Fluorescence confocal microscopy for evaluation of fresh surgical specimens and consecutive tumor cell isolation in rare pediatric tumors.

Fluorescence confocal microscopy (FCM) is an optical technique that uses laser light sources of different wavelengths to generate real-time images of fresh, unfixed tissue specimens. FCM allows histological evaluation of fresh tissue samples without the associated cryo artifacts after frozen sectioning. The aim of this study was to prospectively evaluate pediatric tumor specimens and assess their suitability for fresh tumor sampling. In addition, we aimed to determine whether tumor cell isolation for stable cell culture is still feasible after FCM imaging. Pediatric tumor specimens were imaged using FCM. Tumor viability and suitability for tissue sampling were evaluated and compared with H&E staining after paraffin embedding. In addition, FCM-processed and non-FCM-processed tissue samples were sent for tumor cell isolation to evaluate possible effects after FCM processing. When comparing estimated tumor cell viability using FCM and H&E, we found good to excellent correlating estimates (intraclass correlation coefficient = 0.891, p < 0.001), as well as substantial agreement in whether the tissue appeared adequate for fresh tissue collection (κ = 0.762, p < 0.001). After FCM, seven out of eight samples yielded passable cell cultures, compared to eight out of eight for non-FCM processed samples. Our study suggests that the use of FCM in tumor sampling can increase the yield of suitable fresh tumor samples by identifying viable tumor areas and ensuring that sufficient tissue remains for diagnosis. Our study also provides first evidence that the isolation and growth of tumor cells in culture are not compromised by the FCM technique.

[p53 suppresses type II endometrial carcinomas in mice and governs endometrial tumor aggressiveness in humans]. / p53 unterdrückt die Bildung von Typ-II-Endometriumkarzinomen im Mausmodell und ist ein unabhängiger negativer Prognosefaktor bei Patientinnen mit Endometriumkarzinom.

Type II endometrial carcinomas are a highly aggressive group of tumor subtypes that are frequently associated with inactivation of the TP53 tumor suppressor gene. We show that mice with endometrium-specific deletion of the Trp53 gene initially exhibited histological changes that are identical to known precursor lesions of type II endometrial carcinomas in humans and later developed carcinomas representing all type II subtypes. The mTORC1 signalling pathway was frequently activated in these precursor lesions and tumors, suggesting a genetic cooperation between this pathway and Trp53 deficiency in tumor initiation. Consistent with this idea, analyses of 521 human endometrial carcinomas identified frequent mTORC1 pathway activation in type I as well as type II endometrial carcinoma subtypes. The mTORC1 pathway activation and p53 expression or mutation status each independently predicted poor patient survival. We suggest that molecular alterations in p53 and the mTORC1 pathway play different roles in the initiation of the different endometrial cancer subtypes but combined p53 inactivation and mTORC1 pathway activation are unifying pathogenic features among histologically diverse subtypes of late stage aggressive endometrial tumors.

The NeuroSAFE frozen section technique during radical prostatectomy - Implementation and optimization of technical aspects in a routine pathology workflow.

BACKGROUND AND AIMS: In prostate cancer patients, application of the NeuroSAFE frozen section technique during radical prostatectomy has been shown to increase the rate of nerve sparing surgery and to improve functional outcome for the patients. The aim of this study is to report on technical and organizational optimization opportunities of the procedure. MATERIAL AND METHODS: All patients submitted to bilateral intraoperative frozen section from January 2018 until December 2020 (n = 452) were retrospectively analyzed and parameters such as turnaround time, staff situation in the laboratory and histologic properties of the tumors were assessed. RESULTS: The median turnaround time per case was 40.3 ( ± 10.5) min. In 2020 the average time needed from accessioning to diagnosis was 38.1 min. Multivariate linear regression suggested that the number of technical assistants/cryotomes (46.1 min vs. 39.13 min; p < 0.001), the place of microscopic examination (43.0 min vs. 38.7 min; p < 0.001) and the presence of a positive margin (38.0 vs. 44.0 min; p < 0.001) were significant influential factors. The turnaround time was independent of the uropathological expertize of the consultant (39.84 min vs. 40.7 min; p = 0.09), the tumor grade (42.3 vs 39.8 min; p = 0.493) and the presence of extraprostatic extension (44.0 vs 39.8 min; p = 0.099). CONCLUSION: The implementation of simple optimization measures in the workflow as well as structured training of all pathology staff involved in the examination leads to a significant increase in the efficiency of the examination while maintaining the same level of resources. The results could thus be a contribution to the broader application of the procedure.

