Jan Budczies

Tumour-infiltrating lymphocytes and prognosis in different subtypes of breast cancer: a pooled analysis of 3771 patients treated with neoadjuvant therapy

BACKGROUNDnTumour-infiltrating lymphocytes (TILs) are predictive for response to neoadjuvant chemotherapy in triple-negative breast cancer (TNBC) and HER2-positive breast cancer, but their role in luminal breast cancer and the effect of TILs on prognosis in all subtypes is less clear. Here, we assessed the relevance of TILs for chemotherapy response and prognosis in patients with TNBC, HER2-positive breast cancer, and luminal-HER2-negative breast cancer.nnnMETHODSnPatients with primary breast cancer who were treated with neoadjuvant combination chemotherapy were included from six randomised trials done by the German Breast Cancer Group. Pretherapeutic core biopsies from 3771 patients included in these studies were assessed for the number of stromal TILs by standardised methods according to the guidelines of the International TIL working group. TILs were analysed both as a continuous parameter and in three predefined groups of low (0-10% immune cells in stromal tissue within the tumour), intermediate (11-59%), and high TILs (≥60%). We used these data in univariable and multivariable statistical models to assess the association between TIL concentration and pathological complete response in all patients, and between the amount of TILs and disease-free survival and overall survival in 2560 patients from five of the six clinical trial cohorts.nnnFINDINGSnIn the luminal-HER2-negative breast cancer subtype, a pathological complete response (pCR) was achieved in 45 (6%) of 759 patients with low TILs, 48 (11%) of 435 with intermediate TILs, and 49 (28%) of 172 with high TILs. In the HER2-positive subtype, pCR was observed in 194 (32%) of 605 patients with low TILs, 198 (39%) of 512 with intermediate TILs, and 127 (48%) of 262 with high TILs. Finally, in the TNBC subtype, pCR was achieved in 80 (31%) of 260 patients with low TILs, 117 (31%) of 373 with intermediate TILs, and 136 (50%) of 273 with high TILs (p<0·0001 for each subtype, χ2 test for trend). In the univariable analysis, a 10% increase in TILs was associated with longer disease-free survival in TNBC (hazard ratio [HR] 0·93 [95% CI 0·87-0·98], p=0·011) and HER2-positive breast cancer (0·94 [0·89-0·99], p=0·017), but not in luminal-HER2-negative tumours (1·02 [0·96-1·09], p=0·46). The increase in TILs was also associated with longer overall survival in TNBC (0·92 [0·86-0·99], p=0·032), but had no association in HER2-positive breast cancer (0·94 [0·86-1·02], p=0·11), and was associated with shorter overall survival in luminal-HER2-negative tumours (1·10 [1·02-1·19], p=0·011).nnnINTERPRETATIONnIncreased TIL concentration predicted response to neoadjuvant chemotherapy in all molecular subtypes assessed, and was also associated with a survival benefit in HER2-positive breast cancer and TNBC. By contrast, increased TILs were an adverse prognostic factor for survival in luminal-HER2-negative breast cancer, suggesting a different biology of the immunological infiltrate in this subtype. Our data support the hypothesis that breast cancer is immunogenic and might be targetable by immune-modulating therapies. In light of the results in luminal breast cancer, further research investigating the interaction of the immune system with different types of endocrine therapy is warranted.nnnFUNDINGnDeutsche Krebshilfe and European Commission.

Abstract S4-5: Ki67 levels in pretherapeutic core biopsies as predictive and prognostic parameters in the neoadjuvant GeparTrio trial

