T. Karn

Predictive value of HER2 serum levels in patients treated with lapatinib or trastuzumab – a translational project in the neoadjuvant GeparQuinto trial

Background:We were able to demonstrate a predictive value of serum HER2 (sHER2) in patients receiving trastuzumab in the neoadjuvant GeparQuattro trial. However, the role of sHER2 in patients receiving neoadjuvant therapy (NT) with lapatinib is still unclear.Methods:The neoadjuvant GeparQuinto trial compared trastuzumab vs lapatinib in addition to chemotherapy in HER2-positive primary breast cancer patients. The sHER2 levels were measured by enzyme-linked immunosorbant assay in 210 patients, of whom 109 (52%) patients received trastuzumab and 101 (48%) lapatinib at three different time points.Results:Twenty-two percent of patients had elevated baseline sHER2 levels (>15u2009ngu2009ml−1). A decrease of sHER2 levels (>20%) in the trastuzumab and lapatinib-treated group during NT was seen in 44% and 24% of the patients, an increase of sHER2 levels (>20%) was seen in 6% and 41% of patients, respectively. Higher pre-chemotherapy sHER2 levels were associated with higher pathological complete remission (pCR) rates in the entire study cohort (OR 1.8, 95% CI 1.02–3.2, P=0.043). A decline of sHER2 levels (>20%) during NT was a predictor for pCR in the lapatinib-treated patient group (OR: 11.7, 95% CI 1.3–110, P=0.031).Conclusion:Results of this study demonstrate that sHER2 levels change differently during NT depending on the anti-HER2 treatment strategy. Elevated baseline sHER2 levels (>15u2009ngu2009ml−1) and a decrease of sHER2 levels (>20%) early after therapy initiation are both relevant criteria to predict response to lapatinib-based treatment.

Thymosin beta 15A (TMSB15A) is a predictor of chemotherapy response in triple-negative breast cancer

Background:Biomarkers predictive of pathological complete response (pCR) to neoadjuvant chemotherapy (NACT) of breast cancer are urgently needed.Methods:Using a training/validation approach for detection of predictive biomarkers in HER2-negative breast cancer, pre-therapeutic core biopsies from four independent cohorts were investigated: Gene array data were analysed in fresh frozen samples of two cohorts (n=86 and n=55). Quantitative reverse transcription polymerase chain reaction (qRT–PCR) was performed in formalin-fixed, paraffin-embedded (FFPE) samples from two neoadjuvant phase III trials (GeparTrio, n=212, and GeparQuattro, n=383).Results:A strong predictive capacity of thymosin beta 15 (TMSB15A) gene expression was evident in both fresh frozen cohorts (P<0.0001; P<0.0042). In the GeparTrio FFPE training cohort, a significant linear correlation between TMSB15A expression and pCR was apparent in triple-negative breast cancer (TNBC) (n=61, P=0.040). A cutoff point was then defined that divided TNBC into a low and a high expression group (pCR rate 16.0% vs 47.2%). Both linear correlation of TMSB15A mRNA levels (P=0.017) and the pre-defined cutoff point were validated in 134 TNBC from GeparQuattro (pCR rate 36.8% vs 17.0%, P=0.020). No significant predictive capacity was observed in luminal carcinomas from GeparTrio and GeparQuattro.Conclusion:In TNBC, TMSB15A gene expression analysis might help to select patients with a high chance for pCR after NACT.

Abstract P6-06-44: Role of uPA and PAI-1 mRNA expression as prognostic factors in molecular subtypes of breast cancer

Background: Urokinase plasminogen activator (uPA) and its inhibitor (PAI-1) determined by ELISA from fresh-frozen tumor tissue have been evaluated as prognostic factors in prospectively randomized trials in node-negative breast cancer. However, uPA and PAI-1 mRNA expression might reveal new clinically relevant information using paraffin-embedded tumor tissue. Methods: We evaluated uPA and PAI-1 mRNA expression with the Affymetrix HG-U 133A array also within molecular subgroups of breast cancer in a finding cohort of breast cancer patients (cohort A; n = 548). We validated mRNA expression in a cohort of HER2 positive breast cancer patients (cohort B, n = 347). For uPA and PAI-1, mRNA values below and above the median were compared in two different probesets. “Luminal”, “triple-negative” and “HER2 positive” subcohorts were defined by ESR1 and ERBB2 mRNA expression using pre-defined cut-offs. Results: In cohort A, elevated PAI-1 mRNA expression was associated with shorter disease-free survival (DFS) and overall survival (OS), elevated uPA mRNA expression was associated with shorter OS. Regarding different molecular subgroups, 71% (n = 388) of tumors had a luminal, 13% (n = 69) a HER2 positive and 17% (n = 91) a triple-negative subtype. Only in the HER2 positive subgroup, elevated PAI-1 mRNA expression was associated with shorter disease-free survival and overall survival (p = 0.003 for DFS and p = 0.012 for OS). Same results were found for uPA in HER2 positive patients (DFS, p = 0.027 and OS, p = 0.011). In contrast, no association between both markers and DFS and OS could be seen in the luminal or triple-negative subgroups. In the HER2 positive validation cohort B elevated uPA and PAI-1 mRNA expression showed a strong association with shorter DFS (p = 0.013 for PAI-1, p = 0.001 for uPA). Conclusion: Results of this study demonstrate that the prognostic impact of uPA and PAI-1 mRNA expression was observed mainly in patients with HER2 positive tumors. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-06-44.

