Search

CHK1 is an important regulator of the cell cycle and DNA damage response, and its altered expression has been identified in various tumors. Chk1 inhibitors are currently being evaluated as monotherapy and as potentiators of chemotherapy in clinical settings. However, to our knowledge, no previous study has investigated either the activation status or the therapeutic potential of CHK1 targeting in vulvar cancer. Therefore, we examined the expression status of activated CHK1 forms pCHK1Ser345 , pCHK1Ser317 , pCHK1Ser296 , and pCHK1Ser280 in 294 vulvar squamous cell carcinomas (VSCC) using immunohistochemistry and analyzed their relationships with various clinicopathological variables and clinical outcome. To aid translation of preclinical studies, we also assessed cell sensitivity to the Chk1 inhibition in two vulvar cancer cell lines. Compared to the levels of pCHK1Ser345 , pCHK1Ser317 , pCHK1Ser296 , and pCHK1Ser280 in normal vulvar squamous epithelium, high nuclear pCHK1Ser345 expression was found in 57% of vulvar carcinomas, whereas low nuclear pCHK1Ser317 , pCHK1Ser296 , and pCHK1Ser280 expressions were observed in 58%, 64%, and 40% of the cases, respectively. Low levels of pCHK1Ser317 and pCHK1Ser280 in the nucleus correlated significantly with advanced tumor behaviors and aggressive features. None of pCHK1Ser345 , pCHK1Ser317 , pCHK1Ser296 , and pCHK1Ser280 forms were identified as prognostic factors. In vitro inhibition of CHK1 by small molecular inhibitors or siRNA reduced viability by inducing DNA damage and apoptosis of vulvar cancer cell lines. In summary, we conclude that cellular functions regulated by CHK1 are phosphorylation/localization-dependent and deregulation of CHK1 function occurs in VSCC and might contribute to tumorigenesis. Targeting CHK1 might represent as a useful antitumor strategy for the subgroup of VSCC harboring p53 mutations.

// Xiaoli Li 1, 2, * , Gaoyang Han 3, * , Xiaoran Li 2 , Quancheng Kan 4 , Zhirui Fan 1 , Yaqing Li 1, 2 , Yasai Ji 1 , Jing Zhao 1 , Mingzhi Zhang 1 , Mantas Grigalavicius 2 , Viktor Berge 5 , Mariusz Adam Goscinski 6 , Jahn M. Nesland 2 and Zhenhe Suo 1, 2 1 Department of Oncology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan Province, 450000, China 2 Department of Pathology, The Norwegian Radium Hospital, Oslo University Hospital, Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, Oslo, 0379, Norway 3 Department of Thoracic Surgery, The First Affiliated Hospital of Xinxiang Medical University, Weihui City, Henan Province, 453000, China 4 Department of Clinical Pharmacology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan Province, 450000, China 5 Department of Urology, The Norwegian Radium Hospital, Oslo University Hospital, Oslo, 0379, Norway 6 Department of Surgery, The Norwegian Radium Hospital, Oslo University Hospital, Oslo, 0379, Norway * These authors have contributed equally to this work Correspondence to: Zhenhe Suo, email: zhenhes@medisin.uio.no , zhenhesuo@aliyun.com Keywords: MPC, OXPHOS, TCA, glycolysis, stem cell Received: November 09, 2016 Accepted: May 05, 2017 Published: May 25, 2017 ABSTRACT One of the remarkable features of cancer cells is aerobic glycolysis, a phenomenon known as the “Warburg Effect”, in which cells rely preferentially on glycolysis instead of oxidative phosphorylation (OXPHOS) as the main energy source even in the presence of high oxygen tension. Cells with dysfunctional mitochondria are unable to generate sufficient ATP from mitochondrial OXPHOS, and then are forced to rely on glycolysis for ATP generation. Here we report our results in a prostate cancer cell line in which the mitochondrial pyruvate carrier 1 (MPC1) gene was knockout. It was discovered that the MPC1 gene knockout cells revealed a metabolism reprogramming to aerobic glycolysis with reduced ATP production, and the cells became more migratory and resistant to both chemotherapy and radiotherapy. In addition, the MPC1 knockout cells expressed significantly higher levels of the stemness markers Nanog, Hif1α, Notch1, CD44 and ALDH. To further verify the correlation of MPC gene function and cell stemness/metabolic reprogramming, MPC inhibitor UK5099 was applied in two ovarian cancer cell lines and similar results were obtained. Taken together, our results reveal that functional MPC may determine the fate of metabolic program and the stemness status of cancer cells in vitro .

BACKGROUND Intraductal carcinoma of the prostate (IDC-P) is a distinct histopathologic feature associated with high-grade, advanced prostate cancer. Although studies have shown that IDC-P is a predictor of progression following surgical or radiation treatment for prostate cancer, there are sparse data regarding IDC-P on diagnostic needle biopsy as a prognosticator of prostate cancer mortality. MATERIALS AND METHODS This was a population-based study of all prostate cancer patients diagnosed using needle biopsy and without evidence of systemic disease between 1991 and 1999 within a defined geographic region of Norway. Patients were identified by cross-referencing the Norwegian Cancer Registry. Of 318 eligible patients, 283 had biopsy specimens available for central pathology review. Clinical data were obtained from medical charts. We examined whether IDC-P on diagnostic needle biopsy was associated with adverse clinicopathological features and prostate cancer mortality. RESULTS Patients with IDC-P on diagnostic needle biopsy had a more advanced stage and a higher Gleason score compared to patients without IDC-P. IDC-P was also associated with an intensively reactive stroma. The 10-year prostate cancer-specific survival was 69% for patients with IDC-P on diagnostic needle biopsy and 89% for patients without IDC-P (Log rank P-value

// Xiaoli Li 1, 2 , Yaqing Li 1, 2 , Gaoyang Han 3 , Xiaoran Li 2 , Yasai Ji 1 , Zhirui Fan 1 , Yali Zhong 1 , Jing Cao 1, 4 , Jing Zhao 1 , Goscinski Mariusz 5 , Mingzhi Zhang 1 , Jianguo Wen 6 , Jahn M. Nesland 2 , Zhenhe Suo 1, 2 1 Department of Oncology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan Province, China 2 Department of Pathology, The Norwegian Radium Hospital, Oslo University Hospital, Institute of Clinical Medicine, University of Oslo, Montebello, Oslo, Norway 3 Department of Thoracic Surgery, The First Affiliated Hospital of Xinxiang Medical University, Weihui City, Henan Province, China 4 Department of Pathology, The Third Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan Province, China 5 Department of Surgery, The Norwegian Radium Hospital, Oslo University Hospital, Institute for Clinical Medicine, Faculty of Medicine, University of Oslo, Norway 6 The Institute of Clinical Medicine, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan Province, China Correspondence to: Zhenhe Suo, email: zhenhes@medisin.uio.no, zhenhesuo@aliyun.com Keywords: MPC1, CRISPR/Cas9, diabetes, gene knockout, reproductive capability Received: June 13, 2016 Accepted: October 19, 2016 Published: November 08, 2016 ABSTRACT Pyruvate plays a critical role in the mitochondrial tricarboxylic acid (TCA) cycle, and it is the center product for the synthesis of amino acids, carbohydrates and fatty acids. Pyruvate transported across the inner mitochondrial membrane appears to be essential in anabolic and catabolic intermediary metabolism. The mitochondrial pyruvate carrier (MPC) mounted in the inner membrane of mitochondria serves as the channel to facilitate pyruvate permeating. In mammals, the MPC is formed by two paralogous subunits, MPC1 and MPC2. It is known that complete ablation of MPC2 in mice causes death on the 11th or 12th day of the embryonic period. However, MPC1 deletion and the knowledge of gene function in vivo are lacking. Using the new technology of gene manipulation known as Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated 9 (CRISPR/Cas9) systems, we gained stable MPC1 gene heterozygous mutation mice models, and the heterozygous mutations could be stably maintained in their offsprings. Only one line with homozygous 27 bases deletion in the first exon was established, but no offsprings could be obtained after four months of mating experiments, indicating infertility of the mice with such homozygous deletion. The other line of MPC1 knockout (KO) mice was only heterozygous, which mutated in the first exon with a terminator shortly afterwards. These two lines of MPC1 KO mice showed lower fertility and significantly higher bodyweight in the females. We concluded that heterozygous MPC1 KO weakens fertility and influences the metabolism of glucose and fatty acid and bodyweight in mice.

