Current Evidence in the Management of Premalignant Cystic Lesions of the Pancreas in Patients Undergoing Liver Transplantation.

ABSTRACT: Incidental premalignant pancreatic cystic lesions (pPCLs) are increasingly being detected in patients undergoing orthotopic liver transplantation (OLT). The impact of chronic immunosuppression upon pPCLs may elevate risk of progression to pancreatic cancer. This systematic review assesses prevalence, outcome, and management of pPCLs in patients undergoing OLT. Systematic literature searches were performed in accordance with Cochrane review guidelines. Data on 658 patients were identified from 13 articles. Median age was 59 years with a prevalence of 6.2%. Most studies focused on branch-duct intraductal papillary mucinous neoplasms. Average cyst size at diagnosis was 10.3 mm. Six patients (0.9%) underwent pancreatic resection, post-OLT, for suspected "worrisome features" on imaging. One death was due to pancreatic-related cancer, post-OLT. Based on the review, the authors suggest the following: (1) patients with pPCLs undergoing OLT, without "worrisome features," should be followed conservatively; (2) presence of pPCLs alone should not preclude eligibility for OLT, nor should chronic immunosuppression be altered; (3) follow-up should parallel standard approach applied in immunocompetent patients, as development of "worrisome features" of cancer is rare and does not appear to be hastened by immunosuppression; (4) resection is recommended for surgically fit patients without portal hypertension that develop "worrisome features."

Liver Transplantation in Locally Unresectable, Undifferentiated Embryonal Cell Sarcoma.

BACKGROUND: Undifferentiated embryonal cell sarcoma (UESL) of the liver is the third most common malignant liver disease of childhood presenting as a rapidly enlarging intraabdominal mass. This systematic review explores the practicality of liver transplantation as a viable option in the treatment armamentarium for locally advanced undifferentiated embryonal cell sarcoma. METHODS: A systematic review of the literature was performed using Medline and Embase, from inception of databases to December 31, 2018. Keywords and MeSH headings used were embryonal sarcoma, mesenchymal sarcoma, and liver transplant. Reviews and manuscripts with incomplete data were excluded. RESULTS: Twenty-eight patients had orthotopic liver transplantation (OLT) as a curative treatment option. The median age at presentation was 8 and 27 years in the pediatric and adult population, respectively, with a similar male to female ratio. A majority of the patients presented with abdominal pain, palpable mass, and a normal alpha-feto-protein. The median tumor size was 15 cm mainly affecting the right lobe (62%) of the liver. Eighty-two percent of the patients underwent primary OLT and 5 patients had salvage OLT. One death (3.6%) was due to initial misdiagnosis and management for hepatoblastoma. Recurrence was noted in 7.1% of the population. The median follow-up was noted to be 28.5 months. The documented survival rate post-liver transplant for UESL was 96%. CONCLUSIONS: Based on available data and the very positive results therein, liver transplantation is a practical and justifiable use of a scarce resource as a treatment option for locally unresectable, undifferentiated embryonal cell sarcoma. The authors propose (accepting existence of different proposals) neoadjuvant therapy before curative resection, and if not achievable, then liver transplantation followed by adjuvant chemotherapy is an option for suitable candidates. For recurrent tumors after surgical resection, adjuvant therapy with salvage liver transplantation is an option.

Using the VALGENT-3 framework to assess the clinical and analytical performance of the RIATOL qPCR HPV genotyping assay.

