Mitochondrial Function of CKS2 Oncoprotein Links Oxidative Phosphorylation with Cell Division in Chemoradioresistant Cervical Cancer.

CDK regulatory subunit 2 (CKS2) has a nuclear function that promotes cell division and is a candidate biomarker of chemoradioresistance in cervical cancer. The underlying mechanisms are, however, not completely understood. We investigated whether CKS2 also has a mitochondrial function that augments tumor aggressiveness. Based on global gene expression data of two cervical cancer cohorts of 150 and 135 patients, we identified a set of genes correlated with CKS2 expression. Gene set enrichment analysis showed enrichment of mitochondrial cellular compartments, and the hallmarks oxidative phosphorylation (OXPHOS) and targets of the MYC oncogene in the gene set. By in situ proximity ligation assay, we showed that CKS2 formed complex with the positively correlated MYC target, mitochondrial single-stranded DNA binding protein SSBP1, in the mitochondrion of cervix tumor samples and HeLa and SiHa cervical cancer cell lines, indicating a role in mitochondrial DNA (mtDNA) replication and thereby OXPHOS. CDK1 was found to be part of the complex. Flow cytometry analyses of HeLa cells showed cell cycle regulation of the CKS2-SSBP1 complex consistent with mtDNA replication activity. Moreover, repression of mtDNA replication and OXPHOS by acute hypoxia decreased CKS2-SSBP1 complex abundance and expression of MYC targets. By immunohistochemistry, cytoplasmic CKS2 expression was found to add to the prognostic impact of nuclear CKS2 expression in patients, suggesting that the mitochondrial function promotes tumor aggressiveness. Our study uncovers a novel link between regulation of cell division by nuclear pathways and OXPHOS in the mitochondrion that involves CKS2 and promotes chemoradioresistance of cervical cancer.

Evaluation of CHK1 activation in vulvar squamous cell carcinoma and its potential as a therapeutic target in vitro.

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.

Expression of 14-3-3 sigma and eta proteins is unrelated to survival in metastatic high-grade serous carcinoma.

The objective of this study was to analyze the expression and clinical role of 14-3-3 family proteins in high-grade serous carcinoma (HGSC). Protein expression of 14-3-3 sigma (14-3-3σ) and 14-3-3 eta (14-3-3η) by immunohistochemistry was studied in 298 HGSC specimens (249 peritoneal, 49 pleural) and was analyzed for association with clinicopathologic parameters, chemoresponse and survival. The 14-3-3σ protein was diffusely (>75% of cells) expressed in 100% of carcinomas in analysis of a pilot series and was therefore not further analyzed. The 14-3-3η protein was expressed to a variable extent in 260/298 (87%) effusions. Higher 14-3-3η protein expression was significantly related to higher CA 125 levels at diagnosis (p = 0.004), but was unrelated to other clinicopathologic parameters, chemoresponse or survival. Analysis of the association between 14-3-3η and previously studied proteins regulating mitosis showed positive association with class III ß-tubulin expression (p = 0.025). The present study documents frequent expression of 14-3-3σ and 14-3-3η in HGSC effusions, but does not support a role for these proteins as prognostic markers or predictors of chemotherapy response in metastatic HGSC.

Prognostic and clinicopathological significance of Cacna2d1 expression in epithelial ovarian cancers: a retrospective study.

