Desmoplastic tumour-associated stroma versus neural tissue in central nervous system metastasis: effects of different microenvironments on tumour growth

Chang K‐C, Lu Y‐C, Lin M‐J, Chen H‐Y & Jin Y‐T
(2011) Histopathology 59 , 31–39 Desmoplastic tumour‐associated stroma versus neural tissue in central nervous system metastasis: effects of different microenvironments on tumour growth Aims: Interactions between tumour cells and extracellular matrix (ECM) are critical in the metastatic cascade. We compared effects of desmoplastic stroma versus neural tissue on central nervous system (CNS) metastasis. Methods and results: Using integrins (ECM receptors), ECM (fibronectin, laminin and collagen IV) and CD31 and vascular endothelial growth factor (VEGF) for angiogenesis, this study examined immunohistochemically 69 consecutive cases of CNS metastases. In contrast to low‐level expression in tumour‐embedded neural tissue, ECM [fibronectin (71%), laminin γ‐1 (79%) and collagen IV (92%)] and CD31‐positive microvascular densities (33 versus 4 vessels/field) were significantly richer in desmoplastic tumour stroma, which was present in 90% (53 of 59) of carcinomas, 100% (five of five) of malignant melanomas and 100% (two of two) of sarcomas. Collagen IV expression in tumour stroma was correlated with the expression of fibronectin (P = 0.013) and laminin (P = 0.034) and with infiltrative tumour edges (P = 0.005); fibronectin‐positive tumour stroma was correlated with a higher microvascular density (P = 0.015). In addition, tumour cells expressed integrins (～75%) and laminin (84%) more frequently than VEGF (23%), and tumour expression of laminin was correlated with the presence of desmoplastic stroma (P = 0.006). Interestingly, laminin‐positive tumour stroma was a worse prognosticator (P = 0.072). Conclusions: ECM‐ and vascular‐rich stroma is important in tumour growth, which underlies therapeutic strategies targeting tumour‐associated stroma.     

Expression of vascular endothelial growth factor and vascular endothelial growth factor receptors 1 and 2 in invasive breast carcinoma: prognostic significance and relationship with markers for aggressiveness

Dhakal H P, Naume B, Synnestvedt M, Borgen E, Kaaresen R, Schlichting E, Wiedswang G, Bassarova A, Holm R, Giercksky K‐E & Nesland J M
(2012) Histopathology  61, 350–364 Expression of vascular endothelial growth factor and vascular endothelial growth factor receptors 1 and 2 in invasive breast carcinoma: prognostic significance and relationship with markers for aggressiveness Aims: Vascular endothelial growth factor (VEGF), VEGF receptor 1 (VEGFR‐1) and VEGF receptor 2 (VEGFR‐2) play a role in breast cancer growth and angiogenesis. We examined the expression and relationship with clinical outcome and other prognostic factors. Methods and results: Tumour sections from 468 breast cancer patients were immunostained for VEGF, VEGFR‐1, and VEGFR‐2, and their relationships with tumour vascularity, disseminated tumour cells (DTCs) in bone marrow and other clinicopathological parameters were evaluated. VEGF, VEGFR‐1 and VEGFR‐2 immunoreactivities were observed in invasive breast carcinoma cells. VEGF expression was significantly associated with VEGFR‐1 and VEGFR‐2 expression (P < 0.001). High‐level cytoplasmic expression of VEGFR‐1 was associated with significantly reduced distant disease‐free survival (DDFS) (P = 0.017, log‐rank) and breast cancer‐specific survival (BCSS) (P = 0.005, log‐rank) for all patients, and for node‐negative patients without systemic treatment (DDFS, P = 0.03, log‐rank; BCSS, P = 0.009, log‐rank). VEGFR‐1 expression was significantly associated with histopathological markers of aggressiveness (P < 0.05). Significantly reduced survival was observed in DTC‐positive patients as compared with DTC‐negative patients in the combined moderate/high VEGFR‐1 group (P < 0.001 for DDFS and BCSS), and the same was true for DDFS in the moderate VEGFR‐2 group (P = 0.006). Conclusions: High‐level expression of VEGFR‐1 indicates reduced survival. Higher‐level expression of VEGFR‐1 or VEGFR‐2 in primary breast carcinomas combined with the presence of DTC selects a prognostically unfavourable patient group.     

