy such as endocrine therapy in a patient with an ER + tumor (as defined by the ASCO / CAP guidelines) that is identified as CES- is not recommended. C or high ROR, although in patients whose tumors contain low levels of ER (1% to 10%), the ASCO / CAP recommends discussing the advantages and disadvantages of endocrine therapy. It is unlikely that a large randomized adjuvant trial involving thousands of patients to answer this particular question will be carried out. 30

Although the clinical implications of CES in a low and high risk HR + / HER2- disease are minimal, the observation that an intermediate risk HR + / HER2- disease, representing ∼30% of breast cancers with a recent diagnosis, It is biologically heterogeneous and with a wide range of sensitivities to chemotherapy could have implications in the interpretation of two prospective clinical trials in progress. In the TailorX Phase III trial, 4500 patients with cancer
Early breast HR + / HER2- with negative nodes and with an intermediate recurrence rate have been randomized to receive adjuvant chemotherapy or not to receive chemotherapy. According to our analysis, this intermediate group could be composed of a minimum of 3 groups with different chemotherapy sensitivities. It should be noted that the CES-U group seems to be very poorly defined and decisions regarding the need for chemotherapy could be difficult. A similar situation could occur in the RxPONDER phase III clinical trial in which patients with early breast cancer HR + / HER2- are being randomized, and between 1 and 3 positive lymph nodes, with low or intermediate risk, to receive adjuvant chemotherapy or not. One possible explanation is that the Oncotype DX recurrence index, as well as other prognostic tests based on gene expression, such as PAM50 ROR or MammaPrint43, have been specifically designed or obtained to predict the results and not intrinsic tumor biology or sensitivity to the treatment. Although a high negative correlation is observed between ROR (risk) and CES (drug sensitivity), there are considerable differences between them at the individual level 15 (disc40% mismatch).

There are several caveats in our study. First, it is a retrospective study in populations of heterogeneous patients and the results must be confirmed in one or several prospective clinical trials. Secondly, although the data presented here validate CES from a clinical perspective, it will be necessary to carry out a subsequent analytical validation, given that in the majority of data sets, except for the whole of Malaga, the version of PAM50 has been used for research . However, the fact that the ESC (as a continuous variable and with the 2 cut-off points) predicted the RpC in the whole of Malaga indicates that the analytical validation of this biomarker is feasible. Thirdly, we cannot assess the association of CES with the survival data of a randomized clinical trial of adjuvant chemotherapy against the absence of adjuvant chemotherapy, or adjuvant endocrine therapy versus the absence of adjuvant endocrine therapy. Therefore, the predictive value of these profiles was only evaluated in neoadjuvant therapy 30 where the different tumor response variables were evaluated, most of which have been related to patient survival (Ogston KN, et al. The Breast. 2003, 12: 320-327; Cortazar P, et al. The Lancet. 2014, 384: 164-172). Fourth, some of the profiles evaluated in the MDACC-based data set, such as the Oncotype DX recurrence index or the genomic grade 35 index, came from microarray-based data and, therefore, it is not about the commercialized versions. Fifth, we could not prove a
Constant association of CES with the endocrine response in HR + disease after excluding HER2-positive cases. In the Edinburgh data set, the status of HER2 was not available in all patients. Although we derive a subrogated definition based on the ERBB2 expression of the HER2 state, which showed that the ESC is independently associated with the response, this was not pre-specified and did not meet the REMARK guidelines. In addition, the association of CES with the endocrine response did not reach statistical significance (p = 0.09) in patients with HR + / HER2- disease in the Marsden data set. Finally, the patients of each of the data sets received different regimens, programs and doses of anthracycline / taxane-based chemotherapy and, therefore, the ability of the profiles to predict the response to certain chemotherapeutic drugs could not be assessed. or treatment guidelines.

Another important consideration of this new method is that we have not attempted to identify one or several optimal cut-off points for CES, but rather that we have focused on the association of continuous CES expression with each variable. The main reason is that in each cohort different platforms and protocols based on gene expression were used and, therefore, the normalization of a biomarker cut-off point would have been difficult to reach and probably unreliable. In any case, the fact that the four contrast groups presented 20 very similar associations that were found independently of the platform / protocol used, advocates a strong conclusion.

To conclude, CES is a unique genetic signature capable of measuring the sensitivity to chemotherapy and endocrine therapy in breast cancer HR + / HER2-, beyond the intrinsic subtype, other genetic signatures and standard pathological variables. CES could have considerable clinical value in patients with intermediate risk HR + / HER2- disease in which the benefit of adjuvant multiagent chemotherapy cannot be determined.
 30
According to the information in relation to this new method, a first aspect refers to an in vitro method to predict whether a patient with breast cancer responds to chemotherapy or endocrine therapy in an isolated sample of HR + / HER2- of the classified patient in the intermediate ROR group using the PAM50 kit, whose method consists of:
a) obtain with the PAM50 kit, the correlation coefficient corresponding to the sample classified as intrinsic subtype Luminal A and the coefficient of
correlation corresponding to the sample classified as intrinsic Basal-like subtype, in the isolated sample, and
b) obtain the chemoendocrine index (CES) by subtracting the correlation coefficient corresponding to the sample classified as a Basal-like subtype from the correlation coefficient corresponding to the sample classified as 5 Luminal A subtype (CES = CC Luminal A - CC Basal- Like);
where CES equal to or greater than 0.7 indicates that said patient responds to endocrine treatment (CES-E), and where CES equal to or less than 0.3 indicates that said patient responds to chemotherapy (CES-C).
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In a preferred representation of the first aspect of this new method, the isolated sample is a biopsy sample.

In a second aspect of this new method, it refers to the in vitro use of CES to predict whether a patient with breast cancer responds to chemotherapy or endocrine therapy in an isolated HR + / HER2- sample of the patient classified in the group of intermediate ROR through the PAM50 kit.

In a preferred representation, the ESC is obtained by subtracting the correlation coefficient corresponding to the sample classified as a Basal-like subtype from the correlation coefficient corresponding to the sample classified as a Luminal A subtype using the PAM50 kit.

