Showing papers by "Paul J. van Diest published in 2015"

Identification of the DEAD box RNA helicase DDX3 as a therapeutic target in colorectal cancer.

Abstract: Identifying druggable targets in the Wnt-signaling pathway can optimize colorectal cancer treatment. Recent studies have identified a member of the RNA helicase family DDX3 (DDX3X) as a multilevel activator of Wnt signaling in cells without activating mutations in the Wnt-signaling pathway. In this study, we evaluated whether DDX3 plays a role in the constitutively active Wnt pathway that drives colorectal cancer. We determined DDX3 expression levels in 303 colorectal cancers by immunohistochemistry. 39% of tumors overexpressed DDX3. High cytoplasmic DDX3 expression correlated with nuclear β-catenin expression, a marker of activated Wnt signaling. Functionally, we validated this finding in vitro and found that inhibition of DDX3 with siRNA resulted in reduced TCF4-reporter activity and lowered the mRNA expression levels of downstream TCF4-regulated genes. In addition, DDX3 knockdown in colorectal cancer cell lines reduced proliferation and caused a G1 arrest, supporting a potential oncogenic role of DDX3 in colorectal cancer. RK-33 is a small molecule inhibitor designed to bind to the ATP-binding site of DDX3. Treatment of colorectal cancer cell lines and patient-derived 3D cultures with RK-33 inhibited growth and promoted cell death with IC50 values ranging from 2.5 to 8 μM. The highest RK-33 sensitivity was observed in tumors with wild-type APC-status and a mutation in CTNNB1. Based on these results, we conclude that DDX3 has an oncogenic role in colorectal cancer. Inhibition of DDX3 with the small molecule inhibitor RK-33 causes inhibition of Wnt signaling and may therefore be a promising future treatment strategy for a subset of colorectal cancers.

Prognostic significance of the EGFR pathway in nasopharyngeal carcinoma: a systematic review and meta-analysis.

Abstract: Objective: To evaluate the prognostic impact of the EGF receptor (EGFR) pathway molecules and assess their clinical usefulness. Methods: We conducted a systematic review. Pubmed and EMBASE were searched January 2014. The prognostic relevance of EGFR, JAK, PI3K, PIK3CA, STAT3, STAT5, PTEN, AKT, mTOR, GRB2, SOS, RAF, RAS, MAPK, ERK, MEK and CCND1 in nasopharyngeal carcinoma was assessed. The outcomes considered were overall survival, disease-free survival and tumor-node-metastasis stage. Twenty-two studies were included. Risk of bias was evaluated. Meta-analysis for which pooled hazard ratios and 95% CIs were calculated. Conclusion: EGFR overexpression predicts a worse overall survival and disease-free survival in nasopharyngeal carcinoma, but no specific causal pathway molecule could be identified.

FGFR4 Is a Potential Predictive Biomarker in Oral and Oropharyngeal Squamous Cell Carcinoma

Abstract: Objective: The aim of this study was to investigate whether fibroblast growth factor receptor 4 (FGFR4) could serve as a potential therapeutic target, prognostic biomarker or biomarker predicting radiotherapy sensitivity in oral squamous cell carcinoma (OSCC) and oropharyngeal squamous cell carcinoma (OPSCC). Methods: FGFR4 immunohistochemistry and FGFR4/CEN5q FISH were performed on tissue microarrays from 212 OSCC and 238 OPSCC patients. FGFR4 genotypes were determined by PCR and DNA sequencing in 76 random OPSCC samples. The response to radiotherapy was evaluated 3 months after the last radiotherapy treatment session by a head and neck radiation oncologist and/or surgeon during clinic visits. The results were correlated to overall survival and response to radiotherapy. Results: The FGFR4 protein was overexpressed in 64% (153/238) of OPSCCs and 41% (87/212) of OSCCs. The FGFR4 gene was amplified in 0.47% (1/212) of OSCCs and 0.42% (1/238) of OPSCCs, and the FGFR4 Gly388Arg polymorphism was detected in 62% (47/76) of OPSCCs. FGFR4 protein expression, FGFR4 gene copy numbers and FGFR4 genotypes were not related to overall survival or response to radiotherapy in OSCC or OPSCC. Conclusion: FGFR4 is frequently overexpressed in OSCC and OPSCC in the absence of gene amplification, and may serve as a potential predictive marker for FGFR4-directed targeted therapy in OSCC and OPSCC.