Lymphangiogenesis in regional lymph nodes predicts nodal recurrence in pathological N0 squamous cell carcinoma of the tongue

Hirota K, Wakisaka N, Sawada‐Kitamura S, Kondo S, Endo K, Tsuji A, Murono S & Yoshizaki T
(2012) Histopathology
Lymphangiogenesis in regional lymph nodes predicts nodal recurrence in pathological N0 squamous cell carcinoma of the tongue Aims: Cancer cells induce de‐novo lymphatic vessel growth within draining lymph nodes before they metastasize. The aim of this study was to retrospectively evaluate lymph node lymphangiogenesis before the establishment of nodal recurrence in squamous cell carcinoma (SCC) of the tongue. Methods and results: Surgical specimens from 28 patients with pT2–T3N0M0 SCC of the tongue after local excision with supraomohyoid neck dissection were studied by immunohistochemistry. Intranodal lymphatic endothelium was highlighted by podoplanin staining to evaluate lymphatic vessel counts (LVCs). Primary tumour sections were examined for the expression of lymphangiogenic factors: vascular endothelial growth factor (VEGF)‐C and VEGF‐D. LVCs in regional lymph nodes were significantly increased in the cases with nodal recurrence (P = 0.0013). Simultaneous increases in VEGF‐C and VEGF‐D expression were significantly associated with both an increase in LVC in regional lymph nodes (P = 0.0001) and a decrease in the rate of survival without nodal recurrence (P = 0.016). Conclusions: Knowing the status of lymphangiogenesis in the regional pN0 lymph nodes in tongue cancer would help in predicting which patients will develop nodal recurrence. The use of a therapeutic approach which blocks lymphangiogenic factors, such as VEGF‐C and VEGF‐D, may be beneficial in suppressing the lymphatic spread of tongue cancer with intense intranodal lymphangiogenesis.     

Prognosis in human melanoma: PAR-1 expression is superior to other coagulation components and VEGF

Aims: Two hundred and four accessible cases of malignant melanoma from the Grampian region of Scotland, collected over a period of 4 years, with minimum follow‐up of 2 years, were studied for coagulation factors and vascular endothelial growth factor (VEGF) expression as potential prognostic markers. The aim was to allow comparison with previous work using microvessel density on the same cases. Methods and results: Immunohistochemistry for VEGF, tissue factor (TF), fibrin and protease‐activated thrombin receptor (PAR)‐1 in 204 cases of melanoma was performed, and intensity of expression scored. Chalkley microvessel counts (MVD) were obtained for the tumour edge. TF expression and presence of fibrin correlated well with Breslow thickness and ulceration, reaching statistical significance, but surprisingly not for metastatic recurrence. Fibrin was variably present in over half the cases, located at the invasive edge, ulcerated surface and between tumour cell surfaces. In a few cases fibrin was within tumour cells, typically co‐located with melanin and confirmed by electron microscopy. In contrast, immunohistochemistry for PAR‐1 produced statistically significant results, correlating expression with Breslow thickness (P ≤ 0.001), ulceration (P = 0.001) and recurrence (P ≤ 0.005). Intensity of reactivity of VEGF correlated significantly with Breslow thickness, Clark level, ulceration and MVD, but not for metastatic recurrence. Conclusions: It appears paradoxical that VEGF expression is not more predictive of recurrence, but even low expression may be sufficient for tumour angiogenesis and other factors must govern tumour aggression. Antagonism of VEGF may still prove a successful adjunct in future therapeutic trials. Both MVD and PAR‐1 can be used as adjuncts to Breslow thickness and ulceration as prognostic indicators for melanoma, as they appear to give independent information for all thicknesses. PAR‐1 expression is the best antibody marker of recurrence risk from those studied. It remains to be seen whether this methodology can predict response to novel antiangiogenic therapies currently entering trial.     

Association of mast cell, eosinophil leucocyte and microvessel densities in actinic cheilitis and lip squamous cell carcinoma