In another preferred representation of this aspect of the new method, it should be noted that a CES equal to or greater than 0.7 indicates that said patient is CES-E. In another preferred representation, a CES equal to or less than 0.3 indicates that said patient is CES-C.

In another preferred representation, the isolated sample is a biopsy sample.
 30
Unless otherwise indicated, all technical and scientific terms used here have the same meaning that an expert in the discipline to which this new method corresponds corresponds to. In the practice of this new method, methods and materials similar or equivalent to those described herein may be used. In the description and statements, the word "include" and its variations 35 are not intended to exclude other technical characteristics, additives, components or steps. Other objects, advantages and characteristics of the new method will be
manifest to those skilled in the art after analyzing the description or may be known through the practice of the new method. The following examples and images are provided by way of illustration and are not intended to be restrictive of this new method.
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DESCRIPTION OF THE FIGURES

Fig 1. Association of gene expression with sensitivity to chemotherapy or endocrine therapy. (A) Association between the expression of each gene (n = 542) and the response of Miller and Payne in each group of the GEICAM 2006-03 trial. On the right are certain main genes whose expression is highly associated with the response. (B) Average expression of the 50 main genes associated with sensitivity to endocrine therapy (upper panel) and chemotherapy (lower panel) in the 15 GEICAM 2006-03 trial in the various intrinsic subtypes of breast cancer. Gene expression data based on the RNA sequence have been obtained from the data portal of The Cancer Genome Atlas breast cancer project (https://tcgadata.nci.nih.gov/tcga/). (C) Meaning and score of CES. (D) Association of CES (as a continuous variable) with response to chemotherapy or endocrine therapy in the 4 validation data sets.
Fig. S1. CES association with Miller and Payne after chemotherapy in HR + / HER2- disease of the Malaga cohort.
Fig. S2. Association of CES with sensitivity to endocrine therapy in the Edinburgh data set (n = 120). (A) Changes in the tumor volume of each patient and 25 response classification. (B) Association of CES and other response variables (defined as a decrease of at least 70% in 90 days) in the global population. (C) Association of CES and other response variables in HER2-negative disease.
Fig 2. Prognosis (ROR PAM50), intrinsic subtype and CES in 6007 primary breast cancers. (A) A scatter plot of CES index and ROR index, colored by subtype, is shown. The two horizontal lines indicate the cut-off points of each CES group. The two vertical lines indicate the cut points of each ROR PAM50 group. (B) Number of patients in each CES group based on ROR. Each bar is colored according to the subtype. 35
Fig 3. Survival results in early HR + breast cancer with intermediate ROR. (A) Negative node disease treated without systemic therapy
adjuvant (B) Disease with negative nodes and with positive nodes treated only with adjuvant tamoxifen. (C) Positive node disease treated with adjuvant chemotherapy and endocrine therapy in the GEICAM / 9906 clinical trial. (D) Disease with negative and positive nodes treated with neoadjuvant chemotherapy and adjuvant endocrine therapy. 5

EXAMPLES

Methods and materials 10

Clinical trial GEICAM / 2006-03

Pre-treatment biopsy samples obtained with a thick needle were evaluated in patients recruited from the luminal cohort of the phase II clinical trial with neoadjuvant therapy GEICAM / 2006-03 (NCT00432172) (Alba E, et al. Annals of Oncology. 2012) . In this study, 95 patients with estrogen receptor (ER) positive (Allred 3-8), progesterone receptor (PR) positive (Allred 3-8), HER2- (according to the ASCO / CAP guidelines) (Wolff AC, et al. Journal of Clinical Oncology 25: 118-145, 2006)), and 8/18 positive cytokeratins, were randomized 20 to receive neoadjuvant chemotherapy or endocrine therapy for 24 weeks. Chemotherapy consisted of epirubicin 90 mg / m2 intravenously (i.v.) in combination with cyclophosphamide 600 mg / m2 i.v. on 1 day every 21 days, for 4 cycles, followed by docetaxel 100 mg / m2 administered i.v. on day 1 every 21 days for 4 cycles. Endocrine therapy consisted of exemestane 25 mg administered orally 25 once daily. Premenopausal patients received goserelin 3.6 mg subcutaneously every 28 days in 6 doses. After neoadjuvant treatment, the patients underwent a mastectomy or conservation surgery plus axillary lymph node dissection (except prior biopsy of the sentinel lymph node with a negative result). 30

End point of pathological response in the GEICAM 2006-03 trial

The Miller and Payne histological classification system with a 5-point scale (Ogston KN, et al. The Breast. 2003, 12: 320-327) was used to measure the tumor response. This classification system consists of a 5-point scale that focuses on the reduction of tumor cellularity in the treated breast tumor (in surgery)
compared to the samples before treatment. Grade 1: no changes in global cellularity. Grade 2: up to 30% reduction in cellularity. Grade 3: an estimated 30% to 90% reduction in tumor cells. Grade 4: more than 90% reduction of tumor cells. Grade 5: complete pathological response (RpC). There may be a ductal carcinoma in situ. In this study, Miller and Payne's scale 5 was reduced to a 3-point scale to have a good number of cases in each category and group: absence of response (grades 1 and 2), intermediate response (grade 3) and response high (grades 4 and 5).

Gene expression analysis in GEICAM 2006-03 10

63 of the 95 tumor samples were available prior to treatment for gene expression analyzes. For each sample, a section of formalin fixed and paraffin embedded breast tissue (FFPE) was first examined with hematoxylin and eosin stains to confirm the diagnosis and determine the area of the tumor. Two 1 mm nuclei enriched with tumor tissue from the original tumor block were obtained and a minimum of 100 ng of total RNA was purified to measure the expression of 543 breast cancer related genes using the nCounter platform (Nanostring Technologies, Seattle, WA, USA). A logarithmic transformation was made in base 2 of the data and they were normalized by 5 constitutive genes 20 (ACTB, MRPL19, PSMC4, RPLP0 and SF3A1) and 14 negative and positive controls using the nCounter platform (Nanostring Technologies, Seattle, WA, USA). UU.) (GK Geiss, et al. Nat Biotech. 2008, 26: 317-325).