The accuracy of target delineation in laryngeal and hypopharyngeal cancer

Abstract: Background and purpose. To determine the spatial correspondence between the gross tumor volume (GTV) delineated on computer tomography (CT) and the actual tumor on histopathology.Material and methods. Sixteen patients with T3 or T4 laryngeal or hypopharyngeal cancer underwent a CT scan before total laryngectomy. The GTV was delineated on CT by three independent observers and by consensus between the three observers. After surgery, whole-mount hematoxylin-eosin stained (H&E) sections were obtained. One pathologist delineated the tumor in the H&E sections (tumorH&E). The reconstructed specimen was registered to the CT scan in order to compare the GTV to the tumorH&E in three dimensions. The overlap between the GTV and the tumorH&E was calculated and the distance between the volumes was determined.Results. Tumor tissue was delineated in 203 of 516 H&E sections. For 14 patients a detailed analysis could be performed. The GTV volume was on average 1.7 times larger than the volume of the tumorH&E. The m...

Effects of magnetic resonance-guided high-intensity focused ultrasound ablation on bone mechanical properties and modeling.

Abstract: Magnetic resonance-guided high-intensity focused ultrasound (MR-HIFU) is a promising technique for palliative treatment of bone pain. In this study, the effects of MR-HIFU ablation on bone mechanics and modeling were investigated. A total of 12 healthy rat femurs were ablated using 10 W for 46 ± 4 s per sonication with 4 sonications for each femur. At 7 days after treatments, all animals underwent MR and single photon emission computed tomography/computed tomography (SPECT/CT) imaging. Then, six animals were euthanized. At 1 month following ablations, the remaining six animals were scanned again with MR and SPECT/CT prior to euthanization. Thereafter, both the HIFU-treated and contralateral control bones of three animals from each time interval were processed for histology, whereas the remaining bones were subjected to micro-CT (μCT), three-point bending tests, and micro-finite element (micro-FE) analyses. At 7 days after HIFU ablations, edema formation around the treated bones coupled with bone marrow and cortical bone necrosis was observed on MRI and histological images. SPECT/CT and μCT images revealed presence of bone modeling through an increased uptake of 99mTc-MDP and formation of woven bone, respectively. At 31 days after ablations, as illustrated by imaging and histology, healing of the treated bone and the surrounding soft tissue was noted, marked by decreased in amount of tissue damage, formation of scar tissue, and sub-periosteal reaction. The results of three-point bending tests showed no significant differences in elastic stiffness, ultimate load, and yield load between the HIFU-treated and contralateral control bones at 7 days and 1 month after treatments. Similarly, the elastic stiffness and Young’s moduli determined by micro-FE analyses at both time intervals were not statistically different. Multimodality imaging and histological data illustrated the presence of HIFU-induced bone damage at the cellular level, which activated the bone repair mechanisms. Despite that, these changes did not have a mechanical impact on the bone.

