Perfusion Air Culture of Precision-Cut Tumor Slices: An Ex Vivo System to Evaluate Individual Drug Response under Controlled Culture Conditions.

Precision-cut tumor slices (PCTS) maintain tissue heterogeneity concerning different cell types and preserve the tumor microenvironment (TME). Typically, PCTS are cultured statically on a filter support at an air-liquid interface, which gives rise to intra-slice gradients during culture. To overcome this problem, we developed a perfusion air culture (PAC) system that can provide a continuous and controlled oxygen medium, and drug supply. This makes it an adaptable ex vivo system for evaluating drug responses in a tissue-specific microenvironment. PCTS from mouse xenografts (MCF-7, H1437) and primary human ovarian tumors (primary OV) cultured in the PAC system maintained the morphology, proliferation, and TME for more than 7 days, and no intra-slice gradients were observed. Cultured PCTS were analyzed for DNA damage, apoptosis, and transcriptional biomarkers for the cellular stress response. For the primary OV slices, cisplatin treatment induced a diverse increase in the cleavage of caspase-3 and PD-L1 expression, indicating a heterogeneous response to drug treatment between patients. Immune cells were preserved throughout the culturing period, indicating that immune therapy can be analyzed. The novel PAC system is suitable for assessing individual drug responses and can thus be used as a preclinical model to predict in vivo therapy responses.

The INTREST registry: protocol of a multicenter prospective cohort study of predictors of women's response to integrative breast cancer treatment.

BACKGROUND: Cancer registries usually assess data of conventional treatments and/or patient survival. Beyond that, little is known about the influence of other predictors of treatment response related to the use of complementary therapies (CM) and lifestyle factors affecting patients' quality and quantity of life. METHODS: INTREST is a prospective cohort study collecting register data at multiple German certified cancer centers, which provide individualized, integrative, in- and outpatient breast cancer care. Patient-reported outcomes and clinical cancer data of anticipated N = 715 women with pTNM stage I-III breast cancer are collected using standardized case report forms at the time of diagnosis, after completing neo-/adjuvant chemotherapy, after completing adjuvant therapy (with the exception of endocrine therapy) as well as 1, 2, 5, and 10 years after baseline. Endpoints for multivariable prediction models are quality of life, fatigue, treatment adherence, and progression-based outcomes/survival. Predictors include the study center, sociodemographic characteristics, histologic cancer and comorbidity data, performance status, stress perception, depression, anxiety, sleep quality, spirituality, social support, physical activity, diet behavior, type of conventional treatments, use of and belief in CM treatments, and participation in a clinical trial. Safety is recorded following the Common Terminology Criteria for Adverse Events. DISCUSSION: This trial is currently recruiting participants. Future analyses will allow to identify predictors of short- and long-term response to integrative breast cancer treatment in women, which, in turn, may improve cancer care as well as quality and quantity of life with cancer. TRIAL REGISTRATION: German Clinical Trial Register DRKS00014852 . Retrospectively registered at July 4th, 2018.

Gene Expression Signatures of BRCAness and Tumor Inflammation Define Subgroups of Early-Stage Hormone Receptor-Positive Breast Cancer Patients.