Clinically relevant gene signatures in triple-negative and basal-like breast cancer.

521 Background: The relationship of triple-negative breast cancers (TNBC) to basal-like breast cancer (BLBC) is not fully clear and further understanding is important for identifying new treatment approaches. We aimed to identify principal factors differing among TNBC. Methods: We assembled an Affymetrix dataset of TNBC only to avoid con-founding of expression profiles associated with ER, PgR, and HER2 status. Since TNBC are generally high-grade confounding of proliferation genes should also be reduced. Datasets were excluded based on strict statistical criteria of comparability of the microarrays. Unsupervised methods identified principal metagenes. Results: We started with a n = 579 TNBC from n = 3488 primary breast cancers from 30 datasets of Affymetrix U133A data. 13 datasets (n = 185 TNBC) were excluded from the finding cohort based on criteria of comparability and used for validation. As expected, previously described gene signatures related to ER, PgR, HER2 status and proliferation did not play a r...

Abstract S1-07: Immune sculpting of the triple negative breast cancer genome

Background: Tumors with infiltrating lymphocytes (TIL) demonstrate a better prognosis particularly in TNBC and HER2 positive breast cancer. Two competing hypothesis predict contrasting relationships of TILs and genomic heterogeneity. On one hand, a strong immune response may lead to “pruning” of intratumor heterogeneity by eliminating immunogenic clones resulting in a near equilibrium, hence better prognosis, while cancers that escape the surveillance may evolve towards greater clonal heterogeneity and genomic complexity. In some cancers, the predicted neoantigens are less frequent than expected by chance also suggesting immune mediated elimination of neoplastic clones (Rooney et al. 2015). Studies also showed an inverse association between immune cell infiltration and intratumor clonal heterogeneity (Morris et al. 2016). On the other hand, cancers with greater genomic instability and mutational burden will have larger clonal heterogeneity and therefore more neoantigens and greater immune infiltration. Indeed, a positive correlation between overall mutation load and immune activity in the tumor microenvironment was observed in pooled data across a broad range of cancer types (Brown et al. 2014, Rooney et al. 2015, Schumacher and Schreiber 2015). Methods: We assessed these two competing hypothesis and examined the relationship between genomic complexity and immune gene expression in different breast cancer subtypes. We used previously described immune metagene expression (DNA microarray n=655) as measures of immune infiltration in the TCGA data set (RNA-Seq n=1215). We compared somatic mutations, mutation count, neoantigen load, clonal heterogeneity metrics and the distribution of mutations in 119 canonical cancer genes and 12 cancer pathways between good and poor prognosis TNBC (n=208) corresponding to high and low immune infiltration. Results:A positive but weak correlation between mutation count and immune metagene expression was observed when all breast cancer subtypes were analyzed together (P=0.08). This was driven by the generally higher mutation count and immune infiltration in TNBC. When TNBC was analyzed separately, good prognosis TNBC with high immune infiltration had lower total mutation count (P=0.021) and predicted neo-antigen count (P=0.035). Clonal heterogeneity was also lower in good prognosis TNBC (P=0.001). There was a strong inverse relationship of dispersion in mutation variant allele frequencies and immune metagene expression. CASP8 was the top enriched mutation in TNBC with high immune infiltration (P=0.007 with no adjustment for multiple testing). Conclusions:High immune infiltration is associated with reduced intratumor heterogeneity in TNBC suggesting immune sculpting of the tumor and a near equilibrium between the cancer and immune surveillance. Surgical resection of the primary tumor may tilt the balance towards the immune system resulting in the better prognosis of high-TIL TNBC. TNBC with low immune infiltration has greater clonal heterogeneity and mutation load and may represent the consequence of escape from immune surveillance. Mutation of CASP8 may be one way to evade tumor cell killing in high-TIL TNBC as previously noted. Citation Format: Karn T, Jiang T, Hatzis C, Sanger N, El-Balat A, Holtrich U, Becker S, Bianchini G, Pusztai L. Immune sculpting of the triple negative breast cancer genome [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr S1-07.

