BITES: balanced individual treatment effect for survival data.

MOTIVATION: Estimating the effects of interventions on patient outcome is one of the key aspects of personalized medicine. Their inference is often challenged by the fact that the training data comprises only the outcome for the administered treatment, and not for alternative treatments (the so-called counterfactual outcomes). Several methods were suggested for this scenario based on observational data, i.e. data where the intervention was not applied randomly, for both continuous and binary outcome variables. However, patient outcome is often recorded in terms of time-to-event data, comprising right-censored event times if an event does not occur within the observation period. Albeit their enormous importance, time-to-event data are rarely used for treatment optimization. We suggest an approach named BITES (Balanced Individual Treatment Effect for Survival data), which combines a treatment-specific semi-parametric Cox loss with a treatment-balanced deep neural network; i.e. we regularize differences between treated and non-treated patients using Integral Probability Metrics (IPM). RESULTS: We show in simulation studies that this approach outperforms the state of the art. Furthermore, we demonstrate in an application to a cohort of breast cancer patients that hormone treatment can be optimized based on six routine parameters. We successfully validated this finding in an independent cohort. AVAILABILITY AND IMPLEMENTATION: We provide BITES as an easy-to-use python implementation including scheduled hyper-parameter optimization (https://github.com/sschrod/BITES). The data underlying this article are available in the CRAN repository at https://rdrr.io/cran/survival/man/gbsg.html and https://rdrr.io/cran/survival/man/rotterdam.html. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Long-term prognostic impact of surgical complications in the German Rectal Cancer Trial CAO/ARO/AIO-94.

BACKGROUND: The influence of postoperative complications on survival in patients with locally advanced rectal cancer undergoing combined modality treatment is debatable. This study evaluated the impact of surgical complications on oncological outcomes in patients with locally advanced rectal cancer treated within the randomized CAO/ARO/AIO-94 (Working Group of Surgical Oncology/Working Group of Radiation Oncology/Working Group of Medical Oncology of the Germany Cancer Society) trial. METHODS: Patients were assigned randomly to either preoperative chemoradiotherapy (CRT) followed by total mesorectal excision (TME) or postoperative CRT between 1995 and 2002. Anastomotic leakage and wound healing disorders were evaluated prospectively, and their associations with overall survival, and distant metastasis and local recurrence rates after a long-term follow-up of more than 10 years were determined. Medical complications (such as cardiopulmonary events) were not analysed in this study. RESULTS: A total of 799 patients were included in the analysis. Patients who had anterior or intersphincteric resection had better 10-year overall survival than those treated with abdominoperineal resection (63·1 versus 51·3 per cent; P < 0·001). Anastomotic leakage was associated with worse 10-year overall survival (51 versus 65·2 per cent; P = 0·020). Overall survival was reduced in patients with impaired wound healing (45·7 versus 62·2 per cent; P = 0·009). At 10 years after treatment, patients developing any surgical complication (anastomotic leakage and/or wound healing disorder) had impaired overall survival (46·6 versus 63·8 per cent; P < 0·001), a lower distant metastasis-free survival rate (63·2 versus 72·0 per cent; P = 0·030) and more local recurrences (15·5 versus 6·4 per cent; P < 0·001). In a multivariable Cox regression model, lymph node metastases (P < 0·001) and surgical complications (P = 0·008) were the only independent predictors of reduced overall survival. CONCLUSION: Surgical complications were associated with adverse oncological outcomes in this trial.

Relevance of surgery in patients with non-variceal upper gastrointestinal bleeding.