Background: Ki67 has been suggested as a marker for definition of luminal A and luminal B tumors by the 2011 St. Gallen consensus panel. However, the cutoffs for Ki67 are still under debate. In particular, it is not clear if one single cutoff is useful for prognostic and predictive information in the different molecular subtypes. It is an advantage of the neoadjuvant approach that predictive and prognostic outcome measurements can be separated in the same cohort. In this study, we evaluated a large cohort of core biopsies from the neoadjuvant GeparTrio trial to investigate the impact of pretherapeutic Ki67 levels as a predictive marker for response to neoadjuvant chemotherapy as well as a prognostic marker for progression-free and overall survival. The analysis was stratified for hormone-receptor positive and negative tumors as well as HER2 status. Methods: A total of 1166 pretherapeutic core biopsies from the neoadjuvant Gepartrio trial were evaluated for Ki67 by immunohistochemistry, a total of 200 cells were counted in each sample. Ki67 cutoffs were evaluated using web-based software Cutoff Finder (http://molpath.charite.de/cutoff/). The details of the GeparTrio study design have been described before (von Minckwitz, JNCI 2008). We compared pCR rate as well as the overall and disease free survival in the complete cohort as well as subgroups of patients based on hormone receptor and HER2 expression. Results: Using Ki67 as a continuous parameter, a wide range of cutoffs between 10% and 80% for Ki67 were predictive for pCR. For DFS and OS, a wide range of cutoffs between 10% and 45% was significant. For further analysis, the three groups of Ki67 0–15% vs. Ki67 15.1%–35% vs. Ki67 >35 were defined and were compared for different outcome parameters. The pCR rates in these three groups of Ki67 expression were 4.2%, 12.9% and 29.0% (p Conclusion: Ki67 is a valid predictive and prognostic marker in breast cancer. This marker is significant over a wide range of different cutoffs, which explains the different results of Ki67 cutoffs in different previous studies. Therefore, the variability observed in different studies evaluating Ki67 might reflect A) the wide range of valid cutoffs B) the different clinical endpoints of the studies and C) the different contribution of the molecular subtypes in the study cohorts. Based on our results we suggest three subgroups for Ki67 (0–15% vs. 15.1–35 vs. >35%) as a reasonable approach for further standardization of this marker. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr S4-5.

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.

Dynamics of the Intratumoral Immune Response during Progression of High-Grade Serous Ovarian Cancer

PURPOSE: Tumor-infiltrating lymphocytes (TILs) have an established impact on the prognosis of high-grade serous ovarian carcinoma (HGSOC), however, their role in recurrent ovarian cancer is largely unknown. We therefore systematically investigated TIL densities and MHC class I and II (MHC1, 2) expression in the progression of HGSOC. EXPERIMENTAL DESIGN: CD3+, CD4+, CD8+ TILs and MHC1, 2 expression were evaluated by immunohistochemistry on tissue microarrays in 113 paired primary and recurrent HGSOC. TILs were quantified by image analysis. All patients had been included to the EU-funded OCTIPS FP7 project. RESULTS: CD3+, CD4+, CD8+ TILs and MHC1 and MHC2 expression showed significant correlations between primary and recurrent tumor levels (Spearman rho 0.427, 0.533, 0.361, 0.456, 0.526 respectively; P<.0001 each). Paired testing revealed higher CD4+ densities and MHC1 expression in recurrent tumors (Wilcoxon P=.034 and P=.018). There was also a shift towards higher CD3+ TILs levels in recurrent carcinomas when analyzing platinum-sensitive tumors only (Wilcoxon P=.026) and in pairs with recurrent tumor tissue from first relapse only (Wilcoxon P=.031). High MHC2 expression was the only parameter to be significantly linked to prolonged progression-free survival after first relapse (PFS2, log-rank P=.012). CONCLUSIONS: This is the first study that analyzed the development of TILs density and MHC expression in paired primary and recurrent HGSOC. The level of the antitumoral immune response in recurrent tumors was clearly dependent on the one in the primary tumor. Our data contribute to the understanding of temporal heterogeneity of HGSOC immune microenvironment and have implications for selection of samples for biomarker testing in the setting of immune-targeting therapeutics.

Chromosome 9p copy number gains involving PD-L1 are associated with a specific proliferation and immune-modulating gene expression program active across major cancer types

BackgroundInhibition of the PD-L1/PD-1 immune checkpoint axis represents one of the most promising approaches of immunotherapy for various cancer types. However, immune checkpoint inhibition is successful only in subpopulations of patients emphasizing the need for powerful biomarkers that adequately reflect the complex interaction between the tumor and the immune system. Recently, recurrent copy number gains (CNG) in chromosome 9p involving PD-L1 were detected in many cancer types including lung cancer, melanoma, bladder cancer, head and neck cancer, cervical cancer, soft tissue sarcoma, prostate cancer, gastric cancer, ovarian cancer, and triple-negative breast cancer.MethodsHere, we applied functional genomics to analyze global mRNA expression changes associated with chromosome 9p gains. Using the TCGA data set, we identified a list of 75 genes that were strongly up-regulated in tumors with chromosome 9p gains across many cancer types.ResultsAs expected, the gene set was enriched for chromosome 9p and in particular chromosome 9p24 (36 genes and 23 genes). Furthermore, we found enrichment of two expression programs derived from genes within and beyond 9p: one implicated in cell cycle regulation (22 genes) and the other implicated in modulation of the immune system (16 genes). Among these were specific cytokines and chemokines, e.g. CCL4, CCL8, CXCL10, CXCL11, other immunoregulatory genes such as IFN-G and IDO1 as well as highly expressed proliferation-related kinases and genes including PLK1, TTK, MELK and CDC20 that represent potential drug targets.ConclusionsCollectively, these data shed light on mechanisms of immune escape and stimulation of proliferation in cancer with PD-L1 CNG and highlight additional vulnerabilities that may be therapeutically exploitable.