BRCA1 -like profile is not significantly associated with survival benefit of non-myeloablative intensified chemotherapy in the GAIN randomized controlled trial

PurposeThe BRCA1-like profile identifies tumors with a defect in homologous recombination due to inactivation of BRCA1. This profile has been shown to predict which stage III breast cancer patients benefit from myeloablative, DNA double-strand-break-inducing chemotherapy. We tested the predictive potential of the BRCA1-like profile for adjuvant non-myeloablative, intensified dose-dense chemotherapy in the GAIN trial.MethodsLymph node positive breast cancer patients were randomized to 3xa0×xa03 dose-dense cycles of intensified epirubicin, paclitaxel, and cyclophosphamide (ETC) or 4 cycles concurrent epirubicin and cyclophosphamide followed by 10 cycles of weekly paclitaxel combined with 4 cycles capecitabine (EC-TX). Only triple negative breast cancer patients (TNBC) for whom tissue was available were included in these planned analyses. BRCA1-like or non-BRCA1-like copy number profiles were derived from low coverage sequencing data.Results119 out of 163 TNBC patients (73%) had a BRCA1-like profile. After median follow-up of 83xa0months, disease free survival (DFS) was not significantly different between BRCA1-like and non-BRCA1-like patients [adjusted hazard ratio (adj.HR) 1.02; 95% confidence interval (CI) 0.55–1.86], neither was overall survival (OS; adj.HR 1.26; 95% CI 0.58–2.71). When split by BRCA1-like status, DFS and OS were not significantly different between treatments. However, EC-TX seemed to result in a trend to an improvement in DFS in patients with a BRCA1-like tumor, while the reverse accounted for ETC treatment in patients with a non-BRCA1-like tumor (p for interactionxa0=xa00.094).ConclusionsThe BRCA1-like profile is not associated with survival benefit for a non-myeloablative, intensified regimen in this study population. Considering the limited cohort size, capecitabine might have additional benefit for TNBC patients.

Serum carbonic anhydrase IX as predictive marker for efficacy of bevacizumab: a biomarker analysis from the GeparQuinto phase III neoadjuvant breast cancer trial

T cell stimulation with different cytokines results in distinct phenotypes and cytotoxic activity of CD19-specific CART cells

Abstract P3-07-28: BRCA1-like profile as predictive biomarker in non myeloablative chemotherapy (GAIN study)

Background: The BRCA1-like copy number (CN) profile can be used as a biomarker to predict which stage III breast cancer patients benefit from myeloablative, DNA double-strand-break (DSB)-inducing chemotherapy. In addition, a BRCA1-like gene expression classifier, derived from the BRCA1-like CN profile, can predict which patient group will achieve an increased pathological complete remission rate on a standard neoadjuvant regimen complemented with carboplatin/veliparib. A non-myeloablative, dose-dense schedule of epirubicin, paclitaxel and cyclophosphamide (ETC) is used in the clinic to treat stage III patients. Since ETC contains an intensified dose of DNA DSB-inducing cyclophosphamide, we tested the BRCA1-like CN profile as a predictive biomarker for ETC benefit in the GAIN trial. Methods: The GAIN trial was a prospective, multi-center, non-blinded, randomized phase III trial. Eligibility comprised histologically confirmed invasive breast cancer with at least one positive axillary or internal mammary lymph node and no signs of distant metastases. The allocated adjuvant treatment was intensified chemotherapy with sequential E (150 mg/m2), T (225 mg/m2) and C (2500, after amendment 2000 mg/m2) each 3 cycles every 2 weeks (ETC) or concurrent E (112.5 mg/m2) and C (600 mg/m2) for 4 cycles every 2 weeks followed by 10 cycles of weekly T (67.5 mg/m2) combined with 4 cycles of capecitabine (2000mg/m2) on day 1-14 in a 3-weekly cycle (EC-TX). Only the triple negative patients were used for these analyses. For samples with good quality DNA extracted from formalin-fixed paraffin-embedded tumor tissue, a library was prepared and sequenced on an Illumina HiSeq2000 platform. Copy number estimates were extracted from the sequence data by normalizing GC-content and mappability corrected read counts to the average read count. These CN profiles were classified as either BRCA1-like or non-BRCA1-like using a previously established shrunken centroid classifier with an established cut-off. Disease-free survival (DFS) and overall survival (OS) were estimated using the Kaplan-Meier method, the difference in survival was analysed using log-rank tests. Multivariate analyses were done by generating Cox regression models with important prognostic factors. Results: Out of 424 triple negative patients, 166 patients with available tissue and a tumour cell content of at least 60% were analysed for BRCA1-like status and included in the analyses (classified as BRCA1-like: n=122). Based on clinicopathological characteristics, these patients were similar to the total group of triple negative participants. In the BRCA1-like patients, there was no significant difference in DFS or OS between ETC and EC-TX (log-rank tests n.s.). In accordance, Cox regression models confirmed these findings. Conclusion: In contrast to the predictive value of the BRCA1-like profile in myeloablative chemotherapy, it could not predict survival benefit for a non-myeloablative, non-platinum-/veliparib-containing regimen in this study population. An intensified dose of cyclophosphamide resulted in similar outcomes in BRCA1-like patients as addition of capecitabine to standard chemotherapy. These results help to define the appropriate application of the BRCA1-like profile as a predictive biomarker. Citation Format: van Rossum AGJ, Schouten PC, Weber KE, Nekljudova V, Denkert C, von Minckwitz G, Karn T, Mobus VJ, Linn SC, Loibl S, Marme F. BRCA1-like profile as predictive biomarker in non myeloablative chemotherapy (GAIN study). [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-07-28.

