Niki J. Agnantis

Evidence for lymphangiogenesis and its prognostic implications in head and neck squamous cell carcinoma.

Lymph node metastasis is a frequent reason for adverse clinical outcome in many epithelial neoplasms, including head and neck squamous cell carcinoma. The mechanisms underlying the capability of epithelial neoplasms to metastasize via lymphatic vessels have not yet been fully elucidated. There is great debate about whether cancer cells can metastasize by expansion and invasion of pre‐existing peritumoral lymphatics or by the formation and invasion of new lymphatics within tumours (lymphangiogenesis). In order to investigate this issue, we examined 81 tissue specimens from patients with head and neck squamous cell carcinoma, using immunostaining for the specific lymphatic endothelium marker podoplanin, and assessed intratumoral and peritumoral lymphatic density. We also quantified lymphatic invasion and examined the possible associations of all the above parameters with clinicopathological features and outcome. Finally, we used double staining with podoplanin and the cell proliferation marker Ki‐67 in order to evaluate lymphangiogenesis. High intratumoral and peritumoral lymphatic density were both significantly associated with the presence of lymph node metastasis at the time of diagnosis (χ2 test, p < 0.001 and p = 0.007, respectively) and there was a significant correlation between high intratumoral lymphatic density and lymphatic invasion. Patients with higher intratumoral lymphatic density exhibited shorter overall survival (log rank p < 0.001) and this correlation remained significant after multivariate analysis (Cox p = 0.04), indicating that intratumoral lymphatic density is an independent prognostic factor for mortality. Peritumoral lymphatic density had no influence on outcome. Double staining revealed the existence of proliferating intratumoral lymphatics, in which tumour emboli were occasionally observed. These results indicate that lymphangiogenesis indeed occurs in head and neck squamous cell carcinoma; that newly formed vessels are targets of invasion by cancer cells; and that intratumoral lymphatic density might be used as a criterion to separate patients at higher risk of an adverse clinical outcome. Copyright

Transcription factor E2F‐1 acts as a growth‐promoting factor and is associated with adverse prognosis in non‐small cell lung carcinomas

Numerous upstream stimulatory and inhibitory signals converge to the pRb/E2F pathway, which governs cell‐cycle progression, but the information concerning alterations of E2F‐1 in primary malignancies is very limited. Several in vitro studies report that E2F‐1 can act either as an oncoprotein or as a tumour suppressor protein. In view of this dichotomy in its functions and its critical role in cell cycle control, this study examined the following four aspects of E2F‐1 in a panel of 87 non‐small cell lung carcinomas (NSCLCs), previously analysed for defects in the pRb‐p53‐MDM2 network: firstly, the status of E2F‐1 at the protein, mRNA and DNA levels; secondly, its relationship with the kinetic parameters and genomic instability of the tumours; thirdly, its association with the status of its transcriptional co‐activator CBP, downstream target PCNA and main cell cycle regulatory and E2F‐1‐interacting molecules pRb, p53 and MDM2; and fourthly, its impact on clinical outcome. The protein levels of E2F‐1 and its co‐activator CBP were significantly higher in the tumour area than in the corresponding normal epithelium (p<0.001). E2F‐1 overexpression was associated with increased E2F‐1 mRNA levels in 82% of the cases examined. The latter finding, along with the low frequency of E2F‐1 gene amplification observed (9%), suggests that the main mechanism of E2F‐1 protein overexpression in NSCLCs is deregulation at the transcriptional level. Mutational analysis revealed only one sample with asomatic mutation at codon 371 (Glu→Asp) and one carrying a polymorphism at codon 393 (Gly→Ser). Carcinomas with increased E2F‐1 positivity demonstrated a significant increase in their growth indexes (r=0.402, p=0.001) and were associated with adverse prognosis (p=0.033 by Cox regression analysis). The main determinant of the positive association with growth was the parallel increase between E2F‐1 staining and proliferation (r=0.746, p<0.001), whereas apoptosis was not influenced by the status of E2F‐1. Moreover, correlation with the status of the pRb–p53–MDM2 network showed that the cases with aberrant pRb expression displayed significantly higher E2F‐1 indexes (p=0.033), while a similar association was noticed in the group of carcinomas with deregulation of the p53–MDM2 feedback loop. In conclusion, the results suggest that E2F‐1 overexpression may contribute to the development of NSCLCs by promoting proliferation and provide evidence that this role is further enhanced in a genetic background with deregulated pRb–p53–MDM2 circuitry. Copyright