Ovarian cancer is the most lethal gynecologic malignancy, in which cancer stem cells (CSC) have been reported to be the driving force of relapse and therapy-resistance. It is therefore important to explore CSC markers in ovarian cancer. This project aimed to explore the correlation between the expression of potential CSC maker Cacna2d1 and clinicopathological parameters in 238 epithelial ovarian cancer (EOC) samples. Immunohistochemically, positive Cacna2d1 expression was observed in 83.6% (199/238) of the EOC tumors, among which 107 tumors (44.9%) were highly positive and 92 (38.7%) tumors were weakly positive for the Cacna2d1 protein expression. Among the 158 serous carcinomas, the Cacna2d1 positivity was 148 (93.7%), in which 88 (55.7%) were highly positive, and 60 (38.0%) were weakly positive for the Cacna2d1 protein expression. Most strikingly, the Cacna2d1 was specifically expressed in the infiltration front areas of the EOC tumors. Statistical analyses showed that positive expression of Cacna2d1 was significantly associated with advanced FIGO stage (P<0.001), histological subtype (P=0.017) and tumor differentiation (P=0.015). Positive Cacna2d1 protein expression was significantly associated with poor overall survival (OS) and shorter progression free survival (PFS) in both total EOCs and serous carcinomas, although multivariate analyses did not reach statistical significance. In summary, our results suggest Cacna2d1 protein may play a crucial role in promoting aggressive EOC behavior and progression, and Cacna2d1 may serve as a novel predictive prognostic marker and a potential target for therapeutic intervention in EOCs.

The MYCN-HMGA2-CDKN2A pathway in non-small cell lung carcinoma--differences in histological subtypes.

BACKGROUND: Extensive research has increased our understanding of the molecular alterations needed for non-small cell lung cancer (NSCLC) development. Deregulation of a pathway including MYCN, HMGA2 and CDKN2A, with the participation of DICER1, is of importance in several solid tumours, and may also be of significance in the pathogenesis of NSCLC. METHODS: Gene expression of MYCN, HMGA2, CDKN2A and DICER1 were investigated with RT-qPCR in surgically resected NSCLC tumour tissue from 175 patients. Expression of the let-7 microRNA family was performed in 78 adenocarcinomas and 16 matching normal lung tissue samples using microarrays. The protein levels of HMGA2 were determined by immunohistochemistry in 156 tumour samples and the protein expression was correlated with gene expression. Associations between clinical data, including time to recurrence, and expression of mRNA, protein and microRNAs were analysed. RESULTS: Compared to adenocarcinomas, squamous cell carcinomas had a median 5-fold increase in mRNA expression of HMGA2 (p = 0.003). A positive correlation (r = 0.513, p < 0.010) between HMGA2 mRNA expression and HMGA2 protein expression was seen. At the protein level, 90% of the squamous cell carcinomas expressed high levels of the HMGA2 protein compared to 47% of the adenocarcinomas (p < 0.0001). MYCN was positively correlated with HMGA2 (p < 0.010) and DICER1 mRNA expression (p < 0.010), and the expression of the let-7 microRNAs seemed to be correlated with the genes studied. MYCN expression was associated with time to recurrence in multivariate survival analyses (p = 0.020). CONCLUSIONS: A significant difference in HMGA2 mRNA expression between the histological subtypes of NSCLC was seen with a higher expression in the squamous cell carcinomas. This was also found at the protein level, and we found a good correlation between the mRNA and the protein expression of HMGA2. Moreover, the expression of MYCN, HMGA2, and DICER1 seems to be correlated to each other and the expression of the let7-genes impacted by their expression. MYCN gene expression seems to be of importance in time to recurrence in this patient cohort with resected NSCLC.

The expression of aldehyde dehydrogenase 1 (ALDH1) in ovarian carcinomas and its clinicopathological associations: a retrospective study.

BACKGROUND: Aldehyde dehydrogenase 1 (ALDH1) is widely used as a specific cancer stem cell marker in a variety of cancers, and may become a promising target for cancer therapy. However, the role of its expression in tumor cells and the microenvironment in different cancers is still controversial. METHODS: To clarify the clinicopathological effect of ALDH1 expression in ovarian carcinoma, a series of 248 cases of paraffin-embedded formalin fixed ovarian carcinoma tissues with long term follow-up information were studied by immunohistochemistry. RESULTS: The immunostaining of ALDH1was variably detected in both tumor cells and the stromal cells, although the staining in tumor cells was not as strong as that in stromal cells. Statistical analyses showed that high ALDH1 expression in tumor cells was significantly associated with histological subtypes, early FIGO stage, well differentiation grade and better survival probability (p < 0.05). The expression of ALDH1 in the stromal cells had no clinicopathological associations in the present study (p > 0.05). CONCLUSIOMS: High expression of cancer stem cell marker ALDH1 in ovarian carcinoma cells may thus portend a favorable prognosis, but its expression in tumor microenvironment may have no role in tumor behavior of ovarian carcinomas. More studies are warranted to find out the mechanisms for this.