Independent / contrast data sets 25

Gene expression and response data were evaluated from 4 independent data sets of neoadjuvant therapy (Dunbier AK, et al. Steroids. 2011, 76: 736-740; Hatzis C, et al. Jama. 2011, 305: 1873-1881; Prat A, et al. Clinical Cancer Research. 2015; Dunbier AK, et al. Journal of Clinical Oncology. 2010, 28: 1161-1167; 30 Smith IE, et al. Journal of Clinical Oncology. 2007, 25 : 3816-3822; Turnbull AK, et al. Journal of Clinical Oncology. 2015). Gene expression and survival data were evaluated from 4 independent data sets of patients with early breast cancer (Prat A, et al. Journal of Clinical Oncology. 2013, 31: 203-209; Hatzis C, et al Jama. 2011, 305: 1873-1881; Fan C, et al. BMC Medical Genomics. 35 2011, 4: 1-15; Prat A, et al. Annals of Oncology. 2012, 23: 2866-2873).

Hatzis independent data set

We evaluate the data set, accessible to the public, based on the genetic expression of microarrays (GSE25066) reported by Hatzis et al. (Hatzis C, et al. Jama. 2011, 305: 1873-1881) that includes 508 patients (272 with HR + / HER2- disease) treated 5 with multiagent neoadjuvant chemotherapy in various research protocols: LAB99-402, USO-02- 103, 2003-0321 and I-SPY-1. The vast majority of patients (96.4%) received sequential guidelines based on anthracyclines / taxanes. Gene expression of the pre-treatment samples was analyzed and tumor response data were available after chemotherapy (i.e. RpC in the breast or armpit 10 versus no response) for 488 patients (260 with HR + / HER2-).

Malaga independent data set
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As previously described (Prat A, et al. Clinical Cancer Research, 2015), a total of 216 HR + / HER2- tumor samples conserved in biobank and locally evaluated from a Spanish independent multicenter cohort of patients with breast cancer were selected. All patients were prescribed a standard neoadjuvant chemotherapy regimen consisting of 8-10 cycles of anthracyclines and 20 taxanes. Tumor samples prior to treatment were evaluated by standard PAM50 analysis (PROSIGNA®) through the nCounter diagnostic platform. The gene expression of the pre-treatment samples was analyzed at the University Hospital of Malaga, Nanostring Technologies normalized the data and the CES was applied in the VHIO in a masked way with respect to the clinical data. Tumor response data (ie RpC in the breast / armpit versus absence of response) were available for 180 patients. In addition, Miller and Payne response data were available for 171 patients.

Independent data set Marsden 30

We evaluated the RNA profiles (HumanWG-6 v2 Expression BeadChips [Illumina, San Diego, CA]) obtained from tumor biopsies with thick needle pre-treatment of 103 postmenopausal patients with primary ER + breast cancer treated with neoadjuvant anastrozole for 16 weeks in a Phase II clinical trial (Dunbier 35 AK, et al. Steroids. 2011, 76: 736-740; Dunbier AK, et al. Journal of Clinical Oncology. 2010, 28: 1161-1167; Smith IE, et al. Journal of Clinical Oncology, 2007, 25: 3816-
3822). A subgroup of patients received gefitinib during the first 2 weeks; however, the addition of gefitinib to anastrozole had no additional clinical or biological effect on Ki67. The clinical tumor response (complete and partial response to stable and progressive disease) was used as a variable. The CES was calculated in the IDIBAPS (Barcelona) in a masked way with respect to the clinical data. 5

Edinburgh independent data set

We evaluate the profiles of RNA (HumanWG-6 v2 Expression BeadChips [Illumina, San Diego, CA]) and Affymetrix U133A 2.0 [Santa Clara, California, USA. UU.]) Obtained from 10 tumor biopsies with thick needle pre-treatment of 120 postmenopausal patients with primary ER + breast cancer treated with neoadjuvant anastrozole for at least 12 weeks (Turnbull AK, et al. Journal of Clinical Oncology, 2015). The response was evaluated by ultrasound. The clinical tumor response was defined as a reduction in tumor volume of at least 70% during 90 days of treatment. Raw gene expression data is found in Gene Expression Omnibus (GSE55374 and GSE20181). The clinical status of HER2 was available in 45 cases. The cases of HER2 + showed greater expression of ERBB2 compared to the cases of HER2-. We use the 80th percentile as a cut-off point to define the positivity of HER2 in those cases without clinical status 20 of HER2. A total of 89 cases turned out to be HER2-.

Assignment of intrinsic subtypes

All tumors were assigned to an intrinsic molecular subtype of breast cancer (Luminal A, Luminal B, enriched HER2, Basal-like) and the normal group using the PAM50 subtype predictor for research (Parker JS, et al. J Clin Oncol. 2009, 27: 1160-1167; Nielsen TO, et al. Clin Cancer Res. 2010, 16: 5222-5232), except for the Malaga cohort in which a standardized and commercialized analysis of PAM50 based on nCounter was used . Before subtyping, each data set was normalized according to what was previously reported (Prat A, et al. Br J Cancer. 2014, 111: 1532-1541, 2014), except for the Malaga cohort that was normalized by Nanostring according to your algorithm. It should be noted that the Edinburgh dataset based on microarrays only consists of ER + samples and it was not possible to adequately focus the so-called intrinsic subtyping 35 (Prat A, et al. Nat Rev Clin Oncol. 2012, 9). In this data set, the ESC was evaluated as a continuous variable, since it was not affected by the centering.

