Tumor mutational burden assessed by targeted NGS predicts clinical benefit from immune checkpoint inhibitors in non-small cell lung cancer.

In non-small cell lung cancer (NSCLC), immune checkpoint inhibitors (ICIs) significantly improve overall survival (OS). Tumor mutational burden (TMB) has emerged as a predictive biomarker for patients treated with ICIs. Here, we evaluated the predictive power of TMB measured by the Oncomine™ Tumor Mutational Load targeted sequencing assay in 76 NSCLC patients treated with ICIs. TMB was assessed retrospectively in 76 NSCLC patients receiving ICI therapy. Clinical data (RECIST 1.1) were collected and patients were classified as having either durable clinical benefit (DCB) or no durable benefit (NDB). Additionally, genetic alterations and PD-L1 expression were assessed and compared with TMB and response rate. TMB was significantly higher in patients with DCB than in patients with NDB (median TMB = 8.5 versus 6.0 mutations/Mb, Mann-Whitney p = 0.0244). 64% of patients with high TMB (cut-off = third tertile, TMB ≥ 9) were responders (DCB) compared to 33% and 29% of patients with intermediate and low TMB, respectively (cut-off = second and first tertile, TMB = 5-9 and TMB ≤ 4, respectively). TMB-high patients showed significantly longer progression-free survival (PFS) and OS (log-rank test p = 0.0014 for PFS and 0.0197 for OS). While identifying different subgroups of patients, combining PD-L1 expression and TMB increased the predictive power (from AUC 0.63 to AUC 0.65). Our results show that the TML panel is an effective tool to stratify patients for ICI treatment. A combination of biomarkers might maximize the predictive precision for patient stratification. Our study supports TMB evaluation through targeted NGS in NSCLC patient samples as a tool to predict response to ICI therapy. We offer recommendations for a reliable and cost-effective assessment of TMB in a routine diagnostic setting. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Endothelial cell-derived nidogen-1 inhibits migration of SK-BR-3 breast cancer cells.

BACKGROUND: The tumour microenvironment is a critical regulator of malignant cancer progression. While endothelial cells have been widely studied in the context of tumour angiogenesis, their role as modulators of cancer cell invasion and migration is poorly understood. METHODS: We have investigated the influence of endothelial cells on the invasive and migratory behaviour of human cancer cells in vitro. RESULTS: Upon exposure to culture supernatants of endothelial cells, distinct cancer cells, such as SK-BR-3 cells, showed significantly increased invasion and cell migration concomitant with changes in cell morphology and gene expression reminiscent of an epithelial-mesenchymal transition (EMT). Interestingly, the pro-migratory effect on SK-BR-3 cells was significantly enhanced by supernatants obtained from subconfluent, proliferative endothelial cells rather than from confluent, quiescent endothelial cells. Systematically comparing the supernatants of subconfluent and confluent endothelial cells by quantitative MS proteomics revealed eight candidate proteins that were secreted at significantly higher levels by confluent endothelial cells representing potential inhibitors of cancer cell migration. Among these proteins, nidogen-1 was exclusively expressed in confluent endothelial cells and was found to be necessary and sufficient for the inhibition of SK-BR-3 cell migration. Indeed, SK-BR-3 cells exposed to nidogen-1-depleted endothelial supernatants showed increased promigratory STAT3 phosphorylation along with increased cell migration. This reflects the situation of enhanced SK-BR-3 migration upon stimulation with conditioned medium from subconfluent endothelial cells with inherent absence of nidogen-1 expression. CONCLUSION: The identification of nidogen-1 as an endothelial-derived inhibitor of migration of distinct cancer cell types reveals a novel mechanism of endothelial control over cancer progression.

Results of concurrent radio-chemotherapy for the treatment of head and neck squamous cell carcinoma in everyday clinical practice with special reference to early mortality.