INTRODUCTION: Upper GI bleeding remains one of the most common emergencies with a substantial overall mortality rate of up to 30%. In severe ill patients, death does not occur due to failure of hemostasis, either medical or surgical, but mainly from comorbidities, treatment complications, and decreased tolerated blood loss. Management strategies have changed dramatically over the last two decades and include primarily endoscopic intervention in combination with acid-suppressive therapy and decrease in surgical intervention. Herein, we present one of the largest patient-based analysis assessing clinical parameters and outcome in patients undergoing endoscopy with an upper GI bleeding. Data were further analyzed to identify potential new risk factors and to investigate the role of surgery. PATIENTS AND METHODS: In this retrospective study, we aimed to analyze outcome of patients with an UGIB and data were analyzed to identify potential new risk factors and the role of surgery. Data collection included demographic data, laboratory results, endoscopy reports, and details of management including blood administration, and surgery was carried out. Patient events were grouped and defined as "overall" events and "operated," "non-operated," and "operated and death" as well as "non-operated and death" where appropriate. Blatchford, clinical as well as complete Rockall-score analysis, risk stratification, and disease-related mortality rate were calculated for each group for comparison. RESULTS: Overall, 253 patients were eligible for analysis: endoscopy was carried out in 96% of all patients, 17% needed surgical intervention after endoscopic failure of bleeding control due to persistent bleeding, and the remaining 4% of patients were subjected directly to surgery. The median length of stay to discharge was 26 days. Overall mortality was 22%; out of them, almost 5% were operated and died. Anticoagulation was associated with a high in-hospital mortality risk (23%) and was increased once patients were taken to surgery (43%). Patients taking steroids presented with a risk of death of 26%, once taken to surgery the risk increased to 80%. Patients with liver cirrhosis had a risk of death of 42%; we observed a better outcome for these patients once taken to theater. Clinically, once scored with Blatchford score, statistical correlation was found for initial need for blood transfusion and surgical intervention. Clinical as well as complete Rockall score revealed a correlation between need for blood transfusion as well as surgical intervention in addition with a decreased outcome with increasing Rockall scores. Risk factor analysis including comorbidity, drug administration, and anticoagulation therapy introduced the combination of tumor and non-steroidal antirheumatic medication as independent risk factors for increased disease-related mortality. CONCLUSION: UGIB remains challenging and endoscopy is the first choice of intervention. Care must be taken once a patient is taking antirheumatic non-steroidal pain medication and suffers from cancer. In patients with presence of liver cirrhosis, an earlier surgical intervention may be considered, in particular for patients with recurrent bleeding. Embolization is not widely available and carries the risk of necrosis of the affected organ and should be restricted to a subgroup of patients not primarily eligible for surgery once endoscopy has failed. Taken together, an interdisciplinary approach including gastroenterologists as well as surgeons should be used once the patient is admitted to the hospital to define the best treatment option.

Microenvironmental interactions between endothelial and lymphoma cells: a role for the canonical WNT pathway in Hodgkin lymphoma.

The interaction between vascular endothelial cells (ECs) and cancer cells is of vital importance to understand tumor dissemination. A paradigmatic cancer to study cell-cell interactions is classical Hodgkin Lymphoma (cHL) owing to its complex microenvironment. The role of the interplay between cHL and ECs remains poorly understood. Here we identify canonical WNT pathway activity as important for the mutual interactions between cHL cells and ECs. We demonstrate that local canonical WNT signaling activates cHL cell chemotaxis toward ECs, adhesion to EC layers and cell invasion using not only the Wnt-inhibitor Dickkopf, tankyrases and casein kinase 1 inhibitors but also knockdown of the lymphocyte enhancer binding-factor 1 (LEF-1) and ß-catenin in cHL cells. Furthermore, LEF-1- and ß-catenin-regulated cHL secretome promoted EC migration, sprouting and vascular tube formation involving vascular endothelial growth factor A (VEGF-A). Importantly, high VEGFA expression is associated with a worse overall survival of cHL patients. These findings strongly support the concept that WNTs might function as a regulator of lymphoma dissemination by affecting cHL cell chemotaxis and promoting EC behavior and thus angiogenesis through paracrine interactions.

WNT5A: a motility-promoting factor in Hodgkin lymphoma.