Computed Tomography Radiomics to Differentiate Intrahepatic Cholangiocarcinoma and Hepatocellular Carcinoma.

AIMS: Intrahepatic cholangiocarcinoma (iCCA) and hepatocellular carcinoma (HCC) differ in prognosis and treatment. We aimed to non-invasively differentiate iCCA and HCC by means of radiomics extracted from contrast-enhanced standard-of-care computed tomography (CT). MATERIALS AND METHODS: In total, 94 patients (male, n = 68, mean age 63.3 ± 12.4 years) with histologically confirmed iCCA (n = 47) or HCC (n = 47) who underwent contrast-enhanced abdominal CT between August 2014 and November 2021 were retrospectively included. The enhancing tumour border was manually segmented in a clinically feasible way by defining three three-dimensional volumes of interest per tumour. Radiomics features were extracted. Intraclass correlation analysis and Pearson metrics were used to stratify robust and non-redundant features with further feature reduction by LASSO (least absolute shrinkage and selection operator). Independent training and testing datasets were used to build four different machine learning models. Performance metrics and feature importance values were computed to increase the models' interpretability. RESULTS: The patient population was split into 65 patients for training (iCCA, n = 32) and 29 patients for testing (iCCA, n = 15). A final combined feature set of three radiomics features and the clinical features age and sex revealed a top test model performance of receiver operating characteristic (ROC) area under the curve (AUC) = 0.82 (95% confidence interval =0.66-0.98; train ROC AUC = 0.82) using a logistic regression classifier. The model was well calibrated, and the Youden J Index suggested an optimal cut-off of 0.501 to discriminate between iCCA and HCC with a sensitivity of 0.733 and a specificity of 0.857. CONCLUSIONS: Radiomics-based imaging biomarkers can potentially help to non-invasively discriminate between iCCA and HCC.

The peri- and intratumoral immune cell infiltrate and PD-L1 status in invasive squamous cell carcinomas of the penis.

INTRODUCTION: Penile carcinomas are rare tumors throughout Europe. Therefore, little attention is drawn to this disease. That makes it important to study tumor-associated key metrics and relate these to known data on penile neoplasias. MATERIALS AND METHODS: A cohort of 60 well-defined penile invasive carcinomas with known human papillomavirus (HPV) infection status was investigated. Data on tumor type, grading and staging were recorded. Additionally, data on the peri- and intratumoral immune cell infiltrate in a semiquanititave manner applying an HE stain were assessed. RESULTS: Our study showed a significant correlation of immune cell infiltrate and pT stage with overall survival. Therefore, in a subset of tumors, PD-L1 staining was applied. For tumor proportion score (TPS), 26 of 30 samples (87%) were scored >0%. For the immune cell score (IC), 28 of 30 samples (93%) were defined as >0% and for CPS, 29 of 30 samples (97%) scored >0. PD-L1 expression was not associated with overall survival. CONCLUSION: PD-L1 is expressed in penile carcinomas, providing a rationale for targeted therapy with checkpoint inhibitors. We were able to show that immune reaction appears to be prognostically relevant. These data enhance the need for further studies on the immune cell infiltrate in penile neoplasias and show that PD-L1 expression is existent in our cohort, which may be a potential target for checkpoint inhibitor therapy.

Collecting system invasion and Fuhrman grade but not tumor size facilitate prognostic stratification of patients with pT2 renal cell carcinoma.