BACKGROUND: Randomized controlled trials have established concurrent chemo-radiotherapy as the preferred treatment option for inoperable local-regionally advanced head and neck squamous cell carcinomas (HNSCCs). Because many patients have multiple co-morbidities and would not fulfill the eligibility criteria of clinical trials, the results need to be re-evaluated in daily clinical practice with special reference to early mortality. METHODS: 167 consecutive patients with HNSCC who received concurrent chemo-radiotherapy at the Basel University Hospital between 1988 and 2006 were analyzed retrospectively with a special focus on early deaths and risk factors for an unfavorable outcome. RESULTS: In our cohort, the 3- and 5-year overall survival rates were 54% and 47%, respectively. The therapy was associated with relevant toxicity and an early mortality rate of 5.4%. Patients dying early were analyzed individually for the cause of death. Patients with elevated white blood cell counts (HR: 2.66 p=0,016) and vascular co-morbidities (HR: 5.3, p=0,047) showed significantly worse survival rates. The same factors were associated with a trend toward increased treatment-related mortality. The 3-year survival rate improved from approximately 43% for patients treated before the year 2000 to 65% for patients treated after the year 2000 (Fisher's exact test p=0.01). CONCLUSIONS: Although many patients who received concurrent chemo-radiotherapy would not have qualified for clinical trials, the outcome was favorable and has significantly improved in recent years. However the early mortality was slightly worse than what is described in the literature.

Analysis of the EGFR mutation status in head and neck squamous cell carcinoma before treatment with Gefitinib.

BACKGROUND: The efficacy of chemotherapy in metastatic and recurrent squamous cell carcinomas of the head and neck (HNSCC) remains unsatisfactory. Gefitinib offers a new therapeutic option with comparable results and better tolerability than chemotherapy. We conducted this study to see if mutations in the epidermal growth factor receptor (EGFR) might predict the therapeutic benefit in HNSCC patients. PATIENTS AND METHODS: In a pilot trial, 8 patients with metastatic or recurrent HNSCC were treated palliatively with gefitinib (500 mg/day orally). Forceps biopsies were taken to confirm tumor recurrence and to perform an EGFR mutation analysis. RESULTS: The EGFR status could be determined in 6 of the 8 patients. 5 patients had no EGFR gene mutation, and 1 patient showed a silent guanine-to-adenosine mutation in position 2607. Even without any relevant mutation in the EGFR, we observed partial remission in 3 of 6 patients treated with gefitinib. We also observed that an additional 4 patients had stable disease for at least 10 weeks. The median progression-free survival was 6.25 months, and the median overall survival was 7.39 months. CONCLUSION: In HNSCC, there are tumor responses to gefitinib without protein-altering mutations in the EGFR gene.

Androgen Deprivation Therapy for Intracranial Metastasis of a Salivary Duct Carcinoma: Case Report.

Salivary duct carcinoma (SDC) is a rare subtype of salivary cancers associated with androgen receptor and human epidermal growth factor receptor 2 (HER2/neu) overexpression. It shows a high propensity to give rise to distant metastases mainly to the lung, the bone, and the liver. Intracranial metastases are rare. We report the case of a 61-year-old male patient with SDC developing intracranial metastases. Unresponsive to radiotherapy and anti-HER/neu targeted therapy the intracranial metastases showed a very good partial remission to androgen deprivation therapy with goserelin acetate. This case demonstrates the potential of a highly targeted therapy with a relatively cheap and well-known drug in a patient with a rare disease without other good therapeutic options, which is a good example of modern, personalized medicine.

Outcome of Hospitalized Cancer Patients with Hypernatremia: A Retrospective Case-Control Study.

Hypernatremia (>145 mmol/L) is a relatively rare event, and the data regarding its role in the outcome of inpatients on an oncology ward are weak. The aim of this study was to describe the prevalence, prognosis, and outcome of hospitalized cancer patients with hypernatremia. We performed a retrospective case-control study of data obtained from inpatients with a solid tumor at the St. Claraspital, Basel, Switzerland, who were admitted between 2017 and 2020. The primary endpoint was overall survival. Hypernatremia was found in 93 (3.16%) of 2945 inpatients bearing cancer or lymphoma. From 991 eligible normonatremic control patients, 93 were matched according to diagnosis, age, and sex. The median overall survival time (OS) of patients with hypernatremia was 1.5 months compared to 11.7 months of the normonatremic controls (HR 2.69, 95% CI 1.85-3.90, p < 0.0001). OS of patients with irreversible compared to reversible hypernatremia was significantly shorter (23 versus 88 days, HR 4.0, 95% CI 2.04-7.70, p < 0.0001). The length of hospital stay was significantly longer for the hypernatremic than for the normonatremic group (p < 0.0001). Significantly more patients with hypernatremia died in the hospital (30.1% versus 8.6%, p < 0.001). These results suggest hypernatremia to be associated with an unfavorable outcome and a very short OS.

Gene profiling of clinical routine biopsies and prediction of survival in non-small cell lung cancer.