Souza L R, Fonseca‐Silva T, Santos C C O, Oliveira M V M, Corrêa‐Oliveira R, Guimarães A L S & De Paula A M B
(2010) Histopathology 57, 796–805 Association of mast cell, eosinophil leucocyte and microvessel densities in actinic cheilitis and lip squamous cell carcinoma Aims: To determine the contributions of mast cells (MC), eosinophil leucocytes (EL) and microvessel density (MVD) in lip carcinogenesis, and to establish the relationships between these biomarkers and their possible prognostic value in lip squamous cell carcinoma (LSCC). Methods and results: Archived specimens of lip mucosa (n = 13), actinic cheilitis (n = 29) and LSCC (n = 29) were formalin‐fixed, paraffin‐embedded, sectioned and stained with toluidine blue and haematoxylin and eosin (H&E) in order to identify MC and EL and to measure their densities. Tumour angiogenesis was estimated by determining, with the use of CD31 antibody MVD in areas with the highest number of stained microvessels (‘hot spots’). Progressive increases of MC, EL and MVD were observed during lip tumour development. Correlation analysis revealed positive associations between the biomarkers during tumour progression. In LSCC samples, significant associations were found between MVD values and metastatic disease. On multivariate analysis, MVD was a predictor of risk of cervical metastasis. Conclusions: The densities of MC, EL and microvessels increase during lip carcinogenesis, and for MC and EL this may be related to the stimulation of tumour angiogenesis. MVD could be a useful predictor of cervical metastasis in LSCC.     

Podoplanin expression by cancer-associated fibroblasts predicts poor outcome in invasive ductal breast carcinoma

Pula B, Jethon A, Piotrowska A, Gomulkiewicz A, Owczarek T, Calik J, Wojnar A, Witkiewicz W, Rys J, Ugorski M, Dziegiel P & Podhorska‐Okolow M
(2011) Histopathology  59, 1249–1260
Podoplanin expression by cancer‐associated fibroblasts predicts poor outcome in invasive ductal breast carcinoma Aims: It has recently been shown that podoplanin, a mucin‐type glycoprotein, is expressed by cancer cells and cancer‐associated fibroblasts (CAFs), and promotes cancer cell migration and invasiveness. The biological role of podoplanin expression in tumour stroma of invasive ductal carcinoma of the breast (IDC) has not been determined. Methods and results: Podoplanin expression was analysed in 117 cases of IDC and 27 cases of fibrocystic change, as well as in breast cancer cell lines, with the use of immunohistochemistry and real‐time polymerase chain reaction. In 82.1% of analysed tumours, podoplanin was found only in CAFs. Only two of 117 IDC cases (1.7%) were characterized by expression of this glycoprotein in cancer cells. None of the fibrocystic changes or stroma surrounding normal ducts showed podoplanin expression. Podoplanin‐positive CAFs correlated with tumour size (P = 0.0125), grade of malignancy (P = 0.0058), lymph node metastasis (P = 0.0149), lymphovascular invasion (LVI) (P = 0.0486) and Ki67 expression in cancer cells (P = 0.0128). High‐level podoplanin expression (>50% of positive stroma) in the tumour stroma was significantly associated with a negative oestrogen status (P = 0.0201). Univariate, but not multivariate, analysis showed that podoplanin expression by CAFs was associated with poor patient outcome (P = 0.0202). Conclusions: Our results suggest that podoplanin expression by CAFs could be an unfavourable prognostic marker for IDC.     

Papillary thyroid carcinomas with and without BRAF V600E mutations are morphologically distinct

Finkelstein A, Levy G H, Hui P, Prasad A, Virk R, Chhieng D C, Carling T, Roman S A, Sosa J A, Udelsman R, Theoharis C G & Prasad M L
(2012) Histopathology  60, 1052–1059 Papillary thyroid carcinomas with and without BRAF V600E mutations are morphologically distinct Aims: The BRAF V600E mutation resulting in the production of an abnormal BRAF protein has emerged as the most frequent genetic alteration in papillary thyroid carcinomas (PTCs). This study was aimed at identifying distinctive features in tumours with and without the mutation. Methods and results: Thirty‐four mutation‐positive and 22 mutation‐negative tumours were identified by single‐strand conformation polymorphism of the amplified BRAF V600E region in the tumour DNA. Mutation‐positive tumours were more common in patients older than 45 years (24/33, P = 0.05), in classic (23/30, P = 0.01), tall cell (4/5) and oncocytic/Warthin‐like (2/2) variants of PTC, and in subcapsular sclerosing microcarcinomas (4/4). In contrast, all 12 follicular variants (P < 0.0001) and two diffuse sclerosing variants were negative for the mutation. Mutation‐positive tumours displayed infiltrative growth (32/34, P = 0.02), stromal fibrosis (33/34, P < 0.001), psammoma bodies (17/34, P = 0.05), plump eosinophilic tumour cells (22/34, P = 0.01), and classic fully developed nuclear features of PTC (33/34, P = 0.0001). Encapsulation was significantly associated with mutation‐negative tumours (15/22, P = 0.02). Conclusions: BRAF V600E mutation‐positive and negative PTCs are morphologically different. Recognition of their morphology may help in the selection of appropriate tumours for genetic testing.     