PURPOSE: The 7th edition of TNM for renal cell carcinoma introduced a subdivision of pT2 tumors at a 10 cm cutoff. In the present multicenter study the influence of tumor size as well as further clinical and histopathological parameters on cancer specific survival in patients with pT2 tumors was evaluated. MATERIALS AND METHODS: A total of 670 consecutive patients with pT2 tumors (10.4%) of 6,442 surgically treated patients with all tumor stages were pooled (mean followup 71.4 months). Tumors were reclassified according to the current TNM classification, and subdivided in stages pT2a and pT2b. Cancer specific survival was analyzed using the Kaplan-Meier method, and univariable and multivariable analyses were used to assess the influence of several parameters on survival. RESULTS: Tumor size continuously applied and subdivided at 10 cm or alternative cutoffs did not significantly influence cancer specific survival. In addition to N/M stage, Fuhrman grade and collecting system invasion also had an independent influence on survival. Integration of a dichotomous variable subsuming Fuhrman grade and collecting system invasion (grade 3/4 and/or collecting system invasion present vs grade 1/2 and collecting system invasion absent) into multivariate models including established prognostic parameters resulted in improvement of predictive abilities by 11% (HR 2.3, p <0.001) for all pT2 cases and 151% (HR 3.1, p <0.001) for stage pT2N0M0 cases. CONCLUSIONS: Tumor size did not have a significant influence on cancer specific survival in pT2 tumors, neither continuously applied nor based on various cutoff values. To enhance prognostic discrimination, multifactorial staging systems including pathological features should be implemented. The prognostic relevance of the variable subsuming Fuhrman grade and collecting system invasion should be considered for future evaluation.

Expression of the lipid transporters ABCA3 and ABCA1 is diminished in human breast cancer tissue.

ATP-binding cassette transporters ABCA3 and ABCA1 are related to a differentiated, lipid-secreting phenotype of type II pneumocytes. Since mammary gland epithelial cells also show pronounced lipid metabolism and secretion, we investigated the expression of these proteins in normal as well as in neoplastic breast tissue. Normal human breast tissue, breast cancer cell lines, and 162 tumor samples of patients with primary unilateral invasive breast cancer were analyzed for ABCA3 and ABCA1 protein expression by immunohistochemistry using tissue microarrays. Strong ABCA3 and ABCA1 expression was found in the inner layer of normal mammary gland epithelium. Concurrent cytoplasmic ABCA3 and ABCA1 immunoreactivity was found in 9 of 11 breast cancer cell lines. ABCA3 and ABCA1 were shown to be differentially expressed in human breast cancer. Loss of ABCA3 staining was significantly associated with positive nodal status and negative progesterone receptor expression. In multivariate analysis, diminished ABCA3 expression proved to be a significant, independent and adverse risk factor for tumor recurrence. ABCA1 expression was associated with positive lymph nodes, but not significantly associated with tumor recurrence or breast cancer-specific survival. ABCA3 and ABCA1 are strongly expressed in normal mammary gland epithelium. Decreased ABCA3 expression in breast cancer seems to be associated with poor prognosis.

Frequent loss of SFRP1 expression in multiple human solid tumours: association with aberrant promoter methylation in renal cell carcinoma.

Oncogenic wingless-related mouse mammary tumour virus (Wnt) signalling, caused by epigenetic inactivation of specific pathway regulators like the putative tumour suppressor secreted frizzled-related protein 1 (SFRP1), may be causally involved in the carcinogenesis of many human solid tumours including breast, colon and kidney cancer. To evaluate the incidence of SFRP1 deficiency in human tumours, we performed a large-scale SFRP1 expression analysis using immunohistochemistry on a comprehensive tissue microarray (TMA) comprising 3448 tumours from 36 organs. This TMA contained 132 different tumour subtypes as well as 26 different normal tissues. Although tumour precursor stages of, for example kidney, colon, endometrium or adrenal gland still exhibited moderate to abundant SFRP1 expression, this expression was frequently lost in the corresponding genuine tumours. We defined nine novel tumour entities with apparent loss of SFRP1 expression, i.e., cancers of the kidney, stomach, small intestine, pancreas, parathyroid, adrenal gland, gall bladder, endometrium and testis. Renal cell carcinoma (RCC) exhibited the highest frequency of SFRP1 loss (89% on mRNA level; 75% on protein level) and was selected for further analysis to investigate the cause of SFRP1 loss in human tumours. We performed expression, mutation and methylation analysis in RCC and their matching normal kidney tissues. SFRP1 promoter methylation was frequently found in RCC (68%, n=38) and was correlated with loss of SFRP1 mRNA expression (p<0.05). Although loss of heterozygosity was found in 16% of RCC, structural mutations in the coding or promoter region of the SFRP1 gene were not observed. Our results indicate that loss of SFRP1 expression is a very common event in human cancer, arguing for a fundamental role of aberrant Wnt signalling in the development of solid tumours. In RCC, promoter hypermethylation seems to be the predominant mechanism of SFRP1 gene silencing and may contribute to initiation and progression of this disease.