PURPOSE: Patients with estrogen receptor- and/or progesterone receptor-positive, early breast cancer benefit from hormonal treatment, yet high global death burdens due to high prevalence and long-term recurrence risk call for biomarkers to guide additional treatment approaches. EXPERIMENTAL DESIGN: From a prospective, observational study of postmenopausal early breast cancer patients treated with tamoxifen or aromatase inhibitors, gene expression analyses of 612 tumors was performed using the NanoString Breast Cancer 360 panel to interrogate 23 breast cancer pathways. Candidate signatures associated with disease subtype and event-free survival (EFS) were obtained by cluster analysis, Cox modeling, and conditional inference trees, and were independently tested in 613 patients from BreastMark. Tumor-infiltrating lymphocytes (TIL) were assessed on tissue sections, and mutational burden was assessed in 36 tumors by whole-exome sequencing. RESULTS: PAM50-derived classification distinguished lower-risk (Luminal A) from higher-risk subtypes (Luminal B, P = 0.04; HER2, P = 0.006; Basal, P = 0.008). In higher-risk patients, shorter EFS was associated with low androgen receptor [HR = 3.61; 95% confidence interval (CI), 1.72-7.56; P = 0.001] or high BRCAness signature expression (HR = 3.58; 95% CI, 1.19-10.7; P = 0.023). BRCAness was independently confirmed as a predictor of shorter EFS (HR = 2.64; 95% CI, 1.31-5.34; P = 0.007). About 13%-15% of patients, enriched for high-grade, higher-risk subtypes (P ≤ 0.0001), had strong expression of the Tumor Inflammation Signature (TIS) suggestive of an inhibited antitumor immune response. TIS scores were strongly associated with TIL numbers (P < 1e-30) but not with tumor mutation status. CONCLUSIONS: BRCA-related DNA repair deficiency and suppressed tumor immune responses may be clinically relevant predictors of endocrine therapy complementing treatment options in subgroups of hormone-sensitive early breast cancer.

Hyperthermia Synergizes with Chemotherapy by Inhibiting PARP1-Dependent DNA Replication Arrest.

Although hyperthermia offers clinical appeal to sensitize cells to chemotherapy, this approach has been limited in terms of long-term outcome as well as economic and technical burden. Thus, a more detailed knowledge about how hyperthermia exerts its effects on chemotherapy may illuminate ways to improve the approach. Here, we asked whether hyperthermia alters the response to chemotherapy-induced DNA damage and whether this mechanism is involved in its sensitizing effect in BRCA-competent models of ovarian and colon cancer. Notably, we found that hyperthermia delayed the repair of DNA damage caused by cisplatin or doxorubicin, acting upstream of different repair pathways to block histone polyADP-ribosylation (PARylation), a known effect of chemotherapy. Furthermore, hyperthermia blocked this histone modification as efficiently as pharmacologic inhibitors of PARP (PARPi), producing comparable delay in DNA repair, induction of double-strand breaks (DSB), and cell cytotoxicity after chemotherapy. Mechanistic investigations indicated that inhibiting PARylation by either hyperthermia or PARPi induced lethal DSB upon chemotherapy treatment not only by reducing DNA repair but also by preventing replication fork slowing. Overall, our work reveals how PARP blockade, either by hyperthermia or small-molecule inhibition, can increase chemotherapy-induced damage in BRCA-competent cells. Cancer Res; 76(10); 2868-75. ©2016 AACR.

Identification of alternative transcripts of rat CD9 expressed by tumorigenic neural cell lines and in normal tissues.

CD9 is the best-studied member of the tetraspanin family of transmembrane proteins. It is involved in various fundamental cellular processes and its altered expression is a characteristic of malignant cells of different origins. Despite numerous investigations confirming its fundamental role, the heterogeneity of CD9 or other tetraspanin proteins was considered only to be caused by posttranslational modification, rather than alternative splicing. Here we describe the first identification of CD9 transcript variants expressed by cell lines derived from fetal rat brain cells. Variant mRNA-B lacks a potential translation initiation codon in the alternative exon 1 and seems to be characteristic of the tumorigenic BT cell lines. In contrast, variant mRNA-C can be translated from a functional initiation codon located in its extended exon 2, and substantial amounts of this form detected in various tissues suggest a contribution to CD9 functions. From the alternative sequence of variant C, a different membrane topology (5 transmembrane domains) and a deviating spectrum of functions can be expected.

Molecular indicators of non-sentinel node status in breast cancer determined in preoperative biopsies by multiplexed sandwich immunoassays.