// Yaqing Li 1, 2, 3 , Xiaoran Li 2, 3 , Xiaoli Li 1, 2 , Yali Zhong 1 , Yasai Ji 1 , Dandan Yu 1, 2 , Mingzhi Zhang 1 , Jian-Guo Wen 4 , Hongquan Zhang 5 , Mariusz Adam Goscinski 6 , Jahn M. Nesland 2, 3 , Zhenhe Suo 1, 2, 3 1 Department of Oncology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan Province, China 2 Department of Pathology, The Norwegian Radium Hospital, Oslo University Hospital, University of Oslo, Montebello, Oslo, Norway 3 Department of Pathology, The Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, Montebello, Oslo, Norway 4 Institute of Clinical Medicine, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan Province, China 5 Department of Anatomy, Histology and Embryology, Peking University Health Science Center, Peking University, Beijing, China 6 Department of Surgery, the Norwegian Radium Hospital, Oslo University Hospital, University of Oslo, Oslo, Norway Correspondence to: Zhenhe Suo, email: zhenhes@medisin.uio.no Keywords: PDHA1 gene knockout, metabolic reprogramming, glutamine dependence, prognosis, GC-MS analysis Received: April 06, 2016 Accepted: July 10, 2016 Published: July 22, 2016 ABSTRACT Alternative pathways of metabolism endowed cancer cells with metabolic stress. Inhibiting the related compensatory pathways might achieve synergistic anticancer results. This study demonstrated that pyruvate dehydrogenase E1α gene knockout (PDHA1 KO) resulted in alterations in tumor cell metabolism by rendering the cells with increased expression of glutaminase1 (GLS1) and glutamate dehydrogenase1 (GLUD1), leading to an increase in glutamine-dependent cell survival. Deprivation of glutamine induced cell growth inhibition, increased reactive oxygen species and decreased ATP production. Pharmacological blockade of the glutaminolysis pathway resulted in massive tumor cells apoptosis and dysfunction of ROS scavenge in the LNCaP PDHA1 KO cells. Further examination of the key glutaminolysis enzymes in human prostate cancer samples also revealed that higher levels of GLS1 and GLUD1 expression were significantly associated with aggressive clinicopathological features and poor clinical outcome. These insights supply evidence that glutaminolysis plays a compensatory role for cell survival upon alternative energy metabolism and targeting the glutamine anaplerosis of energy metabolism via GLS1 and GLUD1 in cancer cells may offer a potential novel therapeutic strategy.

BACKGROUND Previous studies suggest that lymphovascular invasion (LVI) has a weak and variable effect on prognosis. It is uncertain whether LVI, determined by diagnostic prostate biopsy, predicts prostate cancer death. Data from experimental studies have indicated that carcinoma-associated fibroblasts in the reactive stroma could promote LVI and progression to metastasis. Thus, combining LVI with reactive stromal grade may identify prostate cancer patients at high risk of an unfavorable outcome. The purpose of the present study was to examine if LVI, determined by diagnostic biopsy, alone and in combination with reactive stromal grade could predict prostate cancer death. METHODS This population-based study included 283 patients with prostate cancer diagnosed by needle biopsy in Aust-Agder County (Norway) from 1991 to 1999. Clinical data were obtained by medical charts review. Two uropathologists evaluated LVI and reactive stromal grade. The endpoint was prostate cancer death. RESULTS Patients with LVI had marginally higher risk of prostate cancer death compared to patients without LVI (hazard ratio: 1.8, P-value = 0.04). LVI had a stronger effect on prostate cancer death risk when a high reactive stromal grade was present (hazard ratio: 16.0, P-value

Wee1 is a nuclear kinase regulating cell cycle progression, and has emerged as a promising therapeutic target in cancer. Expression of Wee1 has been associated with poor outcome in certain tumor types, but the prognostic impact and clinical significance in colorectal cancer is unknown. The expression of Wee1 was examined by immunohistochemistry in primary colorectal carcinomas from a prospectively collected patient cohort, and associations with clinicopathological parameters and outcome were investigated. Cell culture experiments were performed using the cell lines RKO and SW620, and the relationship with the metastasis-promoting protein S100A4 was investigated. Nuclear expression was detected in 229 of the 258 tumors analyzed (89 %). Wee1 staining was associated with low pT stage, but no other significant associations with demographic or histopathological variables were found. Moderate Wee1 staining intensity was a predictor of favorable metastasis-free and overall survival compared to strong intensity and no or weak staining. The fraction of positive cells was not a prognostic factor in the present cohort. Inhibition of Wee1 expression using siRNA or treatment with the Wee1 inhibitor MK-1775 reduced expression of the metastasis-promoting protein S100A4, but no relationship between Wee1 and S100A4 was found in the patient samples. In conclusion, Wee1 is highly expressed in primary colorectal carcinomas, but few relevant associations with clinicopathological parameters or outcome were found. The lack of clinical significance of Wee1 expression could indicate that other tumor types might be better suited for further development of Wee1 inhibitors.

// Caroline E. Nunes-Xavier 1 , Karine Flem Karlsen 1 , Christina Tekle 1 , Cathrine Pedersen 2 , Tove Oyjord 1 , Vesa Hongisto 3 , Jahn M. Nesland 4 , Ming Tan 5 , Kristine Kleivi Sahlberg 6, 7 , Oystein Fodstad 1, 8 1 Department of Tumor Biology, Institute for Cancer Research, Oslo University Hospital Radiumhospitalet, Oslo, Norway 2 Department of Genetics, Institute for Cancer Research, Oslo University Hospital Radiumhospitalet, Oslo, Norway 3 Misvik Biology Oy, Turku, Finland 4 Department of Pathology, Oslo University Hospital Radiumhospitalet, Oslo, Norway 5 Department of Oncologic Sciences, Mitchell Cancer Institute, University of South Alabama, Mobile, AL, USA 6 Department of Research, Vestre Viken, Drammen, Norway 7 K.G. Jebsen Centre for Breast Cancer Research, Institute for Clinical Medicine, Faculty of Medicine, University of Oslo, Oslo, Norway 8 Institute for Clinical Medicine, Faculty of Medicine, University of Oslo, Oslo, Norway Correspondence to: Caroline E. Nunes-Xavier, e-mail: carnun@rr-research.no Keywords: CD276/B7-H3, breast cancer cells, API-2, everolimus, glycolysis Received: June 19, 2015 Accepted: December 29, 2015 Published: January 12, 2016 ABSTRACT B7 family proteins are important immune response regulators, and can mediate oncogenic signaling and cancer development. We have used human triple-negative breast cancer cell lines with different expression levels of B7-H3 to evaluate its effects on the sensitivity to 22 different anticancer compounds in a drug screen. API-2 (triciribidine) and everolimus (RAD-001), two inhibitors that target the PI3K/AKT/mTOR pathway, showed enhanced inhibition of cell viability and proliferation in B7-H3 knockdown tumor cells compared to their B7-H3 expressing counterparts. Similar inhibition was seen in control cells treated with an anti-B7-H3 monoclonal antibody. In B7-H3 overexpressing cells, the effects of the two drugs were reduced, supported also by in vivo experiments in which B7-H3 overexpressing xenografts were less sensitive to everolimus than control tumors. In API-2 and everolimus-treated B7-H3 overexpressing cells, phospho-mTOR levels were decreased. However, phosphorylation of p70S6K was differentially regulated in B7-H3 cells treated with API-2 or everolimus, suggesting a different B7-H3-mediated mechanism downstream of mTOR. Both API-2 and everolimus decreased the glycolysis of the cells, whereas knockdown of B7-H3 decreased and B7-H3 overexpression increased the glycolytic capacity. In conclusion, we have unveiled a previously unknown relationship between B7-H3 expression and glycolytic capacity in tumor cells, and found that B7-H3 confers resistance to API-2 and everolimus. The results provide novel insights into the function of B7-H3 in cancer, and suggest that targeting of B7-H3 may be a novel alternative to improve current anticancer therapies.

BACKGROUND In vitro and in vivo studies have shown that nerves, tumor epithelium, and stroma interact and promote prostate cancer (PC) progression. Perineural invasion (PNI) is established amidst these interactions and may therefore indicate an aggressive PC phenotype. The purpose of the present study was to determine the relationship between PNI, tumor grade, reactive stroma, and PC-specific mortality. METHODS A population-based study on 318 patients, encompassing all cases of PC diagnosed by needle biopsies and without evidence of systemic metastasis at the time of diagnosis in Aust–Agder County in the period of 1991–1999. Patients were identified by cross-referencing the Cancer Registry of Norway. Clinical data were obtained by review of medical charts. Diagnostic prostate needle biopsies were reviewed with respect to presence of PNI, percentage of biopsy cores with PNI, Gleason score (GS), and reactive stromal grade (RSG). The endpoint was PC-specific mortality. RESULTS The presence of PNI was significantly associated with high tumor grade and abundant reactive stroma. The 10-year PC-specific survival for patients with and without PNI was 72% and 91%, respectively (P = 0.001, log rank). PNI predicted PC-specific mortality independently of clinical factors, though the effect of PNI was attenuated when adjusting for GS and RSG. However, a percentage of biopsy cores with PNI >50% was found to predict PC-specific mortality independently of other clinicopathologic parameters. CONCLUSIONS The present population-based study shows that PNI on diagnostic prostate needle biopsy is associated with increased risk of PC-specific mortality. Our findings demonstrate that the prognostic effect of PNI is dependent on an association with high grade carcinoma and reactive stroma. However; the impact of PNI on clinical outcome becomes stronger and independent of other clinicopathologic factors upon increased percentage of PNI positive biopsy cores. Thus, our study highlights the importance of PNI and microenvironmental interactions for the long-term outcome of PC. Prostate 76:207–214, 2016. © 2015 Wiley Periodicals, Inc.

A growing body of evidence indicates that aberrant activation of epithelial-to-mesenchymal transition (EMT) plays a key role in tumor cell invasion and metastasis. Zinc finger E-box-binding homeobox factor 1 (ZEB1), as a crucial mediator of EMT, contributes to the malignant progression of various epithelial tumors. To determine whether ZEB1 is involved in the progression of ovarian cancer, we immunohistochemically evaluated the expression of ZEB1 in 238 cases of epithelial ovarian cancer (EOC) and analyzed its associations with clinicopathological parameters. Positive expression of ZEB1 was observed in 32.8% (78/238) of EOCs and it was found to be significantly associated with advanced tumor stage (P=0.001). The survival analysis indicated that the expression of ZEB1 was associated with a poor 5-year progression-free survival (PFS) (P=0.021). A similar tendency was also observed between the expression of ZEB1 and 5-year overall survival, although it did not reach statistical significance (P=0.118). Moreover, the multivariate analysis demonstrated that ZEB1 expression was an independent risk factor for 5-year PFS in ovarian cancer. Taken together, our data provide evidence that ZEB1 may play a crucial role in promoting aggressive ovarian carcinoma progression. Therefore, ZEB1 may serve as an effectively predictive marker and a potential target for therapeutic intervention in EOC.