Tracing differences between male and female breast cancer : Both diseases own a different biology

Abstract: Aims: Male breast cancer (MBC) is a rare and poorly characterized disease. In the present study we used a novel biomathematical model to further characterize MBC and to identify differences between male and female breast cancer (FBC). Methods and results: A total of 134 cases of MBC were stained immunohistochemically for 13 key oncoproteins, and staining percentages were used in a mathematical model to identify dependency patterns between these proteins. The results were compared with a large group of FBC (n = 728). MBC and FBC clearly differed on the molecular level. In detail, the results suggest a different role for progesterone receptor (PR) compared to oestrogen receptor (ER) in MBC, while in FBC ER and PR show a similar pattern. In addition, Androgen receptor (AR) seems to be a more powerful effector in MBC. Grades 1 and 2 tumours were clearly separated from grade 3 tumours, and luminal types A and B tumours also showed a different pattern. Conclusions: Defined morphological and molecular phenotypes can be identified in MBC, but these seem to be the result of different molecular mechanisms and perhaps multiple genetic pathways, as characterized previously in FBC, emphasizing the rising concept that MBC and FBC should be regarded as different and unique diseases.

Promoter hypermethylation profiling of distant breast cancer metastases

Abstract: Promoter hypermethylation of tumor suppressor genes seems to be an early event in breast carcinogenesis and is potentially reversible. This makes methylation a possible therapeutic target, a marker for treatment response and/or a prognostic factor. Methylation status of 40 tumor suppressor genes was compared between 53 primary breast tumors and their corresponding metastases to brain, lung, liver, or skin. In paired analyses, a significant decrease in methylation values was seen in distant metastases compared to their primaries in 21/40 individual tumor suppressor genes. Furthermore, primary tumors that metastasized to the liver clustered together, in line with the finding that primary breast carcinomas that metastasized to the brain, skin, or lung, showed higher methylation values in up to 27.5 % of tumor suppressor genes than primary carcinomas that metastasized to the liver. Conversion in methylation status of several genes from the primary tumor to the metastasis had prognostic value, and methylation status of some genes in the metastases predicted survival after onset of metastases. Methylation levels for most of the analyzed tumor suppressor genes were lower in distant metastases compared to their primaries, pointing to the dynamic aspect of methylation of these tumor suppressor genes during cancer progression. Also, specific distant metastatic sites seem to show differences in methylation patterns, implying that hypermethylation profiles of the primaries may steer site-specific metastatic spread. Lastly, methylation status of the metastases seems to have prognostic value. These promising findings warrant further validation in larger patient cohorts and more tumor suppressor genes.

Expression of Connective Tissue Growth Factor in Male Breast Cancer : Clinicopathologic Correlations and Prognostic Value

Abstract: Connective tissue growth factor (CTGF/CCN2) is a member of the CCN family of secreted proteins that are believed to play an important role in the development of neoplasia. In particular, CTGF has been reported to play an important role in mammary tumorigenesis and to have prognostic value in female breast cancer (FBC). The aim of the present study was to investigate clinicopathologic correlations and prognostic value of CTGF in male breast cancer (MBC) and to compare these findings with FBC. For this, we studied CTGF protein expression by immunohistochemistry in 109 MBC cases and 75 FBC cases. In MBC, stromal CTGF expression was seen in the majority of the cases 78% (85/109) with high expression in 31/109 cases (28.4%), but expression in tumor cells was only seen in 9.2% (10/109) of cases. High stromal CTGF expression correlated with high grade and high proliferation index (>15%) assessed by MIB-1 immunohistochemical staining. CTGF expression in tumor epithelial cells did not correlate with any of the clinicopathologic features. In FBC, stromal CTGF expression positively correlated with mitotic count and tumor CTGF expression was associated with triple negative status of the tumor (p = 0.002). Neither stromal nor tumor epithelial cell CTGF expression had prognostic value in MBC and FBC. In conclusion, stromal CTGF expression was seen in a high percentage of MBC and was correlated with high grade and high proliferation index. In view of the important role of the microenvironment in cancer progression, this might suggest that stromal CTGF could be an interesting target for novel therapies and molecular imaging. However, the lack of association with prognosis warrants caution. The potential role of CTGF as a therapeutic target for triple negative FBC deserves to be further studied.

Improved cytodiagnostics and quality of patient care through double reading of selected cases by an expert cytopathologist.

