Matthias Christgen

Trastuzumab emtansine (T-DM1) renders HER2+ breast cancer highly susceptible to CTLA-4/PD-1 blockade

An antibody-drug conjugate overcomes resistance to immune checkpoint blockade in HER2-positive breast cancer. Targeted therapy with more punch Overexpression of the human epidermal growth factor receptor 2 (HER2) is common in breast cancer, and it is associated with poor outcomes despite the availability of trastuzumab, an antibody against HER2, and other HER2-targeted agents. The reason for the poor outcomes is that many patients develop resistance to the targeted drugs. Müller et al. have now shown that this resistance can be overcome with trastuzumab emtansine, an antibody-drug conjugate that combines the HER2-targeting ability of trastuzumab with a cytotoxic drug, which the antibody delivers directly to the tumor. In addition to its cytotoxic effects, treatment with trastuzumab emtansine activated a strong antitumor immune response and effectively combined with immune checkpoint inhibitors, suggesting that it can be used in combination therapy. Targeted drug delivery with antibody-drug conjugates such as the HER2-directed ado-trastuzumab emtansine (T-DM1) has emerged as a powerful strategy for cancer therapy. We show that T-DM1 is particularly effective in eliciting antitumor immunity in patients with early breast cancer (WSG-ADAPT trial) and in a HER2-expressing orthotopic tumor model. In the latter, despite primary resistance to immunotherapy, combined treatment with T-DM1 and anti–CTLA-4/PD-1 (cytotoxic T lymphocyte–associated protein-4/programmed cell death protein-1) was curative because it triggered innate and adaptive immunity. Tumor rejection was accompanied by massive T cell infiltration, TH1 (T helper 1) cell polarization, and, notably, a substantial increase in regulatory T cells. Depletion of regulatory T cells resulted in inflammation and tissue damage, implying their essential role in protecting the host during therapy. This study provides insights into the mechanisms of T-DM1’s therapeutic activity and a rationale for potential therapeutic combination strategies with immunotherapy.

Standardized evaluation of tumor-infiltrating lymphocytes in breast cancer : results of the ring studies of the international immuno-oncology biomarker working group

Multiple independent studies have shown that tumor-infiltrating lymphocytes (TIL) are prognostic in breast cancer with potential relevance for response to immune-checkpoint inhibitor therapy. Although many groups are currently evaluating TIL, there is no standardized system for diagnostic applications. This study reports the results of two ring studies investigating TIL conducted by the International Working Group on Immuno-oncology Biomarkers. The study aim was to determine the intraclass correlation coefficient (ICC) for evaluation of TIL by different pathologists. A total of 120 slides were evaluated by a large group of pathologists with a web-based system in ring study 1 and a more advanced software-system in ring study 2 that included an integrated feedback with standardized reference images. The predefined aim for successful ring studies 1 and 2 was an ICC above 0.7 (lower limit of 95% confidence interval (CI)). In ring study 1 the prespecified endpoint was not reached (ICC: 0.70; 95% CI: 0.62–0.78). On the basis of an analysis of sources of variation, we developed a more advanced digital image evaluation system for ring study 2, which improved the ICC to 0.89 (95% CI: 0.85–0.92). The Fleiss’ kappa value for <60 vs ≥60% TIL improved from 0.45 (ring study 1) to 0.63 in RS2 and the mean concordance improved from 88 to 92%. This large international standardization project shows that reproducible evaluation of TIL is feasible in breast cancer. This opens the way for standardized reporting of tumor immunological parameters in clinical studies and diagnostic practice. The software-guided image evaluation approach used in ring study 2 may be of value as a tool for evaluation of TIL in clinical trials and diagnostic practice. The experience gained from this approach might be applicable to the standardization of other diagnostic parameters in histopathology.