Expression of classical NF‐κB pathway effectors in human ovarian carcinoma

Darb‐Esfahani S, Sinn B V, Weichert W, Budczies J, Lehmann A, Noske A, Buckendahl A‐C, Müller B M, Sehouli J, Koensgen D, Györffy B, Dietel M & Denkert C
(2010) Histopathology 56. 727–739
Expression of classical NF‐κB pathway effectors in human ovarian carcinoma Aims: Functional studies have demonstrated that nuclear factor (NF)‐κB promotes tumour progression in ovarian cancer cells. However, surprisingly little is known of the expression of effectors of the NF‐κB pathway in human ovarian cancer in vivo. Methods and results: Immunohistochemistry and in situ hybridization revealed that in a cohort of 85 primary ovarian carcinomas, total p65 expression was inversely correlated to nuclear and cytoplasmic phospho‐IκBα (P = 0.002 and P = 0.05, respectively), and IκBα mRNA expression (P = 0.032). In contrast, phospho‐p65 expression was paralleled by the expression of nuclear (P = 0.027) and cytoplasmic phospho‐IκBα (P = 0.01). Total p65 expression was an adverse prognostic factor for overall survival (P = 0.018). In contrast, total IκBα and phosphorylated nuclear and cytoplasmic IκBα expression were favourable prognostic markers (P = 0.001, P = 0.031, P = 0.001, respectively). Cytoplasmic phospho‐IκBα expression remained a significant prognostic factor on multivariate analysis (P = 0.010). In cultured, stimulated OVCAR‐3 ovarian cancer cells the cytoplasmic retranslocation of p65 was delayed by inhibition of the nuclear membrane transporter chromosomal region maintenance/exportin1 protein (CRM1). A positive association of p65 and CRM1 expression was demonstrated in ovarian cancer tissue (P < 0.0001). Conclusions: Total and phosphorylated IκBα protein expression might serve as markers for NF‐κB activation in human ovarian carcinoma. Cytoplasmic localization of p65 may be related to deregulated nucleocytoplasmic transport in carcinomas overexpressing CRM1.     

COX-2 expression correlates with microvessel density in non-melanoma skin cancer from renal transplant recipients and immunocompetent individuals

Angiogenesis, the generation of a new vascular network, is regulated in part by inducers of endothelial cell migration and proliferation, such as cyclooxygenase-2 (COX-2). Microvessel density (MVD) measurement is widely used to quantify angiogenesis in tissue sections of tumors, including cutaneous malignancies. The increasing number of successful renal transplantations worldwide is producing a progressive increase in patients at risk for non-melanoma skin cancers, such as squamous cell carcinoma (SCC), basal cell carcinoma (BCC), and Bowen's disease (BD), and at significantly increased risk for metastatic SCC. The aim of this study was to investigate whether there was any difference in angiogenesis between these tumor types in renal transplant recipients (RTRs) and immunocompetent individuals (ICIs) and whether angiogenesis in these tumors was related to COX-2 expression. The study measured angiogenesis and COX-2 expression in BD, SCC, BCC, and normal skin from both RTRs and ICIs. Vessel counts were performed, and COX-2 immunoexpression was assessed semiquantitatively. The MVD counts differed significantly between normal skin and all tumor types. Significant differences in MVD density were found between all SCCs and BCCs. BCCs from RTRs had significantly greater MVD at the invasive front of the tumor than BCCs from ICIs. Increased COX-2 expression correlated with increased MVD in all tumors examined. These findings indicate a difference in vascular profiles between RTRs and ICIs in BCCs and suggest a relationship between COX-2 and angiogenesis that may provide a possible treatment target for skin tumors in these 2 patient populations.     