Abstract: Double reading may be a valuable tool for improving the quality of patient care by restoring diagnostic errors before final sign-out, but standard double reading would significantly increase costs of pathology. The aim of this study was to assess the added value of routine double reading of defined categories of clinical cytology specimens by specialized cytopathologists. Specialized cytopathologists routinely re-diagnosed blinded defined categories of clinical cytology specimens that had been signed out by routine pathologists from January 2012 up to December 2013. Major and minor discordance rates between initial and expert diagnoses were determined, and both diagnoses were validated by comparison with same-site histological follow-up. Initial and expert diagnoses were concordant in 131/218 specimens (60.1 %). Major and minor discordances were present in 28 (12.8 %) and 59 (27.1 %) specimens, respectively. Pleural fluid, thyroid and urine specimens showed the highest major discordance rates (19.4, 19.2 and 16.7 %, respectively). Histological follow-up (where possible) supported the expert diagnosis in 95.5 % of specimens. Our implemented double reading strategy of defined categories of cytology specimens showed major discordance in 12.8 % of specimens. The expert diagnosis was supported in 95.5 % of discordant cases where histological follow-up was available. This indicates that this double reading strategy is worthwhile and contributes to better cytodiagnostics and quality of patient care, especially for suspicious pleural fluid, thyroid and urine specimens. Our results emphasize that cytopathology is a subspecialization of pathology and requires specialized cytopathologists.

Copy number profiling by array comparative genomic hybridization identifies frequently occurring BRCA2-like male breast cancer

Abstract: Genomic aberrations can be used to subtype breast cancer. In this study, we investigated DNA copy number (CN) profiles of 69 cases of male breast cancer (MBC) by array comparative genomic hybridization (aCGH) to detect recurrent gains and losses in comparison with female breast cancers (FBC). Further, we classified these profiles as BRCA1-like, BRCA2-like or non-BRCA-like profiles using previous classifiers derived from FBC, and correlated these profiles with pathological characteristics. We observed large CN gains on chromosome arms 1q, 5p, 8q, 10p, 16p, 17q, and chromosomes 20 and X. Large losses were seen on chromosomes/chromosome arms 1p, 6p, 8p, 9, 11q, 13, 14q, 16q, 17p, and 22. The pattern of gains and losses in estrogen receptor positive (ER+) MBC was largely similar to ER+ FBC, except for gains on chromosome X in MBC, which were uncommon in FBC. Out of 69 MBC patients, 15 patients (22%) had a BRCA2-like profile, of which 2 (3%) were also BRCA1-like. One patient (1%) was only BRCA1-like; the remaining 53 (77%) patients were classified as non-BRCA-like. BRCA2-like cases were more often p53 accumulated than non-BRCA-like cases (P = 0.014). In conclusion, the pattern of gains and losses in ER+ MBC was largely similar to that of its ER+ FBC counterpart, except for gains on chromosome X in MBC, which are uncommon in FBC. A significant proportion of MBC has a BRCA2-like aCGH profile, pointing to a potentially hereditary nature, and indicating that they could benefit from a drug regimen targeting BRCA defects as in FBC.

Methylation biomarkers for pleomorphic lobular breast cancer - a short report.

Abstract: Background Pleomorphic invasive lobular cancer (pleomorphic ILC) is a rare variant of ILC that is characterized by a classic ILC-like growth pattern combined with an infiltrative ductal cancer (IDC)-like high nuclear atypicality. There is an ongoing discussion whether pleomorphic ILC is a dedifferentiated form of ILC or in origin an IDC with a secondary loss of cohesion. Since gene promoter hypermethylation is an early event in breast carcinogenesis and thus may provide information on tumor progression, we set out to compare the methylation patterns of pleomorphic ILC, classic ILC and IDC. In addition, we aimed at analyzing the methylation status of pleomorphic ILC.