BACKGROUND AND OBJECTIVE: The VALGENT framework is developed to assess the clinical performance of HPV tests that offer genotyping capability. Samples from the VALGENT-3 panel are used to identify an optimal viral concentration threshold for the RIATOL qPCR HPV genotyping assay (RIATOL qPCR) to assure non-inferior accuracy to detect high-grade cervical intraepithelial neoplasia (CIN), compared to Qiagen Hybrid Capture 2 (HC2), a standard comparator test validated for cervical cancer screening. STUDY DESIGN: The VALGENT-3 panel comprised 1300 samples from women participating in the Slovenian cervical cancer screening programme, enriched with 300 samples from women with abnormal cytology. In follow- up, 126 women were diagnosed with CIN2+ (defined as diseased) and 1167 women had two consecutive negative Pap smears (defined as non-diseased). All 1600 samples were analyzed with the RIATOL qPCR. Viral concentration was expressed as viral log10 of the number of copies/ml. A zone of viral concentration cut-offs was defined by relative ROC analysis where the sensitivity and specificity were not inferior to HC2. RESULTS: The RIATOL qPCR had a sensitivity and specificity for CIN2+ of 97.6% (CI: 93.2-99.5%) and 85.1% (CI: 82.9-87.1%), respectively, when the analytical cut off was used. At a cut off of 6.5, RIATOL qPCR had a sensitivity of 96.0% (CI: 91.0-98.7%) and a specificity of 89.5% (87.6-91.2%). At optimized cut off, accuracy of the qPCR was non-inferior to the HC2 with a relative sensitivity of 1.00 [CI: 0.95-1.05 (p = 0.006)] and relative specificity of 1.00 [CI: 0.98-1.01 (p = 0.0069)]. CONCLUSIONS: The RIATOL qPCR has a high sensitivity and specificity for the detection of CIN2 + . By using a fixed cut-off based on viral concentration, the test is non-inferior to HC2. HPV tests that provide viral concentration measurements or other quantifiable signals allow flexibility to optimize accuracy required for cervical cancer screening.

VALHUDES: A protocol for validation of human papillomavirus assays and collection devices for HPV testing on self-samples and urine samples.

BACK GROUND: Systematic reviews have concluded that hrHPV DNA testing using target-amplification tests is as accurate on vaginal self-samples as on clinician-taken specimens for the detection of cervical precancer. However, insufficient evidence is available for specific HPV assay/self-sample device combinations. OBJECTIVES: The VALHUDES protocol is designed as a diagnostic test accuracy study that aims to compare the clinical sensitivity and specificity of particular hrHPV assay(s) on vaginal self-samples and first-void-urine, collected in agreement with standardized protocols, with hrHPV testing on matched clinician-taken samples. STUDY DESIGN: Five hundred enrolled women referred to a colposcopy clinic are invited to collect a first-void urine sample and one or more vaginal self-samples with particular devices before collection of a cervical sample by a clinician. Sample sets are subsequently analysed in a laboratory accredited for HPV testing. Disease verification for all enrolled patients is provided by colposcopy combined with histological assessment of biopsies. RESULTS: A first VALHUDES study has started in Belgium in December 2017 with enrolment from four colposcopy centres. The following assays are foreseen to be evaluated: RealTime High Risk HPV assay (Abbott), cobas-4800 and -6800 (Roche), Onclarity (BD), Xpert HPV (Cepheid) and Anyplex II HPV HR (Seegene). CONCLUSION: Given empirical evidence that the relative accuracy of HPV-testing on self- vs clinician-samples is robust across clinical settings, the VALHUDES protocol offers a framework for validation of HPV assay/self-sample device combinations that can be translated to a primary screening setting.

Management and diagnosis of gallbladder polyps: a systematic review.