Adjuvant data set

Gene expression and survival data were evaluated from 4 independent data sets of patients with early breast cancer with intermediate ROR 5 (Wolff AC, et al. Journal of Clinical Oncology, 2006, 25: 118-145; Hatzis C , et al. Jama. 2011, 305: 1873-1881; Fan C, et al. BMC Medical Genomics. 2011, 4: 1-15; Prat A, et al. Annals of Oncology. 2012, 23: 2866-2873) . The first is the MDACC-based data set described above, in which survival without distant recurrence was recorded (Hatzis C, et al. Jama. 2011, 305: 1873-1881, 2011). 10 All patients received neoadjuvant chemotherapy and endocrine therapy. The second is a combined and previously published cohort of 1318 patients with HR + disease treated with adjuvant tamoxifen alone (Prat A, et al. Annals of Oncology. 2012, 23: 2866-2873). The third is a combined and previously published cohort of patients who did not receive any adjuvant systemic therapy 15 (Fan C, et al. BMC Medical Genomics. 2011, 4: 3). Finally, we analyzed the samples, as described previously (Prat A, et al. Br J Cancer. 2014; 111: 1532-1541), of the GEICAM / 9906 clinical trial, in which all patients received multiagent adjuvant chemotherapy and endocrine therapy (Prat A, et al. J Clin Oncol. 2013, 31: 203-9). twenty

Combined cohort of primary breast cancer

To assess the relationship between the subtypes of PAM50, prognosis (ROR-P) and CES, we have combined the PAM50 data from 7 independent cohorts and described above (TCGA: Comprehensive molecular portraits of human breast tumors. Nature, 2012 490: 61 -70; Prat A, et al. Journal of Clinical Oncology. 2013, 31: 203-209; Prat A, et al. Breast Cancer Research and Treatment. 2012, 135: 301-306; Hatzis C, et al. Jama. 2011, 305: 1873-1881; Curtis C, et al. Nature. 2012, 486: 346-352; Horak CE, et al. Clinical Cancer Research. 2013, 19: 1587-1595; Fan C, et al. BMC Medical 30 Genomics. 2011, 4: 3), representing a total of 6007 primary tumor samples. The ESC was evaluated in each cohort, and a combined matrix was created.

Statistic analysis
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The biological analysis of the gene lists was performed with the annotation tool DAVID 6.7 (Dennis G, et al. Genome Biol. 2003, 4: R60-543), using the list of 543
genes as a reference. The association between the expression of each gene and the response of Miller and Payne (3 categories) was evaluated by means of a quantitative microarray significance analysis (SAM) (Tusher VG, et al. Proc Natl Acad Sci USA. 2001, 98: 5116 -5121). In both sets of contrast data, the association between each variable and the RpC or clinical / radiological response was evaluated by simple and multivariate logistic regression analysis. The predictive performance of the ESC was evaluated by analyzing the "receiver operating characteristic" (ROC) curve. Survival estimates were obtained from the Kaplan-Meier curves and the existence of differences was verified by the log rank test. Simple and multivariate Cox models were used to determine the significance of the independent prognostic character of each variable. The p-values notified are bilateral.

Results
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GEICAM data set / 2006-03

63 premenopausal and postmenopausal patients were evaluated in this study (Table 1). The majority of patients presented with ductal carcinomas (83%), tumors with a size of 2-5 cm (76%), histological tumors of grade 3 (59%), 20 negative nodes (54%) and luminal subtype by PAM50 ( 84%) After chemotherapy, the Luminal B subtype tumors showed a higher Miller and Payne response than the Luminal A subtype tumors (mean of 2.0 versus 1.4, P = 0.048). However, no difference in response was observed between the two luminal subtypes after endocrine therapy (P = 0.407). In addition, no statistically significant interaction (P = 0.429) between the subtype and the treatment (endocrine therapy versus chemotherapy) in the tumor response was observed. It should be noted that the only patient who achieved an RpC (that is, Miller and Payne grade 5) had a Basal-like tumor and was included in the chemotherapy group.
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Table 1. Clinical-pathological characteristics and distribution of subtypes in the GEICAM 2006-03 * study.
 QT% TE% P-value

 No.  32 - 31 - -
 Middle Ages)  53.7 - 52.3 - 0.596
 Menopausal status
 QT% TE% P-value

 Pre-menopausal  14 44% 14 45% 1,000
 Post-menopausal  18 56% 17 55%
 Tumor stage
 T1  1 3% 2 6% 0.420
 T2  23 72% 25 81%
 T3  8 25% 4 13%
 Average tumor size (cm)  4.2 3.8 0.278
 Ganglia
 N0  15 47% 19 61% 0.501
 N1  16 50% 11 35%
 N2  1 3% 1 3%
 Grade
 G1  0 0% 0 0%
 G2  8 25% 6 19% 0.862
 G3  18 56% 19 61%
 G4  6 19% 6 19%
 Histological type
 Ductal  26 81% 26 84% 1,000
 Lobular  2 6% 2 6%
 Others  4 13% 3 10%
 Ki-67 IHQ (average)  31.1 33.5 0.720
 Miller-Payne response (average)  2.6 2.2 0.124
 PAM50
 Luminal A  16 50% 13 42% 0.564
 Luminal B  11 34% 13 42%
 HER2-E  0 0% 1 3%
 Basal-like  2 6% 0 0%
 Normal-like  3 9% 4 13%
* QT, chemotherapeutic arm; TE, endocrine therapy arm

Association of gene expression with treatment sensitivity

To understand the biology associated with sensitivity to chemotherapy or endocrine therapy in HR + / HER2- disease, we investigated the association between the expression of 543 genes related to breast cancer and the response of Miller and Payne in each treatment group. The high expression of 70 (12.9%) and 17 (3.1%) genes was significantly related (P <0.05 not corrected for multiple comparisons) with the response after endocrine therapy and chemotherapy, 10 respectively. The list of genes associated with the response to endocrine therapy was enriched in the following biological processes: vascularization (e.g., AKT1 and beta 1 catenin), duct development (e.g., FOXA1 and gremlin 1) and increase
cellular (eg, androgen receptor and fibroblast growth factor receptor 1). On the other hand, the list of genes associated with the response to chemotherapy was enriched in the cell cycle (e.g., EXO1 and MKI67) and the extracellular matrix (e.g., netrin 4 and thrombospondin 1). Next, the interaction between the expressions of each gene with the response to therapy (endocrine therapy versus chemotherapy) was evaluated. It should be noted that 41 of the 70 genes associated with the response to endocrine therapy and 8 of the 17 genes associated with the response to chemotherapy showed a significant interaction with the treatment (P <0.05 not corrected in multiple comparisons). Therefore, the biological factors associated with endocrine sensitivity seemed to be associated also with chemotherapy resistance, and vice versa. In fact, a global inverse pattern was observed between the expression of most genes and the response to treatment (Fig. 1A).