Aerobic glycolysis is one of the important hallmarks of cancer cells and eukaryotic cells. In this study, we have investigated the relationship between blocking mitochondrial pyruvate carrier (MPC) with UK5099 and the metabolic alteration as well as stemness phenotype of prostatic cancer cells. It was found that blocking pyruvate transportation into mitochondrial attenuated mitochondrial oxidative phosphorylation (OXPHOS) and increased glycolysis. The UK5099 treated cells showed significantly higher proportion of side population (SP) fraction and expressed higher levels of stemness markers Oct3/4 and Nanog. Chemosensitivity examinations revealed that the UK5099 treated cells became more resistant to chemotherapy compared to the non-treated cells. These results demonstrate probably an intimate connection between metabolic reprogram and stem-like phenotype of LnCap cells in vitro. We propose that MPC blocker (UK5099) application may be an ideal model for Warburg effect studies, since it attenuates mitochondrial OXPHOS and increases aerobic glycolysis, a phenomenon typically reflected in the Warburg effect. We conclude that impaired mitochondrial OXPHOS and upregulated glycolysis are related with stem-like phenotype shift in prostatic cancer cells.

BACKGROUND Reactive tumor stroma has been shown to play an active role in prostatic carcinogenesis. A grading system for reactive stroma in prostate cancer (PC) has recently been established and found to predict biochemical recurrence and prostate cancer-specific mortality (PCSM) in prostatectomized patients. To the best of our knowledge, there has been no study investigating the prognostic value of reactive stromal grading (RSG) with regard to PCSM when evaluated in diagnostic prostate needle biopsies. METHODS A population-based study on 318 patients, encompassing all cases of PC diagnosed by needle biopsies and without evidence of systemic metastasis at the time of diagnosis in Aust–Agder County in the period 1991–1999. Patients were identified by cross-referencing the Cancer Registry of Norway. Clinical data were obtained by review of medical charts. The endpoint was PCSM. RSG was evaluated on haematoxylin and eosin stained sections according to previously described criteria; grade 0, 0–5% reactive stroma; grade 1, 6–15%; grade 2, 16–50%; grade 3, 51–100%. RESULTS RSG could be evaluated in 278 patients. The median follow- up time was 110 months (interquartile range: 51–171). The 10-year PC - specific survival rate for RSGs of 0, 1, 2, and 3 was 96%, 81%, 69%, and 63%, respectively (P

Background:B7-H3, an immunoregulatory protein, is overexpressed in several cancers and is often associated with metastasis and poor prognosis. Here, our aim was to identify microRNAs (miRNAs) regulating B7-H3 and assess their potential prognostic implications in breast cancer. Methods:MicroRNAs targeting B7-H3 were identified by transfecting two breast cancer cell lines with a library of 810 miRNA mimics and quantifying changes of B7-H3 protein levels using protein lysate microarrays. For validations we used western immunoblotting and 3'-UTR luciferase assays. Clinical significance of the miRNAs was assayed by analysing whether their expression levels correlated with outcome in two cohorts of breast cancer patients (142 and 81 patients). Results:We identified nearly 50 miRNAs that downregulated B7-H3 protein levels. Western immunoblotting validated the impact of the 20 most effective miRNAs. Thirteen miRNAs (miR-214, miR-363*, miR-326, miR-940, miR-29c, miR-665, miR-34b*, miR-708, miR-601, miR-124a, miR-380-5p, miR-885-3p, and miR-593) targeted B7-H3 directly by binding to its 3'-UTR region. Finally, high expression of miR-29c was associated with a significant reduced risk of dying from breast cancer in both cohorts. Conclusions:We identified miRNAs efficiently downregulating B7-H3 expression. The expression of miR-29c correlated with survival in breast cancer patients, suggesting a tumour suppressive role for this miRNA.British Journal of Cancer advance online publication, 27 February 2014; doi:10.1038/bjc.2014.113 www.bjcancer.com

Purpose We explored changes in hypoxia-inducible factor 1 (HIF1) signaling during androgen deprivation therapy (ADT) of androgen-sensitive prostate cancer xenografts under conditions in which no significant change in immunostaining of the hypoxia marker pimonidazole had occurred. Methods and Materials Gene expression profiles of volume-matched androgen-exposed and androgen-deprived CWR22 xenografts, with similar pimonidazole-positive fractions, were compared. Direct targets of androgen receptor (AR) and HIF1 transcription factors were identified among the differentially expressed genes by using published lists. Biological processes affected by ADT were determined by gene ontology analysis. HIF1α protein expression in xenografts and biopsy samples from 35 patients receiving neoadjuvant ADT was assessed by immunohistochemistry. Results A total of 1344 genes showed more than 2-fold change in expression by ADT, including 35 downregulated and 5 upregulated HIF1 targets. Six genes were shared HIF1 and AR targets, and their downregulation was confirmed with quantitative RT-PCR. Significant suppression of the biological processes proliferation, metabolism, and stress response in androgen-deprived xenografts was found, consistent with tumor regression. Nineteen downregulated HIF1 targets were involved in those significant biological processes, most of them in metabolism. Four of these were shared AR and HIF1 targets, including genes encoding the regulatory glycolytic proteins HK2, PFKFB3, and SLC2A1. Most of the downregulated HIF1 targets were induced by hypoxia in androgen-responsive prostate cancer cell lines, confirming their role as hypoxia-responsive HIF1 targets in prostate cancer. Downregulation of HIF1 targets was consistent with the absence of HIF1α protein in xenografts and downregulation in patients by ADT ( P Conclusions AR repression by ADT may lead to downregulation of HIF1 signaling independently of hypoxic fraction, and this may contribute to tumor regression. HIF1α expression is probably not a useful hypoxia biomarker during ADT in prostate cancer.

Photodynamic therapy (PDT) with a light-activated drug is an approved modality for cancer treatment. Hexaminolevulinate (HAL), a hexylester of 5-aminolevulinic acid as the photosensitising protoporphyrin IX (PpIX) precursor, is clinically used for both PDT and photodetection. Our previous studies have shown that HAL–PDT can effectively induce apoptosis in several human blood malignant cell lines. However, the mechanisms involved in the apoptotic induction are still not fully elucidated. In this study we have focused on the role of cellular lamin A/C in the apoptotic induction. HAL–PDT-mediated apoptosis was confirmed by various techniques including fluorescence microscopy and electron microscopy in both human B-cell lymphoma Ramos and Daudi cell lines. The lamin A/C, together with caspases-6 and -3, was cleaved during the apoptosis. Western blots, immunocytochemistry, fluorescence microscopy and electron microscopy demonstrated that the specific caspase-6inhibitor abrogated the HAL-PDT-mediated cleavages of both caspase-6 and lamin A/C and subsequent apoptosis in these two cell lines, suggesting that the cleavage of lamin A/C by the caspase-6 activation is crucial for such apoptotic induction.

Aims To investigate the aberrant expression of CD117 in oesophageal squamous cell carcinoma (SCC) and its prognostic significance. Methods and results Immunohistochemical staining for CD117 was performed on tissue microarray and routine tissue sections from 157 oesophageal SCC patients and 10 normal oesophageal epithelia adjacent to tumour. The positive rate of CD117 expression was 29.9% in oesophageal SCC tissues, whereas no CD117 expression was detected in the 10 normal oesophageal epithelia. CD117 expression was significantly associated with T stage (P

Detection of disseminated tumour cells (DTCs) in bone marrow by immunocytochemistry (ICC) includes morphological evaluation of cytokeratin immunopositive cells. The aim of this study was to disclose the prognostic significance of different morphological categories of ICC-positive cells according to treatment status and tumour subtype. Bone marrow samples (at surgery) were analysed for the presence of cytokeratin-positive DTCs by a standard immunocytochemical method. The immunopositive cells were classified into the following categories, prior to any analysis of the association between DTCs and clinical outcome: tumour cells (TC), uninterpretable cells (UIC), hematopoietic cells (HC), and questionable HC (QHC). The analysis included 747 early breast cancer patients. Median follow-up was 84 months for relapse, and 99 months for death. The categorisation of the ICC positive cells revealed TC in 13.3 % of the patients, whereas 13.1, 17.8, and 21.4 % of the cases were positive for UIC, QHC, and HC, respectively. Analysing all patients, only TC and UIC predicted systemic relapse. Separate analysis of all patients not receiving adjuvant systemic treatment (No-Adj; n = 389) showed that only QHC were associated with reduced survival (DDFS: p = 0.008; BCSS: p = 0.004, log rank) and the presence of QHC also remained significant in multivariate analysis. Primary tumour subgroup analysis (of all patients) by hormone receptors (HR) and HER2, demonstrated that only TC/UIC had prognostic impact in the HR+/HER2- patients, whereas presence of QHC was associated with unfavourable outcome only in triple negative patients (DDFS: p = 0.004; BCSS: p = 0.024). Patients with ≥3HC had improved outcome compared to those with fewer/no HC (DDFS: p = 0.005; BCSS: p = 0.009). Hence, morphological DTC subgroups may differ in clinical significance according to primary tumour subtype and treatment status. This emphasises the importance of DTC characterisation, and separate analyses of DTC categories according to tumour subtype. Hematopoietic ("false positive") cells might predict an immune-related favorable clinical outcome.