RATIONALE: Global gene expression analysis provides a comprehensive molecular characterization of non-small cell lung cancer (NSCLC). OBJECTIVES: To evaluate the feasibility of integrating expression profiling into routine clinical work-up by including both surgical and minute bronchoscopic biopsies and to develop a robust prognostic gene expression signature. METHODS: Tissue samples from 41 chemotherapy-naive patients with NSCLC and 15 control patients with inflammatory lung diseases were obtained during routine clinical work-up and gene expression profiles were gained using an oligonucleotide array platform (NovaChip; 34'207 transcripts). Gene expression signatures were analyzed for correlation with histological and clinical parameters and validated on independent published data sets and immunohistochemistry. MEASUREMENTS AND MAIN RESULTS: Diagnostic signatures for adenocarcinoma and squamous cell carcinoma reached a sensitivity of 80%/80% and a specificity of 83%/94%, respectively, dependent on the proportion of tumor cells. Sixty-seven of the 100 most discriminating genes were validated with independent observations from the literature. A 13-gene metagene refined on four external data sets was built and validated on an independent data set. The metagene was a strong predictor of survival in our data set (hazard ratio = 7.7, 95% CI [2.8-21.2]) and in the independent data set (hazard ratio = 1.6, 95% CI [1.2-2.2]) and in both cases independent of the International Union against Cancer staging. Vascular endothelial growth factor-beta, one of the key prognostic genes, was further validated by immunohistochemistry on 508 independent tumor samples. CONCLUSIONS: Integration of functional genomics from small bronchoscopic biopsies allows molecular tumor classification and prediction of survival in NSCLC and might become a powerful adjunct for the daily clinical practice.

SAKK 16/14: Durvalumab in Addition to Neoadjuvant Chemotherapy in Patients With Stage IIIA(N2) Non-Small-Cell Lung Cancer-A Multicenter Single-Arm Phase II Trial.

PURPOSE: For patients with resectable stage IIIA(N2) non-small-cell lung cancer, neoadjuvant chemotherapy with cisplatin and docetaxel followed by surgery resulted in a 1-year event-free survival (EFS) rate of 48% in the SAKK 16/00 trial and is an accepted standard of care. We investigated the additional benefit of perioperative treatment with durvalumab. METHODS: Neoadjuvant treatment consisted of three cycles of cisplatin 100 mg/m2 and docetaxel 85 mg/m2 once every 3 weeks followed by two doses of durvalumab 750 mg once every 2 weeks. Durvalumab was continued for 1 year after surgery. The primary end point was 1-year EFS. The hypothesis for statistical considerations was an improvement of 1-year EFS from 48% to 65%. RESULTS: Sixty-eight patients were enrolled, 67 were included in the full analysis set. Radiographic response rate was 43% (95% CI, 31 to 56) after neoadjuvant chemotherapy and 58% (95% CI, 45 to 71) after sequential neoadjuvant immunotherapy. Fifty-five patients were resected, of which 34 (62%) achieved a major pathologic response (MPR; ≤ 10% viable tumor cells) and 10 (18%) among them a complete pathologic response. Postoperative nodal downstaging (ypN0-1) was observed in 37 patients (67%). Fifty-one (93%) resected patients had an R0 resection. There was no significant effect of pretreatment PD-L1 expression on MPR or nodal downstaging. The 1-year EFS rate was 73% (two-sided 90% CI, 63 to 82). Median EFS and overall survival were not reached after 28.6 months of median follow-up. Fifty-nine (88%) patients had an adverse event grade ≥ 3 including two fatal adverse events that were judged not to be treatment-related. CONCLUSION: The addition of perioperative durvalumab to neoadjuvant chemotherapy in patients with stage IIIA(N2) non-small-cell lung cancer is safe and exceeds historical data of chemotherapy alone with a high MPR and an encouraging 1-year EFS rate of 73%.

IGF-I induced genes in stromal fibroblasts predict the clinical outcome of breast and lung cancer patients.