To better understand the biological factors associated with the response to treatment, we evaluated the average expression of genes associated with a high sensitivity to endocrine therapy, but low to chemotherapy or a low sensitivity to endocrine therapy, but high to chemotherapy, in 1034 primary tumors representing all intrinsic molecular subtypes of breast cancer (Fig. 1B). The results revealed that the biology associated with sensitivity to chemotherapy and endocrine therapy is mainly based on Luminal A biology (i.e. high sensitivity to endocrine therapy, but low to chemotherapy) versus Basal-like biology (i.e., low sensitivity to endocrine therapy, but high to chemotherapy).

Development of an ESC based on PAM50 25

Reflecting the relative differences between Luminal A and Basal-like biology in HR + / HER2- could help to better predict sensitivity to endocrine therapy and chemotherapy. To reflect this biological state in each tumor, we have obtained, from the PAM50 classification algorithm, the correlation coefficients (CC) of each sample at the PAM50 centroids of the Luminal A subtype and the Basal-like and then We have subtracted the 2 values to obtain the chemoendocrine index (CES = CC to Luminal A - CC to Basal-like). Thus, the samples with a positive score had a greater sensitivity to endocrine therapy than to chemotherapy, while the samples with a negative score had a greater sensitivity to chemotherapy (CES-C) than to endocrine therapy (CES). -E) (Fig. 1C). From the results with samples of the GEICAM 2006-03 trial,
determined cut-off points based on tertiles (CES-E group against uncertain CES [CES-U], cut-off point = 0.70; CES-U group against CES-C, cut-off point = 0.30). The interaction of the CES index (as a continuous variable) with the treatment in the GEICAM 2006-03 trial provides some evidence of association (P = 0.059).
 5
MDACC based data set

We evaluated a combined data set of 272 patients with HR + / HER2- disease treated with neoadjuvant chemotherapy based on anthracyclines / taxanes in several trials with neoadjuvant therapies (Table 2). 10



Table 2. Clinical-pathological characteristics and distribution of subtypes in the 4 contrast groups *. fifteen
 MDACC Málaga Marsden Edinburgh
 N% N% N% N%

 Treatment  QT QT TE TE
 N  272 180 103 120
 HER2 status ¥
 HER2-negative  272 100% 180 100% 89 86% 31 69%
 HER2-positive  0 0% 0 0% 14 14% 14 31%
 Middle Ages)  50.1 50.0 53.7 76.1
 Menopausal status
 Pre-menopausal  ND 108 60% 0 0% 0 0%
 Post-menopausal  ND 72 40% 103 100% 120 100%
 Tumor stage
 T0-T1  19 7% 18 10% 60 58% 10 9%
 T2  142 52% 115 67% 42 36%
 T3-T4  111 41% 39 23% 43 42% 63 55%
 Ganglia
 N0  96 35% 67 37% 61 59% 86 72%
 N1  133 49% 61 34% 39 38% 34 28%
 N2-N3  43 16% 52 29% 3 3%
 Grade
 G1  28 11% 27 16% 15 15% 13 11%
 G2  136 53% 96 57% 63 62% 82 68%
 G3  91 36% 46 27% 24 23% 25 21%
 TE response rate§  ND ND 53% 72%
 QT response rate
 RpC breast / armpit  8.8% 6.7% ND ND
 PAM50
 Luminal A  141 52% 54 30% 37 36% - -
 Luminal B  102 38% 105 58% 20 19% - -
 HER2-E  6 2% 7 4% 12 12% - -
 Basal-like  7 2% 14 8% 4 4% - -
 Normal-like  16 6% - - 30 29% - -
*, TE, Endocrine Therapy; QT, Chemotherapy. ¥, In the Edinburgh data set there are 75 patients without determination of the clinical status of Her2. § The definition of response to TE is different between the Marsden and Edinburgh sets. The clinical tumor response (complete and partial response to a stable and progressive disease) has been used as an endpoint in the Marsden set. The response was evaluated by ultrasound in the Edinburgh set. The clinical tumor response was defined as a decrease in tumor volume by at least 70% after 90 days of treatment
In this data set, 51.5%, 25.8% and 22.7% of the samples were identified as CES-E, CES-U and CES-C, respectively. The RpC rates in 10 groups CES-E, CES-U and CES-C were 2.4%, 9.0% and 23.7%, respectively (P <0.0001), and it was observed that they were similar, although non-luminal tumors are eliminated (2.2%, 8.8% and 25.0%). The predictive capacity of neoadjuvant chemotherapy at CES was independent of the clinical-pathological variables and the intrinsic subtype (Table 3 and Table S1). Similar results were obtained when residual tumor load was used as a variable (Tables S2-S3).


Table 3. Association of CES with sensitivity to chemotherapy in the MDACC-based data set. twenty







Table S1. Association of CES with chemotherapy sensitivity (measured as RpC) in the MDACC-based data set.