PURPOSE: Purpose of this study was to determine the accuracy of prediction of non-sentinel lymph node (NSLN) involvement in sentinel node (SLN)-positive breast cancer patients based on protein concentrations measured in lysates from initially taken breast biopsies. METHODS: Data on protein expression, previously generated by multiplexed bead-based immunoassays, were analysed by multivariate logistic regression to define parameter sets of value to predict NSLN involvement. Receiver-operator characteristics (ROCs) were calculated as indicators of diagnostic significance. RESULTS: Analyses of data from all patients (n = 99) resulted in parameter sets that allowed direct prediction of the NSLN status with a ROC area under the curve (AUC) of 0.83. The clinically most relevant prediction of NSLN status in SLN-positive patients (n = 37) based on only seven parameters (including TIMP-2 as the most relevant single value) was possible with high accuracy indicated by an AUC of 0.89. CONCLUSIONS: Parallel assessment of protein concentrations in breast biopsies is a highly promising approach to predict nodal involvement and even the NSLN status in SLN-negative breast cancer patients. Such diagnostic information could substantially reduce the number of completion axillary lymph node dissections in clinical practice.

Multiplexed immunoassays for the analysis of breast cancer biopsies.

Within the last decade, protein microarray technology has been successfully used for the simultaneous quantification of target proteins from minimal amounts of samples in basic and applied proteome research. The robustness and appropriate sensitivity of these miniaturized assays have been demonstrated and thus the transfer to routine and high-throughput applications is now possible. In this study, multiplexed bead-based sandwich immunoassays were used to determine the concentrations of 54 protein analytes, including HER 2 and the estrogen receptor, from ultrasound-guided large-core needle biopsies (LCNBs) from breast cancer patients. Expression levels for HER 2, estrogen receptors and progesterone receptors were also assessed by immunohistochemical routine staining, performed in the clinic on corresponding biopsy samples. The high concordance of the data sets generated with the bead-based protein arrays and by conventional immunohistochemical assessment of HER 2 and the estrogen receptor expressed by breast cancer cells present in the biopsies was demonstrated.

Differential expression of tetraspanin CD9 in basal cell and squamous cell carcinomas of the skin and actinic keratosis.

Tetraspanins are potentially useful molecular markers that differentiate between tumour classes and subtypes, since members of this protein family were often found to be altered during malignant conversion and tumour progression. In this study, we analysed expression of the tetraspanin CD9 in the frequent cutaneous neoplasms basal cell carcinoma (BCC), squamous cell carcinoma (SCC) and actinic keratosis (AK), which is considered a precursor lesion (carcinoma in situ) from which an invasive SCC can develop. A moderate to strong CD9-specific staining of the tumour cells' plasma membranes was uniquely observed in all BCCs, SCCs and AKs. All SCCs showed additional intracellular CD9 which was rarely (20%) seen in AKs. Semi-quantitative assessment of CD9 present in the plasma membranes of tumour cells of BCCs (mean staining intensity 1.91) and SCCs (3.64) reflected the different CD9 expression of normal precursor cells from which these tumours most likely originate. Although considered an intermediate stage in the development of SCCs, AKs did not show intense staining of the plasma membranes typical of normal keratinocytes or invasive SCCs (p=0.011) but only moderate intensity (mean 1.63). In BCCs, significantly (p=0.0005, n=56) stronger CD9-specific immunoreactivity was seen in the inner regions of the tumours than at their sites of expansion. In summary, our results point to an important role of CD9 at the front of tumour expansion in BCCs and SCCs, and in the pathogenesis of invasive SCCs.

Glycosylation of tetraspanin Tspan-1 at four distinct sites promotes its transition through the endoplasmic reticulum.

We showed that Tspan-1, a tetraspanin overexpressed in many human cancers, harbours oligosaccharides at all four potential N-glycosylation sites. Its most abundant form contained only mannose-rich sugar chains but two distinct glycosylation sites could also contain complex carbohydrates. Glycosylation seemed to be required for correct folding and subsequent transition through the endoplasmic reticulum.

Tspan-1 is a tetraspanin preferentially expressed by mucinous and endometrioid subtypes of human ovarian carcinomas.

In many human cancers, tumor progression was found to be associated with an altered expression of tetraspanins, a group of transmembrane adaptor proteins that are implicated in fundamental cellular processes. Although recognized as a characteristic of malignant cells of various origins, Tspan-1 has not yet been characterized in detail due to lack of specific antibodies. We describe the generation of Tspan-1-specific antibodies and immunohistochemical staining of different subtypes of ovarian carcinomas (n=72) that revealed significant differences in Tspan-1 expression that was pronounced in mucinous and endometrioid tumors. The observation that immunoreactivity was focused in intracellular vesicles often concentrated at the luminal sides of glandular structures further supported the assumption that Tspan-1 is involved in secretory pathways. In the group of serous ovarian carcinomas, pronounced expression of Tspan-1 was observed in FIGO stage III C-classified tumors of advanced stages. In summary, our results show that Tspan-1 is an important characteristic of malignant ovarian cancer cells and a potential therapeutic target.