BACKGROUND: Insulin-like growth factor-1 (IGF-I) signalling is important for cancer initiation and progression. Given the emerging evidence for the role of the stroma in these processes, we aimed to characterize the effects of IGF-I on cancer cells and stromal cells separately. METHODS: We used an ex vivo culture model and measured gene expression changes after IGF-I stimulation with cDNA microarrays. In vitro data were correlated with in vivo findings by comparing the results with published expression datasets on human cancer biopsies. RESULTS: Upon stimulation with IGF-I, breast cancer cells and stromal fibroblasts show some common and other distinct response patterns. Among the up-regulated genes in the stromal fibroblasts we observed a significant enrichment in proliferation associated genes. The expression of the IGF-I induced genes was coherent and it provided a basis for the segregation of the patients into two groups. Patients with tumours with highly expressed IGF-I induced genes had a significantly lower survival rate than patients whose tumours showed lower levels of IGF-I induced gene expression (P = 0.029 - Norway/Stanford and P = 7.96e-09 - NKI dataset). Furthermore, based on an IGF-I induced gene expression signature derived from primary lung fibroblasts, a separation of prognostically different lung cancers was possible (P = 0.007 - Bhattacharjee and P = 0.008 - Garber dataset). CONCLUSION: Expression patterns of genes induced by IGF-I in primary breast and lung fibroblasts accurately predict outcomes in breast and lung cancer patients. Furthermore, these IGF-I induced gene signatures derived from stromal fibroblasts might be promising predictors for the response to IGF-I targeted therapies. See the related commentary by Werner and Bruchim: http://www.biomedcentral.com/1741-7015/8/2.

Much has changed in the last decade except overall survival: A Swiss single center analysis of treatment and survival in patients with stage IV non-small cell lung cancer.

BACKGROUND: Molecular therapies for cancers with targetable driver mutations and immunotherapies have revolutionized treatment of non-small cell lung cancer (NSCLC) during the last decade. International treatment guidelines began integrating targeted therapies in 2009 and immunotherapies in 2015. The aim of this study was to examine whether the benefits described for these new therapies in pivotal phase III trials have been translated to a real world patient population. PATIENTS AND METHODS: Data from all consecutive patients diagnosed with stage IV NSCLC diagnosed at a community hospital in Switzerland between 2007 and 2018 were analyzed. Three groups of patients were compared, namely patients diagnosed before 2009 (group 1), between 2009 and 2015 (introduction of targeted therapies, group 2) and after 2015 (introduction of immunotherapies, group 3). The primary outcome was overall survival (OS). Time to treatment failure was a secondary outcome. Both endpoints were estimated using the Kaplan Meyer method and compared by log-rank test. RESULTS: 408 patients were included. Patient characteristics were similar in the three groups. Median OS in groups 1, 2, and 3 was 9.8 (95% CI, 6.2 to 13.4), 9.9 (95% CI, 7.6 to 12.1) and 8.6 (95% CI, 6.6 to 10.5) months, respectively (p = 0.5). Across groups patients treated with targeted- and immunotherapies had a significantly better outcome than those treated with chemotherapy or best supportive care (p<0.001). Nevertheless, OS remained unchanged between groups despite adequate molecular testing and integration of targeted- and immunotherapies. Over time, the patient population got more morbid with respect to tumor burden (p = 0.02) and co-morbidities (p = 0.02). CONCLUSIONS: While selected subgroups of patients may benefit from new therapies, outcome in this unselected population of patients with stage IV NSCLC treated in daily practice has not improved over the last decade.

BMP2 response pattern in human lung fibroblasts predicts outcome in lung adenocarcinomas.

BACKGROUND: Bone morphogenetic proteins play important roles in development, morphogenesis and cancer. With this study we aimed to characterize the response of lung stromal fibroblasts to BMPs and their antagonists on a genome wide level and investigate its potential role in human lung adenocarcinomas. METHODS: We used an ex vivo culture model and measured gene expression changes in human lung fibroblasts after stimulation with BMPs and their antagonists using HEEBO microarrays. The in vitro data were correlated with in vivo observations in published expression datasets of human lung adenocarcinomas. RESULTS: We have systematically analyzed the response to BMP2, BMP4, BMP7 and their antagonists, Gremlin and Noggin, to define common and specific gene expression patterns. A BMP2 induced gene expression signature was defined, which is specific for stromal fibroblasts. Gene expression profiles from lung adenocarcinoma biopsies were analyzed to determine the prognostic significance of the "Fibroblast specific BMP2 induced gene list". This gene list successfully segregated patients with different prognostic outcome in 3 datasets. In a small dataset (Garber et al.) there was a strong trend for a worse prognosis of patients with adenocarcinomas of all stages over-expressing the "Fibroblast specific BMP2 induced gene list". In two larger datasets with stage I adenocarcinomas we observed a significantly worse disease-free (p = 0.002, Lee et al. and p = 0.002, Bhattacharjee et al.) and overall survival (p = 0.0002). CONCLUSIONS: The effects of BMPs and their antagonists are heterogeneous in different cell types. The gene expression pattern induced by BMP2 in primary lung fibroblasts may predict outcomes of patients with lung adenocarcinomas.