Prognostic significance of VEGF immunohistochemical expression and tumor angiogenesis in head and neck squamous cell carcinoma

Purpose: Tumor angiogenesis is crucial for both the growth of the primary tumor and the development of metastases. Among the factors causing tumor angiogenesis, vascular endothelial growth factor (VEGF) is considered as a leading candidate. We aimed to assess the prognostic significance of VEGF and tumor angiogenesis in head and neck squamous cell carcinoma (HNSCC). Methods: We performed a retrospective study of 69 patients with HNSCC, in order to investigate whether VEGF immunohistochemical expression and tumor angiogenesis correlate with clinicopathological parameters and outcome. Tumor angiogenesis was estimated by determining microvessel density (MVD), and VEGF expression was assessed quantitatively. Results: Vascular endothelial growth factor and MVD correlated statistically significant with the clinical stage, but not with the presence of lymph node metastasis at the time of diagnosis. Tumors located in the oral cavity and larynx more often expressed high VEGF immunostaining compared with tumors located in the lower lip. High VEGF expression was associated with higher clinical stage and worse overall survival in this cohort of patients. Conclusions: Vascular endothelial growth factor expression may have prognostic significance for patients with HNSCC.

Perspectives and Risks of Breast-Conservation Therapy for Breast Cancer

Although breast cancer, with 211,300 new cases expected in 2003 in the United States, continues to be the most common malignant tumor among women, it is a highly treatable disease with a 5-year survival rate of 97% for localized disease in the United States.1 Logically, scientific efforts have been focused on the improvement of quality of life of these women. As a result, the concept of a less radical surgery has been developed toward replacement of total mastectomy considering the strong desire of women for breast conservation and advances in the understanding of breast cancer spread mechanisms. Total mastectomy has been the standardized primary treatment in all stages, early or late, of breast cancer although there is little or no evidence that it is superior to breast-conservation treatment. Earlier results from randomized controlled trials have suggested that there is no significant difference in survival after breast-conservation treatment or total mastectomy.2a However, because of the long natural history of breast cancer and the lack of long-term follow-up results, breast-conserving treatment has not yet been established the treatment of choice. Therefore, the recently published 20-year follow-up results of two landmark studies3,4 are relevant for treatment decision-making. They confirm earlier results that total mastectomy is not superior to breast-conservation treatment with respect to survival, but the rate of local failure was 14.3%3 and 8.8%,4 respectively. In an accompanying editorial, it was pointed out that it is time to declare the case against breast-conserving therapy closed and to increase the current low rate of breast-conserving surgery.5 These reports and the better quality of life after breast conservation will lead to wider clinical use of breast conservation treatment increasingly replacing total mastectomy in the management of early breast cancer during the next years. What will be the consequences of this expected trend in the treatment strategy of early-stage breast cancer? Is breast-conservation treatment beneficial in all women with stage I/II breast cancer or are there risks from an over simplicity in decision-making? Can these risks be minimized and how? Randomized controlled trials (RCTs) provide the highest level of evidence but they are limited by the lack of generalizability in each individual patient. What lessons can we take from RCTs available for breast-conserving treatment with respect to their pitfalls and the risks for each individual woman enrolled in these studies? Scientific data report that breast-conserving surgery is associated with higher rates of close or positive margins than mastectomy. The rate of positive margin on the final excision has been consistently high and ranges from 10% in the National Surgical Adjuvant Breast and Bowel Project (NSABP B-06) trial3 to 48% in the European Organization for Research and Treatment of Cancer (EORTC) trial.6 In recent nonrandomized studies a similar high overall rate of close ( 2 mm) or positive margin has also been reported ranging from 22%7 to 41%.8 This variation is attributable to the selection criteria, definition of margin status, extent of conservative surgery (lumpectomy, quadrantectomy, local/wide excision), tumor size, adjuvant treatment, and institution. Close or positive margin on final resection specimens is clearly associated with increased risk of local failure although the magnitude of this risk cannot accurately be estimated. Long-term data on the use of breast-conserving therapy in patients with positive margins is obviously limited and recurrence rate varies considerably.9 In most Received May 23, 2003; accepted June 10, 2003. From the Departments of Surgery (DHR) and Pathology (NJA), Ioannina University School of Medicine, Greece. Address correspondence to: Dimitrios H. Roukos, MD, Department of Surgery, Ioannina University School of Medicine, GR-451 10 Ioannina, Greece; Fax: 30-26510-97094; E-mail: droukos@cc.uoi.gr.

