Silvia Gisfredi

Mutational Analysis in Cytological Specimens of Advanced Lung Adenocarcinoma: A Sensitive Method for Molecular Diagnosis

Introduction: The discovery that somatic mutations in the epidermal growth factor receptor (EGFR) gene are associated with sensitivity to the EGFR tyrosine kinase inhibitors (TKIs) in lung adenocarcinomas, whereas Kras mutations are associated with resistance, has generated excitement among both clinicians and researchers studying non-small cell lung cancer (NSCLC). Mutational analysis may soon be very useful in choosing among a wide range of targeted therapies to individualize treatment to tumor characteristics. This analysis would be even more useful in patients with advanced NSCLC, in whom cytological specimens are often the only material available. Methods: We analyzed 23 archived cytologic specimens of advanced/metastatic lung adenocarcinomas for mutations in EGFR exons 18 to 21, and Kras exon 2. Results: Our data show that our cytological specimens were perfectly adequate for the molecular analysis of EGFR and Kras mutations. EGFR TK domain mutations were found in three cases (13.04%) and were associated with both female gender (p = 0.02) and a nonsmoking history (p = 0.008). Moreover, we explored the relationship between EGFR mutation status and the presence of Kras mutations. Kras mutations involving codon 12 in exon 2 were found in 5 (21.73%) of the 23 adenocarcinomas and were associated, where known, with smoking habits. We never found EGFR alterations in tumors with Kras mutations. Conclusions: Our results provide oncologists with a highly accurate laboratory method to identify biological predictors of the efficacy of different therapies, and they may have an important impact on clinical practice. This method may be particularly useful in patients with advanced/metastatic NSCLC.

Expression and Mutational Status of c-kit in Small-Cell Lung Cancer Prognostic Relevance

Purpose: The c-kit protein, also known as CD117, is a member of the type III receptor tyrosine kinase family. Kinase activity has been implicated in the pathophysiology of many tumors, including small-cell lung carcinoma (SCLC). Autocrine or paracrine activation of c-kit by its ligand has been postulated for lung cancer, but this receptor can also be activated by mutations of the c-kit gene. We examined c-kit expression and mutational status in SCLC to verify its putative expression and genetic alterations, as well as its eventual prognostic impact. Experimental Design: We studied 60 SCLC samples to determine the mutations of the coding region of the gene; the exons 9 and 11 were analyzed by PCR-single-strand conformational polymorphism and automated sequencing. Moreover, c-kit expression was evaluated in 55 samples by immunohistochemical method. Results: Expression of c-kit was demonstrated in about 40% of SCLC samples. Two mutations in exon 9 and three mutations in exon 11 were found. Kaplan-Meier analysis revealed no prognostic significance of c-kit expression for survival. Conclusions: In our series, the expression of c-kit and its mutational status failed to appear relevant or to have a significant impact on survival; this makes the therapeutic approach with an inhibitor of tyrosine kinase more difficult in SCLC until a sure demonstration of c-kit implication is obtained for this tumor.

Angiogenesis in intracranial meningiomas: immunohistochemical and molecular study

Much of the morbidity of intracranial meningiomas is related to the degree of tumour vascularity and the extent of peritumoural vasogenic oedema. Several studies have shown that vascular endothelial growth factor (VEGF) is up‐regulated in meningiomas, although its relationship with tumour vasculature is still unclear. In order to better understand the angiogenic assessment of intracranial meningiomas, we analysed its vascular pattern, both as number and as morphologic configuration of microvessels. Moreover, we investigated the mRNA‐VEGF expression, relating this expression to vascular pattern. A total of 40 intracranial meningiomas, classified as benign (31 cases), atypical (7 cases), and anaplastic (2 cases) were analysed. RT‐PCR analyses of mRNA‐VEGF and competitive‐PCR were performed. VEGF expression and microvessel density (MVD) were also immunohistochemically investigated. Grade II–III meningiomas showed numerous small microvessels (mean: 34), while the majority of Grade I showed few larger vessels (mean: 13.09) (P = 0.000003). A microvessel pattern overlapping into atypical subtype was found in eignt of the 31 (25.8%) Grade I meningiomas. A significant association was found between grading and vascular pattern (P = 0.0002), as well as between the MVD and the immunohistochemical expression of VEGF (P = 0.0005). The expression of mRNA agreed with the immunohistochemical expression of the protein (P < 0.0001). A total of 39 cases expressed the 121 VEGF isoform and, among these, 28 cases also expressed the 165 isoform. Only 9 cases expressed both isoforms 165 and 189. Grade II and III meningiomas showed a preponderant expression of soluble isoforms (121 and 165). These results prompt us to speculate that the microvessel pattern could underlie a higher metabolic demand, probably due to a rapid growth with a consequent worse clinical behaviour of the tumour. In this sense, the vascular pattern may be used as a prognostic factor, in order to mostly focus attention on those Grade I meningiomas which have a higher likelihood of either recurrence or development of perilesional oedema. The pattern of vasculature itself seems to be dependent on the types of VEGF isoforms: the Grade II–III meningiomas (that presented numerous microvessels) expressed the soluble isoforms 121 and 165, while the isoform 189 was more frequently detected in Grade I meningiomas.