Table S2. Association of CES with sensitivity to chemotherapy (measured as residual tumor load [RCB]) in the MDACC-based data set. Model A



Table S3. Association of CES to chemotherapy sensitivity (measured as residual tumor load [RCB]) in the MDACC-based data set. Model B



Seven genetic signatures based on expression (ie, proliferation index of PAM50, ROR-P, genomic grade index, SET index, chemo-predictor, DLDA30 and residual tumor burden predictor [RCB]) were previously described in this data set. (Hatzis C, et al. Jama. 2011, 305: 1873-1881). In addition, we have applied a microarray-based version of the Oncotype DX recurrence index (Fan C, et al. New England Journal of Medicine. 2006, 355: 560-569; Paik S, et al. New England Journal of Medicine. 2004, 351: 2817-2826). Here, we evaluate the ability to
CES in predicting RpC in HR + / HER2- disease compared to these seven genetic signatures. Interestingly, the ESC provided the highest aROC (Table S4-S12) either as a continuous variable (aROC = 0.770) or as group categories (aROC = 0.765). The second most predictive profile was the RCB predictor (aROC = 0.740). It should be noted that the RCB predictor was obtained using 165 of 272 5 samples (60.7%) HR + / HER2- from this data set (that is, the training data set). When these training samples were not considered, CES showed higher performance, either as a continuous variable (aROC = 0.805) or as group categories (aROC = 0.786) than the RCB predictor (aROC = 0.640). 10


Table S4. Univariate association of CES and several firms with sensitivity to chemotherapy in HR + / HER2- disease in the MDACC-based data set. fifteen
 Univariate Analysis
 Firms  N RpC Index AUC OR Lower 95% Higher 95% p-value
 CES
 CESSATION  134 2% 0.765 1.0 - - -
 CES-U  67 9% 4.3 1.04 17.75 0.044
 CES-C  59 24% 13.6 3.73 49.46 <0.001
 CES  - - 0.770 0.17 0.08 0.40 <0.001
 GHI  - - 0.648 1.31 1.06 1.63 0.013
 GHI PROLIF  - - 0.663 1.33 1.08 1.62 0.007
 ROR-P
 Low  75 5% 0.659 1.0 - - -
 Med  133 6% 1.1 0.33 3.91 0.840
 Tall  52 21% 4.8 1.42 15.93 0.011
 PAM50 PROLIF  - - 0.700 0.2 0.08 0.40 <0.001
 CHEMOPRED
 RxInsensitive  159 6% 0.597 1.0 - - -
 RxSensitive  101 13% 2.2 0.93 5.23 0.074
 GGI
 Low  126 3% 0.670 1.0 - - -
 Tall  134 14% 5.0 1.66 15.26 0.004
 SET
 TALL  21 5% 0.525 1.0 - - -
 INTERM  35 9% 1.9 0.18 19.29 0.597
 LOW  204 9% 2.1 0.26 16.16 0.494
 Univariate Analysis
 Firms  N RpC Index AUC OR Lower 95% Higher 95% p-value
 CTRPRED
 CTR II / III  159 3% 0.740 1.0 - - -
 CTR 0 / I  101 19% 9.0 2.96 27.27 <0.001
 DLDA30
 RD  254 2% 0.511 1.0 - - -
 RpC  6 17% 2.1 0.24 18.87 0.504


Table S5. Association of CES and the proliferation profile of PAM50 with chemotherapy sensitivity in HR + / HER2- disease from the MDACC-based data set. 5
 Bivariate Analysis

 Firms  N RpC Index OR Lower 95% Higher 95% p-value
 CES
 CESSATION  134 2% 1.0 - - -
 CES-U  67 9% 4.6 1.02 20.99 0.047
 CES-C  59 24% 15.8 2.93 85.36 0.001
 PAM50 PROLIF  - - 0.82 0.21 3.28 0.783


Table S6. Association of CES and CHEMOPRED profile with sensitivity to chemotherapy in HR + / HER2- disease from the MDACC-based data set.
 Bivariate Analysis

 Firms  N RpC Index OR Lower 95% Higher 95% p-value
 CES
 CESSATION  134 2% 1.0 - - -
 CES-U  67 9% 5.0 1.18 20.83 0.029
 CES-C  59 24% 15.3 4.12 56.72 <0.001
 CHEMOPRED
 RxInsensitive  159 6% 1.00 - - -
 RxSensitive  101 13% 2.73 1.09 6.87 0.032
 10

Table S7. Association of CES and the proliferation component of the Genomic Health Index (GHI; Oncotype DX recurrence index) with chemotherapy sensitivity in HR + / HER2- disease from the MDACC-based data set.
 Bivariate Analysis

 Firms  N RpC Index OR Lower 95% Higher 95% p-value
 CES
 CESSATION  134 2% 1.0 - - -
 CES-U  67 9% 5.5 1.19 25.18 0.029
 CES-C  59 24% 22.7 3.93 131.45 <0.001
 GHI Proliferation  - - 0.87 0.65 1.18 0.387

 5
Table S8. Association of CES and Genomic Grade Index (GGI) profile with chemotherapy sensitivity in HR + / HER2- disease from the MDACC-based data set.
 Bivariate Analysis

 Firms  N RpC Index OR Lower 95% Higher 95% p-value
 CES
 CESSATION  134 2% 1.0 - - -
 CES-U  67 9% 3.7 0.79 17.16 0.097
 CES-C  59 24% 10.6 2.15 52.00 0.004
 GGI
 Low  126 3% 1.00 - - -
 Tall  134 14% 1.43 0.35 5.77 0.615

 10
Table S9. Association of CES and SET Index Signature with sensitivity to chemotherapy in HR + / HER2- disease from the MDACC-based data set.
 Bivariate Analysis

 Firms  N RpC Index OR Lower 95% Higher 95% p-value
 CES
 CESSATION  134 2% 1.0 - - -
 CES-U  67 9% 4.7 1.09 19.85 0.038
 CES-C  59 24% 15.3 3.99 59.02 <0.001
 SET
 TALL  21 5% 1.00 - - -
 INTERM  35 9% 2,37 0.20 27.94 0.494
 Bivariate Analysis

 Firms  N RpC Index OR Lower 95% Higher 95% p-value
 LOW  204 9% 1.20 0.14 10.62 0.868


Table S10. Association of CES and the RCBPRED firm with sensitivity to chemotherapy in HR + / HER2- disease from the MDACC-based data set.
 Bivariate Analysis