Current Treatment of Isolated Locoregional Breast Cancer Recurrences.

Patients with isolated locoregional breast cancer recurrences should be treated with curative intent. Mastectomy is regarded as the standard of care for patients with ipsilateral breast tumor recurrence. In a selected group of patients, partial breast irradiation after second breast-conserving surgery is a viable alternative to mastectomy. If a second breast conservation is performed, additional irradiation should be mandatory, especially in patients who had not been irradiated previously. In case of re-irradiation, the largest experience exists for multi-catheter brachytherapy. Prospective clinical trials are needed to clearly define selection criteria, long-term local control, and toxicity. In patients with resectable locoregional breast cancer recurrences after mastectomy, multi-modal therapy comprising complete resection, radiation therapy in previously unirradiated patients, and systemic therapy results in 5-year disease-free and overall survival rates of 69% and 88%, respectively. In radiation-naive patients with unresectable, isolated locoregional recurrences, radiation therapy is mandatory. In selected patients with previous irradiations and unresectable locoregional recurrences, a second irradiation as part of an individual treatment concept can be applied. The increased risk of severe toxicity should always be weighed up against the potential clinical benefit. A combination therapy with hyperthermia can further improve the treatment results.

Amplification of SKI is a prognostic marker in early colorectal cancer.

BACKGROUND: Improved risk stratification of early colorectal cancer might help to better select patients for adjuvant treatment. Alterations in the transforming growth factor-beta (TGF-beta) pathway have frequently been found in colorectal cancer, but their impact on prognosis remains controversial. We therefore analyzed two transcriptional corepressors of the TGF-beta signaling pathway with respect to prognosis and prediction of chemotherapy benefit in early colorectal cancer. METHODS: The gene copy status of SKI and SNON was analyzed by use of quantitative real-time polymerase chain reaction in 179 colorectal tumor biopsies, which had been collected from a randomized multicenter trial of the Swiss Group for Clinical Cancer Research (SAKK). RESULTS: Partial or complete allelic loss was found in 41.5% and 55.2% for SKI and SNON, whereas amplification was found in 10.1% and 15.1%, respectively. Multivariate Cox analysis showed that gene amplification of SKI independently predicted reduced relapse-free [hazard ratio (HR) for relapse 2.08, P =.049] and overall survival (HR for death 2.62, P =.012). In contrast, deletion of SKI and the gene copy status of SNON were not significantly correlated with prognosis. CONCLUSION: Amplification of SKI is a negative prognostic marker in early-stage colorectal cancer. This marker should help to improve risk stratification to better select patients for adjuvant therapy. Confirmatory investigations are warranted.

STRAP is a strong predictive marker of adjuvant chemotherapy benefit in colorectal cancer.

BACKGROUND: Molecular predictors for the effectiveness of adjuvant chemotherapy in colorectal cancer are of considerable clinical interest. To this aim, we analyzed the serine threonine receptor-associated protein (STRAP), an inhibitor of TGF-beta signaling, with regard to prognosis and prediction of adjuvant 5-FU chemotherapy benefit. METHODS: The gene copy status of STRAP was determined using quantitative real-time polymerase chain reaction in 166 colorectal tumor biopsies, which had been collected from a randomized multicenter trial of 5-fluorouracil (5-FU)/mitomycin C (MMC) adjuvant chemotherapy of the Swiss Group for Clinical Cancer Research (SAKK). RESULTS: Amplification of STRAP was found in 22.8% of the tumors. When left without adjuvant chemotherapy, patients bearing tumors with a STRAP amplification had a significantly better prognosis (hazard ratio for death: 0.26; P=.004). Interestingly, these patients, when receiving adjuvant treatment, had a worse survival (hazard ratio for death: 3.48; P=.019) than without chemotherapy, whereas patients carrying tumors with diploidy or deletion of STRAP benefited from the treatment (hazard ratio for death: 0.44; P=.052). This suggests the amplification of STRAP as a strong predictor of an unfavorable effect of 5-FU-based adjuvant chemotherapy. CONCLUSION: If confirmed, the STRAP gene copy status might provide a parameter to decide about the use of 5-FU-based adjuvant chemotherapy.