Renal cell carcinoma marker reliably discriminates central nervous system haemangioblastoma from brain metastases of renal cell carcinoma.

AIMS: The distinction between central nervous system (CNS) metastases of clear cell renal cell carcinoma (RCC) and CNS haemangioblastoma still poses a challenge to the pathologist. Since both entities occur in von Hippel-Lindau disease, this aggravates the issue. The antibody renal cell carcinoma marker (RCC-ma) has been suggested to identify primary RCCs specifically, but its value for diagnosing metastases of RCC is controversial. The aim was to assess two distinct clones of the RCC-ma for their potential to: (i) identify primary RCCs and (ii) differentiate between CNS metastases of clear cell RCC and CNS haemangioblastomas. METHODS AND RESULTS: Using tissue microarrays, 77% (n = 363; PN-15) and 66% (n = 355; 66.4C2) of clear cell RCCs, and 93% (PN-15) and 74% (66.4C2) of papillary RCCs (n = 46) were immunopositive for RCC-ma, whereas none of the investigated chromophobe RCCs (n = 22) or any of the oncocytomas (n = 15) showed immunoreactivity. Importantly, 50.9% of CNS metastases of clear cell RCCs (n = 55) exhibited RCC-ma expression, whereas all CNS haemangioblastomas (71) were negative. CONCLUSIONS: Both RCC-ma clones, despite some variation in their sensitivity to detect clear cell and papillary RCCs, are of value in differentiating subtypes of primary RCC and are excellent markers for discriminating clear cell lesions in the brain.

Nutritional depletion and its relationship to respiratory impairment in patients with chronic respiratory failure due to COPD or restrictive thoracic diseases.

OBJECTIVE: While malnutrition, especially fat-free mass index (FFMI), is a predictor for mortality in chronic obstructive pulmonary disease (COPD), less information on prevalence and mechanisms is available in patients with chronic respiratory failure (CRF) due to restrictive thoracic diseases (RTD). DESIGN AND SETTING: Cross-sectional study of patients consecutively admitted to an in-patient primary pulmonary centre. SUBJECTS: One hundred and thirty-two patients (30% RTD; 70% COPD) with CRF and intermittent non-invasive positive pressure ventilation. INTERVENTIONS: Malnutrition was quantified by bioelectrical impedance analysis or body mass index (BMI), and its relationship to laboratory, lung function, inspiratory muscle and blood gas parameters and 6-min walking distance (6-MWD) was assessed. RESULTS: Malnutrition in terms of BMI<20 kg/m(2) occurred in 16.1% of patients with COPD but none of those with RTD. FFMI<17.4 (<15.0 in female patients) kg/m(2) was found in 35.4 and 30.7%, respectively. FFMI was correlated with airway obstruction (sR(aw), r = -0.50; FEV(1)/VC, r = -0.28; P< or = 0.01 each) and lung hyperinflation (intrathoracic gas volume, r = -0.41; total lung capacity (TLC), r = -0.50; P< or = 0.001 each) in COPD, and with lung restriction in RTD (TLC, r=0.40; P=0.011). Furthermore, malnourished patients showed a higher inspiratory load (P (0.1)) and reduced 6-MWD in both groups. In COPD, only hyperinflation and P (0.1) were independently related to FFMI. CONCLUSIONS: Malnutrition as indicated by low FFMI was similarly prevalent in patients with CRF and COPD or RTD, but inadequately represented by BMI. The correlations between lung function impairments specific for the disease and FFMI emphasized the link between malnutrition and respiratory mechanical load irrespective of its aetiology.

Biobased alkylphenols from lignins via a two-step pyrolysis - Hydrodeoxygenation approach.

Five technical lignins (three organosolv, Kraft and soda lignin) were depolymerised to produce monomeric biobased aromatics, particularly alkylphenols, by a new two-stage thermochemical approach consisting of dedicated pyrolysis followed by catalytic hydrodeoxygenation (HDO) of the resulting pyrolysis oils. Pyrolysis yielded a mixture of guaiacols, catechols and, optionally, syringols in addition to alkylphenols. HDO with heterogeneous catalysts (Ru/C, CoMo/alumina, phosphided NiMO/C) effectively directed the product mixture towards alkylphenols by, among others, demethoxylation. Up to 15wt% monomeric aromatics of which 11wt% alkylphenols was obtained (on the lignin intake) with limited solid formation (<3wt% on lignin oil intake). For comparison, solid Kraft lignin was also directly hydrotreated for simultaneous depolymerisation and deoxygenation resulting in two times more alkylphenols. However, the alkylphenols concentration in the product oil is higher for the two-stage approach. Future research should compare direct hydrotreatment and the two-stage approach in more detail by techno-economic assessments.