Detection of pretreatment disseminated cells (pre-DTC) reflecting its homing to bone marrow (BM) in prostate cancer (PCa) might improve the current model to predict recurrence or survival in men with nonmetastatic disease despite of primary treatment. Thereby, pre-DTC may serve as an early prognostic biomarker. Post-treatment DTCs (post-DTC) finding may supply the clinician with additional predictive information about the possible course of PCa. To assess the prognostic impact of DTCs in BM aspirates sampled before initiation of primary therapy (pre-DTC) and at least 2 years after (post-DTC) to established prognostic factors and survival in patients with PCa. Available BM of 129 long-term follow-up patients with T1-3N0M0 PCa was assessed in addition to 100 BM of those in whom a pretreatment BM was sampled. Patients received either combined therapy [n = 81 (63%)], radiotherapy (RT) with different duration of hormone treatment (HT) or monotherapy with RT or HT alone [n = 48 (37%)] adapted to the criteria of the SPCG-7 trial. Mononuclear cells were deposited on slides according to the cytospin methodology and DTCs were identified by immunocytochemistry using the pancytokeratin antibodies AE1/AE3. The median age of men at diagnosis was 64.5 years (range 49.5–73.4 years). The median long-term follow-up from first BM sampling to last observation was 11 years. Categorized clinically relevant factors in PCa showed only pre-DTC status as the statistically independent parameter for survival in the multivariate analysis. Pre-DTCs homing to BM are significantly associated with clinically relevant outcome independent to the patient's treatment at diagnosis with nonmetastatic PCa.

The purpose of this study was to determine the expression and potential clinical role of epithelial-to-mesenchymal transition (EMT)-related factors in malignant ovarian germ cell tumors (MOGCT). Protein expression of E-cadherin, N-cadherin, P-cadherin, Zeb1, HMGA2, and vimentin by immunohistochemistry was analyzed in 42 MOGCT from patients treated in Norway during the period 1981-2001. Expression was analyzed for association with clinicopathologic parameters. E-cadherin (p = 0.016) and HMGA2 (p = 0.002) expression was significantly higher in immature teratomas and yolk sac tumors compared with dysgerminomas. Vimentin (p < 0.001) and Zeb1 (p = 0.029) staining was significantly higher in immature teratomas compared with yolk sac tumors and dysgerminomas, whereas no significant differences were observed for N-cadherin and P-cadherin. EMT-associated markers were not significantly related to clinicopathologic parameters including age, tumor diameter, and FIGO stage. In conclusion, based on this limited series, EMT-associated markers are not associated with clinical parameters in MOGCT, in contrast to ovarian carcinoma. EMT-related proteins are differentially expressed among various MOGCT subtypes, suggesting differences in biological characteristics associated with invasion and metastasis.

// Yali Zhong 1, 2, 3, 4 , Xiaoli Li 1 , Yasai Ji 1 , Xiaoran Li 3, 4 , Yaqing Li 1 , Dandan Yu 1 , Yuan Yuan 5 , Jian Liu 6 , Huixiang Li 7 , Mingzhi Zhang 1 , Zhenyu Ji 8 , Dandan Fan 8 , Jianguo Wen 9 , Mariusz Adam Goscinski 10 , Long Yuan 11 , Bin Hao 12 , Jahn M Nesland 3, 4 , Zhenhe Suo 1, 3, 4 1 Department of Oncology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan Province, China 2 Department of Gastroenterology, The Second Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan Province, China 3 Department of Pathology, The Norwegian Radium Hospital, Oslo University Hospital, University of Oslo, Montebello, Oslo, Norway 4 Department of Pathology, Institute for Clinical Medicine, Faculty of Medicine, University of Oslo, Oslo, Norway 5 Department of Pathology, Capital Medical University, Beijing, China 6 Institute of Health Quarantine, Chinese Academy of Inspection and Quarantine, Beijing, China 7 Department of Pathology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan Province, China 8 Henan Academy of Medical and Pharmaceutical Sciences, Zhengzhou University, Zhengzhou 9 Institute of Clinical Medicine, The First Affiliated Hospital of Zhengzhou University, Zhengzhou University, Henan, China 10 Department of Surgery, The Norwegian Radium Hospital, Oslo University Hospital, Oslo, Norway 11 Department of Surgery, The Affiliated Cancer Hospital of Zhengzhou University, Zhengzhou, Henan, China 12 Department of Urology, The Second Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan, China Correspondence to: Zhenhe Suo, email: zhenhes@medisin.uio.no Keywords: PDHA1, glycolysis, stemness, prostate cancer Received: September 30, 2016 Accepted: December 28, 2016 Published: January 05, 2017 ABSTRACT Cells generate adenosine-5′-triphosphate (ATP), the major currency for energy-consuming reactions, through mitochondrial oxidative phosphorylation (OXPHOS) and glycolysis. One of the remarkable features of cancer cells is aerobic glycolysis, also known as the “Warburg Effect”, in which cancer cells rely preferentially on glycolysis instead of mitochondrial OXPHOS as the main energy source even in the presence of high oxygen tension. One of the main players in controlling OXPHOS is the mitochondrial gatekeeperpyruvate dehydrogenase complex (PDHc) and its major subunit is E1α (PDHA1). To further analyze the function of PDHA1 in cancer cells, it was knock out (KO) in the human prostate cancer cell line LnCap and a stable KO cell line was established. We demonstrated that PDHA1 gene KO significantly decreased mitochondrial OXPHOS and promoted anaerobic glycolysis, accompanied with higher stemness phenotype including resistance to chemotherapy, enhanced migration ability and increased expression of cancer stem cell markers. We also examined PDHA1 protein expression in prostate cancer tissues by immunohistochemistry and observed that reduced PDHA1 protein expression in clinical prostate carcinomas was significantly correlated with poor prognosis. Collectively, our results show that negative PDHA1 gene expressionis associated with significantly higher cell stemness in prostate cancer cells and reduced protein expression of this gene is associated with shorter clinical outcome in prostate cancers.

Aerobic glycolysis is one of the emerging hallmarks of cancer cells. In this study, we investigated the relationship between blocking mitochondrial pyruvate carrier (MPC) with MPC blocker UK5099 and the metabolic alteration as well as aggressive features of esophageal squamous carcinoma. It was found that blocking pyruvate transportation into mitochondria attenuated mitochondrial oxidative phosphorylation (OXPHOS) and triggered aerobic glycolysis, a feature of Warburg effect. In addition, the HIF-1α expression and ROS production were also activated upon UK5099 application. It was further revealed that the UK5099-treated cells became significantly more resistant to chemotherapy and radiotherapy, and the UK5099-treated tumor cells also exhibited stronger invasive capacity compared to the parental cells. In contrast to esophageal squamous epithelium cells, decreased MPC protein expression was observed in a series of 157 human squamous cell carcinomas, and low/negative MPC1 expression predicted an unfavorable clinical outcome. All these results together revealed the potential connection of altered MPC expression/activity with the Warburg metabolic reprogramming and tumor aggressiveness in cell lines and clinical samples. Collectively, our findings highlighted a therapeutic strategy targeting Warburg reprogramming of human esophageal squamous cell carcinomas.

Over-treatment of low-risk early breast cancer patients with adjuvant systemic therapies is an important clinical challenge. Better techniques are required which can be used to distinguish between the large group of patients with no residual disease after surgery and consequently no benefit of adjuvant treatment, from the smaller group with high relapse risk. A better integration of available prognostic factors might contribute to improved prediction of clinical outcome.The current study included 346 unselected pT1pN0 patients who did not receive adjuvant systemic treatment. In Norway, no patients with this stage were recommended systemic treatment at the time of the study (1995-1998). Histological type, tumour size, grade, vascular invasion (VI), hormone receptor (HR) status, HER2 and Ki67 (cut-off 10%) were analysed. Median follow-up was 86 months for relapse and 101 months for death.Thirty-eight patients experienced relapse, 31 with distant metastasis. Twenty-one patients died of breast cancer. In univariate analysis grade, HER2, HR, VI and Ki67 had impact on clinical outcome (p0.005, log rank). In multivariate analysis, only grade 1-2 vs. grade 3, HER2, VI, and Ki67 status were significant for disease free survival, distant disease free survival, and/or breast cancer specific survival. These factors were used in combination, to separate patients into groups based on the number of unfavourable factors present [combined prognostic score (CPS) 0-4]. Close to 2/3 of the patients (61.4%) had no unfavourable factor (CPS0), whilst 18.4% had CPS ≥ 2. Only 3.6% of those with CPS0 developed metastasis (p0.001). The outcome was clearly worse for patients with CPS ≥ 2 (p0.001), systemic relapse was detected in approximately 40%.This study indicates that the combined use of grade, VI, HER2 and Ki67 identifies a subgroup of breast cancer patients with a relapse risk that may question the benefit of adjuvant systemic therapy.