Subgroup-specific immune and stromal microenvironment in medulloblastoma

ABSTRACT Knowledge on immune and stromal cells in medulloblastoma microenvironment is still limited as previous work was frequently restricted by low sample size and the lack of molecular subgroup information. We characterized 10 microenvironment cell populations as well as PD-L1 from gene expression in 1422 brain tumors and 763 medulloblastomas. All in all, medulloblastomas showed low expression of immune markers. Still, there were substantial differences with a clustering of medulloblastoma subgroups according to their microenvironment profile. Specifically, SHH medulloblastomas displayed strong signatures of fibroblasts, T cells and macrophages, while markers of cytotoxic lymphocytes were enriched in Group 4 tumors. PD-L1 gene expression appeared to be relatively high in single SHH and WNT cases but was undetectable by immunohistochemistry. In addition, two diverse immuno-stromal patterns were identified, indicating distinct types of local tumor immunosuppression, which were primarily controlled by either macrophage and regulatory T cell-mediated mechanisms or immunosuppressive cytokines and checkpoints, respectively. None of the immune cell signatures had an independent prognostic value in the present dataset after multiple testing correction. These results suggest a mild, but subgroup-specific infiltration of immune cells in medulloblastoma.

A multi-omics analysis reveals metabolic reprogramming in THP-1 cells upon treatment with the contact allergen DNCB

ABSTRACT Dendritic cell (DC) activation by contact allergens is one of the key steps in the development of allergic contact dermatitis (ACD). Recent evidence suggests that metabolic reprogramming is a prerequisite for the activation of DCs, macrophages and monocytes. Therefore, we used an integrated approach by combining proteomics and metabolomics to investigate the metabolism of human THP‐1 cells in response to the strong contact allergen, 2,4‐dinitrochlorobenzene (DNCB). Cells were treated with 5, 10 and 20 &mgr;M DNCB for 4, 8, and 24 h, respectively. Using a targeted metabolomics approach, we quantified levels of 188 endogenous metabolites, among them phospholipids, acylcarnitines, amino acids and hexoses. In addition, proteomic changes were analyzed using an untargeted quantitative approach based on stable isotope labeling with amino acids in cell culture (SILAC). We detected several alterations in the metabolome and consistently in the proteome indicating metabolic reprogramming of THP‐1 cells by DNCB. In particular, we found an increase in phospholipids that was accompanied by an up‐regulation of fatty acid synthase (FAS), a key enzyme in lipid synthesis. HighlightsMetabolomics and proteomics reveal consistent findings in DNCB‐treated THP‐1 cells.Findings indicate metabolic reprogramming of THP‐1 cells activated by DNCB.THP‐1 cells activated by DNCB induce fatty acid synthase and phosphatidylcholines.

Abstract P2-03-09: Comparison of the mutational landscape of breast cancer during pregnancy and non-pregnant controls