Classical Hodgkin lymphoma (cHL) has a typical clinical manifestation, with dissemination involving functionally neighboring lymph nodes. The factors involved in the spread of lymphoma cells are poorly understood. Here we show that cHL cell lines migrate with higher rates compared with non-Hodgkin lymphoma cell lines. cHL cell migration, invasion and adhesion depend on autocrine WNT signaling as revealed by the inhibition of WNT secretion with the porcupine inhibitors Wnt-C59/IWP-2, but did not affect cell proliferation. While application of recombinant WNT5A or WNT5A overexpression stimulates HL cell migration, neither WNT10A, WNT10B nor WNT16 did so. Time-lapse studies revealed an amoeboid type of cell migration modulated by WNT5A. Reduced migration distances and velocity of cHL cells, as well as altered movement patterns, were observed using porcupine inhibitor or WNT5A antagonist. Knockdown of Frizzled5 and Dishevelled3 disrupted the WNT5A-mediated RHOA activation and cell migration. Overexpression of DVL3-K435M or inhibition of ROCK (Rho-associated protein kinase) by Y-27632/H1152P disrupted cHL cell migration. In addition to these mechanistic insights into the role of WNT5A in vitro, global gene expression data revealed an increased WNT5A expression in primary HL cells in comparison with normal B-cell subsets and other lymphomas. Furthermore, the activity of both porcupine and WNT5A in cHL cells had an impact on lymphoma development in the chick chorionallantoic membrane assay. Massive bleeding of these lymphomas was significantly reduced after inhibition of WNT secretion by Wnt-C59. Therefore, a model is proposed where WNT signaling has an important role in regulating tumor-promoting processes.

[Lymph node metastases in ypT1/2 rectal cancer after neoadjuvant chemoradiotherapy : The Achilles heel of organ-preserving operative procedures?]. / Lymphknotenmetastasen beim ypT1/2-Rektumkarzinom nach neoadjuvanter Radiochemotherapie : Die Achillesferse organerhaltender Operationsverfahren?

BACKGROUND: For patients with rectal cancer and complete remission (ypT0) or with good response and residual tumor restricted only to the bowel wall (ypT1-2) after neoadjuvant chemoradiotherapy (CRT), local excision has been suggested as an alternative to avoid the significant morbidity and functional deficits associated with total mesorectal excision (TME). The aim of this investigation was to investigate the incidence, distribution and tumor-related localization of mesorectal lymph node (LN) metastases in TME specimens with complete remission (ypT0), intramural (ypT1-2) and extramural (ypT3-4) residual tumor tissue. PATIENTS AND METHODS: Specimens of TME from 81 patients with locally advanced rectal cancer (UICC II-III) undergoing neoadjuvant CRT within the phase III German rectal cancer trial CAO/ARO/AIO-04 were prospectively evaluated. The entire mesorectal compartment was microscopically screened after complete paraffin embedding. The number and localization of all detectable LN metastases were documented in relation to the primary tumor. RESULTS: Whereas 50 patients (62 %) had ypT3-4 rectal cancer after neoadjuvant CRT, 20 patients (25 %) presented with residual tumor within the bowel wall (ypT1-2), 11 patients (14 %) had pathological complete remission (ypT0), an average of 28 ± 13.7 LN were detected per specimen and 25 patients (31 %) had residual LN metastases after CRT. Although the incidence of LN metastases was higher in the ypT3-4 group (40 %), 25 % of patients in the ypT1-2 group with intramural residual tumor had a mean number of 2.2 residual LN metastases of which 55 % were located far from the primary lesion in the proximal mesorectum. None of the patients with ypT0 status (complete response) had residual LN metastases. CONCLUSION: Even in patients with good response and post-CRT tumor tissue restricted only to the bowel wall (ypT1-2), there is still a considerable risk for residual LN metastases. Local excision of residual rectal cancer was accompanied by a higher rate of local failure and radical surgery with TME should remain the standard treatment in these patients. To date, valid selection criteria for patients eligible for organ-sparing surgery are still lacking.

The putative oncogene CEP72 inhibits the mitotic function of BRCA1 and induces chromosomal instability.