PURPOSE: Recommendation for management of gallbladder polyps (GBPs) >1 cm is cholecystectomy. No consensus exists on management of GBPs <1 cm. This systematic review examines current evidence on management of GBPs. METHODS: MEDLINE, EMBASE and Cochrane library databases were searched from January 1991 to June 2013 using specified terms. A predefined protocol for data extraction was used to retrieve specified end points. RESULTS: Literature search yielded 43 manuscripts with a dataset of 11,685 patients with GBPs. M:F ratio was 1.3:1. Average age (range) was 49 years (32-83). Patients with malignant GBPs had an average (range) age of 58 (50-66) years with M:F ratio of 0.78:1. Cholesterol polyps constituted 60.5% of GBPs followed by adenomas (15.2%) and cancer (11.6%). Malignant GBPs ≥1 cm, <1 cm and <5 mm constituted 8.5, 1.2 and 0% of GBPs, respectively. Majority of patients requiring surgical intervention had laparoscopic cholecystectomy. CONCLUSIONS: Presently employed policy of cholecystectomy for GBPs >1 cm is appropriate. For GBPs <1 cm, the authors propose (accepting existence of differing proposals) the following: 1. Surveillance may not be needed for GBPs <5 mm. 2. For GBPs between 5 and 10 mm, two scans at six monthly intervals is suggested and after that, tailor surveillance to age, growth and ethnicity. In the non-Asian population, if GBP remains the same size or number, discontinuation of surveillance may be considered. In the Asian population, if GBPs remain the same, yearly surveillance is continued for a suggested period of 3 years. 3. Discontinue surveillance if GBPs is/are smaller/ disappeared. Cholecystectomy is advised where size increases to >10 mm.

Clinical validation of a type-specific real-time quantitative human papillomavirus PCR against the performance of hybrid capture 2 for the purpose of cervical cancer screening.

To be acceptable for use in cervical cancer screening, a new assay that detects DNA of high-risk human papillomavirus (hrHPV) types must demonstrate high reproducibility and performance not inferior to that of a clinically validated HPV test. In the present study, a real-time quantitative PCR (qPCR) assay targeting the E6 and E7 genes of hrHPV was compared with Hybrid Capture 2 (hc2) in a Belgian cervical cancer screening setting. In women >30 years old, the sensitivity and specificity for intraepithelial neoplasias of grade 2 or worse (93 cases of cervical intraepithelial neoplasias of grade 2 or worse (CIN2+) and 1,207 cases of no CIN or CIN1) were 93.6% and 95.6%, respectively, and those of hc2 were 83.9% and 94.5%, respectively {relative sensitivity of qPCR/hc2 = 1.12 [95% confidence interval (CI), 1.01 to 1.23]; relative specificity = 1.01 [95% CI, 0.99 to 1.03]}. A score test showed that the sensitivity (P < 0.0001) and specificity (P < 0.0001) of the qPCR assay were not inferior to those of hc2 at the required thresholds of 90% and 98%, respectively. The overall agreement of hrHPV positivity between the two runs of the qPCR tests was 98.7% (95% CI, 97.5 to 99.4%), with a kappa value of 0.96 (95% CI, 0.83 to 1.00). The qPCR assay used in this study can be considered a reliable HPV assay that fulfills the clinical validation criteria defined for use in cervical cancer screening.

Circulating tumour cells in the central and the peripheral venous compartment in patients with metastatic breast cancer.

BACKGROUND: The enumeration of circulating tumour cells (CTC) has prognostic significance in patients with metastatic breast cancer (MBC) and monitoring of CTC levels over time has considerable potential to guide treatment decisions. However, little is known on CTC kinetics in the human bloodstream. METHODS: In this study, we compared the number of CTC in both 7.5 ml central venous blood (CVB) and 7.5 ml peripheral venous blood (PVB) from 30 patients with MBC starting with a new line of chemotherapy. RESULTS: The number of CTC was found to be significantly higher in CVB (median: 43.5; range: 0-4036) than in PVB (median: 33; range: 0-4013) (P=0.001). When analysing samples pairwise, CTC counts were found to be significantly higher in CVB than in PVB in 12 out of 26 patients with detectable CTC. In contrast, only 2 out of 26 patients had higher CTC counts in PVB as compared with CVB, whereas in 12 remaining patients no significant difference was seen. The pattern of CTC distribution was independent of the sites of metastatic involvement. CONCLUSION: A substantial difference in the number of CTC was observed between CVB and PVB of patients with MBC. Registration of the site of blood collection is warranted in studies evaluating the role of CTC assessment in these patients.