Obstructive jaundice caused by enteropathy-associated T-cell lymphoma in a patient with celiac sprue.

Patients with celiac sprue carry a considerable risk of gastrointestinal malignancies; in particular, non-Hodgkin's lymphoma. These malignancies represent the most serious complications of celiac disease. Commonly, patients present with deteriorating symptoms of the underlying disease, which makes an early diagnosis difficult. We report a patient with a 13-year history of celiac sprue presenting with painless jaundice and a Courvoisier gallbladder. Abdominal computed tomography (CT) scan showed thickening of the duodenal wall, suggesting a neoplastic infiltration of the papilla of Vater, causing biliary obstruction. Biopsies taken on endoscopy revealed enteropathy-associated T-cell lymphoma of the duodenum. Biliary obstruction is a rare clinical finding in enteropathy-associated T-cell lymphoma. To our knowledge, this is the first reported case of this unusual manifestation in celiac disease.

Global gene expression analysis of the interaction between cancer cells and osteoblasts to predict bone metastasis in breast cancer.

BACKGROUND: Bone metastasis is a main cause of morbidity in breast cancer. Since breast cancer is a heterogeneous disease, the interactions of cancer cells with the skeletal host cells might also be diverse. We hypothesized that gene expression signatures induced by heterotypic interaction of breast cancer cells and osteoblasts might be of clinical relevance. METHODOLOGY/PRINCIPAL FINDINGS: We established an ex vivo co-culture model using benign breast epithelial cells or a panel of 5 malignant breast epithelial cells in combination with primary human osteoblasts and determined associated gene expression changes with HEEBO microarrays. Pretreatment gene expression profiles of 295 early stage breast cancers published from the Netherlands Cancer Institute with a median follow up of 12.6 years allowed evaluating in vitro effects in the in vivo situation.The effects of the interaction between osteoblasts and breast cancer cell lines of different origin were very heterogeneous. Hs578T cells started to proliferate in co-culture with osteoblasts, SKBR-3 induced a TGF-ß response and MDA-MB231 cells showed two distinct sets of up-regulated genes: A set of interferon response genes associated with an up-regulation of STAT1 was in vivo remarkably coherent providing a basis for segregation of tumors into two groups. In a uni-variate analysis, early stage tumors with high expression levels (n = 136) of this gene set had a significantly lower overall survival rate (p = 0.005) (63% at 10 years) than tumors with low expression levels (n = 159) (overall survival: 77% at 10 years). The second gene set was associated with IL-6 and did not significantly change the overall survival rate (p = 0.165), but was significantly associated with a shorter time to bone metastasis (p = 0.049; 74% vs. 83% at 10 years). CONCLUSION/SIGNIFICANCE: An IL-6 gene expression pattern induced by heterotypic interaction of breast cancer cells with osteoblasts in vitro is associated with a higher rate of bone metastasis in vivo.

Tumor-endothelial interaction links the CD44(+)/CD24(-) phenotype with poor prognosis in early-stage breast cancer.

MATERIALS AND METHODS: The genomic effects of tumor-endothelial interactions in cancer are not yet well characterized. To study this interaction in breast cancer, we set up an ex vivo coculture model with human benign and malignant breast epithelial cells with endothelial cells to determine the associated gene expression changes using DNA microarrays. RESULTS: The most prominent response to coculture was the induction of the M-phase cell cycle genes in a subset of breast cancer cocultures that were absent in cocultures with normal breast epithelial cells. In monoculture, tumor cells that contained the stem cell-like CD44(+)/CD24(-) signature had a lower expression of the M-phase cell cycle genes than the CD44(-)/CD24(+) cells, and in the CD44(+)/CD24(-) cocultures, these genes were induced. Pretreatment gene expression profiles of early-stage breast cancers allowed evaluating in vitro effects in vivo. The expression of the gene set derived from the coculture provided a basis for the segregation of the tumors into two groups. In a univariate analysis, early-stage tumors with high expression levels (n = 137) of the M-phase cell cycle genes had a significantly lower metastasis-free survival rate (P = 1.8e - 5, 50% at 10 years) and overall survival rate (P = 5e - 9, 52% at 10 years) than tumors with low expression (n = 158; metastasis-free survival, 73%; overall survival, 84%). CONCLUSIONS: Our results suggest that the interaction of endothelial cells with tumor cells that express the CD44(+)/CD24(-) signature, which indicates a low proliferative potential, might explain the unexpected and paradoxical association of the CD44(+)/CD24(-) signature with highly proliferative tumors that have an unfavorable prognosis.