Expression of key hypoxia sensing prolyl‐hydroxylases PHD1, ‐2 and ‐3 in pancreaticobiliary cancer

Gossage L, Zaitoun A, Fareed K R, Turley H, Aloysius M, Lobo D N, Harris A L & Madhusudan S
(2010) Histopathology 56, 908–920
Expression of key hypoxia sensing prolyl‐hydroxylases PHD1, ‐2 and ‐3 in pancreaticobiliary cancer Aims: Tumour hypoxia is associated with an aggressive phenotype and resistance to chemotherapy and radiotherapy. The aim was to investigate whether key hypoxia sensing prolyl hydroxylases PHD1, PHD2 and PHD3 are dysregulated in pancreaticobiliary cancers, and to evaluate their potential clinical significance. Methods and results: Formalin‐fixed human pancreatic tissue from 120 consecutive patients undergoing pancreatic resections between June 2001 and June 2006 was constructed into tissue microarrays. Expression of PHD1, PHD2 and PHD3 was analysed using immunohistochemistry and correlated with clinicopathological variables and disease‐specific overall survival. PHD1, PHD2 and PHD3 were significantly overexpressed in pancreaticobiliary tumours compared with normal pancreatic ductal tissues (P = 0.03, P < 0.0001 and P < 0.0001, respectively). PHD3 expression in tumour tissue was associated with a trend towards worse overall disease‐specific survival in ampullary adenocarcinomas (P = 0.035) and pancreatic adenocarcinomas (P = 0.084). Absence of PHD1 expression was significantly associated with perineural invasion in pancreatic adenocarcinomas (P = 0.02) and a trend towards significance was also seen for absence of PHD2 expression in pancreatic adenocarcinomas (P = 0.04). Conclusions: Our results provide the first clinical evidence that PHD1, PHD2 and PHD3 may be involved in pancreaticobiliary tumorigenesis.     

Angiopoietins 1 and 2 and Tie-2 receptor expression in human ductal breast disease

Staton C A, Hoh L, Baldwin A, Shaw L, Globe J, Cross S S, Reed M W & Brown N J
(2011) Histopathology 59 , 256–263 Angiopoietins 1 and 2 and Tie‐2 receptor expression in human ductal breast disease Aims: This study aimed to identify the involvement of the angiopoietin/Tie‐2 receptor system in breast cancer development, progression, metastasis and angiogenesis. Methods and results: We quantified and correlated angiopoietin‐1 (Ang‐1), Ang‐2 and Tie‐2 expression in sections of normal human breast, benign and premalignant hyperplastic tissue, pre‐invasive and invasive cancer, and compared these findings with our previously published data on vascular endothelial growth factor (VEGF) and microvessel density (MVD) in the same samples. A breast cancer tissue microarray was used to evaluate the prognostic value of these factors. Histological analysis revealed a significant decrease in Ang‐1 expression (P = 0.001) and an inverse correlation with MVD (r = ?0.442, P = 0.008) and VEGF (r = ?0.510, P = 0.002) in the non‐invasive lesions. In contrast Ang‐2 expression increased significantly (P = 0.0004) with increasing severity of lesion and correlated with MVD (r = 0.570; P = 0.0002), while Tie‐2 expression remained relatively unchanged. Expression of all three factors was reduced in invasive breast cancer and did not correlate with oestrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2), lymph node status or tumour grade. Conclusions: These data suggest that a change in the angiopoietin balance in favour of Ang‐2 is associated with the angiogenic switch at the onset of hyperplasia in the breast. However, angiopoietins and the Tie‐2 receptor are not related to known prognostic indicators in invasive breast cancer.     

Immunohistochemical features of post-radiation vaginal recurrences of endometrioid carcinomas of the endometrium: role for proteins involved in resistance to apoptosis and hypoxia

Santacana M, Yeramian A, Velasco A, Bergada L, Gatius S, García V, Azueta A, Palacios J, Dolcet X, Oliva E & Matias‐Guiu X
(2012) Histopathology  60, 460–471
Immunohistochemical features of post‐radiation vaginal recurrences of endometrioid carcinomas of the endometrium: role for proteins involved in resistance to apoptosis and hypoxia Aims: Endometrioid carcinoma of the endometrium (EEC) is treated with surgery and radiotherapy. Post‐radiation recurrences are associated with increased risk of metastases. Comparison of the expression of genes important in the development and progression of EEC, and others involved in resistance to apoptosis and hypoxia and adaptation to radiation, was performed between post‐radiation vaginal recurrences (PVRs) and primary EECs. We tried to reproduce the results by exposing an EEC cell line to hypoxia and radiation. Methods and results: Immunohistochemistry and tissue microarrays were used to compare 24 PVRs with 82 primary EECs. PVRs exhibited increased expression of p53 (P < 0.0001), cytoplasmic FLICE‐inhibitory protein (FLIP) (P < 0.0001), and Ki67 (P < 0.0001), and nuclear staining for FLIP, nuclear factor kappaB (NF‐κB) family members (p50, P < 0.0001; c‐Rel, P = 0.0077; RelB, P = 0.0157), and β‐catenin (P = 0.0001). Differences regarding p50, hypoxia‐inducible factor 1α (HIF‐1α), and cytoplasmic FLIP were statistically significant when PVRs and primary EECs were matched for histological grade. Exposure of the EEC cell line to hypoxia induced nuclear expression of β‐catenin, FLIP, and HIF‐1α, as well as increased NF‐κB activity. No changes in FLIP, HIF‐1α or NF‐κB were seen when cells were exposed to radiation. Nuclear expression of β‐catenin was seen at 3 Gy, but not at 1 Gy. Conclusions: Genes involved in resistance to hypoxia are expressed in PVRs, and may play a role in the development of post‐radiation recurrences.     

