Simone Muenst

Increased programmed death-1+ tumor-infiltrating lymphocytes in classical Hodgkin lymphoma substantiate reduced overall survival

Programmed death-1 (PD-1), a protein that is physiologically expressed by germinal center-associated helper T cells, has an inhibitory function on T-cell activity. The distribution of PD-1+ lymphocytes in the microenvironment of Hodgkin lymphoma is not random and can serve as a diagnostic marker. We measured the number of PD-1+ lymphocytes in Hodgkin lymphoma and correlated it with the remaining background lymphocyte populations and known biological and clinical key data on a tissue microarray platform encompassing 280 cases of classical Hodgkin lymphoma and 3 cases of nodular lymphocyte-predominant Hodgkin lymphoma. Prognostic cutoff scores were determined by receiver operating curve analysis. The number of PD-1+ tumor-infiltrating lymphocytes in 189 evaluable cases was median of 27 and mean of 269 cells/mm(2), being higher in lymphocyte-rich classical Hodgkin lymphoma and lower in the mixed cellularity variant. Rimming of tumor cells by PD-1+ cells was observed in all cases of nodular lymphocyte-predominant Hodgkin lymphoma but only in 1% of classical Hodgkin lymphomas, particularly in lymphocyte-rich and -mixed cellularity variants. Thus, the presence of PD-1+ rosettes around neoplastic cells is typical but not exclusive for nodular lymphocyte-predominant Hodgkin lymphoma because it may be encountered in classical Hodgkin lymphoma. The PD-1+ cell amount was lower in classical Hodgkin lymphoma cases with 9p24 gains (PD-1 ligand 2 locus) and in cases with higher numbers of FOXP3+ regulatory T cells. An increased amount of PD-1+ tumor-infiltrating lymphocytes above the prognostic cutoff score (23 cells/mm(2)) was a stage-independent negative prognostic factor of overall survival as opposed to the number of FOXP3+ regulatory T cells. Along with the latter, PD-1+ cells might represent important lymphoma/host microenvironment modulators.

The immune system and cancer evasion strategies: therapeutic concepts

The complicated interplay between cancer and the host immune system has been studied for decades. New insights into the human immune system as well as the mechanisms by which tumours evade immune control have led to the new and innovative therapeutic strategies that are considered amongst the medical breakthroughs of the last few years. Here, we will review the current understanding of cancer immunology in general, including immune surveillance and immunoediting, with a detailed look at immune cells (T cells, B cells, natural killer cells, macrophages and dendritic cells), immune checkpoints and regulators, sialic acid‐binding immunoglobulin‐like lectins (Siglecs) and other mechanisms. We will also present examples of new immune therapies able to reverse immune evasion strategies of tumour cells. Finally, we will focus on therapies that are already used in daily oncological practice such as the blockade of immune checkpoints cytotoxic T‐lymphocyte antigen 4 (CTLA‐4) and programmed death‐1 (PD‐1) in patients with metastatic melanoma or advanced lung cancer, or therapies currently being tested in clinical trials such as adoptive T‐cell transfer.

EpCAM expression varies significantly and is differentially associated with prognosis in the luminal B HER2 + , basal-like, and HER2 intrinsic subtypes of breast cancer

Background:Epithelial cell adhesion molecule (EpCAM) is frequently expressed in breast cancer, and its expression has been associated with poor prognosis. Breast cancer can be subdivided into intrinsic subtypes, differing in prognosis and response to therapy.Methods:To investigate the association between EpCAM expression and prognosis in the intrinsic subtypes of breast cancer, we performed immunohistochemical studies on a tissue microarray encompassing a total of 1365 breast cancers with detailed clinicopathological annotation and outcomes data.Results:We observed EpCAM expression in 660 out of 1365 (48%) cases. EpCAM expression varied significantly in the different intrinsic subtypes. In univariate analyses of all cases, EpCAM expression was associated with a significantly worse overall survival. In the intrinsic subtypes, EpCAM expression was associated with an unfavourable prognosis in the basal-like and luminal B HER2+ subtypes but associated with a favourable prognosis in the HER2 subtype. Consistently, specific ablation of EpCAM resulted in increased cell viability in the breast cancer cell line SKBR3 (ER−, PR−, and HER2+) but decreased viability in the breast cancer cell line MDA-MB-231 (ER−, PR−, and HER2− ).Conclusion:The differential association of EpCAM expression with prognosis in intrinsic subtypes has important implications for the development of EpCAM-targeted therapies in breast cancer.