BRCA1 is a tumor-suppressor gene associated with, but not restricted to, breast and ovarian cancer and implicated in various biological functions. During mitosis, BRCA1 and its positive regulator Chk2 are localized at centrosomes and are required for the regulation of microtubule plus end assembly, thereby ensuring faithful mitosis and numerical chromosome stability. However, the function of BRCA1 during mitosis has not been defined mechanistically. To gain insights into the mitotic role of BRCA1 in regulating microtubule assembly, we systematically identified proteins interacting with BRCA1 during mitosis and found the centrosomal protein Cep72 as a novel BRCA1-interacting protein. CEP72 is frequently upregulated in colorectal cancer tissues and overexpression of CEP72 mirrors the consequences of BRCA1 loss during mitosis. In detail, the overexpression of CEP72 causes an increase in microtubule plus end assembly, abnormal mitotic spindle formation and the induction of chromosomal instability. Moreover, we show that high levels of Cep72 counteract Chk2 as a positive regulator of BRCA1 to ensure proper mitotic microtubule assembly. Thus, CEP72 represents a putative oncogene in colorectal cancer that might negatively regulate the mitotic function of BRCA1 to ensure chromosomal stability.

UDP glucuronosyltransferase 1A expression levels determine the response of colorectal cancer cells to the heat shock protein 90 inhibitor ganetespib.

HSP90 inhibition represents a promising route to cancer therapy, taking advantage of cancer cell-inherent proteotoxic stress. The HSP90-inhibitor ganetespib showed benefit in advanced clinical trials. This raises the need to identify the molecular determinants of treatment response. We tested the efficacy of ganetespib on a series of colorectal cancer (CRC)-derived cell lines and correlated their sensitivities with comprehensive gene expression analysis. Notably, the drug concentration required for 50% growth inhibition (IC50) varied up to 70-fold (from 36 to 2500 nM) between different cell lines. Correlating cell line-specific IC50s with the corresponding gene expression patterns revealed a strong association between ganetespib resistance (IC50>500 nM) and high expression of the UDP glucuronosyltransferase 1A (UGT1A) gene cluster. Moreover, CRC tumor samples showed a comparable distribution of UGT1A expression levels. The members of the UGT1A gene family are known as drug-conjugating liver enzymes involved in drug excretion, but their function in tumor cells is hardly understood. Chemically unrelated HSP90 inhibitors, for example, 17-N-allylamino-17-demethoxygeldanamycin (17-AAG), did not show correlation of drug sensitivities with UGT1A levels, whereas the ganetespib-related compound NVP-AUY922 did. When the most ganetespib-resistant cell line, HT29, was treated with ganetespib, the levels of HSP90 clients were unaffected. However, HT29 cells became sensitized to the drug, and HSP90 client proteins were destabilized by ganetespib upon siRNA-mediated UGT1A knockdown. Conversely, the most ganetespib-sensitive cell lines HCT116 and SW480 became more tolerant toward ganetespib upon UGT1A overexpression. Mechanistically, ganetespib was rapidly glucuronidated and excreted in resistant but not in sensitive CRC lines. We conclude that CRC cell-expressed UGT1A inactivates ganetespib and other resorcinolic Hsp90 inhibitors by glucuronidation, which renders the drugs unable to inhibit Hsp90 and thereby abrogates their biological activity. UGT1A levels in tumor tissues may be a suitable predictive biomarker to stratify CRC patients for ganetespib treatment.

TNF-α mediates mitochondrial uncoupling and enhances ROS-dependent cell migration via NF-κB activation in liver cells.

Development of hepatocellular carcinoma (HCC) is accompanied by a continuous increase in reactive oxygen species (ROS) levels. To investigate the primary source of ROS in liver cells, we used tumor necrosis factor-alpha (TNF-α) as stimulus. Applying inhibitors against the respiratory chain complexes, we identified mitochondria as primary source of ROS production. TNF-α altered mitochondrial integrity by mimicking a mild uncoupling effect in liver cells, as indicated by a 40% reduction in membrane potential and ATP depletion (35%). TNF-α-induced ROS production activated NF-κB 3.5-fold and subsequently enhanced migration up to 12.7-fold. This study identifies complex I and complex III of the mitochondrial respiratory chain as point of release of ROS upon TNF-α stimulation of liver cells, which enhances cell migration by activating NF-κB signalling.

Nuclear LEF1/TCF4 correlate with poor prognosis but not with nuclear ß-catenin in cerebral metastasis of lung adenocarcinomas.