Chromosome 17 copy number changes in male breast cancer

Abstract: Background Overall, HER2-amplified female breast cancer (FBC) is associated with a high grade, an aggressive phenotype and a poor prognosis. In male breast cancer (MBC) amplification of HER2, located on chromosome 17, occurs at a lower frequency than in FBC, where it is part of complex rearrangements. So far, only few studies have addressed the occurrence of chromosome 17 alterations in small MBC cohorts.

Brief fixation does not hamper the reliability of Ki67 analysis in breast cancer core-needle biopsies: a double-centre study

Abstract: Aims: Immunohistochemical assessment of Ki67 expression in core-needle biopsies (CNBs) is increasingly playing a role in therapeutic decision-making for breast cancer patients. Within the framework of same-day diagnosis of breast lesions, fixation times are markedly decreased. We therefore attempted to validate Ki67 analysis in briefly fixed breast cancer CNBs. Methods and results: CNBs of 136 consecutive patients with invasive breast cancer diagnosed through the same-day diagnosis programme of both the University Medical Centre Utrecht (UMCU) and the Radboud University Medical Centre (RUMC) were included. CNBs were fixed in formaldehyde for approximately 45 min at the UMCU and for between 60 and 90 min at the RUMC. Immunohistochemistry for Ki67 expression was compared between the briefly fixed CNBs and conventionally fixed resection specimens of the same tumour. The overall agreement between CNBs and resections was 122 of 142 (85.9%) (κ = 0.71; 95% CI 0.60-0.83). Positive and negative predictive values were 79.7% (95% CI 67-88%) and 91.0% (95% CI 82-96%), respectively. Conclusions: Overall agreement for Ki67 expression was good between briefly fixed CNBs and conventionally fixed resection specimens, and within the range of studies comparing conventionally fixed CNBs and resections.

Abstract 3570: Identification of the DEAD box RNA helicase DDX3 as a therapeutic target in colorectal cancer

Abstract: Over 85% of colorectal cancers is driven by aberrations in the Wnt-signaling pathway. Thus, identifying druggable targets in this pathway can be beneficial for optimizing colorectal cancer treatment. Within this context, a member of the RNA helicase gene family, DDX3, has been identified to exhibit oncogenic properties in breast and lung carcinomas as well as medulloblastomas. Notably, recent studies have identified DDX3 as a multilevel activator of Wnt-signaling in both normal and transformed cells without activating mutations in the Wnt signaling pathway. In this study, we evaluated whether DDX3 also plays a role in the constitutionally activated Wnt-signaling that drives colorectal cancer and therefore could be a potential therapeutic target in this cancer type. To determine if DDX3 is expressed in colorectal cancers, we immunohistochemically stained a cohort of 303 Dutch and German colorectal cancer patients. We found 40.4% of these tumors to overexpress DDX3 in comparison to the surrounding normal tissue. DDX3 expression was found predominantly in the cytoplasm and occasionally in the nucleus. High cytoplasmic DDX3 expression correlated with nuclear Beta-catenin expression, a marker of activated Wnt-signaling. The presence of nuclear DDX3 expression correlated with shorter overall survival (HR = 2.38, 95% CI 1.45-3.93, p With respect to targeting DDX3, we developed a small molecule inhibitor of DDX3, referred to as RK-33. RK-33 is designed to bind to the ATP-binding site of DDX3 and abrogate its functional activity. As proof of principle, we demonstrated that RK-33 binds preferentially to DDX3 and not to DDX5 and DDX17, other members of the RNA helicase family. Moreover, RK-33 inhibited the helicase activity in an in vitro assay. Furthermore, treatment of colorectal cancer cell lines and patient derived 3D- tumor cell cultures indicated that RK-33 inhibits growth and promotes cell death with IC-50 values ranging from 2.5 to 8 uM. To further elucidate the mechanism of RK-33, we studied if inhibition of DDX3 with RK-33 could cause inhibition of Wnt-signaling in colorectal cancer cell lines. Treatment with RK-33 indeed resulted in reduced TCF-reporter activity and lowered the mRNA expression levels of the Wnt-signaling downstream target genes AXIN-2, C-MYC, CCND1 and BIRC5A. Overall, we conclude that DDX3 has an oncogenic role in colorectal cancer. Inhibition of DDX3 with the small molecule inhibitor RK-33 causes potent inhibition of Wnt-signaling and is a promising future treatment strategy in colorectal cancer. Citation Format: Marise R. Heerma van Voss, Farhad Vesuna, Kari Trumpi, Justin Brilliant, Liudmila L. Kodach, Folkert H.M. Morsink, G. Johan A. Offerhaus, Horst Buerger, Elsken van der Wall, Paul J. van Diest, Venu Raman. Identification of the DEAD box RNA helicase DDX3 as a therapeutic target in colorectal cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3570. doi:10.1158/1538-7445.AM2015-3570