Background: Proteolytic enzymes and their regulators have important biological roles in colorectal cancer by stimulating invasion and metastasis, which makes these factors attractive as potential prognostic biomarkers. Methods: The expression of extracellular matrix metalloproteinase inducer (EMMPRIN) was characterised using immunohistochemistry in primary tumours from a cohort of 277 prospectively recruited colorectal cancer patients, and associations with expression of S100A4, clinicopathological parameters and patient outcome were investigated. Results: One hundred and ninety-eight samples (72%) displayed positive membrane staining of the tumour cells, whereas 10 cases (4%) were borderline positive. EMMPRIN expression was associated with shorter metastasis-free, disease-specific and overall survival in both univariate and multivariate analyses. The prognostic impact was largely confined to TNM stage III, and EMMPRIN-negative stage III patients had an excellent prognosis. Furthermore, EMMPRIN was significantly associated with expression of S100A4, and the combined expression of these biomarkers conferred an even poorer prognosis. However, there was no evidence of direct regulation between the two proteins in the colorectal cancer cell lines HCT116 and SW620 in siRNA knockdown experiments. Conclusion: EMMPRIN is a promising prognostic biomarker in colorectal cancer, and our findings suggest that it could be used in the selection of stage III patients for adjuvant therapy.

Objectives: To assess the expression of the cell surface protein B7-H3 in prostate cancer, and its association to clinically relevant parameters after radical prostatectomy and to the proliferation marker Ki-67. Methods: Radical prostatectomy specimens from a cohort of 130 patients with a median clinical follow up of 8 years were used for the analysis. The expression of B7-H3 and the proliferation marker Ki-67, as well as other standard clinicopathological parameters, were evaluated. Results: A high expression of B7-H3 was associated with pathological stage T3a and T3b, high Gleason score, extraprostatic extension, seminal vesicle invasion and high proliferative activity. Univariable analysis showed that a high expression level of B7-H3 was also correlated with biochemical failure and clinical relapse, and with the expression of Ki-67. A high expression level of Ki-67 was associated with clinical progression and a tendency towards higher rates of prostate-specific antigen relapse in multivariate analyses. Conclusions: Our findings show that a high expression level of B7-H3 in prostate cancer correlates with the expression of the proliferation marker Ki-67, biochemical failure and clinical relapse. Thus, expression of the cell surface molecule B7-H3 adds to the malignant phenotype of prostate cancer cells expressing high levels of Ki-67. The impact of B7-H3 function on prostate cancer and its potential role in immunotherapy should be explored further.

Introduction: Detection of disseminated tumor cells (DTC) after completion of systemic adjuvant treatment is a strong predictor of early systemic relapse and death. This analysis can discover early failure of a chosen adjuvant systemic treatment. In this study, we wanted to evaluate the value of DTC detection in bone marrow (BM) as a surrogate marker for response to docetaxel rescue treatment, to predict the effect of this treatment. Further, we wanted to compare disease free survival between patients treated with docetaxel resulting in eradication of DTC after treatment and patients treated with docetaxel where DTC persists after treatment. The follow up of the study is still ongoing. Here, we present the preliminary descriptive data from the study. Materials and Methods: A total of 1128 pts with node positive or high risk node negative disease (T1c/T2GII-IIIN0) was enrolled in the period from October 2003 to May 2008. All patients had completed primary surgery and received 6 cycles of adjuvant antracycline containing chemotherapy. The first BM aspiration was performed 8–12 weeks after termination of adjuvant chemotherapy (BM1). A second BM aspiration was performed 6 months later (BM2). The processing of BM and DTC analysis (by ICC) were performed as previsously described (Wiedswang G et al, J Clin Oncol 2003). If BM2 was positive (+) for DTC, the patient was treated with docetaxel (3qw, 6 courses) Docetaxel-treated patients were reexamined at the inclusion hospital with new BM analysis at approximately 1 month (BM3) and 13 months (BM4) after the last docetaxel infusion. Results: Of 997 patients with conclusive DTC results for both BM1 and BM2, 83 patients (8.3%) were BM1 positive and 78 (7.8%) BM2 positive. Among the BM1+ patients, 15 (18.1%) were BM2+. The concordance between BM1 and BM2 were 87%. Of the patients positive at one or both time points, the concordance was 10% (15/146). The BM1 was not significantly associated with primary tumor characteristics (although borderline significance for Grade and ER status), whereas for BM2, DTC+ patients had increased frequency of node positive disease and pN2-3 stage (p=0.001, chi-square), and were positively associated with lobular carcinoma (p=0.01, chi-square). At BM1 24.7% of the DTC+ patients had >1 DTC, and 9.9% had ≥3 DTC. For BM2, 48.2% had >1 DTC, and 25.7% had ≥3 DTC. For patients with positive BM2 receiving docetaxel, the BM3 turned DTC negative in 55 of 66 evaluable cases (83.3%), and 48 of 59 were negative in BM4 (81.4%). Of 67 patients with a conclusive BM result at either BM3 or BM4, 12 were BM positive. Of 16 patients with ≥3 DTC before docetaxel treatment, only 4 patients were positive after treatment (25%). Conclusions: DTC status after adjuvant antracycline containing chemotherapy changes during the first 9 months of FU, with increased DTC positivity among patients with pN+ disease and lobular carcinomas. After docetaxel rescue treatment, the majority of patients experience disappearance of the DTCs. The clinical significance of these results awaits mature FU data, but the present results may indicate possibility for eradication of residual disease by alternative chemotherapy. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-18-07.

B7-H3, an immunoregulatory protein, is known to play a role in tumor progression. In many cancer types, observed correlations between high B7-H3 expression and poor prognosis have been attributed to involvement in antitumor immunity. However, here we demonstrate a nonimmunological alternative function of B7-H3 in cancer metastasis. Since advanced malignant melanoma is a disease with a poor survival rate and a broad pattern of metastasis, we used this disease as a model in our studies. We found that shRNA silencing of B7-H3 reduced the in vitro migratory potential and matrigel invasiveness of MDA-MB-435 and FEMX-I melanoma cells. In an experimental metastasis model in vivo, B7-H3 silencing of MDA-MB-435 cells resulted in reduced metastatic capacity and significantly increased the median symptom-free survival of nude mice (147 vs. 65 days, p < 0.001) and rats (53 vs. 42 days, p = 0.025) injected with MDA-MB-435 cells. Furthermore, a smaller fraction of mice had microscopically detectable metastases compared to control animals, and the pattern of metastases was slightly different between the two groups but with the brain as the predominant organ. Immunohistochemistry on samples from two melanoma patients showed strong B7-H3 staining in both a primary tumor and metastases. Notably, the metastasis-associated proteins, matrix metalloproteinase (MMP)-2, signal transducer and activator of transcription 3 (Stat3), and the level of secreted interleukin-8 (IL-8) were reduced in the B7-H3 knock-down cell variants, whereas tissue inhibitor of metalloproteinase (TIMP)-1 and-2 levels were increased. Taken together, our findings indicate a novel role for B7-H3 in the regulation of the metastatic capacity of melanoma cells and it might be a potential therapeutic target for anti-metastasis therapy.

In many types of cancer, the expression of the immunoregulatory protein B7-H3 has been associated with poor prognosis. Previously, we observed a link between B7-H3 and tumor cell migration and invasion, and in present study, we have investigated the role of B7-H3 in chemoresistance in breast cancer. We observed that silencing of B7-H3, via stable short hairpin RNA or transient short interfering RNA transfection, increased the sensitivity of multiple human breast cancer cell lines to paclitaxel as a result of enhanced drug-induced apoptosis. Overexpression of B7-H3 made the cancer cells more resistant to the drug. Next, we investigated the mechanisms behind B7-H3–mediated paclitaxel resistance and found that the level of Stat3 Tyr705 phosphorylation was decreased in B7-H3 knockdown cells along with the expression of its direct downstream targets Mcl-1 and survivin. The phosphorylation of Janus kinase 2 (Jak2), an upstream molecule of Stat3, was also significantly decreased. In contrast, reexpression of B7-H3 in B7-H3 knockdown and low B7-H3 expressing cells increased the phosphorylation of Jak2 and Stat3. In vivo animal experiments showed that B7-H3 knockdown tumors displayed a slower growth rate than the control xenografts. Importantly, paclitaxel treatment showed a strong antitumor activity in the mice with B7-H3 knockdown tumors, but only a marginal effect in the control group. Taken together, our data show that in breast cancer cells, B7-H3 induces paclitaxel resistance, at least partially by interfering with Jak2/Stat3 pathway. These results provide novel insight into the function of B7-H3 and encourage the design and testing of approaches targeting this protein and its partners. Mol Cancer Ther; 10(6); 960–71. ©2011 AACR.

Background: The prognostic significance of disseminated tumor cells (DTC) in bone marrow (BM) of breast cancer patients at the time of primary diagnosis has been confirmed by a large pooled analysis. In view of the lack of early indicators for secondary adjuvant treatment, we here evaluated whether the persistence of DTCs after adjuvant therapy increases the risk of subsequent relapse and death. Patients and Methods: Individual patient data from 676 women with primary diagnosis of early breast cancer stages I–III from 3 follow-up studies were pooled. During clinical follow-up, patients underwent BM aspiration (BMA) to determine the presence of DTC. Tumor cells were detected by the standardized immunoassays. Univariate and multivariable proportional hazards models were estimated to assess the prognostic significance of DTC for disease-free survival (DFS) and overall survival (OS). Results: Patients were followed for a median of 89 months. BMA was performed at median 37 months after diagnosis of breast cancer. At follow-up BMA, 15.5% of patients had DTCs. The presence of DTC was an independent indicator of poor prognosis for DFS, distant DFS (DDFS), cancer-specific survival, and OS during the first 5 years following cancer diagnosis (log-rank test P < 0.001 values for all investigated endpoints). Conclusion: Among breast cancer patients, persistent DTCs during follow-up significantly predicted the increased risk for subsequent relapse and death. Analysis of DTC might serve as a clinically useful monitoring tool and should be tested as an indicator for secondary adjuvant treatment intervention within clinical trials. Clin Cancer Res; 17(9); 2967–76. ©2011 AACR.