 Firms  N RpC Index OR Lower 95% Higher 95% p-value
 CES
 CESSATION  134 2% 1.0 - - -
 CES-U  67 9% 4.3 1.01 18.35 0.048
 CES-C  59 24% 11.8 3.14 44.47 <0.001
 CTRPRED
 CTR II / III  159 3% 1.00 - - -
 CTR 0 / I  101 19% 7.97 2.55 24.94 <0.001
 5

Table S11. Association of CES and DLDA30 with chemotherapy sensitivity in HR + / HER2- disease from the MDACC-based data set.
 Bivariate Analysis

 Firms  N RpC Index OR Lower 95% Higher 95% p-value
 CES
 CESSATION  134 2% 1.0 - - -
 CES-U  67 9% 4.3 1.04 17.83 0.044
 CES-C  59 24% 13.9 3.79 51.25 <0.001
 DLDA30
 RD  254 2% 1.00 - - -
 RpC  6 17% 0.72 0.08 6.75 0.773

 10




Table S12. Association of CES and the ROR-P firm with chemotherapy sensitivity 15 in the HR + / HER2- disease of the MDACC-based data set.
             Bivariate Analysis

 Firms  N RpC Index OR Lower 95% Higher 95% p-value
 CES
 CESSATION  134 2% 1.0 - - -
 CES-U  67 9% 5.38 1.15 25.06 0.032
 CES-C  59 24% 17.29 3.22 92.88 <0.001
 ROR-P
 Low  75 5% 1.00 - - -
 Med  133 6% 0.44 0.10 1.86 0.264
 Tall  52 21% 0.60 0.12 3.12 0.546


Malaga data set

We evaluated a data set of 180 patients with HR + / HER2-5 disease treated with neoadjuvant chemotherapy based on anthracyclines / taxanes (Table 2). In this data set, 46.1%, 16.1% and 37.8% of the samples were identified as CES-E, CES-U and CES-C, respectively. The rates of RpC and RCB 0/1 in the CES-E, CES-U and CES-C groups were 2.4% / 9.6%, 3.4% / 17.2% and 13.2% / 30.9%, respectively (P = 0.022 and 0.004). 10

To assess the ability of the ESC to predict the response to chemotherapy, regardless of the known clinical-pathological variables and the intrinsic subtype, we performed a multivariate logistic regression analysis using RCB (0/1 vs. 2/3) as a variable, since Only 12 samples reached an RCB 0 (ie 15 RpC) in this data set. The results revealed that the ESC provided independent predictive information, above the intrinsic subtype (Table 4), of Ki-67 determined by IHQ (Table S13) and the ROR index PAM50 (Table S14). The AROC of CES to predict a RCB 0/1 was 0.746. Finally, a significant association was observed between CES and Miller and 20 Payne response data (Fig. S1).



Table 4. Association of CES with sensitivity to chemotherapy in the data set 25 Málaga.






Table S13. Association of CES and Ki-67 determined by IHQ with sensitivity to chemotherapy in HR + / HER2- disease from the Málaga data set.
 Bivariate Analysis

 Firms  N CTR Index 0/1 OR Lower 95% Higher 95% p-value
 CES  - - 0.46 0.23 0.89 0.022
 Ki67 IHQ  - - 1.02 0.99 1.04 0.194


Table S14. Association of CES and ROR PAM50 with sensitivity to chemotherapy in HR + / HER2- disease from the Malaga data set.
 Bivariate Analysis

 Firms  N CTR Index 0/1 OR Lower 95% Higher 95% p-value
 CES  - - 0.27 0.14 0.51 <0.001
 PAM50 ROR  - - 0.98 0.95 1.02 0.308
 5
Marsden-based data set: CES and sensitivity to endocrine therapy

We evaluated a data set of 103 postmenopausal patients with HR + disease treated with anastrozole for 16 weeks with neoadjuvant therapy (Table 2). In this data set, 23.5%, 34.3% and 42.2% of the samples were identified as CES-E, CES-U and CES-C, respectively. The clinical tumor response (complete and partial response to stable and progressive disease) was used as a variable. In this data set no RpC was observed. The clinical tumor response rates in the CES-E, CES-U and CES-C groups were 75.0%, 48.6% and 44.2%, respectively (P = 0.043). CES was the only variable 15 with a significant association to the response (Table S15), regardless of the status of HER2 (Tables S15-S16).


Table S15. Association of CES with sensitivity to endocrine therapy in the set of 20 Marsden data (n = 103).



Table S16. Association of CES with sensitivity to endocrine therapy in the Marsden data set in HER2- disease (n = 89).
 Univariate Analysis

 Firms  N Index Response OR Lower 95% Higher 95% p-value
 Age (cont. Variable)  89 - 1.0 0.18 293 0.38
 Tumor size
 T0-T2  54 60% 1.0 - - -
 T3-T4  35 54% 0.8 0.35 2.00 0.697
 ARM
 Arm B  38 50% 1.0 - - -
 Arm C  51 60% 1.5 0.62 3.48 0.379
 Grade
 one  14 57% 1.0 - - -
 2  54 59% 1.1 0.32 3.57 0.886
 3  20 45% 0.7 0.16 2.69 0.579
 PAM50
 Basal-like  4 50% 1.0 - - -
 Luminal A  35 66% 2.09 0.23 19.39 0.489
 Luminal B  17 41% 0.70 0.07 7.00 0.750
 HER2-E  7 29% 0.40 0.03 5.32 0.480
 Normal-like  26 61% 1.60 0.17 15.16 0.660
 CES (cont. Variable)  89 - 2.00 0.90 4.89 0.090


Edinburgh-based data set: CES and sensitivity to endocrine therapy 5

We evaluated a data set of 120 postmenopausal patients with HR + disease treated with letrozole for at least 12 weeks with neoadjuvant therapy (Fig. S2A). Two patients out of 120 achieved a complete response. Similar to previous results, CES as a continuous variable was the only 10 variable with a significant association to a ≥70% reduction in tumor volume before 90 days (Fig. S2B), even in HER2- disease (Fig. S2C ).