Prognostic relevance of P-cadherin expression in melanocytic skin tumours analysed by high-throughput tissue microarrays.

AIM: To investigate whether protein expression or cellular localisation of P-cadherin is associated with clinicopathological characteristics in benign and malignant melanocytic skin tumours. EXPERIMENTAL DESIGN: P-cadherin expression and the Ki-67 labelling index were analysed immunohistochemically by using tissue microarrays (TMAs). Membranous and cytoplasmic expression was scored semiquantitatively (0 to 2+). RESULTS: P-cadherin protein expression of any intensity (1+ to 2+) was detected in the membrane in 41.5% (132/318) and in the cytoplasm in 64.2% (204/318) of patients. In general, P-cadherin expression was significantly reduced in malignant melanomas (p<0.001) and melanoma metastases (p<0.001), compared with benign nevi. Additionally, loss of membranous P-cadherin was associated with Clark level (p = 0.011) and tumour thickness (p<0.001). Interestingly, a significantly lower P-cadherin expression was shown by dermal nevi than by compound and junctional nevi (p = 0.005; p = 0.025). In primary melanomas, a Ki-67 labelling index <5% was not associated with P-cadherin protein expression, suggesting that loss of P-cadherin expression was not associated with proliferation. None of the other clinical and histological factors analysed was significantly related to P-cadherin expression. Low cytoplasmic P-cadherin expression was associated with tumour recurrence (p = 0.03) in all the patients who were analysed. After testing various multivariate Cox regression models, loss of cytoplasmic P-cadherin expression remained a highly significant adverse risk factor for tumour recurrence in patients with tumours <2 mm. CONCLUSIONS: Loss of cytoplasmic P-cadherin expression is common in advanced melanomas and can be a prognostic marker of progression in patients with melanoma, most useful in patients with primary tumours <2 mm in thickness.

Exclusion of deep venous thrombosis with D-dimer testing--comparison of 13 D-dimer methods in 99 outpatients suspected of deep venous thrombosis using venography as reference standard.

In a direct assay comparison we evaluated the diagnostic performance of 10 novel D-Dimer assays for the exclusion of deep venous thrombosis (DVT). In addition, 3 conventional ELISA D-Dimer assays were included as reference tests. The study was performed in 99 consecutive outpatients referred to the emergency department for clinical suspicion of DVT. Venography was used as reference standard and demonstrated the presence of DVT in 50 patients (6 patients with isolated distal DVT and 44 patients with proximal DVT). The qualitative D-Dimer assays Minutex and SimpliRED and the quantitative BC DD showed overall sensitivities (for proximal and distal DVT) of only 80-83% with specificities that ranged from 87 to 94%. Overall sensitivity was 94% for the qualitative INSTANT I.A. and 98% for the quantitative Turbiquant at a cut-off level equal to the detection limit. Using different cut-off levels a sensitivity of 100% for proximal DVT and for proximal as well as distal DVT could be obtained for NycoCard, IL DD, Liatest, Tinaquant and VIDAS D-Dimer assays with specificities that ranged from 31% (NycoCard) to 71% (VIDAS) for proximal DVT and from 12% (NycoCard) to 47% (IL DD) for overall DVT. At a cut-off level equal to the upper limit of the reference range only Tinaquant and VIDAS showed a sensitivity of 100% for proximal as well as for distal DVT with a specificity of 39% and 41% respectively. The results of this study suggest that the VIDAS and Tinaquant D-Dimer assays have the highest sensitivity for the exclusion of DVT in outpatients. In outpatients that have a low or moderate pretest probability for DVT, these tests may be used in management studies where anticoagulation is withheld on the basis of D-Dimer testing alone.

The influence of thermochemical treatments on the lignocellulosic structure of wheat straw as studied by natural abundance 13C NMR.