Hypoxia-inducible factor 1-α in chronic gastrointestinal ischemia

Abstract: Chronic gastrointestinal ischemia (CGI) is the result of decreased mucosal perfusion. Typical histological characteristics are lacking which hamper its early diagnosis. Hypoxia-inducible factor-1α (HIF-1α) is expressed under acute hypoxia. We investigated HIF-1α expression in chronic ischemic and inflammatory conditions of the human gastrointestinal (GI) tract. Immunohistochemical expression of HIF-1α was analyzed in 61 patients, including patients with CGI, Helicobacter pylori gastritis, ischemic colitis (IC), infectious colitis, and inflammatory bowel disease (IBD), and 22 controls. HIF-1α expression in >10 % of the cells was regarded as positive staining, and expression <10 % of the cells was considered as negative staining. In the upper GI tract, HIF-1α expression was found in 5/20 CGI patients, but not in controls (p = 0.08). The sensitivity and specificity of HIF-1α expression for diagnosing CGI were 25 and 84%, respectively. In the lower GI tract, HIF-1α was expressed in all patients with IC and infectious colitis and in a majority of IBD patients as well as in 7/12 controls. The sensitivity and specificity of HIF-1α for diagnosing IC were 100 and 51%, respectively. HIF-1α expression was more often (p = 0.02) observed in patients with histological signs of inflammation in the lower GI tract. HIF-1α is expressed in acute and chronic ischemic tissue, but also in normal colon tissue and inflammatory disorders.

Brief fixation and hormone receptor expression in breast cancer: authors' reply.

Abstract: To the Editor: Estrogen receptor (ER) expression has been shown to be comparable in needle core biopsy and surgical specimens of breast cancer. Currently, a minimum of 6 hours of fixation is recommended.1 Kalkman et al2 compared ER expression in breast cancers in core biopsies fixed for about 45 minutes with that in the corresponding surgical specimen. For ER there was agreement in 73 of 74 tumors. The 1 discordant case showed strong staining in the core and weak staining in the surgical specimen, which would probably be scored as positive with the 1% threshold recommended by ASCO/CAP.1 We suggest that important questions need to be addressed before this result can be translated into routine practice. The tumors may not be representative. The overall positive rate for ER in the study is 96%. The average positive rate in the United Kingdom is 82%.3 Weakly positive tumors are a source of potential discordance between core and excision specimens,4 and this study does not include any. The previous studies by Apple et al5 and Ibarra et al6 only investigated strongly positive tumors. Kalkman and colleagues found that the mean percentage of positive cells was slightly lower in the surgical than in the core specimen (87% vs. 91%). Were there any tumors with a marked discordance? It would also be useful to know whether the surgical specimens were incised immediately to ensure good fixation. We agree with the authors’ suggestion that ER and PR expression in briefly fixed cores should be compared with cores fixed for at least 6 hours. It is important that such a study includes a reasonable number of weakly positive tumors. Similar studies for HER2 should also be performed. We suggest that the current practice of fixation for at least 6 hours based on the study of Goldstein et al should not be changed until there is further evidence to support a shorter duration of fixation.