Open pancreatic necrosectomy in the multidisciplinary management of postinflammatory necrosis.

OBJECTIVE: To examine clinical outcome in a consecutive cohort of patients undergoing open necrosectomy for postinflammatory necrosis. BACKGROUND INFORMATION: The last decade has witnessed major developments in the surgical management of pancreatic necrosis. Minimally invasive approaches have become established. However, there are limited data from contemporary open necrosectomy, in particular where multidisciplinary care and aggressive interventional radiology are used. This report provides data on outcome from open necrosectomy in a tertiary referral Hepatobiliary unit over the last decade. METHODS: During the period January 1, 2000 to July 31, 2008, 1535 patients were admitted with a final discharge code of acute pancreatitis. Twenty-eight (1.8%) of all admissions underwent open surgical necrosectomy. Twenty-four (86%) were tertiary referral patients. RESULTS: The median APACHE II score on admission was 10.5 (5-26). Median logistic organ dysfunction score on admission was 3 (0-10). Median LODS score after surgery was 2 (0-8). Twenty patients (71%) underwent radiologically guided drainage of collections before surgery. Thirty-day mortality occurred in 2 (7%), 4 further deaths occurred in patients after discharge from intensive care resulting in a total of 6 (22%) episode-related deaths. CONCLUSIONS: Modern open necrosectomy can be performed without the procedure-related deterioration in organ dysfunction associated with major debridement. Multidisciplinary care with an emphasis on aggressive radiologic intervention before and after surgery results in acceptable outcomes in this cohort of critically ill patients. Newer laparoscopic techniques must demonstrate similar outcomes in the setting of stage-matched severity before wider acceptance.

Circulating tumour cell detection: a direct comparison between the CellSearch System, the AdnaTest and CK-19/mammaglobin RT-PCR in patients with metastatic breast cancer.

BACKGROUND: The detection, enumeration and isolation of circulating tumour cells (CTCs) have considerable potential to influence the clinical management of patients with breast cancer. There is, however, substantial variability in the rates of positive samples using existing detection techniques. The lack of standardisation of technology hampers the implementation of CTC measurement in clinical routine practice. METHODS: This study was designed to directly compare three techniques for detecting CTCs in blood samples taken from 76 patients with metastatic breast cancer (MBC) and from 20 healthy controls: the CellSearch CTC System, the AdnaTest Breast Cancer Select/Detect and a previously developed real-time qRT-PCR assay for the detection of CK-19 and mammaglobin transcripts. RESULTS: As a result, 36% of patients with MBC were positive by the CellSearch System, 22% by the AdnaTest, 26% using RT-PCR for CK-19 and 54% using RT-PCR for mammaglobin. Samples were significantly more likely to be positive for at least one mRNA marker using RT-PCR than using the CellSearch System (P=0.001) or the AdnaTest (P<0.001). CONCLUSION: We observed a substantial variation in the detection rates of CTCs in blood from breast cancer patients using three different techniques. A higher rate of positive samples was observed using a combined qRT-PCR approach for CK-19 and mammaglobin, which suggests that this is currently the most sensitive technique for detecting CTCs.

Green tea polyphenols ameliorate pancreatic injury in cerulein-induced murine acute pancreatitis.