Background:Currently, breast cancer during pregnancy (BCP) is not believed to be biologically different from breast cancer unrelated to pregnancy based on limited datasets mainly obtained by immunohistochemistry. However, some groups report that BCP patients have an inferior survival compared to young non-pregnant breast cancer patients. The largest analysis based on the BCP registry by the German Breast Group (GBG) revealed however, no difference between pregnant and non-pregnant breast cancer patients, indicating that treatment rather than biology might be the reason for the inferior survival reported by others. Methods: The BCP study (GBG 29/BIG 03-02) is a multicentre observational study for breast cancer during pregnancy. In tumour tissue collected within this study from pregnant M0 patients we investigated the following genes: AKT1, ATM, BRAF, CBFB, CCND1, CDH1, CDKN2A, CTCF, EGFR, ERBB2, ESR1, FGFR2, GATA3, KRAS, MAP2K4, MAP3K1, MDM2, MED12, MYC, PIK3CA, PIK3R1, PTEN, RB1, RUNX1, and TP53 by massive parallel sequencing (MPS). This included patients with all molecular subtypes: HR+/-, HER2+/-. Sequencing was done on an IonTorrent Proton using a custom designed Breast Cancer Panel (BCPv2). This panel comprises 236 amplicons split into two primer pools and covers hotspot regions of 138 exons of the 25 genes. To test the hypothesis that breast cancer diagnosed during pregnancy is biologically not different from breast cancer diagnosed in young non-pregnant women, we compared the molecular profiles obtained, with genetic data from M0 patients not known to be pregnant from TCGA with age Results: Material from 141 patients from the BCP study was available from which ultimately 109 fully evaluable MPS datasets could be obtained. In the TCGA data set 114 breast cancer patients Exact matching by variables age, HR, HER2 and grade yielded 40 patients from both datasets. In these subcohorts, still divergent mutational rates for TP53 and PIK3CA between pregnant and non-pregnant women were noted, however, the differences failed to reach statistical significance. Conclusions: Overall the mutational landscapes do not seem to be overtly different between pregnant patients and no-pregnant controls, although slight imbalances in mutational rates occurred, which might be partly explained by a selection bias and a small sample size after matching. Further comparisons using other datasets, looking into survival and regarding copy number variation are currently conducted. This research is been funded by the German Cancer Consortium-DKTK and the BANSS Foundation. Citation Format: Loibl S, Pfarr N, Weber K, Neunhoffer T, Villegas S, Stenzinger A, Furlanetto J, Aktas B, Budczies J, Marme F, Kahmann L, Denkert C, Weichert W. Comparison of the mutational landscape of breast cancer during pregnancy and non-pregnant controls [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 P2-03-09.

Abstract P3-07-03: PIK3CA mutations predict resistance to trastuzumab/pertuzumab and nab-paclitaxel in primary HER2-positive breast cancer – Massive parallel sequencing analysis of 293 pretherapeutic core biopsies of the GeparSepto study

Background: Phosphatidylinositol 3-kinase mutations (PIK3CA) are common in breast cancer (BC). Mutations are predominantly found in hot-spots located in the helical and kinase domains (exons 9 and 20). We recently demonstrated that PIK3CA mutations predict lower pathological complete response (pCR) to double blockade with trastuzumab/lapatinib in HER2+ve primary BC. Methods: We evaluated PIK3CA mutations in 293/403 HER2+ve tumors of participants of the neoadjuvant GeparSepto (G7) study (Untch et al. SABCS 2014). The G7 study investigated the effect of exchanging paclitaxel for nab-paclitaxel prior to EC. All patients received trastuzumab and pertuzumab. The G7 study showed a significantly higher pCR rate in patients receiving nab-paclitaxel. HER2, hormone receptors (HR), Ki67 and tumor infiltrating lymphocytes (TILs) were centrally assessed prior to randomization. PIK3CA mutations in exons 9 and 20 were evaluated in formalin-fixed, paraffin embedded core biopsies taken before therapy using deep targeted massive parallel sequencing with a minimum coverage of 500 and a mean coverage of 6520 and 6346 per amplicon, (exon9 and exon 20). Only non-synonymous mutations in the coding region that were called at variant allele frequency ≥10% were taken into consideration. Only cases with a tumor cell content of ≥20% were included. Results: In the G7 study, 396 patients with HER2+ve BC have been randomized from 06/2012 to 01/2014 and started treatment. From these 293 could be sequenced. Median age in the analyzed cohort was 49 years (range 22-75); most tumors were cT1-2 (89.9%); cN0 (54.4%); ductal invasive (88.7%), grade 3 (53.9%), HR+ve (69.6%), Ki67>20% (69.3%), LPBC-negative (83.2%). Overall, 22.2% of the tumors were found to have a PIK3CA mutation, 20.1% in HR+ve and 27.0% in HR-ve. Overall, the pCR rate was significantly lower in the PIK3CA mutant tumors compared to the wild type (wt) group (47.7% vs. 66.7%; p=0.009). This effect was seen both in the HR+ve (43.9% vs. 61.3%; p=0.052) and the HR-ve population (54.2% vs. 80.0%; p=0.029). There was also a significant difference in pCR according to PIK3CA mutation status dependant on the taxane. In the nab-paclitaxel group, pCR rates were significantly lower in patients with PIK3CA mutations compared to those without PIK3CA mutations (38.7% vs. 72.0%; p=0.001), whereas in the paclitaxel group, there was no significant difference between patients with and without a PIK3CA mutation (55.9% vs. 60.9%; p=0.690). The respective interaction could be demonstrated in univariate (p=0.039) as well as multivariate regression analysis (p=0.010) after adjusting for known baseline factors. Conclusion: Patients with PIK3CA mutant HER2+ve BC have a significantly lower pCR rate compared to patients with wt tumors. In contrast to the results with double anti-HER2 blockade consisting of trastuzumab/lapatinib, the effect was evident irrespective of the HR status. In addition, PIK3CA mutation status was significantly associated with higher pCR following nab-paclitaxel. The project has partly been funded within the EU-FP7 project RESPONSIFY No 278659 and the German Cancer Consortium (DKTK). Citation Format: Loibl S, Budczies J, Weichert W, Furlanetto J, Stenzinger A, Pfarr N, von Minckwitz G, Jackisch C, Schneeweiss A, Fasching P, Schmatloch S, Aktas B, Nekljudova V, Weber K, Untch M, Denkert C. PIK3CA mutations predict resistance to trastuzumab/pertuzumab and nab-paclitaxel in primary HER2-positive breast cancer – Massive parallel sequencing analysis of 293 pretherapeutic core biopsies of the GeparSepto 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 P3-07-03.