Prognosis, intrinsic subtype and sensitivity to chemotherapy and endocrine therapy
 fifteen
To better understand the relationship between prognosis, intrinsic biology and sensitivity to chemotherapy and endocrine therapy, we grouped PAM50 data from numerous different data sets to obtain a total of 6007 primary breast cancers representing all subtypes (Fig. 2). The results revealed
that in the low ROR group, 94.9% of the cases were identified as CES-E and 100% were of the Luminal A subtype. In the high ROR group, 92.1% of the samples were identified as CES-C; the non-luminal and Luminal B subtypes represented 64.3% and 35.7% of the cases of high ROR / CES-C, respectively. 5

In the intermediate ROR group, high heterogeneity was observed. In terms of intrinsic biology, the Luminal A, Luminal B and non-luminal subtypes represented 44.4%, 31.5% and 24.1%, respectively. In terms of sensitivity to chemotherapy and endocrine therapy, CES-E, CES-U and CES-C represented 40.6%, 30.3% and 29.1%, respectively. As expected, the vast majority of intermediate ROR / CES-E samples (77.3%) were of the Luminal A subtype.

CES survival results in intermediate HR + / ROR disease
 fifteen
To continue studying the value of CES in intermediate HR + / ROR disease, we have evaluated the association of CES with survival outcomes in early HR + / ROR breast cancer in 4 independent data sets of patients treated without any adjuvant systemic therapy. (n = 189), only with adjuvant tamoxifen (n = 846) or with adjuvant endocrine chemotherapy and therapy (n = 322 and n = 148). In patients with negative node disease treated without adjuvant systemic therapy, ESC (as a continuous variable or as group categories) was found to have a significant association with survival without distant recurrence (Fig. 3A). The hazard ratio between the CES-C and CES-E groups was 2.68 (95% confidence interval of 0.163-0.858). 25 similar results were obtained in the data set where patients were treated with adjuvant tamoxifen alone (Fig. 3B).

However, CES (as a continuous variable or as group categories) did not turn out to have a significant association with survival outcomes in 2 independent cohorts of patients treated with (neo) adjuvant chemotherapy and endocrine therapy (Fig. 3C and D) .

权利要求:
Claims (7)
[1]

1. An in vitro method to predict whether a patient with breast cancer responds to chemotherapy or endocrine therapy in an isolated sample of HR + / HER2- of the patient classified in the intermediate ROR group using kit 5 PAM50, whose method comprises :
(a) obtain the PAM50 kit, the correlation coefficient corresponding to the sample classified as intrinsic subtype Luminal A and the correlation coefficient corresponding to the sample classified as intrinsic subtype Basal-like, in the isolated sample, and 10
(b) obtain the chemoendocrine index (CES) by subtracting the correlation coefficient corresponding to the sample classified as a Basal-like subtype from the correlation coefficient corresponding to the sample classified as a Luminal A subtype;
where CES equal to or greater than 0.7 indicates that said patient responds to endocrine treatment (CES-E), and where CES equal to or less than 0.3 indicates that said patient responds to chemotherapy (CES-C).
[2]
2. An in vitro method according to claim 1 wherein the isolated sample is a biopsy sample.
[3]
3. The in vitro use of CES to predict whether a patient with breast cancer 20 responds to chemotherapy or endocrine treatment in an isolated HR + / HER2- sample of the patient classified in the intermediate ROR group using the PAM50 kit.
[4]
4. The in vitro use according to claim 3 wherein the ESC is obtained by subtracting the correlation coefficient corresponding to the sample classified as a Basal-like subtype from the correlation coefficient corresponding to the sample classified as a Luminal A subtype, by the PAM50 kit.
[5]
5. The in vitro use according to any of claims 3 or 4 wherein a CES equal to or greater than 0.7 indicates that said patient is CES-E.
[6]
6. The in vitro use according to any of claims 3 or 4 wherein a CES 30 equal to or less than 0.3 indicates that said patient is CES-C.
[7]
7. The in vitro use according to any of claims 3 to 6 wherein the isolated sample is a biopsy sample.

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同族专利:

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EP3546593A4|2020-07-01|

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EP3546593A1|2019-10-02|

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WO2018096191A1|2018-05-31|

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ES2674327B2|2018-12-17|

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US20200392581A1|2020-12-17|

引用文献:

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公开号 | 申请日 | 公开日 | 申请人 | 专利标题

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EP2664679B1|2008-05-30|2017-11-08|The University of North Carolina At Chapel Hill|Gene expression profiles to predict breast cancer outcomes|

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CA2801588A1|2010-06-04|2011-12-08|Bioarray Therapeutics, Inc.|Gene expression signature as a predictor of chemotherapeutic response in breast cancer|

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US20140086911A1|2012-09-21|2014-03-27|University Health Network|Methods and Products for Predicting CMTC Class and Prognosis in Breast Cancer Patients|

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ES201601012A|ES2674327B2|2016-11-28|2016-11-28|CES: a chemoendocrine index based on PAM50 for breast cancer with positive hormone receptors with an intermediate risk of recurrence|ES201601012A| ES2674327B2|2016-11-28|2016-11-28|CES: a chemoendocrine index based on PAM50 for breast cancer with positive hormone receptors with an intermediate risk of recurrence|

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PCT/ES2017/000148| WO2018096191A1|2016-11-28|2017-11-27|Chemoendocrine scorebased on pam50 for breast cancer with positive hormone receptors with an intermediate risk of recurrence|

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US16/464,194| US20200392581A1|2016-11-28|2017-11-27|Chemoendocrine scorebased on pam50 for breast cancer with positive hormone receptors with an intermediate risk of recurrence|

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EP17874745.7A| EP3546593A4|2016-11-28|2017-11-27|Chemoendocrine scorebased on pam50 for breast cancer with positive hormone receptors with an intermediate risk of recurrence|