Preventing Breast, Ovarian Cancer in BRCA Carriers: Rational of Prophylactic Surgery and Promises of Surveillance

Women with inherited mutations in the BRCA1 or BRCA2 (BRCA) genes face a high lifetime risk of developing breast and ovarian cancer. Breast cancer is the most common female malignancy in the Western world; 275,000 women will receive a new diagnosis of breast cancer this year in the United States.1 Given that BRCA carriers make up 5 to 10% (13,750-27,500 women in the USA) of all breast cancer cases, increasing is the interest for the management of these women. As genetic test, with the power to identify these high-risk women, has been widely available and preventive surgical and nonsurgical choices abound, there is growing controversy and uncertainty about prevention choice. Ideally, prevention strategy should provide the best combination of cancer protection, survival and quality of life (QoL). Goal of prophylactic surgery is to eliminate risk of cancer, by removing the organ(s) targeted by the BRCA mutated genes, namely breasts and ovaries, before the disease clinically occurs. By contrast, preservation or surgical resection of these target organs only when screening-detected early-stage cancer occurs represents the principal aim of close surveillance. In the absence of data from randomized controlled trials (RCTs) for apparent reasons, prevention management should be guided by other nonrandomized reports.2 The evident high risk of cancer for BRCA carriers urgently suggests the need for intervention. But for which prevention option are there the most convincing data? Wide variation in risk estimates, diverse impacts of surgical and nonsurgical prevention measures on survival and QoL and insufficient data, make decision for prevention of BRCA mutation carriers too complicated and challenging. As new data become available over the last year regarding lifetime risk,3 the efficacy and limitations of surgical and nonsurgical procedures,4–6 and decision analysis,7 critical analysis on the light of these new findings may help women and their physician to deal with this dilemma. Ten years after the discovery of BRCA genes an explosion of research on understanding the molecular mechanisms of BRCA pathway has been noted. But the expectation that this elucidation could lead to effective chemoprevention of the most common noninherited (sporadic) cancer has been proven elusive. Although the BRCA genes themselves appear unconnected to common, nonhereditary cancers, advances in understanding BRCA genes’ biological function,8–10 suggest that defects in other parts of the BRCA pathway might be critical not only driving breast cancer but other cancers as well.9,10 Uncertainty about risk magnitude is a major obstacle in making decision. Most, but not all, women with inherited mutations that affect one allele of either BRCA1 or BRCA2, will develop breast and/or ovarian cancer. This risk varies considerably between 40% to 85% for breast cancer and 11% to 65% for ovarian cancer.3,8,11,12 This variation is depended on mutated gene (BRCA1 or BRCA2 and different mutations in the same gene), family history (strong or weak), as well as genetic and external factors that modify genetic risk.8 Data available suggest that among women with BRCA mutations, those with a strong family history have a higher risk13 than those without such a family history.14,15 However, a recent study published in Science3 may overturn this widely accepted status.16 In the New York Breast Cancer Study (NYBCS) on 1,008 New York-area Ashkenazi Jewish women diagnosed with incident, breast cancer, 104 (10.3%) female probands carried an Received August 24, 2004; accepted October 11, 2004. From the Departments of Pathology (NJA), Gynecology and Obstetrics (EP) and Surgery (DR), at the Ioannina University School of Medicine, GR – 45110, Ioannina, Greece. Address correspondence to: Dimitrios H. Roukos, MD, Department of Surgery, Ioannina University School of Medicine, GR – 45110, Ioannina, Greece; Fax: 3