OBJECTIVE: Green tea polyphenols (GTPs) are naturally occurring antioxidants acting through pathways that include reactive oxygen species and nuclear factor kappa B (NF-kappaB). This study investigates the effect of GTPs in a cerulein-induced murine model of acute pancreatitis (AP). METHODS: Male CD mice (median weight, 37.7 g) were divided into 4 groups: mice administered with cerulein alone, cerulein and GTP, saline alone (sham), and GTP alone. Acute pancreatitis was induced by serial intraperitoneal administration of cerulein (50 microg/kg, x6). Green tea polyphenol was administered intraperitoneally at 25 mg/kg on the first, third, and sixth hours after pancreatitis induction.We analyzed histologic and biochemical features of AP, NF-kappaB pathway activity, leukocyte-mediated damage, cytokine levels, oxidative stress injury, lipid peroxidation, expression of poly-(adenosine diphosphate-ribose) synthetase, and presence of apoptosis. RESULTS: Treatment with GTP reduced the histologic and biochemical features of AP. Western blot revealed significant NF-kappaB inactivation. Immunostaining for P selectin and intercellular adhesion molecule 1, tumor necrosis factor alpha, transforming growth factor beta, vascular endothelial growth factor, nitrotirosine, poly-(adenosine diphosphate ribose) synthetase, and malondialdheide levels were significantly reduced. There was a significant down-regulation of apoptotic markers. CONCLUSIONS: Our results demonstrated that GTP significantly ameliorated the effects of cerulein-induced AP in mice. These effects of GTP are mediated by actions at the NF-kappaB/IkB (inhibitor kB) proteins and oxidative stress pathways.

Comparison of MY09/11 consensus PCR and type-specific PCRs in the detection of oncogenic HPV types.

The causal relationship between persistent infection with high-risk HPV and cervical cancer has resulted in the development of HPV DNA detection systems. The widely used MY09/11 consensus PCR targets a 450bp conserved sequence in the HPV L1 gene, and can therefore amplify a broad spectrum of HPV types. However, limitations of these consensus primers are evident, particularly in regard to the variability in detection sensitivity among different HPV types. This study compared MY09/11 PCR with type-specific PCRs in the detection of oncogenic HPV types. The study population comprised 15, 774 patients. Consensus PCR failed to detect 522 (10.9%) HPV infections indicated by type-specific PCRs. A significant correlation between failure of consensus PCR and HPV type was found. HPV types 51, 68 and 45 were missed most frequently. The clinical relevance of the HPV infections missed by MY09/11 PCR was reflected in the fraction of cases with cytological abnormalities and in follow-up, showing 104 (25.4%) CIN2+ cases. The MY09/11 false negativity could be the result of poor sensitivity, mismatch of MY09/11 primers or disruption of L1 target by HPV integration or DNA degradation. Furthermore, MY09/11 PCR lacked specificity for oncogenic HPVs. Diagnostic accuracy of the PCR systems, in terms of sensitivity (MY09/11 PCR: 87.9%; type-specific PCRs: 98.3%) and specificity (MY09/11 PCR: 38.7%; type-specific PCRs: 76.14%), and predictive values for histologically confirmed CIN2+, suggest that type-specific PCRs could be used in a clinical setting as a reliable screening tool.

Improved endocervical sampling and HPV viral load detection by Cervex-Brush Combi.

OBJECTIVE: Liquid-based cytology (LBC) for cervical screening is becoming increasingly used. Together with SurePath LBC, various collecting devices can be utilized, among which the Cervex-Brush is the most widely used. The new Rovers Cervex-Brush Combi combines the advantages of the Cervex-Brush with the EndoCervex-Brush increasing sampling of the endocervical canal. The objective of this study was to analyse and to compare the Cervex-Brush Combi with the Cervex-Brush for the collection of squamous and endocervical cells, human papillomavirus (HPV) typing/quantification and disease detection in SurePath LBC. METHODS: Using either the Cervex-Brush or the Cervex-Brush Combi 100 consecutive SurePath LBC samples were collected using each brush type. All 200 slides were read by the FocalPoint and screened by guided screening using slide wizards. The viral load of HPV type 16 E7, 18 E7, 31 E6, 33 L1, 33 E6, 35 E4, 39 E7, 45 E7, 51 E6, 52 L1, 52 E7, 53 E6, 56 E7, 58 L1, 58 E6, 59 E7, 66 E6 and 68 E7 was determined using a TaqMan-based real-time quantitative PCR analysis. RESULTS: The mean number of sampled squamous cells did not differ between the two brush types (54 963 versus 54 595 cells). The use of the Cervex-Brush Combi, however, resulted in a two- to threefold increase in the number of sampled endocervical cells (P < 0.00001). Using the Cervex-Brush Combi slightly more lesions were detected (three versus two low-grade squamous intraepithelial lesions), and resulted in the detection of more atypical squamous cells of undetermined significance (six versus three). In the Cervex-Brush group, 60% (3/5) of abnormal smears were positive for oncogenic HPV types, whereas 66.7% (6/9) of abnormal smears in the Cervex-Brush Combi group tested positive. The median HPV viral load for samples taken with the Cervex-Brush Combi was 0.1825 copies/cell and was significantly higher than in samples taken with the Cervex-Brush (0.0042 copies/cell) (P = 0.02). CONCLUSION: Sampling with the Cervex-Brush Combi resulted in the collection of the same amount of squamous cells, but in a two to threefold harvest of endocervical cells. This led to the detection of a higher viral load for oncogenic HPV and an increase in the number of detected abnormal smears.