Abstract P5-07-02: Systematic analysis and modulation of Ki67 interobserver variance in 9069 patients from three clinical trials – How much pathologist concordance is needed for meaningful biomarker results?

Background: Ki67 has been suggested as a marker for diagnosis of luminal A and B breast carcinomas. Interestingly, on one hand a multitude of studies have described significant results for Ki67 as a prognostic marker, while on the other hand the analytical validation and standardization of this marker has been a challenge. The best parameter for Ki67 interobserver performance is the interclass correlation coefficient (ICC). ICC values between 0.59 and 0.92 have been reported. Recently a minimum ICC of 0.8 has been suggested as a goal for the international ring trial and as a prerequisite for introduction of Ki67 into clinical practice. However, this suggested ICC is not derived from analysis of data, and the amount of pathologist variance that is allowed for meaningful biomarker results is still not defined. Methods: This study is based on a total of 9069 tumor samples from three large clinical cohorts (IBCSG VIII+IX, BIG1-98, and GeparTrio). In a systematic modeling approach, we introduced different amounts of variance to previously generated central pathology Ki67 datasets by simulation of a total of 1800 different pathologist evaluations for each study cohort. These evaluations were grouped into groups with defined ICCs, ranging from very good concordance (ICC=0.9) to extremely poor concordance (ICC=0.1). For each of the simulated pathologist evaluations, all possible Ki67 cutoffs were systematically evaluated using the web-based software Cutoff Finder (http://molpath.charite.de/cutoff/). As endpoints, we used DFS for all three study cohorts as well as pCR for the neoadjuvant cohort. Results: For the neoadjuvant GeparTrio study, the different groups with ICCs of 0.8, 0.6 and 0.4 showed a very similar performance resulting in significant analyses for prediction of pCR across a wide range of cutoffs. The odd ratios for pCR were slightly lower with lower ICC. Even with an extremely low ICC of 0.2, 99% of the analyses had one or more significant cutpoints. The survival endpoint DFS was shown to be very stable despite increased interpathologist variance in all three clinical cohorts. Even with a poor ICC of 0.4, the majority of cutpoints were significant for DFS. For IBCSG VIII+IX 85% of the analyses with an ICC of 0.4 had one or more significant cutpoints for Ki67. In the large BIG 1-98 dataset (n=6090) even an ICC of 0.2 resulted in one or more significant DFS cutpoints in 100% of the analyses. Comparable results were obtained if the analysis was restricted to luminal tumors. Conclusion: Our results suggest that Ki67 is extremely robust to pathologist variation. Even if less than 40% of the variance is attributable to true Ki67-based proliferation (ICC Citation Format: Denkert C, Budczies J, Regan M, Loibl S, Dell9Orto P, von Minckwitz G, Mastropasqua M, Mehta K, Muller V, Kammler R, Pfitzner BM, Fasching PA, Viale G. Systematic analysis and modulation of Ki67 interobserver variance in 9069 patients from three clinical trials – How much pathologist concordance is needed for meaningful biomarker results?. [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 P5-07-02.