COX-2 expression correlates with VEGF-C and lymph node metastases in patients with head and neck squamous cell carcinoma

Recent observations suggest an implication of the cyclooxygenase-2 (COX-2) in tumor lymphangiogenesis through an upregulation of vascular endothelial growth factor-C expression. It is unknown whether this mechanism also acts in squamous cell carcinoma of the head and neck region. We performed a retrospective study of 70 patients with head and neck squamous cell carcinoma in order to investigate whether COX-2 immunohistochemical expression correlates with vascular endothelial growth factor-C expression. We also examined the association of the expression of these molecules with clinicopathologic parameters (especially lymph node status) and outcome for these patients. We performed immunostaining on formalin-fixed, paraffin-embedded tissue sections by the routine streptavidin–biotin peroxidase labeled procedure. Increased cyclooxygenase-2 expression was observed in 30 of the 68 tumor samples (44%), while high vascular endothelial growth factor-C expression occurred in 26 of the 68 tumor samples (38%). High expression of the two proteins was correlated with the presence of lymph node metastasis at the time of diagnosis, and the observed association was even stronger when there was overexpression for both the antibodies (P<0.001). High expression of vascular endothelial growth factor-C, but not of COX-2 was correlated with increased mortality in patients with oral–larynx squamous cell carcinoma. When multivariate Cox regression model was applied, the presence of lymph node metastasis at the time of diagnosis, combined with overexpression of both the antibodies, was the only independent prognostic factor for mortality of these patients. Our results suggest that a lymphangiogenic pathway, in which COX-2 overexpression stimulates vascular endothelial growth factor-C upregulation, probably exists in head and neck squamous cell carcinoma. Also, the predictive ability for mortality of regional lymph node metastasis can be improved with the combined evaluation of the immunohistochemical expression of these two proteins.

C-MYC and IGF-II mRNA-binding protein (CRD-BP/IMP-1) in benign and malignant mesenchymal tumors

Mouse coding region determinant–binding (mCRD‐BP) and human IGF‐II mRNA–binding 1 (hIMP‐1) proteins are orthologous mRNA‐binding proteins that recognize c‐myc and IGF‐II mRNA, respectively, and regulate their expression posttranscriptionally. Here, we confirm that human CRD‐BP/IMP‐1 binds to c‐myc mRNA and that it is predominantly expressed in fetal tissues. Moreover, hCRD‐BP/IMP‐1 expression was detected in cell lines of neoplastic origin and in selected primary tumors. In a series of 33 malignant and 10 benign mesenchymal tumors, 73% and 40%, respectively, were found to express hCRD‐BP/IMP‐1. In particular, expression was significant in 14 Ewings sarcomas, all of which were positive. The data suggest that hCRD‐BP/IMP‐1 plays a role in abnormal cell proliferation in mesenchymal tumors.