BACKGROUND: The study was performed to determine detection rate and prognostic relevance of disseminated tumour cells (DTC) in patients receiving curatively intended surgery for colorectal cancer (CRC). METHODS: The study population consisted of 235 patients with CRC prospectively recruited from five hospitals in the Oslo region. Bone marrow (BM) aspirates were collected at the time of surgery and the presence of DTC was determined by two immunological methods; immunomagnetic selection (using an anti-EpCAM antibody) and immunocytochemistry (using a pan-cytokeratin antibody). Associations between the presence of DTC and metastasis-free, disease-specific and overall survival were analysed using univariate and multivariate methods. RESULTS: Disseminated tumour cells were detected in 41 (17%) and 28 (12%) of the 235 examined BM samples by immunomagnetic selection and immunocytochemistry, respectively, with only five samples being positive with both methods. The presence of DTC was associated with adverse outcome (metastasis-free, disease-specific and overall survival) in univariate and multivariate analyses. CONCLUSION: The presence of DTC was associated with adverse prognosis in this cohort of patients curatively resected for CRC, suggesting that DTC detection still holds promise as a biomarker in CRC.

Purpose Tumor response of rectal cancer to preoperative chemoradiotherapy (CRT) varies considerably. In experimental tumor models and clinical radiotherapy, activity of particular subsets of kinase signaling pathways seems to predict radiation response. This study aimed to determine whether tumor kinase activity profiles might predict tumor response to preoperative CRT in locally advanced rectal cancer (LARC). Methods and Materials Sixty-seven LARC patients were treated with a CRT regimen consisting of radiotherapy, fluorouracil, and, where possible, oxaliplatin. Pretreatment tumor biopsy specimens were analyzed using microarrays with kinase substrates, and the resulting substrate phosphorylation patterns were correlated with tumor response to preoperative treatment as assessed by histomorphologic tumor regression grade (TRG). A predictive model for TRG scores from phosphosubstrate signatures was obtained by partial-least-squares discriminant analysis. Prediction performance was evaluated by leave-one-out cross-validation and use of an independent test set. Results In the patient population, 73% and 15% were scored as good responders (TRG 1–2) or intermediate responders (TRG 3), whereas 12% were assessed as poor responders (TRG 4–5). In a subset of 7 poor responders and 12 good responders, treatment outcome was correctly predicted for 95%. Application of the prediction model on the remaining patient samples resulted in correct prediction for 85%. Phosphosubstrate signatures generated by poor-responding tumors indicated high kinase activity, which was inhibited by the kinase inhibitor sunitinib, and several discriminating phosphosubstrates represented proteins derived from signaling pathways implicated in radioresistance. Conclusions Multiplex kinase activity profiling may identify functional biomarkers predictive of tumor response to preoperative CRT in LARC.

International audience; The factors determining the clinical relevance of disseminated tumor cells (DTC) in breast cancer patients are largely unknown. Here we compared the specificity and clinical performance of two antibodies frequently used for DTC detection. Reactivities of antibodies A45-B/B3 (A45) and AE1/AE3 (AE) for selected cytokeratins (CK) were assessed by 2-DE Western Blot analysis. Using these antibodies bone marrow aspirates from 391 breast cancer patients (M, pT1-3, pN0-3) were screened for the presence of DTC. To obtain prognostic information, patients were followed up over a median of 83 months for time to relapse and 99 months for time to death. Among the analyzed CK, AE detected CK5, CK7, CK8, and CK19, whereas A45 recognized CK7 and CK18. In total, 24 of 391 patients (6.1%) were DTC-positive for A45, and 41 (10.5%) for AE. Although concordance between the two antibodies was 84.4%, overlap among positive cases was only 3.2%. DTC-positivity with AE and A45 was more frequent in patients of higher nodal status ( = 0.019 and = 0.036, respectively). Nearly all patients with A45-positive DTC had hormone receptor-positive tumors (23/24), while detection of AE-positive DTC was more frequent among hormone receptor negative patients ( = 0.006). Survival analyses of all patients revealed shorter distant disease-free survival ( = 0.039) for patients with A45-positive DTC, whereas the prognostic relevance of AE-positive DTC was restricted to node-positive patients. The clinical utility of immunocytochemical (ICC) DTC detection depends on the anti-CK antibody used, which may reflect the complex CK composition of DTC.

With the exception of patients with tumors smaller than 2cm and infiltration less than 1mm, standard treatment for squamous cell carcinomas of the vulva includes ipsi- or bilateral inguinofemoral lymph node dissection. However, with only 20% of early stage patients presenting with lymph node metastases in the groin, the majority of these patients do not gain from the procedure, but are at risk of its complications and detriments. The sentinel lymph node biopsy (SLNB) method targets the lymph nodes most likely to contain metastasis and has proven high accuracy in predicting the absence of metastasis in non-sentinel lymph nodes when found negative on pathologic examination. The SLNB further provides for a more thorough examination of the harvested lymph nodes and hence increases the detection of micrometastases. Although the clinical significance of micrometastases is controversial, reports on patients with micrometastasis suffering recurrence emerge, making the importance of detecting micrometastases in the pathologic examination of the sentinel lymph nodes evident. Appreciating its limitations, the sentinel lymph node procedure shows evidence of evolving into a feasible and safe procedure in the hands of experienced surgeons, pathologists and nuclear medicine physicians in early stage vulvar carcinoma patients. Still, larger multicenter trials are needed to assess its accuracy and safety.

The aim of this study was to determine the prevalence and outcome of radiation-induced sarcomas (RISs) among sarcoma patients referred to the Norwegian Radium Hospital (NRH).Ninety patients were identified from the institutional sarcoma data base. Medical records and histological and cytological material from both primary and secondary tumours were reviewed.RIS represented 3.0 % of the sarcomas in the data base. The median latency time from radiotherapy of the primary tumour to the diagnosis of RIS was 13.6 years (range 2.5-57.8 years). Gynaecological, breast and testicular cancers were the most common primary diagnoses. For the RISs 13 different histological types were identified including 25 malignant fibrous histiocytomas (28% of all) and 22 osteosarcomas (24%). The sarcoma-related 5-year crude survival was 33% (95 % CI 23-43 %). Unfavourable prognostic factors were metastases at presentation, incomplete surgery and presence of tumour necrosis.Radiation-induced sarcoma is rare and harbours an aggressive clinical behaviour. Complete surgical resection is mandatory for cure.

Objective: Seprase, dipeptidyl peptidase IV (DPPIV) and urokinase-type plasminogen activator (uPA) play a crucial role in the degradation of the extracellular matrix and in the progression of various human tumors. However, their pathophysiologic significance in esophageal carcinoma has not yet been fully elucidated. Methods: The expression of seprase, DPPIV and uPA in esophageal dysplasia, squamous cell carcinoma (SCC) and normal epithelium was examined by immunohistochemistry. Results: Seprase, DPPIV and uPA immunoreactivity was found in dysplastic and cancer cells as well as in stromal cells adjacent to dysplasia and cancer sites, but not in normal epithelium. We found a significant association between uPA expression and sex, tumor size and histological classification in carcinomas. High expression of DPPIV in cancer cells correlated with longer survival of the patients. No significant associations between seprase and clinicopathological features either in dysplasia or in carcinomas were found. Finally, we demonstrated higher levels of seprase, DPPIV and uPA in SCC cell lines than in normal esophageal epithelial cell lines. Conclusions: Our results showed that seprase, DPPIV and uPA are expressed in both premalignant and malignant forms of SCC, but are lacking in normal esophageal epithelia, suggesting that they are involved in SCC neoplastic progression.

Published version. Source at http://dx.doi.org/10.1002/cam4.766 Current clinical algorithms are unable to precisely predict which colorectal cancer patients would benefit from adjuvant chemotherapy, and there is a need for novel biomarkers to improve the selection of patients. The metastasis-promoting protein S100A4 predicts poor outcome in colorectal cancer, but whether it could be used to guide clinical decision making remains to be resolved. S100A4 expression was analyzed by immunohistochemistry in primary colorectal carcinomas from a consecutively collected, population-representative cohort and a randomized phase III study on adjuvant 5-fluorouracil/ levamisole. Sensitivity to treatment with 5-fluorouracil in S100A4 knockdown cells was investigated using 2D and 3D cell culture assays. Strong nuclear expression of S100A4 was detected in 19% and 23% of the tumors in the two study cohorts, respectively. In both cohorts, nuclear immunoreactivity was associated with reduced relapse-free (P < 0.001 and P = 0.010) and overall survival (P = 0.046 and P = 0.006) in univariate analysis. In multivariate analysis, nuclear S100A4 was a predictor of poor relapse-free survival in the consecutive series (P = 0.002; HR 1.9), but not in the randomized study. Sensitivity to treatment with 5-fluorouracil was not affected by S100A4 expression in in vitro cell culture assays, and there was no indication from subgroup analyses in the randomized study that S100A4 expression was associated with increased benefit of adjuvant treatment with 5-fluorouracil/ levamisole. The present study confirms that nuclear S100A4 expression is a negative prognostic biomarker in colorectal cancer, but the clinical utility in selection of patients for adjuvant fluoropyrimidine-based chemotherapy is limited.