Expression of CDK1(Tyr15), pCDK1(Thr161), Cyclin B1 (total) and pCyclin B1(Ser126) in vulvar squamous cell carcinoma and their relations with clinicopatological features and prognosis.

Cyclin B1-CDK1 complex plays an important role in the regulation of cell cycle. Activation of Cyclin B1 and CDK1 and the formation of the complex in G2/M are under multiple regulations involving many regulators such as isoforms of 14-3-3 and CDC25 and Wee1. Abnormal expression of Cyclin B1 and CDK1 has been detected in various tumors. However, to our knowledge no previous study has investigated Cyclin B1 and CDK1 in vulvar cancer. Therefore, we evaluated the statuses of CDK1Tyr15, pCDK1Thr161, Cyclin B1 (total) and pCyclin B1Ser126 in 297 cases of vulvar squamous cell carcinomas by immunohistochemistry. Statistical analyses were performed to explore their clinicopathological and prognostic values. In at least 25% of tumor cases high expression of CDK1Tyr15, pCDK1Thr161, Cyclin B1 (total) and pCyclin B1Ser126 was observed, compared to the low levels in normal vulvar squamous epithelium. Elevated levels of CDK1Tyr15, pCDK1Thr161, Cyclin B1 (total) and pCyclin B1Ser126 were correlated with advanced tumor behaviors and aggressive features. Although CDK1Tyr15, pCDK1Thr161, Cyclin B1 (total) and pCyclin B1Ser126 could not be identified as prognostic factors, combinations of (pCDK1Thr161 C+N + 14-3-3σN), (pCDK1Thr161 C+N + 14-3-3ηC), (pCDK1Thr161 C+N + Wee1C) and (pCDK1Thr161 C+N + 14-3-3σN + 14-3-3ηC + Wee1C) were correlated with disease-specific survival (p = 0.036, p = 0.029, p = 0.042 and p = 0.007, respectively) in univariate analysis. The independent prognostic significance of (pCDK1Thr161 C+N + 14-3-3σN + 14-3-3ηC + Wee1C) was confirmed by multivariate analysis. In conclusion, CDK1Tyr15, pCDK1Thr161, Cyclin B1 (total) and pCyclin B1Ser126 may be involved in progression of vulvar squamous cell carcinoma. The combination of pCDK1Thr161, 14-3-3σ, 14-3-3η and Wee1 was a statistically independent prognostic factor.

Cellular localization of CIP2A determines its prognostic impact in superficial spreading and nodular melanoma.

Cancerous inhibitor of protein phosphatase 2A (CIP2A) is an important oncogene contributing to cancer progression partially by regulating cMYC and AKT. We examined CIP2A expression in cutaneous melanomas, its association with clinicopathological parameters and mapped molecular mechanisms regulated by CIP2A in vitro. CIP2A expression was analyzed by immunohistochemistry in 17 nevi, 132 primary melanomas and 49 metastases. Effects of siRNA-mediated down-regulation on proliferation, apoptosis and signaling pathways were assessed in melanoma cell lines. In superficial spreading melanomas (SSM), high nuclear CIP2A expression was associated with poor overall survival (OS) (P = 0.0018). Surprisingly, high cytoplasmic expression was related to improved relapse-free (P = 0.031) and OS (P = 0.014) in nodular melanomas (NM). In vitro experiments revealed that CIP2A can regulate proliferation and/or apoptosis partially through the PI3K/AKT pathway but also independently. In summary, CIP2A could represent a potential therapeutic target in SSM. However, in NM cytoplasmic CIP2A is associated with improved prognosis indicating that CIP2A has distinct, complex functions dependent on the molecular context and histological subtype. As seen in other cancer types, CIP2A can influence cMYC and AKT, but our data also suggest that in melanoma it has additional targets which need to be identified.

CD117 expression in fibroblasts-like stromal cells indicates unfavorable clinical outcomes in ovarian carcinoma patients.