Potential autocrine function of vascular endothelial growth factor in head and neck cancer via vascular endothelial growth factor receptor-2

Vascular endothelial growth factor is a peptide with well-defined actions on the vasculature and fundamental role in tumor angiogenesis. Its action in vascular endothelium is exerted in a paracrine manner. The immunohistochemical expression of this protein by cancer cells in head and neck squamous cell carcinoma was correlated with increased tumor aggressiveness and poor survival in previous studies. In the past years, an increasing amount of studies demonstrated potential autocrine action of vascular endothelial growth factor in various neoplasms. However, the existence and the impact of such autocrine action in head and neck cancer have not been demonstrated yet. In this retrospective study, we evaluated the expression of vascular endothelial growth factor and its receptors in neoplastic cells, in a cohort of patients with head and neck squamous cell carcinoma, and compared this expression with tumor aggressiveness, clinicopathologic parameters and outcome. High expression of vascular endothelial growth factor was strongly correlated with high expression of vascular endothelial growth factor receptor-2 (but not vascular endothelial growth factor receptor-1) on the cancer cells (P<0.001). The co-overexpression of both the protein and vascular endothelial growth factor receptor-2 was associated with higher tumor proliferation rate (P<0.001). The above co-overexpression also correlated with worse survival (log rank P<0.05) in patients with oral–larynx squamous cell carcinoma. Our results suggest that an autocrine vascular endothelial growth factor loop, mediated via vascular endothelial growth factor receptor-2, probably exists in head and neck squamous cell carcinoma. These observations support the hypothesis that the use of vascular endothelial growth factor receptor-2 inhibitors as adjuvant antiangiogenic therapy might have beneficial effects for these patients, by disrupting both paracrine (endothelial-dependent) and autocrine actions of vascular endothelial growth factor.

Endoglin (CD105) as a prognostic factor in head and neck squamous cell carcinoma

Endoglin (CD105) is a proliferation-associated protein abundantly expressed in angiogenic endothelial cells. Recent studies revealed that CD105 is intensively expressed in tumor vasculature, whereas intratumoral microvessel density (MVD) determined with the use of antibodies to CD105 has been found to be an important prognostic indicator for the outcome in a number of malignancies. In the current study, we investigated endoglin expression and evaluated MVD in 108 patients with head and neck squamous cell carcinoma. Endoglin was intensively expressed in intratumoral blood vessels, whilst lymphatics were rarely positive for CD105. High microvessel density was associated with a more aggressive tumor phenotype, including advanced clinical stage (p=0.008) and the presence of lymph node metastasis at the time of diagnosis (p=0.02). When microvessel counts were assessed for their prognostic values (high vs low MVD), there was a statistically significant difference in the overall survival among patients with tumors of the oral cavity and larynx (p<0.001) and in the disease-free survival among patients with tumors of the lower lip (p=0.01). The prognostic impact of microvessel density was not dependent on clinical stage or lymph node status. The results of the current study suggest that CD105 is a promising target for tumor imaging and prognosis.

Estrogen receptor beta (ERβ) is expressed in brain astrocytic tumors and declines with dedifferentiation of the neoplasm

Purpose Estrogen receptor β (ERβ) is the second identified receptor mediating the effects of estrogen on target tissues. The role of ERβ in cancer pathobiology is largely unknown, because specific antibodies have not been available until recently. Initial studies have shown that ERβ expression declines in breast, ovarian, prostatic, and colon carcinomas. Tamoxifen, a synthetic anti-estrogen compound that is a mixed agonist/antagonist of estrogen receptor α (ERα) and a pure antagonist of ERβ, has moderate beneficial effects in human astrocytic neoplasms. However, most published studies agree that glial tumors do not express ERα. The purpose of this study was to explore the expression of ERβ in astrocytic neoplasms.Methods ERβ expression was monitored immunohistochemically in 56 cases of astrocytomas of all grades (grade I–IV) and in adjacent non-neoplastic brain tissue.Results Moderate or strong nuclear immunopositivity was obtained in non-neoplastic astrocytes and in low-grade astrocytomas, whereas the majority of high-grade tumors were immunonegative or displayed weak immunoreactivity. The progressive decline in ERβ expression paralleled the increase in tumor grade.Conclusions In as much as ERβ is possibly the only ER expressed in astrocytes, its decreased expression may play an important role in astrocytic tumor initiation and in the potential response of glial neoplasms to tamoxifen.