Immunohistochemical Expression of Vascular Endothelial Growth Factor and Vascular Endothelial Growth Factor Receptor in Canine Cutaneous Fibrosarcomas

The expression of five markers associated with tumour angiogenesis, proliferation and apoptosis was studied in 24 canine cutaneous fibrosarcomas. Tumours were assigned histological grades and were immunohistochemically evaluated for the expression of vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptor-2 (VEGFR-2). Additionally, intra-tumour microvessel density (iMVD) was assessed by immunohistochemical labelling for expression of von Willebrand factor (vWf) and tumour proliferation index (PI) was measured following labelling of Ki-67 antigen. Finally, tumour apoptotic index (AI) was determined by application of the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP end-labelling method (TUNEL). VEGF and VEGFR-2 expression were detected in 22/24 (92%) and 24/24 (100%) of fibrosarcomas, respectively. There was correlation between VEGF and VEGFR-2 expression (r = 0.51) and between histological grade and PI (r = 0.82). A significant difference in PI between tumours of different histological grade was found (P < 0.05). The median PI in grade 2 and 3 tumours (30.6 and 54.7, respectively) was significantly higher than in grade 1 tumours (6.4). Therefore, only PI correlates significantly with the histological grade of canine cutaneous fibrosarcomas. The potential for autocrine activity for VEGF exists in canine cutaneous fibrosarcomas, as VEGF and VEGFR-2 expression was found in most tumours.     

Systematic assessment of protein phenotypes characterizing high‐grade tumour budding in mismatch repair‐proficient colorectal cancer

Karamitopoulou E, Lugli A, Panayiotides I, Karakitsos P, Peros G, Rallis G, Patsouris E S, Terracciano L & Zlobec I (2010) Histopathology 57 , 233–243
Systematic assessment of protein phenotypes characterizing high‐grade tumour budding in mismatch repair‐proficient colorectal cancer Aims: A tumour bud is defined as a single tumour cell or tumour cell cluster of up to five cells at the invasive tumour front. Significant differences in survival have been detected in colorectal cancer patients with low‐ compared to high‐grade budding. The aim of this study was to identify potential multi‐marker phenotypes characterizing low‐ and high‐grade budding in mismatch repair (MMR)‐proficient colorectal cancer. Methods and results: Established and promising prognostic proteins such as epidermal growth factor receptor (EGFR), pERK, RHAMM, RKIP, β‐catenin, E‐cadherin, pAKT, p16, p21, Ki67, Bcl‐2, vascular endothelial growth factor (VEGF), apoptotic protease activating factor‐1 (APAF‐1), MUC1, EphB2, matrix metalloproteinase 7, pSMAD2, CDX2, laminin5γ2 and MST1 were analysed on 208 MMR‐proficient colorectal cancers with complete clinicopathological data. The most accurate markers for predicting high‐grade budding (more than six tumour buds) were EphB2 (P < 0.001), Bcl‐2 (P < 0.001), RKIP (P < 0.001), E‐cadherin (P = 0.004), laminin5γ2 (P = 0.004) and APAF‐1 (P = 0.005). On multivariable analysis, only loss of Bcl‐2 (P < 0.001) and EphB2 (P < 0.001) were independent predictors of high‐grade budding. Bcl‐2?/EphB2? tumours were more frequently poorly differentiated (P < 0.001), of advanced pT stage (P = 0.002), lymph node positive (P = 0.023), presented vascular (P = 0.053) and lymphatic invasion (P = 0.005) and had a negative impact on patient survival (P = 0.012). Conclusions: The multi‐marker phenotype EphB2?/Bcl‐2? is an independent predictor of high‐grade budding and implies increased aggressive behaviour in MMR‐proficient colorectal cancer.     