Prediction of nodal involvement in breast cancer based on multiparametric protein analyses from preoperative core needle biopsies of the primary lesion.

PURPOSE: Identification of molecular characteristics that are useful to define subgroups of patients fitting into differential treatment schemes is considered a most promising approach in cancer research. In this first study of such type, we therefore investigated the potential of multiplexed sandwich immunoassays to define protein expression profiles indicative of clinically relevant properties of malignant tumors. EXPERIMENTAL DESIGN: Lysates prepared from large core needle biopsies of 113 invasive breast carcinomas were analyzed with bead-based miniaturized sandwich immunoassays specific for 54 preselected proteins. RESULTS: Five protein concentrations [fibroblast growth factor-2 (FGF-2), Fas, Fas ligand, tissue inhibitor of metalloproteinase-1, and RANTES] were significantly different in the groups of patients with or without axillary lymph node metastasis. All 15 protein parameters that resulted in P values <0.2 and other diagnostic information [estrogen receptor (ER) status, tumor size, and histologic grading] were analyzed together by multivariate logistic regression. This yielded sets of five (FGF-2, Fas, Fas ligand, IP10, and PDGF-AB/BB) or six (ER staining intensity, FGF-2, Fas ligand, matrix metalloproteinase-13, PDGF-AB/BB, and IP10) parameters for which receiver-operator characteristic analyses revealed high sensitivities and specificities [area under curve (AUC) = 0.75 and AUC = 0.83] to predict the nodal status. A similar analysis including all identified parameters of potential value (15 proteins, ER staining intensity, T) without selection resulted in a receiver-operator characteristic curve with an AUC of 0.87. CONCLUSION: We clearly showed that this approach can be used to quantify numerous proteins from breast biopsies accurately in parallel and define sets of proteins whose combined analyses allow the prediction of nodal involvement with high specificity and sensitivity.

Increased radiosensitivity as an indicator of genes conferring breast cancer susceptibility.

PURPOSE: This paper briefly summarizes the research on increased radiosensitivity in breast cancer patients measured by the micronucleus test (MNT) and its association to genetic variants in DNA repair genes. More preliminary data are presented on the distribution of chromosomes and chromosome fragments in micronuclei (MN) in order to gain more information on clastogenic and aneugenic effects and better understand the phenotype of increased radiosensitivity. MATERIAL AND METHODS: Reports of relevant studies obtained from a search of PubMed and studies referenced in those reports were reviewed. In four patients with high MN frequency (three cancer patients, one control) and four probands with low MN frequency, the presence of chromosome fragments or whole chromosomes in MN was determined by fluorescence in situ hybridization analysis for chromosomes 1, 7, and 17. RESULTS: An increased MN frequency in breast cancer patients compared to controls has consistently been reported with high significance. Higher MN frequencies were observed in 20-50% of breast cancer patients. Chromosomal fragments of chromosome 17, but not of chromosomes 1 and 7 were more frequent in the probands with high MN frequency than in those with low frequency (p = 0.045). CONCLUSION: The MNT detects a cellular phenotype common to a portion of sporadic breast cancer patients. This phenotype is very likely to be genetically determined. For the genetic dissection of breast cancer susceptibility this phenotype may turn out to be more efficient than breast cancer itself. Additional parameters which can be measured simultaneously with the MN frequency may be able to further enhance its usefulness.

Stage IB endometrial cancer. Does lymphadenectomy replace adjuvant radiotherapy?