An essential function of the transcription factors LEF1/TCF4 in cerebral metastases of lung adenocarcinomas has been described in mouse models, suggesting a WNT/ß-catenin effect as potential mechanism. Their role in humans is still unclear, thus we analyzed LEF1, TCF4, ß-catenin, and early stage prognostic markers in 25 adenocarcinoma brain metastases using immunohistochemistry (IHC). IHC revealed nuclear TCF4 in all adenocarcinoma samples, whereas only 36 % depicted nuclear LEF1 and nuclear ß-catenin signals. Samples with nuclear LEF1 as well as high TCF4 (++++) expression were associated with a shorter survival (p = 0.01, HR = 6.68), while nuclear ß-catenin had no significant impact on prognosis and did not significantly correlate with nuclear LEF1. High proliferation index Ki67 was associated with shorter survival in late-stage disease (p = 0.03, HR 3.27). Additionally, we generated a LEF1/TCF4 as well as an AXIN2 signature, the latter as representative of WNT/ß-catenin activity, following a bioinformatics approach with a gene expression dataset of cerebral metastases in lung adenocarcinoma. To analyze the prognostic relevance in primary lung adenocarcinomas, we applied both signatures to a microarray dataset of 58 primary lung adenocarcinomas. Only the LEF1/TCF4 signature was able to separate clusters with impact on survival (p = 0.01, HR = 0.32). These clusters displayed diverging enrichment patterns of the cell cycle pathway. In conclusion, our data show that LEF1/TCF4, but not ß-catenin, have prognostic relevance in primary and cerebrally metastasized human lung adenocarcinomas. In contrast to the previous in vivo findings, these results indicate that LEF1/TCF4 act independently of ß-catenin in this setting.

Bilobar spreading of colorectal liver metastases does not significantly affect survival after R0 resection in the era of interdisciplinary multimodal treatment.

PURPOSE: Bilobar colorectal liver metastases (CRLM) are often considered incurable or associated with poor prognosis even after R0 resection. In this single-center study, we evaluate the impact of CRLM spreading on recurrence-free survival (RFS) and cancer-specific overall survival (CSS) after R0 resection of CRLM with respect to multimodal treatment strategies including perioperative chemotherapy and multistep resections. METHODS: Between January 2001 and December 2010, R0 resection could be achieved in 70 patients with bilobar and 100 with unilobar CRLM. Extent of disease, perioperative chemotherapy, surgical procedures, adjuvant treatment, histopathological workup, RFS, and CSS were compared between both cohorts. RESULTS: Forty-six (66 %) patients with bilobar and 26 (26 %) patients with unilobar CRLM received preoperative chemotherapy (p < 0.001). For bilobar CRLM, more extended and multistep resection including portal vein occlusion were performed (29 % versus 3 %; p < 0.001). Morbidity (39 % versus 28 %, p = 0.183) and mortality (1 % versus 3 %, p = 0.644) rates were comparable in both patients' cohorts. Postoperative therapy was applied in adjuvant intent to 42 (60 %) versus 51 (51 %) patients (p = 0.275). The 5-year RFS and CSS rates were 24 % versus 31 % (p = 0.169) and 42 % versus 55 % (p = 0.131), respectively. CONCLUSIONS: To our single-center experience, there is no significant effect of CRLM spreading (bilobar versus unilobar) on RFS and CSS rates. Bilobar CRLM are more likely to require extended multimodal efforts to achieve R0 resection.

Strong EGFR signaling in cell line models of ERBB2-amplified breast cancer attenuates response towards ERBB2-targeting drugs.