Abstract P3-06-05: Expression of SPARC in human breast cancer and its predictive value in the GeparTrio neoadjuvant trial.

Background: Secreted protein acidic and rich in cysteine (SPARC) is an albumin-binding protein and associated with poor prognosis in multiple cancers. The aim of this analysis was to determine the frequency of SPARC expression among different molecular breast cancer subtypes and to evaluate its predictive value for therapy response after neoadjuvant anthracycline/taxane based chemotherapy (CTX) in participants of the GeparTrio trial. Methods: We evaluated tumoral SPARC expression by immunohistochemistry on tissue microarrays (TMAs) constructed from formalin-fixed paraffin-embedded (FFPE) pre-treatment core biopsies from 667 patients (pts) of the GeparTrio trial. The details of the GeparTrio study design are described elsewhere (von Minckwitz, JNCI 2008). Cutoffs for SPARC expression were determined using the web-based software Cutoff Finder (http://molpath.charite.de/cutoff/). Results: SPARC protein expression was measurable by IHC on the TMAs and 176 (26.4 %) of 667 tumors were SPARC positive. SPARC expression was increased in pts with triple-negative breast cancer (TNBC) compared to hormone receptor or HER2 positive subtypes (p = 0.039). SPARC positivity was associated with an increased pCR rate in the overall population (p In multivariate logistic regression analysis adjusted for standard clinicopathological factors, SPARC was independently predictive in the overall population (p = 0.010) as well as the subgroups of pts with TNBC (p = 0.036). Conclusions: SPARC is expressed in all biological breast cancer subtypes with TNBC revealing the highest expression rate. Our data suggest that SPARC expression may provide predictive information for response to neoadjuvant CTX. As SPARC is an albumin-binding protein and might mediate intratumoral accumulation of nab -Paclitaxel, prospective analysis of SPARC expression is planned in the GeparSepto trial. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P3-06-05.

Translationale Forschung beim Mammakarzinom – Workshop zur Erarbeitung neuer Forschungsideen im Rahmen des Biedenkopf Symposiums XIII der Banss-Stiftung

Translational Research in oncology links experimental and clinical science with the aim of transferring knowledge from research laboratories and hospitals and translating it into new therapy concepts. The definition of standards for the collection of biomaterials and the continued development of technologies are preconditions for Translational Research. Nevertheless new approaches are required to advance Translational Research. Following the invitation of the Banss Foundation, in September 2007 leading clinicians and scientists from German university hospitals and research facilities met together in Biedenkopf, near Marburg. The symposium aimed to develop new scientific concepts using aspects and features of innovation management. The topic was introduced by presenting general therapy concepts in breast cancer treatment (Prof. C. Thomssen) as well as current clinical breast cancer trials (Prof. G. von Minckwitz). Translational Research was described from a clinical point of view (Prof. N. Harbeck) and from the point of view of the laboratory (Dr. D. Niederacher). The question “what we know and what we really should know” (Prof. B. Groner) was discussed during a presentation on the current state of knowledge in cancer research. This article summarizes the results of the discussion on central aspects of Translational Research in this workshop and offers new approaches.