Alterations of Fas (APO-1/CD 95) gene and its relationship with p53 in non small cell lung cancer

The Fas (APO-1/CD95) system regulates a number of physiological and pathological processes of cell death. The ligand for Fas induces apoptosis by interacting with a transmembrane cell surface Fas receptor. The key role of the Fas system has been studied mostly in the immune system, but Fas mutations, one of the possible mechanisms for resistance to apoptosis signaling, may be involved in the pathogenesis of non-lymphoid malignancies as well. To better understand the potential involvement of Fas system in non-small cell lung cancer (NSCLC) we evaluated Fas and Fas-ligand mRNA expression by polymerase chain reaction in 102 tumor samples and in 44 normal surrounding tissues. Although over 60% of the human NSCLC analysed expressed both genes, they seem to be unable to induce apoptosis in vivo by autocrine suicide. In this regard, we investigated in 79 cases, the promoter and the entire coding region of the Fas gene by polymerase chain reaction, single strand conformation polymorphism and DNA sequencing for detecting putative alterations. Sixteen tumors (20.25 %) were found to have Fas alterations, in promoter and/or exon region. In all cases samples carried heterozygous alterations and mostly showed simultaneous mutations of p53 gene. Moreover, the quantitative analysis of Fas mRNA expression showed high levels of Fas messenger associated with p53 wild-type status alone. Taken together, these findings point to an involvement of Fas/Fas-ligand system in the development of NSCLC, suggesting that the loss of its apoptotic function might be linked to p53 alterations which contribute to the self-maintenance of cancer cells.

Effect of the p53 codon 72 and intron 3 polymorphisms on non-small cell lung cancer (NSCLC) prognosis

A total of 42 polymorphisms have been identified in the TP53 gene. The polymorphic site of p53 at codon 72 in exon 4 and p53PIN3, a 16 bp insertion/duplication in intron 3, are the most studied. We tested p53PIN3 and the combined effect of the p53 codon72 and PIN3 polymorphisms on prognosis in 101 NSCLC cases. This study provides support for the prognostic effects of the multi-variant alleles from p53 exon 4 and intron 3, resulting in a significantly poorer prognosis in NSCLC. This approach highlights the value of examining multiple polymorphisms in genes to improve survival estimates.

CD34 microvessel density and VEGF expression in basal and squamous cell carcinoma.

Angiogenesis is a central process in the growth of solid tumors. The purpose of our study was to analyze the angiogenic pattern in squamous and basal cell carcinomas and to point out differences in microvessel density that could explain their different biological behaviour. Thirty-nine skin tumors (26 basal and 13 squamous cell carcinomas) were analyzed. In all samples, the microvessels density (MVD) and the levels of vascular endothelial growth factor mRNA (VEGFmRNA) were analyzed, together with the inter-relationship between these two variables. Using the median value of the entire series (33 vessels per 2.22 mm2), tumors with low and high MVD were identified. The majority of cancers with high vascularization belonged to the squamous histotype (12 of 39), while 19 of the 26 basal cell carcinomas showed a lower number of microvessels than the median value (p = 0.0001). The median value of VEGFcDNA quantitation allowed us to distinguish tumors with high VEGF expression (> 470 molecules cDNA) from those with low (< or = 470 molecules) VEGF expression: 20 of the 26 basal cell carcinomas showed low VEGF expression, while 11 of the 13 squamous cell carcinomas showed high VEGFcDNA levels (p = 0.0003). Moreover, a significant association between a high microvessel density and high VEGFmRNA levels (p = 0.006) was found. Furthermore, when studying VEGF expression by immunohistochemistry, we obtained similar results and noted a correlation with VEGFmRNA expression (p < 0.0001). The association between high vascularization, high VEGF levels, and squamous cell histotype suggests the possible role of neoangiogenesis in determining the more aggressive biological behaviour of this type of cancer.