The stem cell factor (SCF) receptor CD117 (c-kit), is widely used for identification of hematopoietic stem cells and cancer stem cells. Moreover, CD117 expression in carcinoma cells indicates a poor prognosis in a variety of cancers. However the potential expression in tumor microenvironment and the biological and clinical impact are currently not reported. The expression of CD117 was immunohistochemically evaluated in a serial of 242 epithelial ovarian cancer (EOC) cases. Thirty-eight out of 242 cases were CD117 positive in fibroblast-like stromal cells and 22 cases were positive in EOC cells. Four cases were both positive in fibroblast-like stromal cells and EOC cells for CD117. CD117 expression in fibroblast-like stromal cells in ovarian carcinoma was closely linked to advanced FIGO stage, poor differentiation grade and histological subtype (p<0.05), and it was significantly associated with poor overall survival (OS) and progression free survival (PFS) (Kaplan-Meier analysis; p<0.05, log-rank test). CD117 expression in ovarian carcinoma cells was not associated with these clinicopathological variables. The CD117 positive fibroblast-like stromal cells were all positive for mesenchymal stem/stromal cell (MSC) marker CD73 but negative for fibroblast markers fibroblast activation protein (FAP) and α smooth muscle actin (α-SMA), indicating that the CD117+/CD73+ fibroblast-like stromal cells are a subtype of mesenchymal stem cells in tumor stroma, although further characterization of these cells are needed. It is concluded herewith that the presence of CD117+/CD73+ fibroblast-like stromal cells in ovarian carcinoma is an unfavorable clinical outcome indication.

Differential in vivo tumorigenicity of distinct subpopulations from a luminal-like breast cancer xenograft.

Intratumor heterogeneity caused by genetic, phenotypic or functional differences between cancer cell subpopulations is a considerable clinical challenge. Understanding subpopulation dynamics is therefore central for both optimization of existing therapy and for development of new treatment. The aim of this study was to isolate subpopulations from a primary tumor and by comparing molecular characteristics of these subpopulations, find explanations to their differing tumorigenicity. Cell subpopulations from two patient derived in vivo models of primary breast cancer, ER+ and ER-, were identified. EpCAM+ cells from the ER+ model gave rise to tumors independently of stroma cell support. The tumorigenic fraction was further divided based on SSEA-4 and CD24 expression. Both markers were expressed in ER+ breast cancer biopsies. FAC-sorted cells based on EpCAM, SSEA-4 and CD24 expression were subsequently tested for differences in functionality by in vivo tumorigenicity assay. Three out of four subpopulations of cells were tumorigenic and showed variable ability to recapitulate the marker expression of the original tumor. Whole genome expression analysis of the sorted populations disclosed high similarity in the transcriptional profiles between the tumorigenic populations. Comparing the non-tumorigenic vs the tumorigenic populations, 44 transcripts were, however, significantly differentially expressed. A subset of these, 26 identified and named genes, highly expressed in the non-tumorigenic population, predicted longer overall survival (N = 737, p<0.0001) and distant metastasis free survival (DMFS) (N = 1379, p<0.0001) when performing Kaplan-Meier survival analysis using the GOBO online database. The 26 gene set correlated with longer DMFS in multiple breast cancer subgroups. Copy number profiling revealed no aberrations that could explain the observed differences in tumorigenicity. This study emphasizes the functional variability among cell populations that are otherwise genomically similar, and that the risk of breast cancer recurrence can only be eliminated if the tumorigenic abilities in multiple cancer cell subpopulations are inhibited.

Aldehyde dehydrogenase-1 predicts favorable prognosis in patients with vulvar squamous cell carcinoma.