The PD-1/PD-L1 pathway: biological background and clinical relevance of an emerging treatment target in immunotherapy

Introduction: The co-inhibitory receptor programmed death 1 (PD-1) and its ligands are key regulators in a wide spectrum of immune responses and play a critical role in autoimmunity and self-tolerance as well as in cancer immunology. Emerging evidence suggests that cancer cells might use the PD-1/PD-ligand (PD-L) pathway to escape anti-tumor immunity. Based on this evidence, early phase human clinical trials targeting the PD-1/PD-L pathway are currently underway for multiple human cancers. Areas covered: The role of the PD-1/PD-L pathway in autoimmune disease, viral infections as well as in malignant neoplasms is discussed and an overview of the existing therapeutics as well as the results of clinical trials targeting this pathway in cancer is given. Expert opinion: The PD-1/PD-L pathway represents an important mechanism of immune evasion for malignant neoplasms. Early clinical trials indicate effectiveness of PD-1/PD-L pathway blockade in several solid cancers. However, greater insight into the exact mechanisms by which tumors are able to evade anti-tumor immunity is needed to increase clinical effectiveness, for example by combination blockade of diverse co-inhibitory receptors.

Frequency, phenotype, and genotype of minute gastrointestinal stromal tumors in the stomach: an autopsy study

Gastrointestinal stromal tumors are the most common mesenchymal tumors of the human digestive tract. Up to 85% of these tumors show somatic gain-of-function mutation of the receptor tyrosine kinase c-KIT gene. A recent study has shown a high frequency (22.5%) of minute gastrointestinal stromal tumors in stomachs examined during routine autopsies. The aims of our study were to confirm the previously reported incidence of gastric gastrointestinal stromal tumors in routine autopsies and to investigate their molecular alterations. Gastrointestinal stromal tumors were collected prospectively from 578 autopsies over an 18-month period. After recording the size and location of each lesion, representative tissue samples were processed for hematoxylin and eosin staining and immunohistochemically stained for CD117 and CD34. Microdissected DNA from all identified gastrointestinal stromal tumors was studied for c-KIT and platelet-derived growth factor receptor α mutations. We identified 17 gastrointestinal stromal tumors in 578 consecutive autopsies (2.9%) located in the gastric body (47%) and fundus (47%). One tumor location was not recorded. All tumors were immunohistochemically positive for CD117 and CD34. DNA analysis showed c-KIT mutations in 11 cases. One platelet-derived growth factor receptor α mutation was found. The incidence of gastric minute gastrointestinal stromal tumors (2.9%) is higher than the reported clinical incidence. All are benign tumors, and most, including minute tumors, contain c-KIT mutations. This finding highlights the fact that c-KIT mutations are an early event in the evolution of gastrointestinal stromal tumors but are not sufficient per se for clinically relevant disease.

Role of the Tumor Microenvironment in Breast Cancer

In recent years, it has been shown that breast cancer consists not only of neoplastic cells, but also of significant alterations in the surrounding stroma or tumor microenvironment. These alterations are now recognized as a critical element for breast cancer development and progression, as well as potential therapeutic targets. Various components of the breast cancer microenvironment, such as suppressive immune cells, soluble factors and altered extracellular matrix, act together to impede effective antitumor immunity and promote breast cancer progression and metastasis. Stromal cells in the breast cancer microenvironment are characterized by molecular alterations and aberrant signaling pathways, some of which are prognostic of clinical outcome. Several new therapies targeting stromal components are in development or undergoing clinical trials. We focus herein on the composition of the breast cancer microenvironment and concomitant molecular alterations, the specific interplay between various cell types and cancer cells, and the clinical implications of these findings.

Distribution of PD-1+ lymphocytes in reactive lymphadenopathies.