Increasing the efficacy of targeted cancer therapies requires the identification of robust biomarkers suitable for patient stratification. This study focused on the identification of molecular mechanisms causing resistance against the anti-ERBB2-directed therapeutic antibodies trastuzumab and pertuzumab presently used to treat patients with ERBB2-amplified breast cancer. Immunohistochemistry and clinical data were evaluated and yielded evidence for the existence of ERBB2-amplified breast cancer with high-level epidermal growth-factor receptor (EGFR) expression as a separate tumor entity. Because the proto-oncogene EGFR tightly interacts with ERBB2 on the protein level, the hypothesis that high-level EGFR expression might contribute to resistance against ERBB2-directed therapies was experimentally validated. SKBR3 and HCC1954 cells were chosen as model systems of EGFR-high/ERBB2-amplified breast cancer and exposed to trastuzumab, pertuzumab and erlotinib, respectively, and in combination. Drug impact was quantified in cell viability assays and on the proteomic level using reverse-phase protein arrays. Phosphoprotein dynamics revealed a significant downregulation of AKT signaling after exposure to trastuzumab, pertuzumab or a coapplication of both antibodies in SKBR3 cells but no concomitant impact on ERK1/2, RB or RPS6 phosphorylation. On the other hand, signaling was fully downregulated in SKBR3 cells after coinhibition of EGFR and ERBB2. Inhibitory effects in HCC1954 cells were driven by erlotinib alone, and a significant upregulation of RPS6 and RB phosphorylation was observed after coincubation with pertuzumab and trastuzumab. In summary, proteomic data suggest that high-level expression of EGFR in ERBB2-amplified breast cancer cells attenuates the effect of anti-ERBB2-directed antibodies. In conclusion, EGFR expression may serve as diagnostic and predictive biomarker to advance personalized treatment concepts of patients with ERBB2-amplified breast cancer.

ß-catenin-independent WNT signaling in basal-like breast cancer and brain metastasis.

A role of WNT signaling for primary breast cancers of the basal-like subtype and as a predictor of brain metastasis has been described. However, a responsible WNT ligand has not been identified. To further clarify this question, we comparatively investigated 22 human breast cancer brain metastases as well as the highly invasive human breast cancer cell line MDA-MB-231 and the weakly motile MCF-7 as models for the basal-like and the luminal A subtype. WNT5A and B were found overexpressed in MDA-MB-231 cells as compared with MCF-7. This corresponded to reduction of MDA-MB-231 invasiveness by WNT inhibitors, whereas MCF-7 invasion was enhanced by recombinant WNT5B and abolished by WNT and Jun-N-terminal kinase antagonists. Expression and subcellular distribution of ß-catenin remained uninfluenced. Consistently, ß-catenin was not localized in the nuclei of brain metastases while there was strong nuclear c-Jun staining. Similar to MDA-MB-231, metastases showed expression of WNT5A/B and the alternative WNT receptors ROR1 and 2. These findings were validated using external gene expression datasets (Gene Expression Omnibus) of different breast cancer subtypes and brain metastases. Hierarchical cluster analysis yielded a close relation between basal-like cancers and brain metastases. Gene set enrichment analyses confirmed WNT pathway enrichment not only in basal-like primaries but also in cerebral metastases of all subtypes. In conclusion, WNT signaling seems highly relevant for basal-like and other subtypes of breast cancers metastasizing into the brain. ß-catenin-independent WNT signaling, presumably via ROR1-2, plays a major role in this context.

Increased efficiency of folding and peptide loading of mutant MHC class I molecules.

Class I molecules, encoded by diverse alleles at several loci of the major histocompatibility complex (MHC) are assembled in the endoplasmic reticulum (ER) from heavy chain, beta2 microglobulin and peptide in association with accessory proteins of the peptide loading complex. We show here, that mutations in the alpha2 domain (Q115A; D122A) of the human class I allele HLA-A2 cause a lack of apparent association with the loading complex and a faster assembly. Despite the drastically reduced association with the TAP loading complex, i. e. less than 20 % of HLA-A2 expressed in the cells can be co-precipitated with either TAP, calreticulin or tapasin, the mutant proteins are expressed on the cell surface in a stable conformation, and bind a complex set of peptides almost identical to that of wild-type HLA-A2. Furthermore, the mutant class I molecules are more rapidly exported from the ER than wild-type HLA-A2 and undergo faster maturation. The mutation Q115A does not destroy a binding site for the loading complex as this HLA-A2 mutant associates with the loading complex when peptide supply is limited. The association of class I molecules with the TAP-associated loading complex appears to be a reflection of how quickly the stable conformation is gained.