BACKGROUNDS: Aldehyde dehydrogenase-1 (ALDH1) has been considered as a potential cancer stem cell marker in different types of cancer. In the present study, we investigated the expression of ALDH1 in vulvar squamous cell carcinoma, and evaluated its correlation with clinicopathological factors in patients suffering from this disease. MATERIALS AND METHODS: One hundred and fifty-four patients with vulvar squamous cell carcinoma, together with their verified histopathological and complete clinical data in Norway were included in the study. All paraffin-embedded samples of the primary vulvar carcinoma were recruited. The presence of ALDH1 was detected by immunohistochemistry and compared against commonly recognized prognostic factors. RESULTS: By immunohistochemical staining, the expression of ALDH1 was observed in 10/154 (6.5%) vulvar squamous cell carcinomas, while being extensively expressed in the suprabasal cells in normal vulvar epithelia from patients with benign gynecological disease and non-malignant epithelia adjacent to the tumor cells. In addition, ALDH1 was highly expressed in stromal fibroblasts, blood vessels and keratinized pearl of the carcinoma in all the samples. Patients with ALDH1-positive tumors had a significantly longer disease-specific survival (p=0.042). CONCLUSION: Contrary to the characteristics of cancer stem cells shown in other types of cancer with positive expression of ALDH1, the positive expression of ALDH1 in patients with vulvar squamous cell carcinoma indicates a significantly better prognosis. Furthermore, there is a trend that the expression of ALDH1 is associated with better histological differentiation.

Primary tumor vascularity, HIF-1α and VEGF expression in vulvar squamous cell carcinomas: their relationships with clinicopathological characteristics and prognostic impact.

BACKGROUND: Increased vascularity is a crucial event in the tumor progression and has prognostic significance in various cancers. However, the ultimate role of angiogenesis in the pathogenesis and clinical outcome of vulvar carcinoma patients is still not settled. METHODS: Tumor vascularity using CD34 stained slides measured by Chalkley counting method as well as hypoxia-inducible factor (HIF)-1α and vascular endothelial growth factor (VEGF) immunoexpression was examined in 158 vulvar squamous cell carcinomas. Associations between vascular Chalkley count, HIF-1α and VEGF expression and clinicopathological factors and clinical outcome were evaluated. RESULTS: High CD34 Chalkley count was found to correlate with larger tumor diameter (P = 0.002), deep invasion (P < 0.001) and HIF-1α (P = 0.04), whereas high VEGF expression correlate significantly with poor tumor differentiation (P = 0.007). No significant association between CD34 Chalkley counts and VEGF expression and disease-specific survival was observed. High HIF-1α expression showed better disease specific survival in both univariate and multivariate analyses (P = 0.001). CONCLUSIONS: A significant association between high tumor vascularity and larger tumor size as well as deeper tumor invasion suggests an important role of angiogenesis in the growth and progression of vulvar carcinomas. HIF-1α expression in vulvar carcinomas was a statistically independent prognostic factor.

Hypoxia-independent downregulation of hypoxia-inducible factor 1 targets by androgen deprivation therapy in prostate cancer.

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<.001). 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.

High expression of wee1 is associated with malignancy in vulvar squamous cell carcinoma patients.

BACKGROUND: Vulvar squamous cell carcinoma is a cancer form with increasing incidence rate and few treatment options. Wee1 is a central regulator of the G2/M DNA-damage checkpoint, and has in previous studies been described as a prognostic biomarker and a potential target for therapy in other cancer forms. METHODS: In the present study we analyzed the expression of Wee1 in a panel of 297 vulvar tumors by immunohistochemistry. Furthermore, siRNA transfections were carried out in two vulvar cancer cell lines (SW-954 and CAL-39) in order to study the effect on cell cycle distribution (flow cytometry) and proteins (western blot) involved in DNA damage response and apoptosis. RESULTS: Wee1 kinase is increased in vulvar squamous cell carcinomas, as compared to expression in normal epithelium, and a high Wee1 expression is associated with markers of malignancy, such as lymph node metastasis and poor differentiation. Our in vitro results showed that siRNA mediated Wee1 silencing only led to a modest reduction in viability, when examined in vulvar cancer cell lines. Nonetheless, a marked increase in DNA damages, as assessed by augmented levels of γ-H2AX, was observed in both cell lines in the absence of Wee1. CONCLUSIONS: Our results suggest that Wee1 may be involved in the progression of vulvar carcinomas. Based on our in vitro results, Wee1 is unlikely to function as a target for mono-treatment of these patients.