BACKGROUND: The role of surgical lymph node dissection and adjuvant radiation therapy (RT) in early stage endometrial cancer is no longer clearly defined. The increased appreciation of lymphadenectomy and the absence of survival advantage from adjuvant RT rise controversies how patients should adequately be treated in stage IB endometrial cancer. The aim of this review is to rule out the validity of either treatment option and determine which preference provides the best therapeutic benefit. METHODS: Reports of relevant studies obtained from a search of PubMed and studies referenced in those reports were reviewed. RESULTS: Based on the available data in the literature, for stage IB grade 1 or 2, the risk of pelvic relapse is considered too low to justify pelvic RT. However, intravaginal RT (IVRT) should be recommended for those >or= 60 years old or with lymphovascular invasion (LVI). For patients with stage IB grade 3 (and IC all grades), the treatment recommendation is mainly based on whether surgical lymph node staging was performed. These patients have--without surgical lymph node staging--a high risk of pelvic recurrence and should therefore primarily undergo relaparotomy for lymphadenectomy or pelvic RT as second choice. If these patients had a surgical lymph node staging, then IVRT alone is a reasonable alternative to pelvic RT. CONCLUSION: Overall survival may not be the only ideal endpoint for stage IB endometrial cancer since causes of death are mostly other than endometrial cancer. Conventional pelvic RT may be overtreatment in some patients, in particular in those patients with a large number of negative lymph nodes after lymphadenectomy. However, negative surgical staging should not be understood as adjuvant RT can be omitted in all patients.

CD9 promotes adeno-associated virus type 2 infection of mammary carcinoma cells with low cell surface expression of heparan sulphate proteoglycans.

Recombinant adeno-associated virus (AAV) is a promising non-pathogenic vector in the emerging field of gene therapy. For AAV serotype 2 (AAV-2) infection, experimental evidence points to an involvement of heparan sulphate proteoglycans (HSPG), but also to the existence of additional receptors. We investigated a potential role of the tetraspanin CD9 in AAV-2 infection of breast cancer cells mainly because it binds to the heparin-binding EGF-like growth factor, suggesting that it may also interact with a heparin-binding virus. Among breast cancer cell lines, expression of HSPG or potential AAV-2 (co)-receptors was not found to correlate with transduction efficiency. In complete accordance with the role of CD9, blocking with anti-CD9 antibodies resulted in drastically decreased AAV-2 transduction efficiencies in cell lines with low expression of HSPG. Furthermore, specific inhibition of CD9 expression with siRNA resulted in fewer transgene-positive cells, whereas overexpression of CD9 in the breast cancer cell line T47D as well as in BT8Ca and BT12Ca rat glioma cells (with low background expression of HSPG and CD9) increased the number of AAV-transduced cells. The minimal epitope recognized by antibody 72F6, which most efficiently blocked AAV-mediated transgene expression, was deduced from the specific binding to peptides immobilized on colour-encoded microspheres consisting of the amino acid sequence PKKDV located in the large extracellular loop of CD9. Our results clearly point to an involvement of CD9 in the attachment, uptake or processing of AAV-2 by target cells expressing low amounts of HSPG, which may help to define cell populations accessible in AAV-based therapeutic applications.

Chemotherapeutic agents enhance AAV2-mediated gene transfer into breast cancer cells promoting CD40 ligand-based immunotherapy.

PURPOSE: Supplementing conventional treatment with gene therapy to induce an immune response might be beneficial to cancer patients. In this study, we evaluated the efficiency of transduction of breast cancer cells with recombinant adeno-associated virus (rAAV) and effects of cytotoxic agents used in chemotherapy. Furthermore, the capacity of tumor cells expressing transgenic CD40 ligand (CD40L) to stimulate dendritic cells was measured. METHODS: Breast cancer cell lines were infected with a rAAV encoding the enhanced green fluorescent protein (EGFP) or murine CD40L and transgene expression was analyzed by flow cytometry. Stimulation of isolated human dendritic cells by CD40L-expressing tumor cells was quantified by measuring secreted interleukin 12. RESULTS: Infection with an EGFP-encoding rAAV resulted in variable fractions (14-93%, mean 42%) of transgene-expressing cells. Pre-incubation of MM 157, MM 231, and MCF7 cells with epirubicin or carboplatin substantially increased AAV-mediated transgene expression. rAAV/CD40L was used to generate CD40L-transgenic tumor cells, which specifically activated immature dendritic cells, as confirmed by blocking with an antibody binding to CD40L. CONCLUSIONS: The efficiency of rAAV-mediated gene transfer into breast cancer cells is significantly higher than previously reported and can be further enhanced by co-administration of chemotherapeutic agents. We also confirmed that breast cancer cells can activate human dendritic cells after infection with a CD40L-encoding rAAV.