Characterization of heterotypic interaction effects in vitro to deconvolute global gene expression profiles in cancer.

BACKGROUND: Perturbations in cell-cell interactions are a key feature of cancer. However, little is known about the systematic effects of cell-cell interaction on global gene expression in cancer. RESULTS: We used an ex vivo model to simulate tumor-stroma interaction by systematically co-cultivating breast cancer cells with stromal fibroblasts and determined associated gene expression changes with cDNA microarrays. In the complex picture of epithelial-mesenchymal interaction effects, a prominent characteristic was an induction of interferon-response genes (IRGs) in a subset of cancer cells. In close proximity to these cancer cells, the fibroblasts secreted type I interferons, which, in turn, induced expression of the IRGs in the tumor cells. Paralleling this model, immunohistochemical analysis of human breast cancer tissues showed that STAT1, the key transcriptional activator of the IRGs, and itself an IRG, was expressed in a subset of the cancers, with a striking pattern of elevated expression in the cancer cells in close proximity to the stroma. In vivo, expression of the IRGs was remarkably coherent, providing a basis for segregation of 295 early-stage breast cancers into two groups. Tumors with high compared to low expression levels of IRGs were associated with significantly shorter overall survival; 59% versus 80% at 10 years (log-rank p = 0.001). CONCLUSION: In an effort to deconvolute global gene expression profiles of breast cancer by systematic characterization of heterotypic interaction effects in vitro, we found that an interaction between some breast cancer cells and stromal fibroblasts can induce an interferon-response, and that this response may be associated with a greater propensity for tumor progression.

Early CD30 signaling is critical for adoptively transferred CD4+CD25+ regulatory T cells in prevention of acute graft-versus-host disease.

Murine CD4+CD25+ regulatory T cells (Treg cells) reduce acute graft-versus-host disease (aGvHD). However, surface molecules critical for suppression are unclear. Deficiency of CD30 (CD30-/-) leads to impaired thymic negative selection and augmented T-cell autoreactivity. Therefore, we investigated the role of CD30 signaling in Treg-cell function during aGvHD. Treg cells derived from CD30-/- animals were significantly less effective in preventing aGvHD lethality. Early blockade of the CD30/CD153 pathway with a neutralizing anti-CD153 mAb reduced Treg-mediated protection from proinflammatory cytokine accumulation and donor-type T-cell apoptosis. In vivo bioluminescence imaging demonstrated intact homing but reduced expansion of luciferase-expressing Treg cells when CD153 was blocked during the early phase after adoptive transfer. CD30 surface expression on Treg cells increased with alloantigen exposure, and CD153 expression on recipient-type dendritic cells increased in the presence of a proinflammatory environment. These data demonstrate that early CD30 signaling is critical for Treg-mediated aGvHD protection after major MHC-mismatch bone marrow transplantation.

Inhibition of CD4+CD25+ regulatory T-cell function by calcineurin-dependent interleukin-2 production.

CD4+CD25+ regulatory T (Treg) cells control immunologic tolerance and antitumor immune responses. Therefore, in vivo modification of Treg function by immunosuppressant drugs has broad implications for transplantation biology, autoimmunity, and vaccination strategies. In vivo bioluminescence imaging demonstrated reduced early proliferation of donor-derived luciferase-labeled conventional T cells in animals treated with Treg cells after major histocompatibility complex mismatch bone marrow transplantation. Combining Treg cells with cyclosporine A (CSA), but not rapamycin (RAPA) or mycophenolate mofetil (MMF), suppressed Treg function assessed by increased T-cell proliferation, graft-versus-host disease (GVHD) severity, and reduced survival. Expansion of Treg and FoxP3 expression within this population was lowest in conjunction with CSA, suggesting that calcineurin-dependent interleukin 2 (IL-2) production is critically required for Treg cells in vivo. The functional defect of Treg cells after CSA exposure could be reversed by exogenous IL-2. Further, the Treg plus RAPA combination preserved graft-versus-tumor (GVT) effector function against leukemia cells. Our data indicate that RAPA and MMF rather than CSA preserve function of Treg cells in pathologic immune responses such as GVHD without weakening the GVT effect.