Expression of p15INK4b and p57KIP² and relationship with clinicopathological features and prognosis in patients with vulvar squamous cell carcinoma.

BACKGROUND: The cyclin-dependent kinase inhibitors p15(INK4b) and p57(KIP2) are important regulators of the cell cycle, and their abnormal expression has been detected in various tumors. However, little is known about the role of p15(INK4b) and p57(KIP2) in the pathogenesis of vulvar carcinoma, and the prognostic impact is still unknown. In our current study, we examined the expression of p15(INK4b) and p57(KIP2) in a large series of vulvar squamous cell carcinomas to elucidate the prognostic impact. METHODS: Expression of p15(INK4b) and p57(KIP2) were examined in 297 vulvar squamous cell carcinomas using immunohistochemistry. Both uni- and multivariate analysis of prognostic factors were performed, and correlations with clinicopathologic parameters were examined. RESULTS: Compared to the high levels of p15(INK4b) and p57(KIP2) in normal vulvar squamous epithelium, low levels of p15(INK4b) and p57(KIP2) were found in 82% and 44% of vulvar carcinomas, respectively. Low levels of p15(INK4b) and p57(KIP2) correlated significantly with malignant features, including large tumor diameter (p = 0.03 and p = 0.001, respectively) and increased invasiveness (p = 0.003 and p = 0.04, respectively). Although p15(INK4b) and p57(KIP2) levels could not be identified as prognostic markers, combined analysis of p14(ARF)/p15(INK4b)/p16(INK4a) showed that patients whose tumors expressed low levels of two or three of these INK4 proteins had a worse prognosis than those with only low levels of one or no protein (univariate analysis p = 0.02). The independent prognostic significance of these INK4 proteins was confirmed by multivariate analysis (p = 0.008). CONCLUSIONS: We show for the first time that p15(INK4b) and p57(KIP2) may be involved in the progression of vulvar carcinomas and the combined p14(ARF)/p15(INK4b)/p16(INK4a) status was a statistically independent prognostic factor.

Intermittent and continuous imatinib in a human GIST xenograft model carrying KIT exon 17 resistance mutation D816H.

BACKGROUND: Acquired resistance to imatinib is frequently caused by secondary KIT mutations. We have investigated the effects of imatinib in mice with human gastrointestinal stromal tumour (GIST) xenograft which harbours a primary exon 11 deletion mutation and a secondary imatinib resistance mutation D816H in exon 17. Such mutations are commonly present in imatinib-resistant GIST in humans. MATERIAL AND METHODS: The mice were randomly allocated to receive imatinib either continuously or intermittently. Dynamic (18)F-FDG PET was performed and blood volume fraction (vB), rate transfer constants (k1, k2, k3) and metabolic rate of (18)F-FDG (MRFDG) were computed using a three-compartment model. Tumours were evaluated for the mitotic rate and the expression of HIF-1α , caspase-3 and glucose transporters (GLUTs). RESULTS: Both intermittent and continuous imatinib delayed tumour growth significantly compared to controls, significantly in favour of the latter. k1 (representing perfusion, vascular permeability and binding of (18)F-FDG to the GLUTs) was significantly higher in the intermittent group compared to the continuous group, as was tumour GLUT-3 expression. k3 (representing internalisation of (18)F-FDG to the cells) and MR(FDG) were significantly lower. CONCLUSION: Imatinib delays GIST xenograft growth despite the presence of the D816H resistance mutation. The schedule of imatinib administration may influence tumour glucose uptake rate and metabolic rate.

High levels of genomic aberrations in serous ovarian cancers are associated with better survival.