bcl-2 and c-erbB-2 proteins are involved in the regulation of VEGF and of thymidine phosphorylase angiogenic activity in non-small-cell lung cancer

Tumour angiogenesis has been recently recognised as one of the most important prognostic factors in lung cancer. Although a variety of angiogenic factors have been identified, the angiogenesis process remains poorly understood. Bcl-2, c-erbB-2 and p53 are well-known oncogenes involved in non-small-cell lung cancer pathogenesis. A direct correlation of thymidine phosphorylase (TP) and of vascular endothelial growth factor (VEGF) with intratumoural angiogenesis has been reported. In the present study we investigated the possible regulatory role if bcl-2, c-erB-2 proteins in angiogenesis and in VEGF and TP expression in non-small-cell lung cancer. Two hundred sixteen specimens from T1,2-N0,1 staged patients treated with surgery alone were immunohistochemically examined. Bcl-2 and c-erbB-2 were significantly inversely related to each other (P=0.04) and both were inversely associated with microvessel density (P<0.02). High TP and VEGF reactivity was statistically related to loss of bcl-2 expression (P<0.01). A significant co-expression of c-erbB-2 with TP was noted (P=0.01). However, TP expression was related to high angiogenesis only in cases with absence of c-erB-2 expression (P<0.0001). c-erbB-2 expression in poorly vascularised tumours was linked with poor outcome (P=0.03). The present study provides strong evidence that the bcl-2 gene has a suppressive function over genes involved in both angiogenesis (VEGF and TP) and cell migration (c-erbB-2) in NSCLC. TP and c-erbB-2 proteins are significantly, and often simultaneously, expressed in bcl-2 negative cases. However, expression of the c-erbB-2 abolishes the TP-related angiogenic activity. Whether this is a result of a direct activity of the c-erbB-2 protein or a consequence of a c-erbB-2-related immune response remains to be further investigated. This revised version was published online in July 2006 with corrections to the Cover Date.     

p16INK4A overexpression is frequently detected in tumour‐free tonsil tissue without association with HPV

Klingenberg B, Hafkamp H C, Haesevoets A, Manni J J, Slootweg P J, Weissenborn S J, Klussmann J P & Speel E‐J M
(2010) Histopathology 56, 957–967
p16 ^INK4A overexpression is frequently detected in tumour‐free tonsil tissue without association with HPV Aims: Oncogenic human papillomavirus (HPV) type 16 has been strongly associated with tonsillar squamous cell carcinoma (TSCC) and appears to be of prognostic significance. Because HPV+ TSCC also accumulates p16^INK4A, this cyclin‐dependent kinase inhibitor has been proposed as a potential biomarker for HPV in clinical diagnosis. The aim of this study was to determine the prevalence of HPV in tumour‐free tonsillar tissue and the value of p16^INK4A overexpression in predicting its presence. Methods and results: p16^INK4A overexpression was detected by immunohistochemistry in tissue sections of tumour‐free tonsils of 262 patients. They were treated for non‐oncological reasons (snoring or chronic/recurrent tonsillitis) consisting of tonsillectomy. Genomic DNA isolated from these tissues was subjected to HPV‐specific polymerase chain reaction (PCR) analysis. p16^INK4A immunoreactivity was detected in 28% of samples in both crypt epithelium (49/177) and lymphoid germinal centres (52/187), which correlated with each other (P < 0.0001). No reactivity was observed in superficial squamous cell epithelium. HPV16 and 18 were detected by PCR analysis in 2/195 cases (1%), which, however, were negative on fluorescence in situ hybridization analysis and discrepant on p16^INK4A immunostaining. Conclusions: No proof was found for the presence of HPV in tumour‐free tonsil tissue, despite increased p16^INK4A expression in a quarter of tonsil cases. Other mechanisms than HPV infection are therefore implicated in p16^INK4A up‐regulation.     