Molecular targets of ovarian carcinomas with acquired resistance to platinum/taxane chemotherapy.

Ovarian cancer of epithelial origin remains one of the most lethal malignancies despite response rates of more than 80% in first-line combination chemotherapy with platinum drugs and taxanes following surgery. Poor overall prognosis is mainly due to acquired resistance of the recurring tumor mass to initially used and other chemotherapeutic agents. Therefore, novel therapeutic approaches are based on concepts to prevent (improvement of tumor exposure to drugs) or circumvent drug resistance, e.g. with new drugs structurally related to the currently used cytotoxic agents, other types of cytotoxic substances, or with target-specific novel drugs interfering with signaling and apoptotic pathways. In addition, acquired molecular characteristics of drug resistant ovarian carcinoma cells can be defined by expression profiling at different stages of therapy and might be used as specific targets for tumor-suppressing drugs and prodrugs containing cytotoxic components. Revelation of mechanistic details of drug resistance also provides the basis for the development of therapies with novel or conventional antitumor drugs in combination with specific inhibitors able to re-establish chemosensitivity. In this review, we summarize novel approaches in the treatment of ovarian cancer progressed to drug resistant stages and focus on the discussion of recently reported experimental and early clinical results with potentially useful strategies to overcome or modulate acquired drug resistance.

Immunotherapy and cancer vaccines in the management of breast cancer.

Besides the traditional therapeutic options, treatment with antibodies specific for the receptor tyrosine kinase HER-2/neu has been established as a standard therapy in the clinical management of advanced breast cancer. Ongoing clinical studies focus on the improvement of application protocols in order to minimize side effects and evaluate the potential therapeutic benefit of anti-HER-2/neu antibodies in combination with conventional chemotherapy. Various similar strategies to target other tumour-associated antigens or proangiogenic factors with inhibitory antibodies are currently investigated in promising preclinical and clinical trials. In addition, research efforts are made to develop procedures to generate tumour-specific cellular immune responses in breast cancer patients. Therapeutic vaccination is, however, still at an early stage of development, despite encouraging results of animal studies. We summarise and discuss vaccination strategies with tumour-specific proteins or peptides, pulsed dendritic cells, and modified tumour cells as well as antibody-based therapeutic concepts to target HER-2/neu, EGF receptor, MUC-1, uPA/uPAR, and VEGF.

Partial breast irradiation after breast-conserving surgery.

BACKGROUND: The standard technique of postoperative radiotherapy after breast-conserving surgery is percutaneous irradiation of the entire breast to a total dose of 45-50 Gy which is usually followed by a tumor bed boost. Since the majority of local recurrences in selected patients occur close to the former tumor bed, the question arises whether a sole tumor bed irradiation might be a therapeutic alternative to total breast irradiation. METHODS: A systematic review of relevant literature concerning partial breast irradiation (PBI) up to November 2004 was undertaken. Studies of any design were included for comparison and discussion. RESULTS: Nine unique brachytherapy studies using the multi-catheter technique, one the balloon technique (MammoSite), and eight particular intraoperative radiotherapy (IORT) trials were located of which only one was a randomized trial. Only minor postoperative complications were reported. Preliminary results are similar in terms of local tumor control, disease-free and overall survival. However, the current evidence base of IORT studies is poor. CONCLUSION: Despite controversies regarding PBI after breast-conserving surgery, results of phase I-II trials suggest that sole tumor bed irradiation might be an appropriate therapeutic alternative for selected breast cancer patients. However, more experience and data from ongoing phase III trials are required to define these new methods to be an appropriate treatment option. Therefore, total breast irradiation still remains the standard irradiation modality even in the treatment of early breast cancer, and PBI should be considered investigational.