Nuclear Kaiso Expression Is Associated with High Grade and Triple-Negative Invasive Breast Cancer

Kaiso is a BTB/POZ transcription factor that is ubiquitously expressed in multiple cell types and functions as a transcriptional repressor and activator. Little is known about Kaiso expression and localization in breast cancer. Here, we have related pathological features and molecular subtypes to Kaiso expression in 477 cases of human invasive breast cancer. Nuclear Kaiso was predominantly found in invasive ductal carcinoma (IDC) (p = 0.007), while cytoplasmic Kaiso expression was linked to invasive lobular carcinoma (ILC) (p = 0.006). Although cytoplasmic Kaiso did not correlate to clinicopathological features, we found a significant correlation between nuclear Kaiso, high histological grade (p = 0.023), ERα negativity (p = 0.001), and the HER2-driven and basal/triple-negative breast cancers (p = 0.018). Interestingly, nuclear Kaiso was also abundant in BRCA1-associated breast cancer (p<0.001) and invasive breast cancer overexpressing EGFR (p = 0.019). We observed a correlation between nuclear Kaiso and membrane-localized E-cadherin and p120-catenin (p120) (p<0.01). In contrast, cytoplasmic p120 strongly correlated with loss of E-cadherin and low nuclear Kaiso (p = 0.005). We could confirm these findings in human ILC cells and cell lines derived from conditional mouse models of ILC. Moreover, we present functional data that substantiate a mechanism whereby E-cadherin controls p120-mediated relief of Kaiso-dependent gene repression. In conclusion, our data indicate that nuclear Kaiso is common in clinically aggressive ductal breast cancer, while cytoplasmic Kaiso and a p120-mediated relief of Kaiso-dependent transcriptional repression characterize ILC.

MDA-MB-435: the questionable use of a melanoma cell line as a model for human breast cancer is ongoing.

In September 2006, Rae et al. provided unmistakable evidence that the MDA-MB-435 human breast cancer cell line is identical to the M14 human melanoma cell line. This discovery was no surprise to the breast cancer research community. In fact, the expression of melanocyte specific marker proteins by MDA MB-435 cells was first recognized many years ago. In 2007, the year after MDA-MB-435 cells were officially redefined as melanoma cells, this mix-up has gained a new aspect: We could easily identify 20 novel studies published in the recent issues of leading journals which simply proceeded to employ MDA-MB-435 cells as a model system for human breast cancer. Here we want to draw attention to this questionable practice which should further raise our (self-) skepticism and awareness about the true origin, the identity and molecular properties of any biological materials used in cancer research.

KAI1/CD82 is a novel target of estrogen receptor-mediated gene repression and downregulated in primary human breast cancer

The cell‐surface glycoprotein KAI1 suppresses tumor growth and metastasis in various animal models. Downregulation of KAI1 has been implicated in the progression of cancer. However, the mechanisms of KAI1 inactivation are poorly understood. This is the first study that investigates expression and regulation of KAI1 in human breast cancer. KAI1 expression was analyzed on custom‐made tissue microarrays comprising 209 well‐characterized breast cancers and normal mammary gland tissue. Strong KAI1 immunoreactivity was observed throughout the normal mammary gland epithelium. In breast cancer tissue, KAI1 immunoreactivity was lost in 161/209 (77%) cases. Strikingly, KAI1 was preferentially lost in estrogen receptor (ER)‐positive breast cancers (p < 0.001). This was validated by real‐time RT‐PCR analyses showing a 7.5‐fold downregulation of KAI1 mRNA in ER‐positive relative to ER‐negative tumors (p = 0.028). Notably, this was also corroborated by Affymetrix microarray expression data of an independent cohort of 49 breast cancers. Class comparison analysis identified KAI1 as downregulated in ER‐positive tumors. Subsequently, human breast cancer cell lines were employed to test a potential role of ER‐activity in the downregulation of KAI1, as suggested by our expression analyses. Exposure of ER‐positive breast cancer cells to fulvestrant, a clinically approved ER‐antagonist that reverses ER‐mediated gene repression, induced a significant upregulation of KAI1 and inhibited cell proliferation as well as migration. In summary, we demonstrate for the first time that KAI1 is a target of ER‐mediated gene‐repression, and thus, it is downregulated in ER‐positive breast cancer. Importantly, KAI1 might be reinducible by endocrine therapy with ER‐antagonists in patients suffering from ER‐positive breast cancer.