Background Cancer cells exhibit an altered metabolism, which is characterized by a preference for aerobic glycolysis more than mitochondrial oxidation of pyruvate. Mitochondrial pyruvate carrier 1 (MPC1) and mitochondrial pyruvate carrier 2 (MPC2) play a bottleneck role by transporting pyruvate into mitochondrial through the mitochondrial inner membrane. Therefore, their protein expression in cancers may be of clinical consequences. There are studies showing low levels of MPC1 expression in colon, kidney and lung cancers, and the expression of MPC1 correlates with poor prognosis. However, the expression status of MPC1 and MPC2 in prostate cancer (PCA) is unclear. Methods In this study, expression of MPC1 and MPC2 in LNCaP and DU145 prostate cancer cell lines was examined by immunocytochemistry (ICC) and Western blotting. Compared to the LNCaP cells, lower levels of MPC1 and MPC2 expression in the DU145 cell line was identified. We then extended our study to 88 patients with prostate cancer who underwent transurethral electro-vaporization of prostate or radical prostatectomy at the First Affiliated Hospital of Zhengzhou University, Henan, China. Patient-derived paraffin embedded PCA specimens were collected for immunohistochemistry (IHC). Correlations with clinicopathologic factors were evaluated by Chi-square or Fisher´s exact probability tests. Overall survival (OS) rates were determined using the Kaplan-Meier estimator. The Cox proportional hazard regression model was used in univariate analysis and multivariate analysis to identify factors significantly correlated with prognosis. Results Linear regression analysis revealed that MPC1 expression level was positively correlated with MPC2 expression (r = 0.375, P = 0.006) in the prostate cancers. MPC1 expression was negatively associated with UICC stage (P = 0.031). While UICC stage (P

Expression profiles of primary breast tumors were investigated in relation to disseminated tumor cells (DTCs) in bone marrow (BM) in order to increase our understanding of the dissemination process. Tumors were classified into five pre-defined molecular subtypes, and presence of DTC identified (at median 85 months follow-up) a subgroup of luminal A patients with particular poor outcome ( p ¼ 0.008). This was not apparent for other tumor subtypes. Gene expression profiles associated with DTC and with systemic relapse for luminal A patients were identified. This study suggests that DTC in BM differentially distinguishes clinical outcome in patients with luminal A type tumors and that DTC-associated gene expression analysis may identify genes of potential importance in tumor dissemination. a 2007 Federation of European Biochemical Societies.

Treatment of cancer is currently based on three main modalities: surgery, radiothera‐ py and chemotherapy. Most solid tumours can only be cured at an early stage, due to the lack of effective systemic treatment. Surgery and radiotherapy are highly effective for eradicating localized tumours, but unfortunately cannot target disseminated dis‐ ease. Chemotherapy represents systemic treatment, but the clinical use of current drugs is to a large extent hampered by their limited specificity. Over the last two decades, immunotherapy has emerged as an interesting novel approach. Contrary to the traditional treatment modalities, the immune system combines inherent specificity with a systemic range of action. The term “vaccine” is traditionally used to describe substances that protect against the development of infectious diseases. In cancer therapy, this term refers to both therapeutic and prophylactic approaches for eliciting immunological and anti-tumour responses. Prophylactic vaccines have only been developed in a few cancer forms, mainly cancers related to viral infection, such as cervical and hepatocellular carcinoma. Therapeutic vaccines, given to patients after the development of the disease, are however investigated in a number of cancer forms. Some of the therapeutic vaccines may also be used for prophylaxis, particularly in patients with increased risk of cancer process. This paper throws light and depicts the international experience of a number of distinguished researchers in the field of development and testing of vaccines against some tumours, mainly malignant melanoma and prostate cancer.

Objective. To investigate the activation of mitogen-activated protein kinases (MAPK) in breast carcinoma effusions and to analyze its relationship to anatomic site and clinical parameters. Methods. Activated MAPK (p-ERK, p-JNK, and p-p38) expression was studied in 42 effusions and 51 corresponding solid tumors (23 primary, 28 metastases) using immunohistochemistry (IHC). Hormone receptor and HER2 status, proliferation (Ki-67), and apoptosis (p85-PARP fragment) were assessed. MAPK levels, activity, and activation ratio (phospho/pan-MAPK ratio) were analyzed in 19 effusions using immunoblotting (IB). Results. Nuclear expression of p-p38 and p-JNK was significantly higher in effusions compared with both primary tumors ( P P = 0.011 for p-p38) and lymph node metastases ( P = 0.003 for p-JNK, P = 0.025 for p-p38) but was not accompanied by apoptosis. IB showed pan-ERK and p-ERK in 18/19 effusions, pan-JNK and p-JNK in 18/19 and 17/19 effusions, respectively, and pan-p38 and p-p38 in 19/19 and 17/19 specimens, respectively. In univariate survival analysis of all cases, advanced disease stage ( P = 0.041), previous chemotherapy ( P = 0.004), and radiation ( P = 0.001) and higher Ki-67 scores ( P = 0.01) correlated with worse overall survival (OS). In Cox multivariate analysis, stage ( P = 0.018), chemotherapy ( P = 0.024), radiation ( P = 0.017), and ER status ( P = 0.002) were independent prognosticators of OS. Quantitative analysis of IB data showed that higher p38 activation ratio correlates with shorter OS ( P = 0.01). Conclusions. This study presents first evidence of in vivo activation of MAPK in breast carcinoma effusions. The elevated JNK and p38 activation in effusions may be a stress-related mechanism providing breast carcinoma cells with survival advantages rather than a drive towards apoptosis. p38 and Ki-67 may be new prognostic markers for patients with breast cancer effusions.

Background and Purpose Breast cancer is diagnosed worldwide in approximately one million women annually and radiation therapy is an integral part of treatment. The purpose of this study was to investigate the molecular basis underlying response to radiotherapy in breast cancer tissue. Material and Methods Tumour biopsies were sampled before radiation and after 10 treatments (of 2 Gray (Gy) each) from 19 patients with breast cancer receiving radiation therapy. Gene expression microarray analyses were performed to identify in vivo radiation-responsive genes in tumours from patients diagnosed with breast cancer. The mutation status of the TP53 gene was determined by using direct sequencing. Results and conclusion Several genes involved in cell cycle regulation and DNA repair were found to be significantly induced by radiation treatment. Mutations were found in the TP53 gene in 39% of the tumours and the gene expression profiles observed seemed to be influenced by the TP53 mutation status.

Objectives. To analyze expressions of the EphB2 and EphB4 receptors in ovarian carcinomas and explore their clinicopathological correlations and prognostic value. Methods. 115 patients with advanced ovarian carcinoma FIGO IIB to IV were involved. RT-PCR and immunohistochemistry were used to examine the expressions of EphB2/B4 receptor mRNA and protein. Correlations between protein expression and clinicopathological factors were also analyzed. Results. Ovarian carcinoma patients with age elder than 60 years had higher EphB2 expression than younger patients. Expression of EphB2 and EphB4 protein did not significantly correlate with any other clinical variables, including FIGO stage, residual tumor, histological type and differentiation grade. No significant correlation between mRNA and protein expression level for both of these receptors was seen. It was found that patients with strong immunostaining for EphB2 ( P = 0.03) or EphB4 ( P = 0.003) receptors had poorer survival, and patients with strong immunostaining for EphB4 receptor showed poorer response to chemotherapy ( P = 0.036). Conclusions. These studies suggest that EphB2 and B4 receptors are of prognostic value and EphB4 receptor may be an independent predictor of chemotherapy response in ovarian cancer patients.

Photodynamic therapy (PDT) with endogenous protoporphyrin IX derived from 5-aminolevulinic acid or its derivatives has been established for treatments of several premalignancies and malignancies; however, the mechanism of the modality is not fully elucidated. The mitochondrial permeability transition pore consists mainly of the mitochondrial outer membrane voltage-dependent anion channel and the peripheral benzodiazepine receptor (PBR) and the mitochondrial inner membrane adenine nucleotide translocator (ANT). These mitochondrial proteins are responsible for the permeability transition that leads to apoptosis. In the present study, the human leukemia cell line, Reh, was treated with PDT using hexaminolevulinate (HAL). More than 80% of apoptotic Reh cells were found after HAL-mediated PDT (HAL-PDT) with high-molecular-weight (50 kbp) DNA fragmentation. Addition of PK11195 or Ro5-4864, two ligands of PBR, during HAL-PDT significantly inhibited the apoptotic effect. Bongkrekic acid, a ligand for ANT, also reduced the PDT effect. Although the mitochondrial transmembrane potential collapsed, neither cytosolic translocation of mitochondrial cytochrome c nor activation of caspase-9, caspase-8, caspase-3, and poly(ADP-ribose) polymerase were found. However, nuclear translocation of mitochondrial apoptosis-inducing factor (AIF) was shown by both immunoblotting and immunocytochemistry. Because AIF is the sole one among all proapoptotic factors involved in caspase-dependent and caspase-independent pathways that induces the high-molecular-weight DNA fragmentation, we conclude that HAL-PDT specifically targets PBR, leading to apoptosis of the Reh cells through nuclear translocation of mitochondrial AIF. This study suggests PBR as a possible novel therapeutic target for HAL-based PDT of cancer.