Mammalian target of rapamycin expression and laryngeal squamous cell carcinoma prognosis: novel preliminary evidence

Marioni G, Staffieri A, Giacomelli L, Lionello M, Guzzardo V, Busnardo A & Blandamura S
(2011) Histopathology  58, 1148–1156
Mammalian target of rapamycin expression and laryngeal squamous cell carcinoma prognosis: novel preliminary evidence Aims: The mammalian target of rapamycin (mTOR) has a key role in regulating cancer cell proliferation, apoptosis, cell migration, and angiogenesis. The aim of this study was to assess the relationships between mTOR and clinicopathological and prognostic parameters in laryngeal squamous cell carcinoma (SCC). Methods and results: Mammalian target of rapamycin expression was determined in 103 consecutive operable laryngeal SCCs. Among the mTOR‐positive cases, the locoregional recurrence rate was higher (P = 0.048) and the disease‐free survival (DFS) rate was shorter (P = 0.031) in patients with mTOR expression >50.7%. In the N₀ subgroup, the disease recurrence rate was higher (P = 0.034) and the DFS was shorter (P = 0.009) in patients with mTOR expression >50.7%. In mTOR‐positive patients, multivariate analysis showed that N stage (P = 0.0001) and mTOR status (P = 0.042) were independent indicators of a poor prognosis. Conclusions: mTOR appeared to be a significant predictor of DFS in univariate and multivariate models. mTOR expression in laryngeal SCC may be useful for the detection of patients at higher risk for recurrence, and N₀ patients at higher risk for early locoregional recurrence who might benefit from more aggressive therapy. The role of mTOR inhibitors in multimodality or multitarget strategies against laryngeal SCC warrants investigation.     

Tumour budding and the expression of cancer stem cell marker aldehyde dehydrogenase 1 in nasopharyngeal carcinoma

Luo W‐R, Gao F, Li S‐Y & Yao K‐T
(2012) Histopathology
Tumour budding and the expression of cancer stem cell marker aldehyde dehydrogenase 1 in nasopharyngeal carcinoma Aims: To detect the prognostic significance of tumour budding and its expression of aldehyde dehydrogenase 1 (ALDH1) in nasopharyngeal carcinoma (NPC). Methods and results: Tumour budding was investigated in 105 patients with NPC by immunohistochemistry for pan‐cytokeratin (AE1/AE3). The intensity of budding correlated strongly with T classification (P = 0.008), lymphatic invasion (P < 0.001), vascular invasion (P = 0.029), lymph node metastasis (P < 0.001), and clinical stage (P = 0.010). Univariate analysis revealed that patients with high budding grade had poorer survival than those with low grade (P = 0.002). Multivariate analysis showed that tumour budding was an independent predictor of survival (P = 0.001). Furthermore, budding cells showed high‐level expression of the cancer stem cell (CSC) marker ALDH1. Budding cells with high‐level ALDH1 expression contributed to several aggressive behaviours and poor survival (P = 0.000). Conclusions: We describe, for the first time, the presence of tumour budding and its correlation with aggressive tumour behaviour and poor patient survival in NPC. The degree of tumour budding could be a valuable predictive factor in NPC. In addition, we show, also for the first time, that budding cells in NPC might possess the invasive and metastatic properties of CSCs.     

CMV drives the expansion of highly functional memory T cells expressing NK‐cell receptors in renal transplant recipients

Cytomegalovirus (CMV) is a common opportunistic infection encountered in renal transplant recipients (RTRs) and may be reactivated without symptoms at any time post‐transplant. We describe how active and latent CMV affect T‐cell subsets in RTRs who are stable on maintenance therapy. T‐cell responses to CMV were assessed in RTRs (n = 54) >2 years post‐transplant, and healthy controls (n = 38). Seven RTRs had CMV DNA detectable in plasma. CMV antibody and DNA aligned with increased proportions of CD8⁺ T cells and reduced CD4/CD8 ratios. This paralleled an expansion of effector memory T‐cell (T_EM), terminally differentiated T‐cell (T_EMRA) and CD57⁺ T_EMRA cell populations. Expression of NK‐cell receptors, LIR‐1 and KLRG1 on CD4⁺ and CD8⁺ CD57⁺ T_EM and T_EMRA cells correlated with elevated interferon‐γ and cytotoxic responses to anti‐CD3 and increased cytotoxic responses to CMV phosphoprotein (pp) 65 in RTRs who carried CMV DNA. CD8⁺ T cells from all CMV seropositive RTRs responded efficiently to CMV immediate early (IE) ‐1 peptides. The data show that latent and active CMV infection can alter T‐cell subsets in RTRs many years after transplantation, and up‐regulate T‐cell expression of NK‐cell receptors. This may enhance effector responses of CD4⁺ and CD8⁺ T cells against CMV.