Comprehensive genetic and functional characterization of IPH-926: a novel CDH1-null tumour cell line from human lobular breast cancer†

Infiltrating lobular breast cancer (ILBC) is a clinically and biologically distinct tumour entity defined by a characteristic linear cord invasion pattern and inactivation of the CDH1 tumour suppressor gene encoding for E‐cadherin. ILBCs also lack β‐catenin expression and show aberrant cytoplasmic localization of the E‐cadherin binding protein p120‐catenin. The lack of a well‐characterized ILBC cell line has hampered the functional characterization of ILBC cells in vitro. We report the establishment of a permanent ILBC cell line, named IPH‐926, which was derived from a patient with metastatic ILBC. The DNA fingerprint of IPH‐926 verified genetic identity with the patient and had no match among the human cell line collections of several international biological resource banks. IPH‐926 expressed various epithelial cell markers but lacked expression of E‐cadherin due to a previously unreported, homozygous CDH1 241ins4 frameshift mutation. Detection of the same CDH1 241ins4 mutation in archival tumour tissue of the corresponding primary ILBC proved the clonal origin of IPH‐926 from this particular tumour. IPH‐926 also lacked β‐catenin expression and showed aberrant cytoplasmic localization of p120‐catenin. Array‐CGH analysis of IPH‐926 revealed a profile of genomic imbalances that included many distinct alterations previously observed in primary ILBCs. Spectral karyotyping of IPH‐926 showed a hyperdiploid chromosome complement and numerous clonal, structural aberrations. IPH‐926 cells were anti‐cancer drug‐resistant, clonogenic in soft agar, and tumourigenic in SCID mice. In xenograft tumours, IPH‐926 cells recapitulated the linear cord invasion pattern that defines ILBCs. In summary, IPH‐926 significantly extends the biological spectrum of the established breast cancer cell lines and will facilitate functional analyses of genuine human ILBC cells in vitro and in vivo. Copyright

Oncogenic PIK3CA mutations in lobular breast cancer progression

Infiltrating lobular breast cancer (ILBC) is a tumor‐biologically distinct breast cancer subtype. A high frequency of oncogenic PIK3CA mutations has been reported in ILBC, which may allow for targeted therapy with newly developed PI3K inhibitors. This is of particular clinical relevance for ILBC patients, who have failed to respond to current treatment regimes and suffer from tumor recurrence or dissemination. In anticipation of this therapeutic strategy, we investigated PIK3CA mutations in ILBC with special reference to late stage tumor progression. A total of 88 ILBCs from 73 patients, including primary tumors (PTs, n = 43), ipsilateral locally recurrent tumors (LRTs, n = 15), and distant organ metastases (DOMs, n = 30), were compiled on tissue microarrays. Established ILBC marker proteins were evaluated by immunohistochemistry and PIK3CA hot spot mutations in exons 9 and 20 by direct sequencing. Matched PT/LRT, PT/DOM, and DOM/DOM cases were characterized on a patient‐by‐patient basis. Following correction for redundant patient representations, mutation frequencies were compared in PTs versus LRTs or DOMs. Nearly all specimens were E‐cadherin‐negative (99%), estrogen receptor (ER)‐positive (91%), and lacked basal epithelial markers (100%), demonstrating correct ILBC classification. PIK3CA mutations were detected in 32/88 (36%) specimens. The mutation rate was similar in PTs (33%) and DOMs (26%, P = 0.769), but approximately two‐fold increased in LRTs (69%, P = 0.022). Consistently, matched PT/LRT and LRT/DOM cases showed additional PIK3CA mutations in LRTs. Intriguingly, these findings imply that PIK3CA mutations are positively selected for during ILBC progression to local recurrence but not distant metastasis, which may have clinical implications for PI3K inhibitor‐based therapy.

Lobular breast cancer: molecular basis, mouse and cellular models

Infiltrating lobular breast cancer (ILC) is the most common special breast cancer subtype. With mutational or epigenetic inactivation of the cell adhesion molecule E-cadherin (CDH1) being confined almost exclusively to ILC, this tumor entity stands out from all other types of breast cancers. The molecular basis of ILC is linked to loss of E-cadherin, as evidenced by human CDH1 germline mutations and conditional knockout mouse models. A better understanding of ILC beyond the level of descriptive studies depends on physiologically relevant and functional tools. This review provides a detailed overview on ILC models, including well-characterized cell lines, xenograft tumors and genetically engineered mouse models. We consider advantages and limitations of these models and evaluate their representativeness for human ILC. The still incompletely defined mechanisms by which loss of E-cadherin drives malignant transformation are discussed based on recent findings in these models. Moreover, candidate genes and signaling pathways potentially involved in ILC development and progression as well as anticancer drug and endocrine resistance are highlighted.