Objectives: Programmed death-1 (PD-1) is physiologically expressed by germinal center (GC)-associated helper T cells. It has been proposed that an increase in PD-1+ cells outside GC could indicate pattern I angioimmunoblastic T-cell lymphoma (AITL). Methods: We studied the distribution of PD-1+ cells in reactive lymphadenopathies (LA), including HIV-associated and dermatopathic LA (n = 5, each), Castleman (n = 3), Kikuchi (n = 2) and Rosai-Dorfman diseases (n = 1), sarcoidoses (n = 7) and follicular hyperplasias (n = 8). Results: The highest concentrations of PD-1+ cells in GC were found in Castleman and Rosai-Dorfman diseases (mean 57 and 50% of total cells, respectively), and the lowest in HIV-associated LA (mean 6%). The highest proportions in paracortices were found in Castleman disease, and in HIV-associated and dermatopathic LA (mean 7, 3 and 2%, respectively). PD-1+ cells predominated in the pale zones of GC in follicular hyperplasia. In HIV-associated LA, PD-1+ cells showed marked marginalization in the GC. No paracortical PD-1+ cells were found in sarcoidoses and Rosai-Dorfman disease. Conclusions: The varying distribution of PD-1+ cells in defined LA might indicate a functional relevance of these cells in the respective entities. Since increased numbers of PD-1+ cells outside GC are observed in various LA, this finding might not be entirely specific for pattern I AITL.

Ex-vivo assessment of drug response on breast cancer primary tissue with preserved microenvironments

ABSTRACT Interaction between cancerous, non-transformed cells, and non-cellular components within the tumor microenvironment plays a key role in response to treatment. However, short-term culture or xenotransplantation of cancer specimens in immunodeficient animals results in dramatic modifications of the tumor microenvironment, thus preventing reliable assessment of compounds or biologicals of potential therapeutic relevance. We used a perfusion-based bioreactor developed for tissue engineering purposes to successfully maintain the tumor microenvironment of freshly excised breast cancer tissue obtained from 27 breast cancer patients and used this platform to test the therapeutic effect of antiestrogens as well as checkpoint-inhibitors on the cancer cells. Viability and functions of tumor and immune cells could be maintained for over 2 weeks in perfused bioreactors. Next generation sequencing authenticated cultured tissue specimens as closely matching the original clinical samples. Anti-estrogen treatment of cultured estrogen receptor positive breast cancer tissue as well as administration of pertuzumab to a Her2 positive breast cancer both had an anti-proliferative effect. Treatment with anti-programmed-death-Ligand (PD-L)-1 and anti-cytotoxic T lymphocyte-associated protein (CTLA)-4 antibodies lead to immune activation, evidenced by increased lymphocyte proliferation, increased expression of IFNγ, and decreased expression of IL10, accompanied by a massive cancer cell death in ex vivo triple negative breast cancer specimens. In the era of personalized medicine, the ex vivo culture of breast cancer tissue represents a promising approach for the pre-clinical evaluation of conventional and immune-mediated treatments and provides a platform for testing of innovative treatments.

Author’s response to “Letter to the editor: unvalidated antibodies and misleading results”