Expression of endothelin 1 and its angiogenic role in meningiomas.

Meningiomas are one of the most frequent central nervous system tumours. Although slow-growing at times, they continue to be a cause of morbidity and mortality. The endothelin (ET) family consists of three isoforms: ET-1 is the most abundant one. ET-1 may be involved in meningioma tumourigenesis in concert with other growth factors, in particular with angiogenic agents. We analysed ET-1 expression by immunohistochemistry and its activating system by reverse-transcription–polymerase chain reaction in 56 cases of meningioma. We found an association between high-grade meningiomas and high ET-1 expression levels (p=0.002). Moreover, we evaluated the potential angiogenic role of ET-1, finding an elevated microvessel count in tumours with high ET expression levels (p=0.004). ET-1 may contribute to meningioma growth by inducing formation of new blood vessels. The finding that ET-1 expression positively correlates with vascular endothelial growth factor (VEGF) expression in meningiomas (p=0.03) also supports the hypothesized modulating effect of ET-1 on angiogenesis. Thus, the influence of the ET system on the progression of meningiomas may occur through stimulation of VEGF. The association of ET-1 and meningioma represents a potential area for therapeutic intervention with selective ET inhibitors. Additional clinical studies will be needed before inhibitors can be incorporated in clinical practice.

Expression of Cyclooxygenase-2 and Its Correlation with Vasogenic Brain Edema in Human Intracranial Meningiomas

COX-2 expression was evalueted in intracranial meningiomas, relating this molecule to grade, vasculature, VEGF and brain edema. Fifty-six tumors were evaluated for COX-2 and VEGF expression and for microvessel density. In 34/56 cases, the edema was evaluated by CT scan. COX-2 was detected in 46/56 meningiomas (82.14%), and it resulted as being related to histologic grade (t-test: p = 0.006) and to edema (t-test: p = 0.002). No statistical association between COX-2 and VEGF or MVD was found. In conclusion, COX-2 seems to be related to the more aggressive meningiomas and, somehow, to the development of meningioma-associated brain edema.

Immunohistochemical and molecular study of radiation-induced multiple meningiomas with pleural and pulmonary metastasis

In the present study, the telomerase activity and the putative alterations of genes involved in cell-cycle control (p53, Fas and pRb) were investigated in a radiation-induced meningioma with multiple recurrences and pleural-pulmonary metastases (the patient, a 34-year-old male, had a history of carcinoma of the tongue of testicular lymphocytic lymphoma). Expression of VEGF and vasculature pattern were also studied. Expression of VEGF, pRb and p53 were evaluated by immunohistochemistry on formalin-fixed, paraffin-embedded samples of the tumor. VEGFmRNA was determined by competitive PCR. Fas, FasL and hTERT were evaluated by RT-PCR. Telomerase activity was examined by the TRAP assay. An intense vascularization was observed, supported by high expression of VEGFmRNA (isoforms 121 and 165). pRb and p53 were overexpressed. Fas was undectable with PCR, whereas FasL was positive. Furthermore, the lesion showed an elevated telomerase activity (TPG, 22), according to the high expression of hTERT. These findings emphasized that even among generally benign neoplasms, such as meningiomas, some highly malignant tumors may develop, as in our case, in which several mechanisms were activated in the cancer progression to guarantee the immortalization of cellular clones (angiogenic phenomenon, activation of telomerase and of anti-apoptotic mechanisms) and the blood spread. Thus, the data illustrate the importance of searching for genetic aberrations (which are a hallmark of malignancy) in meningiomas, as predictive and reliable factors of the possibility to recur and to metastasize.