ABCB1/MDR1 contributes to the anticancer drug-resistant phenotype of IPH-926 human lobular breast cancer cells

Contribution of the ABCB1/MDR1/P-glycoprotein drug transporter to breast cancer resistance has been controversial. One issue is that ABCB1-dependent drug-resistance has primarily been investigated in mammary epithelial cell models technically manipulated to overexpress ABCB1, either by gene transfer using appropriate expression vectors or by chronic anticancer drug-selection. However, an unmodified human breast cancer cell line with an endogenous overexpression of ABCB1 has not been described thus far. Using Affymetrix microarray analyses, we identified an endogenous overexpression of several tumor-biologically relevant transcripts including ABCB1, BCAR4, CCL28, SCGB2A2 and PIP in IPH-926, an anticancer drug-resistant human lobular breast cancer cell line derived from a chemo-refractory mammary carcinoma patient. In a panel of twenty breast cancer cell lines examined, overexpression of ABCB1 mRNA and protein was exclusively detected in IPH-926. This was further validated using chronically in vitro drug-selected KB-V-1 cells as a widely used reference model to accurately define an ABCB1 overexpression. IPH-926 and KB-V-1 displayed a similar overexpression of ABCB1. Flow cytometric analyses showed that IPH-926 but not ABCB1-negative breast cancer cells extruded the anticancer agent doxorubicin, a classical substrate of the ABCB1 drug transporter. PSC-833 (valspodar), a selective ABCB1 inhibitor, blocked this efflux, restored apoptotic PARP cleavage and increased doxorubicin sensitivity in IPH-926 and KB-V-1. To our knowledge, IPH-926 represents the first human breast cancer cell line with a genuine, endogenous overexpression of ABCB1. IPH-926 provides evidence that ABCB1 can occasionally cause anticancer drug-resistance in breast cancer patients and offers a new tool for the evaluation of compounds to overcome drug-resistance.

The region-of-interest size impacts on Ki67 quantification by computer-assisted image analysis in breast cancer ☆

Therapeutic decision-making in breast cancer depends on histopathologic biomarkers and is influenced by the Ki67 proliferation index. Computer-assisted image analysis (CAIA) promises to improve Ki67 quantification. Several commercial applications have been developed for semiautomated CAIA-based Ki67 quantification, many of which rely on measurements in user-defined regions of interest (ROIs). Because of intratumoral proliferative heterogeneity, definition of the ROI is an important step in the analytical procedure. This study explores the ROI size impacts on Ki67 quantification. Whole-slide sections of 100 breast cancers were immunostained with the anti-Ki67 antibody 30-9 and were analyzed on the iScan Coreo digital pathology platform using a Food and Drug Administration-cleared Ki67 quantification software version v5.3 (Virtuoso; Ventana, Tucson, TX). For each case, the Ki67 labeling index (LI) was determined in multiple ROIs of gradually increasing size centered around a high-proliferation area. The spatial Ki67 decline was modeled with nonlinear regression. Depending on the ROI size, the median Ki67 LI varied between 55% and 15%. The proportion of tumors classified as Ki67 low according to the St Gallen 2013/2015 cutoff increased from 2% to 56%, as the ROI size increased from 50 to 10,000 cells captured. The interrater reliability of conventional Ki67 assessment versus CAIA-based Ki67 quantification was also dependent on the ROI size and varied between slight and almost perfect agreement (Cohen κ = 0.06-0.85). In conclusion, the ROI size is a critically important parameter for semiautomated Ki67 quantification by CAIA. Ki67 LIs determined on platforms like iScan Coreo/Virtuoso require an ROI size adjustment, for which we offer a downloadable data transformation tool.