Real-time RT-PCR detection of disseminated tumour cells in bone marrow has superior prognostic significance in comparison with circulating tumour cells in patients with breast cancer.

This study assessed the ability of real-time reverse transcription-polymerase chain reaction (RT-PCR) analysis to detect disseminated epithelial cells (DEC) in peripheral blood (PB) and bone marrow (BM) of patients with breast cancer (BC). Detection of DEC in BM is an obvious choice in BC, but blood sampling is more convenient. The aim of this study was to evaluate whether the detection of DEC in either PB or BM predicts overall survival (OS). Peripheral blood and BM samples were collected from 148 patients with primary (stage M0, n=116/78%) and metastatic (stage M+, n=32/21%) BC before the initiation of any local or systemic treatment. Peripheral blood of healthy volunteers and BM of patients with a nonmalignant breast lesion or a haematological malignancy served as the control group. Disseminated epithelial cells was detected by measuring relative gene expression (RGE) for cytokeratin-19 (CK-19) and mammaglobin (MAM), using a quantitative RT-PCR detection method. The mean follow-up time was 786 days (+/- 487). Kaplan-Meier analysis was used for predicting OS. By taking the 95 percentile of the RGE of CK-19 (BM: 26.3 and PB: 58.7) of the control group as cutoff, elevated CK-19 expression was detected in 42 (28%) BM samples and in 22 (15%) PB samples. Mammaglobin expression was elevated in 20% (both PB and BM) of the patients with BC. There was a 68% (CK-19) and 75% (MAM) concordance between PB and BM samples when classifying the results as either positive or negative. Patients with an elevated CK-19 or MAM expression in the BM had a worse prognosis than patients without elevated expression levels (OS: log-rank test, P=0.0045 (CK-19) and P=0.025 (MAM)). For PB survival analysis, no statistical significant difference was observed between patients with or without elevated CK-19 or MAM expression (OS: log-rank test, P=0.551 (CK-19) and P=0.329 (MAM)). Separate analyses of the M0 and M+ patients revealed a marked difference in OS according to the BM CK-19 or MAM status in the M+ patient group, but in the M0 group, only MAM expression was a prognostic marker for OS. Disseminated epithelial cells, measured as elevated CK-19 or MAM mRNA expression, could be detected in both PB and BM of patients with BC. Only the presence of DEC in BM was highly predictive for OS. The occurrence of DEC in the BM is probably less time-dependent and may act as a filter for circulating BC cells. The use of either larger volumes of PB or performing an enrichment step for circulating tumour in blood cells might improve these results.

Real-time RT-PCR correlates with immunocytochemistry for the detection of disseminated epithelial cells in bone marrow aspirates of patients with breast cancer.

Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) is a technique with the potential of improving the quantification of disseminated epithelial cells (DEC) in haematological tissues due to its exquisite sensitivity. This sensitivity may lead to false positivity. Immunocytochemistry (ICC) is regarded as the standard methodology to diagnose DEC. In this study, detection with ICC was compared with quantitative real-time RT-PCR for CK-19 and mammaglobin (hMAM) mRNA in bone marrow (BM) of patients with metastatic breast cancer (MBC). Bone marrow was aspirated from 14 control patients and from 29 patients with MBC. Mononuclear cells (MNC) were isolated. Immunostaining was carried out with the Epimet kit. Quantitative PCR was performed on the ABI Prism 7700. The CK-19 and hMAM mRNA quantities were normalised against beta-Actin and calculated relative to a calibrator sample (relative gene expression). All controls were negative by ICC and for hMAM expression measured by RT-PCR, whereas the median RGE value for CK-19 was 0.57. For the MBC patients, the median RGE for hMAM was 0 and 10 out of 25 (40%) tested positive. Median RGE for CK-19 was 2.9 and 20 out of 25 (80%) tested positive. With ICC, the median value was 1 stained cell per sample, and 15 out of 24 (62%) samples were positive. A correlation was observed between CK-19 and hMAM expression (r=0.7; P=0.0003), and between hMAM expression and ICC (r=0.6; P=0.003). CK-19 expression and ICC (r=0.9; P<0.0001) showed the strongest correlation. Reverse transcriptase-polymerase chain reaction for CK-19 resulted in a higher number of positive BM samples of patients with MBC than ICC. Since an excellent correlation is observed between ICC and RT-PCR, and RT-PCR is probably more sensitive with the advantage of being less observer dependent and thus also more easy to automate, we consider our quantitative real-time RT-PCR method as validated for the detection of DEC in the bone marrow of breast cancer patients.

Circulating basic fibroblast growth factor is partly derived from the tumour in patients with colon, cervical and ovarian cancer.

In order to investigate whether the high bFGF serum levels encountered in cancer patients are derived from the tumour, we analysed serum bFGF levels in 18 untreated randomly selected patients with operable colorectal, cervical and ovarian cancer in the blood draining the tumour, i.e., in mesenteric and uterine veins, and compared these with arterial samples. No significantly elevated bFGF levels were found in the veins draining the tumours compared with arterial samples in our patient population. This suggests that, in contrast to what is generally presumed, serum bFGF levels might also be derived from other sources besides the tumour, e.g., platelets.

Inflammatory breast cancer shows angiogenesis with high endothelial proliferation rate and strong E-cadherin expression.

Inflammatory breast cancer (IBC) is the most aggressive form of breast cancer. Improved understanding of the mechanisms responsible for the differences between IBC and non-IBC might provide novel therapeutic targets. We studied 35 consecutive patients with IBC, biopsied prior to the initiation of chemotherapy. Angiogenesis was evaluated by Chalkley counting and by assessment of endothelial cell proliferation (ECP) and vessel maturity. The presence of fibrin, expression of the hypoxia marker carbonic anhydrase IX (CA IX) and epithelialcadherin (E-cadherin) expression were immunohistochemically detected. The same parameters were obtained in a group of 104 non-IBC patients. Vascular density, assessed by Chalkley counting (P<0.0001), and ECP (P=0.01) were significantly higher in IBC than in non-IBC. Abundant stromal fibrin deposition was observed in 26% of IBC and in only 8% of non-IBC (P=0.02). Expression of CA IX was significantly less frequent in IBC than in non-IBC with early metastasis (P=0.047). There was a significant positive correlation between the expression of CA IX and ECP in IBC (r=0.4, P=0.03), implying that the angiogenesis is partly hypoxia driven. However, the higher ECP in IBC and the less frequent expression of CA IX in IBC vs non-IBC points at a role for other factors than hypoxia in stimulating angiogenesis. Strong, homogeneous E-cadherin expression was found at cell-cell contacts in all but two IBC cases, both in lymphovascular tumour emboli and in infiltrating tumour cells, challenging our current understanding of the metastatic process. Both the intense angiogenesis and the strong E-cadherin expression may contribute to the highly metastatic phenotype of IBC.