The effects of thermochemical treatments (aquathermolysis, pyrolysis, and combinations thereof) on the lignocellulosic structure and composition of wheat straw were studied with (13)C and (1)H solid state NMR spectroscopy and proton T1ρ relaxation measurements. Results show that aquathermolysis removes hemicellulose, acetyl groups, and ash minerals. As a result, the susceptibility of lignocellulose to pyrolysis is reduced most likely due to the removal of catalytically active salts, although recondensation of lignin during aquathermolysis treatment can also play a role. In contrast to pyrolysis of wheat straw, pyrolysis of aquathermolysed wheat straw leaves traces of cellulose in the char as well as more intense lignin methoxy peaks. Finally, it was found that both pyrolysis chars contain aliphatic chains, which were attributed to the presence of cutin or cutin-like materials, a macromolecule that covers the aerial surface of plants, not soluble in water and seemingly stable under the pyrolysis conditions applied.

Fractionation of wheat straw by prehydrolysis, organosolv delignification and enzymatic hydrolysis for production of sugars and lignin.

Wheat straw was fractionated using a three-step biorefining approach: (1) aqueous pretreatment for hemicellulose prehydrolysis into sugars, (2) organosolv delignification, and (3) enzymatic cellulose hydrolysis into glucose. Prehydrolysis was applied to avoid degradation of hemicellulose sugars during organosolv delignification. Maximum xylose yield obtained was 67% or 0.17 kg/kg straw (prehydrolysis: 175 °C, 30 min, 20mM H(2)SO(4)) compared to 4% in case of organosolv without prehydrolysis (organosolv: 200 °C, 60 min, 60% w/w aqueous ethanol). Prehydrolysis was found to reduce the lignin yield by organosolv delignification due to the formation of 'pseudo-lignin' and lignin recondensation during prehydrolysis. This reduction could partly be compensated by increasing the temperature of the organosolv delignification step. Prehydrolysis substantially improved the enzymatic cellulose digestibility from 49% after organosolv without prehydrolysis to 80% (20 FPU/g substrate). Increasing the organosolv delignification temperature to 220 °C resulted in a maximum enzymatic glucose yield of 93% or 0.36 kg/kg straw.

The extracellular matrix protein ITIH5 is a novel prognostic marker in invasive node-negative breast cancer and its aberrant expression is caused by promoter hypermethylation.

Inter-alpha-trypsin inhibitors (ITIs) are protease inhibitors stabilizing the extracellular matrix. ITIs consist of one light (bikunin) and two heavy chains (ITIHs). We have recently characterized ITIH5, a novel member of the ITIH gene family, and showed that its messenger RNA is lost in a high proportion of breast tumours. In the present study, an ITIH5-specific polyclonal antibody was generated, validated with western blot and used for immunohistochemical analysis on a tissue microarray; ITIH5 was strongly expressed in epithelial cells of normal breast (n=11/15), while it was lost or strongly reduced in 42% (92/217) of invasive breast cancers. ITIH5 expression in invasive carcinomas was associated with positive expression of oestrogen receptor (P=0.008) and histological grade (P=0.024). Correlation of ITIH5 expression with clinical outcome revealed that patients with primary tumours retaining abundant ITIH5 expression had longer recurrence-free survival (RFS; P=0.037) and overall survival (OS; P=0.044), compared to those with reduced expression (mean RFS: 102 vs 78 months; mean OS: 120 vs 105 months). Methylation-specific PCR analysis frequently showed strong methylation of the ITIH5 promoter in primary breast tumours (41%, n=109) and breast cancer cell lines (n=6). Methylation was significantly associated with mRNA loss (P<0.001; n=39), and ITIH5 expression was induced after treatment of tumour cell lines with the demethylating agent 5-aza-2'-deoxycytidine. Moreover, ITIH5 promoter methylation was significantly associated with reduced OS (P=0.008). The cellular function of ITIH5 was evaluated by forced expression of a full-length ITIH5 complementary DNA in the breast cancer cell line MDA-MB-231, which does not endogenously express ITIH5. ITIH5-expressing clones showed a 40% reduced proliferation rate compared to mock-transfected cells. Overall, these data show that promoter methylation-mediated loss of ITIH5 expression is associated with unfavourable outcome in breast cancer patients, and thus ITIH5 could be used as a prognostic marker, although this marker is not multivariate independent due to its close association with ER expression. Our data indicate that ITIH5 is a candidate class II tumour suppressor gene and could be involved in tumour progression, invasion and metastasis, as its absence is associated with increased proliferation rates and a prognostic value indicating poor clinical outcome.