Immunocytochemical (ICC) detection of disseminated tumor cells (DTC) in bone marrow (BM) in early breast cancer is an independent prognostic factor. The significance of circulating tumor cells (CTC) in peripheral blood (PB) needs further exploration and comparison to DTC detection. PB and BM were prospectively collected from 341 breast cancer patients median 40 months after operation. PB samples were analyzed for tumor cells by a negative immunomagnetic technique (10x10(6) cells/test). BM aspirates were analyzed by standard ICC (2x10(6) cells/test). CTC were present in 10% of the patients and DTC in 14%. Thirty-seven relapses and 14 breast cancer deaths have occurred at median 66 months after diagnosis. Both CTC-status and DTC-status were significantly associated with disease free survival (DFS) (event rate: CTC-positive 26.5% vs. CTC-negative 9.1%; DTC-positive 29.2% vs. DTC-negative 7.8%) (p

Embryonal carcinoma is a histologic subgroup of testicular germ cell tumors (TGCTs), and its cells may follow differentiation lineages in a manner similar to early embryogenesis. To acquire new knowledge about the transcriptional programs operating in this tumor development model, we used 22k oligo DNA microarrays to analyze normal and neoplastic tissue samples from human testis. Additionally, retinoic acid–induced in vitro differentiation was studied in relevant cell lines. We identified genes characterizing each of the known histologic subtypes, adding up to a total set of 687 differentially expressed genes. Among these, there was a significant overrepresentation of gene categories, such as genomic imprinting and gene transcripts associated to embryonic stem cells. Selection for genes highly expressed in the undifferentiated embryonal carcinomas resulted in the identification of 58 genes, including pluripotency markers, such as the homeobox genes NANOG and POU5F1 (OCT3/4), as well as GAL, DPPA4, and NALP7. Interestingly, abundant expression of several of the pluripotency genes was also detected in precursor lesions and seminomas. By use of tissue microarrays containing 510 clinical testicular samples, GAL and POU5F1 were up-regulated in TGCT also at the protein level and hence validated as diagnostic markers for undifferentiated tumor cells. The present study shows the unique gene expression profiles of each histologic subtype of TGCT from which we have identified deregulated components in selected processes operating in normal development, such as WNT signaling and DNA methylation.

Objective The treatment of vulvar squamous cell carcinoma patients is often mutilating. Effort is being made to individualize treatment in order to reduce negative side effects for patients with good prognosis. Molecular markers have been able to predict patient outcome in several tumors. The aim of this study was to characterize the expression of cyclins D1, D3, E, and A in a comparatively large series of patients with vulvar squamous cell carcinoma and look for prognostic impact. Methods A total of 224 vulvar squamous cell carcinomas were immunohistochemically investigated for expression of cyclins D1, D3, E, and A using the biotin-streptavidin-peroxidase method and the OptiMax Plus automated cell staining system. Results High protein levels of cyclin D1 (any positive nuclei) were found in 58 (26%) cases, cyclin D3 (≥50% positive nuclei) in 61 (27%) cases, cyclin E (≥50% positive nuclei) in 41 (18%) cases, and cyclin A (≥5% positive nuclei) in 156 (70%) cases. No prognostic impact was found for the cyclins D1, D3, E, or A. Conclusions The high number of cases showing increased levels of cyclin A suggests that this protein may be important in the pathogenesis of vulvar squamous cell carcinoma. No prognostic impact was found for the cyclins D1, D3, E, or A.

BACKGROUND It was demonstrated previously that the Snail family of transcription factors and Smad-interacting protein 1 (Sip1) regulate E-cadherin and matrix metalloproteinase 2 (MMP-2) expression, cellular morphology, and invasion in carcinoma. For the current study, the authors analyzed the relation between the expression of Snail, Slug, and Sip1; the expression of MMP-2 and E-cadherin; and clinical parameters in patients with metastatic ovarian and breast carcinoma. METHODS One hundred one fresh-frozen, malignant effusions from patients who were diagnosed with gynecologic carcinomas (78 ovarian carcinomas and 23 breast carcinomas) were studied for mRNA expression of Snail, Slug, Sip1, MMP-2, and E-cadherin using reverse transcriptase-polymerase chain reaction analysis. Snail mRNA and E-cadherin protein expression levels also were studied in ovarian carcinoma effusions using in situ hybridization and immunocytochemistry. The results were analyzed for possible correlation with clinicopathologic parameters in both tumor types. RESULTS E-cadherin mRNA expression was lower in breast carcinoma (P = 0.001), whereas Snail expression was higher (P = 0.003). The Snail/E-cadherin ratio (P < 0.001) and the Sip1/E-cadherin ratio (P = 0.002) were higher in breast carcinomas. Sip1 mRNA expression (P < 0.001) and Slug mRNA expression (P < 0.001) were correlated with the expression of MMP-2 in ovarian carcinomas. The Sip1/E-cadherin ratio was higher in primary ovarian carcinomas at the time of diagnosis compared with postchemotherapy ovarian carcinoma effusions (P = 0.003), higher in Stage IV tumors compared with Stage III tumors (P = 0.049), and higher in pleural effusions compared with peritoneal effusions (P = 0.044). In a univariate survival analysis of patients with ovarian carcinoma, a high Sip1/E-cadherin ratio predicted poor overall survival (P = 0.018). High E-cadherin mRNA expression predicted better disease-free survival (P = 0.023), with a similar trend for a low Slug/E-cadherin ratio (P = 0.07). High Snail mRNA expression predicted shorter effusion-free survival (P = 0.008), disease-free survival (P = 0.03), and overall survival (P = 0.008) in patients with breast carcinoma. CONCLUSIONS Transcription factors that regulate E-cadherin were expressed differentially in metastatic ovarian and breast carcinoma. Snail may predict a poor outcome in patients who have breast carcinoma metastatic to effusions. E-cadherin expression generally was conserved in effusions from patients with ovarian carcinoma, but the subset of patients with postulated Sip1-induced repression of this adhesion molecule had a significantly worse outcome. This finding was in agreement with the stronger suppression of E-cadherin by Snail and Sip1 in breast carcinoma effusions, a clinical condition associated with extremely poor survival. Cancer 2005. © 2005 American Cancer Society.

The protein and mRNA expression of dysadherin was studied in a series of squamous cell cervical carcinomas, and their clinicopathological associations and prognostic value were explored. Immunohisto-chemistry was used to assess protein expression of dysadherin in 206 patients with squamous cell cervical carcinoma, FIGO stage Ia-IVb. Frozen tissues from 20 cases in which the tumors showed variable dysadherin protein expression were used for laser capture microdissection (LCM) and processed for RT-PCR detection of dysadherin mRNA. Immunohisto-chemically, all the dysadherin-positive staining was membranous. Positive cell membranous dysadherin-positive staining was often observed at the edge of tumor nests, although strong immunoreactivity throughout whole tumor nests was also seen in some tumors. Basal cells of the normal cervical epithelia were positive for dysadherin while its expression in the squamous cell cervical carcinomas was variable. Among the 206 tumors, 23 (11.2%) were negative, 53 (25.7%) were scored 1+, 54 (26.2%) were scored 2+ and 76 (36.9%) were scored 3+. In the 20 tumors analyzed, mRNA expression of dysadherin basically corresponded to its protein expression. No significant cor-relation between expression of dysadherin and age, FIGO stage or lymph node status was observed. Higher level of dysadherin expression, however, was significantly associated with shorter overall survival (p=0.04). We conclude that there is dysadherin protein expression in basal and parabasal cells of normal cervical epithelia, and higher level of dysadherin protein expression in squamous cell cervical carcinoma is predictive of a shorter overall survival, indicating that dysadherin may be a valuable prognostic marker in cervical carcinoma.

Purpose: The aim of the study was to explore the value of analyzing bone marrow (BM) for the presence of isolated tumor cell(s) (ITCs) in disease-free breast cancer patients 3 years after diagnosis. Experimental Design: ITCs in BM at operation was found to be an independent prognostic factor in 817 breast cancer patients. Among these, 356 disease-free patients were analyzed with a second BM after 3 years follow-up (median 40 months, SD 3 months, range 29–52). ITC was detected by immunocytochemistry with anticytokeratine antibodies (AE1/AE3). Results: The population consisted of 70% T1 and 72% node-negative patients. ITC in BM was detected in 15%. At a median of 25 months since the second BM aspiration (66 months since diagnosis), 32 patients had developed relapse, 12 local and 20 systemic. Of the patients with ITC in BM, 21% relapsed compared with 7% of the ITC-negative patients (P < 0.001). Ten patients died of breast cancer. Survival analyses showed that ITC in BM predicted reduced distant disease-free survival (DDFS) and breast cancer specific survival (BCSS; P < 0.001, log-rank test). Uni-and multivariate analyses of the prognostic value of N, T, estrogen receptor/progesterone receptor, and BM status, histological grade, vascular invasion, p53-, c-erb-B2-, and cathepsin D expression were performed. BM status was the only independent prognostic factor for both DDFS and BCSS, whereas c-erbB-2 and N status were independent for BCSS and vascular invasion and T status for DDFS. Conclusions: ITC in BM 3 years after diagnosis in disease-free breast cancer patients is an independent prognostic factor. Detection of residual disease by BM analysis at follow-up may unravel insufficient adjuvant treatment. The clinical implications should be further explored.