Rimm et al. challenge our study demonstrating an association between PD-L1 expression and poor prognosis in human breast cancer [1] raising a concern about the antibody we used for immunohistochemistry. Rimm et al. refer to ab58810 (Abcam) as an unvalidated antibody. We share Rimm et al.’s concern about the use of unvalidated antibodies in immunohistochemistry studies, and we performed validation studies of the ab58810 antibody prior to using it in our study. Specifically, we screened several anti-PD-L1 antibodies for tissue specificity using human term placenta and lymphoid tissue, as mentioned in our paper [1]. Human term placenta is commonly used for the validation of anti-PD-L1 antibodies and was used by Rimm et al. for validation of anti-PD-L1 antibodies in their manuscript on PD-L1 expression in nonsmall cell lung cancer (NSCLC) [2]. We tested several antigen retrieval and staining protocols until we were satisfied with the specificity of tissue staining. With an optimized protocol using ab58810, we observed strong staining of the trophoblast layer in human term placenta but the absence of staining in the stromal cells and vessels of the chorionic villi, as can be appreciated in Figs. 1–3. In tonsils, strong granular cytoplasmic staining of PD-L1 was observed in follicular dendritic cells (Fig. 4). It is important to recognize the fact that Rimm et al. report a lack of tissue specificity with ab58810 in their study [2] does not preclude the possibility that a positive result can be obtained with a different staining protocol. The ab58810 antibody also recognizes the PD-L1 antigen in Western blot analysis, as demonstrated on the Abcam website (http://www.abcam.com/cd274-antibody-ab58810.html). Rimm et al. also comment on nuclear staining observed in some breast cancer cells in Fig. 1 of our manuscript [1]. Of note, the majority of breast cancer cells in Fig. 1 clearly show cytoplasmic staining. We have attached additional images from our breast cancer tissue micro array (TMA), confirming that the staining was predominantly cytoplasmic (Figs. 5–7). We acknowledge that staining is observed in some apoptotic nuclei, but this nuclear staining was not considered for the evaluation of PD-L1 expression. As stated in the Material & Methods section of our paper [1], only cytoplasmic and membranous staining was considered specific. Of note, we have previously evaluated our TMA for the presence of PD-1 tumor-infiltrating lymphocytes (TIL) [3]. We observed a statistically significant association between PD-L1 expression by tumor cells and the presence of PD-1 TIL in the same tumors (p \ 0.001). This observation provides additional evidence to support the integrity of immunohistochemical staining in our manuscript. In preparation of this response, we stained our breast cancer TMA with an additional, newly developed anti-PDL1 antibody (clone E1L3 N, Cell Signaling), which has been validated by Rimm et al. according to their recent ASCO This rebuttal letter refers to the article availbale at doi:10.1007/ s10549-014-3061-0.

Abstract 615: Scaffold-based perfusion bioreactor system for in vitro maintenance of primary breast cancer tissue microenvironment suitable for personalized medicine

INTRODUCTION Two-dimensional (2D) in vitro culture systems and in vivo animal models are the primary tools used to test cancer cell responses to drugs but they are not suited for the development of immune-mediate therapies. Here we present an innovative method for in vitro culture primary breast cancer (BrCa) tissues in porous 3D scaffolds by using a perfusion-based bioreactor system (U-CUP). MATERIALS & METHODS Freshly excised breast cancer specimens were fragmented and cultured in a 3D “sandwich-like format” between collagen porous scaffolds under perfusion flow. DMEM/F12, supplemented with 10% autologous human serum, was used as a culture medium. Malignant and non-malignant cells survival, expansion into the scaffold and the ability to recapitulate features of the original BrCa specimens were histologically assessed. For estrogen receptor (ER) positive tissues we tested the response to hormonal therapy by adding the anti-ER drug Fulvestrant. Furthermore, the maintenance of immune-infiltrating cells allowed testing immune blockade therapy in vitro using anti PD-L1 on PD-L1 positive samples. Response to treatment was evaluated by histology and qRT-PCR for markers of immune-response. RESULTS By culturing BrCa using the U-CUP we were able to preserve viability and to promote the expansion of breast cancer cells from surgical specimens together with accompanying stromal and immune cells into the porous scaffold. Expanding cancer cells were viable after 21 days and recapitulating the initial histology with formation of glands. Administration of anti-ER treatment was associated with decreased expansion of cancer tissue into the scaffold after 21 days. After 7 days of anti PD-L1 antibody treatment we observed a reduced number of tumor cells due to the activation of infiltrating lymphocytes, as shown by increased expression of IFNg and decreased expression of IL10. CONCLUSIONS The scaffold-based perfusion bioreactor represents a successful organotypic tumor model allowing in vitro long-term culture of breast cancer specimens. Our findings shed the light on a promising system for selecting personalized treatment based on a patient9s tumor specific microenvironment. Citation Format: Manuele Giuseppe Muraro, Simone Muenst, Valentina Mele, Luca Quagliata, Alexandar Tzankov, Walter P. Weber, Giulio C. Spagnoli, Savas D. Soysal. Scaffold-based perfusion bioreactor system for in vitro maintenance of primary breast cancer tissue microenvironment suitable for personalized medicine. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 615.