Genomic instability and copy number alterations in cancer are generally associated with poor prognosis; however, recent studies have suggested that extreme levels of genomic aberrations may be beneficial for the survival outcome for patients with specific tumour types. We investigated the extent of genomic instability in predominantly high-grade serous ovarian cancers (SOC) using two independent datasets, generated in Norway (n = 74) and Australia (n = 70), respectively. Genomic instability was quantified by the Total Aberration Index (TAI), a measure of the abundance and genomic size of copy number changes in a tumour. In the Norwegian cohort, patients with TAI above the median revealed significantly prolonged overall survival (p<0.001) and progression-free survival (p<0.05). In the Australian cohort, patients with above median TAI showed prolonged overall survival (p<0.05) and moderately, but not significantly, prolonged progression-free survival. Results were confirmed by univariate and multivariate Cox regression analyses with TAI as a continuous variable. Our results provide further evidence supporting an association between high level of genomic instability and prolonged survival of high-grade SOC patients, possibly as disturbed genome integrity may lead to increased sensitivity to chemotherapeutic agents.

Identification of eight candidate target genes of the recurrent 3p12-p14 loss in cervical cancer by integrative genomic profiling.

The pathogenetic role, including its target genes, of the recurrent 3p12-p14 loss in cervical cancer has remained unclear. To determine the onset of the event during carcinogenesis, we used microarray techniques and found that the loss was the most frequent 3p event, occurring in 61% of 92 invasive carcinomas, in only 2% of 43 high-grade intraepithelial lesions (CIN2/3), and in 33% of 6 CIN3 lesions adjacent to invasive carcinomas, suggesting a role in acquisition of invasiveness or early during the invasive phase. We performed an integrative DNA copy number and expression analysis of 77 invasive carcinomas, where all genes within the recurrent region were included. We selected eight genes, THOC7, PSMD6, SLC25A26, TMF1, RYBP, SHQ1, EBLN2, and GBE1, which were highly down-regulated in cases with loss, as confirmed at the protein level for RYBP and TMF1 by immunohistochemistry. The eight genes were subjected to network analysis based on the expression profiles, revealing interaction partners of proteins encoded by the genes that were coordinately regulated in tumours with loss. Several partners were shared among the eight genes, indicating crosstalk in their signalling. Gene ontology analysis showed enrichment of biological processes such as apoptosis, proliferation, and stress response in the network and suggested a relationship between down-regulation of the eight genes and activation of tumourigenic pathways. Survival analysis showed prognostic impact of the eight-gene signature that was confirmed in a validation cohort of 74 patients and was independent of clinical parameters. These results support the role of the eight candidate genes as targets of the 3p12-p14 loss in cervical cancer and suggest that the strong selection advantage of the loss during carcinogenesis might be caused by a synergetic effect of several tumourigenic processes controlled by these targets.

Dynamic (18) F-FDG PET for Assessment of Tumor Physiology in Two Breast Carcinoma Xenografts.

PURPOSE: To compare dynamic 2-deoxy-2-[(18) F]fluoro-D-glucose positron emission tomography ((18) F-FDG PET) parameters in two selected human breast cancer xenografts and to evaluate associations with immunohistochemistry and histology. PROCEDURES: Dynamic (18) F-FDG PET of luminal-like MAS98.06 and basal-like MAS98.12 xenografts was performed, and the compartmental transfer rates (k 1 ,k 2 ,k 3 ), blood volume fraction (v B ) and metabolic rate of (18) F-FDG(MR FDG ) were estimated from pharmacokinetic model analysis. After sacrifice, analyses of hypoxia (pimonidazole), proliferation (Ki-67), vascularization (CD31), glucose transport receptor (GLUT1) and necrosis (HE) was performed. The level of hexokinase 2 (HK2) was estimated from Western blot analysis. RESULTS: The (18) F-FDG uptake curves for the two xenografts were significantly different (p < 0.05). k 1 and v B were higher for MAS98.12 (p < 0.01), while k 3 was higher for MAS98.06 (p < 0.01). MAS98.12 had a higher fraction of stromal tissue and higher microvessel density (MVD), and it was less necrotic and hypoxic than MAS98.06. MAS98.12 had stronger positive GLUT1 staining and lower Ki-67 than MAS98.06. In both models significant correlations were found between k 1 and the GLUT1 score, between k 3 and the level of HK2, and between v B and MVD. CONCLUSIONS: Significant differences in dynamic (18) F-FDG parameters between the two human breast cancer xenografts were found. The differences could be explained by underlying histological and physiological characteristics.