Arterio-venous gradients of IL-6, plasma and serum VEGF and D-dimers in human cancer.

The circulating angiogenic factors vascular endothelial growth factor-A, interleukin-6 and the fibrin D-dimer fragment were measured in the mesenteric vein, the uterine vein, as well as in peripheral venous and arterial samples in 21 randomly selected patients with operable colorectal, ovarian and cervical carcinoma. In addition, immunohistochemistry for vascular endothelial growth factor-A and interleukin-6 was performed on colorectal tumours of such patients. Serum and plasma vascular endothelial growth factor-A were not significantly elevated in the vein draining the tumours, despite tumour cell expression of vascular endothelial growth factor-A. Serum vascular endothelial growth factor-A is therefore not all tumour-derived. In contrast, serum interleukin-6 was highly elevated in the draining veins in agreement with expression of interleukin-6 in the cytoplasm of tumour cells. In the megakaryoblastic cell line MEG-01, the expression of vascular endothelial growth factor-A was found to be regulated by interleukin-6. Thus, the higher platelet vascular endothelial growth factor-A load resulting in higher serum vascular endothelial growth factor levels in cancer patients may partly result from an interleukin-6 mediated up-regulation of the expression of vascular endothelial growth factor-A in the precursor of the platelet, i.e. the megakaryocyte. We also confirmed by immunohistochemistry that platelets adhere and aggregate on tumour endothelium. We propose that interleukin-6 indirectly promotes tumour angiogenesis through its up-regulation of the vascular endothelial growth factor-A load in platelets. In addition, the correlations found between peripheral venous interleukin-6 and peripheral venous fibrinogen and D-dimers levels, and the high D-dimer levels found in the draining vein of the tumour, in agreement with fibrin deposits found in the tumour stroma, suggest an important role for interleukin-6 in extra-vascular fibrinogen metabolism. Our results suggest a pivotal role for interleukin-6 in the intrinsic link between haemostasis and angiogenesis. This might be of importance in the development of anti-angiogenic agents based on interference with haemostasis.

Plasma fibrin D-dimer levels correlate with tumour volume, progression rate and survival in patients with metastatic breast cancer.

Plasma levels of D-dimer are elevated in cancer patients. Activation of the extrinsic coagulation system and the fibrinolytic cascade within a tumour is thought to be related with growth, invasion and metastasis. We have investigated the relationship between these markers of fibrin metabolism, standard clinicopathological variables and serum levels of angiogenic cytokines in three cohorts: group A (n=30) consisted of 30 healthy female volunteers, group B (n=23) of consecutive patients with operable breast cancer and group C (n=84) of patients with untreated or progressive metastatic breast cancer. Plasma D-dimers, fibrinogen, IL-6, vascular endothelial growth factor and calculated vascular endothelial growth factor load in platelets are clearly increased in patients with breast cancer. D-dimers were increased in nearly 89% of patients with progressive metastatic disease. The level of D-dimers was positively correlated with tumour load (P<0.0001), number of metastatic sites (P=0.002), progression kinetics (P<0.0001) and the cytokines related to angiogenesis: serum vascular endothelial growth factor (P=0.0016, Spearman correlation=0.285), calculated vascular endothelial growth factor load in platelets (P<0.0001, Spearman correlation=0.37) and serum interleukin-6 (P<0.0001, Spearman correlation=0.59). Similarly increased D-dimer levels were positively correlated with increased fibrinogen levels (P<0.0001, Spearman correlation=0.38). The association between markers of fibrin degradation in patients with progressive breast cancer suggests that the D-dimer level is a clinically important marker for progression and points towards a relation between haemostasis and tumour progression. A role of interleukin-6, by influencing both angiogenesis and haemostasis, is suggested by these observations.