Orietta Gandini

bcl-2/bax mRNA expression ratio as prognostic factor in low-grade urinary bladder cancer

Apoptotic cell death represents an important mechanism for the precise regulation of cell numbers, and a defence mechanism against tumoral cells. bcl‐2 and bax genes are known to be involved in the control of apoptotic pathways; in particular, the ratio between bcl‐2 and bax represents a cell rheostat that is able to predict a cells response toward life or death to an apoptotic stimulus. In the present study we investigated the role of bcl‐2 and bax gene expression in a panel of 37 low‐grade tumours of the urinary bladder, and correlated the expression of these genes to the prognosis of patients in a follow‐up of more than one year. We found that levels of bax expression higher than bcl‐2 in bladder tumours well correlates to a better outcome for patients. Early relapses are much more frequently observed in those patients whose tumours express more bcl‐2 than bax mRNA. We conclude that the bcl‐2/bax expression ratio may be considered as a marker for disease progression in low grade bladder tumours, independently of clinical staging and histological grading.

Circulating tumour cells lacking cytokeratin in breast cancer: the importance of being mesenchymal

Circulating tumour cells (CTCs) are independent predictor of prognosis in metastatic breast cancer. Nevertheless, in one third of patients, circulating tumour cells are undetected by conventional methods. Aim of the study was to assess the prognostic value of circulating tumour cells expressing mesenchymal markers in metastatic breast cancer patients. We isolated CTC from blood of 55 metastatic breast cancer patients. CTC were characterized for cytokeratins and markers of epithelial mesenchymal transition. The gain of mesenchymal markers in CTC was correlated to prognosis of patients in a follow‐up of 24 months. The presence of mesenchymal markers on CTC more accurately predicted worse prognosis than the expression of cytokeratins alone. Because of the frequent loss of epithelial antigens by CTC, assays targeting epithelial antigens may miss the most invasive cell population. Thus, there is an urgent need to improve detection methods to identify CTC which undergone epithelial mesenchymal transition program.

Prognostic value of circulating tumor cells in nonmuscle invasive bladder cancer: a CellSearch analysis

BACKGROUND Circulating tumor cells (CTCs) provide prognostic information in patients with metastatic tumors. Recent studies have shown that CTCs are released in circulation in an early phase of cancer disease so that their presence is under investigation in the adjuvant setting. Few studies investigated the prognostic significance of CTCs enumeration in patients with metastatic and advanced bladder cancer. The current study has analyzed the presence of CTC in patients with nonmuscle-invasive bladder cancer (NMIBC). PATIENTS AND METHODS Forty-four NMIBC patients were enrolled and included in a 24-month follow-up program. Blood drawings were carried out in all patients at the first diagnosis. CellSearch system (Veridex; LLC, Raritan, NJ) was used for CTCs enumeration. RESULTS CTC were detectable in 8/44 patients (18%). Presence of CTC was found significantly associated to shorter time to first recurrence (6.5 versus 21.7 months, P < 0.001). Median time to progression was not reached, due to the short follow-up period. CTC presence was found associated to concomitant carcinoma in situ and higher T category. CONCLUSION The detection of CTC in this setting of disease may allow to distinguish patients with high risk of recurrence from those with high risk of progression, as well as to early identify patients candidate for adjuvant treatment.BACKGROUND Circulating tumor cells (CTCs) provide prognostic information in patients with metastatic tumors. Recent studies have shown that CTCs are released in circulation in an early phase of cancer disease so that their presence is under investigation in the adjuvant setting. Few studies investigated the prognostic significance of CTCs enumeration in patients with metastatic and advanced bladder cancer. The current study has analyzed the presence of CTC in patients with nonmuscle-invasive bladder cancer (NMIBC). PATIENTS AND METHODS Forty-four NMIBC patients were enrolled and included in a 24-month follow-up program. Blood drawings were carried out in all patients at the first diagnosis. CellSearch system (Veridex; LLC, Raritan, NJ) was used for CTCs enumeration. RESULTS CTC were detectable in 8/44 patients (18%). Presence of CTC was found significantly associated to shorter time to first recurrence (6.5 versus 21.7 months, P<0.001). Median time to progression was not reached, due to the short follow-up period. CTC presence was found associated to concomitant carcinoma in situ and higher T category. CONCLUSION The detection of CTC in this setting of disease may allow to distinguish patients with high risk of recurrence from those with high risk of progression, as well as to early identify patients candidate for adjuvant treatment.

Monitoring PD-L1 positive circulating tumor cells in non-small cell lung cancer patients treated with the PD-1 inhibitor Nivolumab

Controversial results on the predictive value of programmed death ligand 1 (PD-L1) status in lung tumor tissue for response to immune checkpoint inhibitors do not allow for any conclusive consideration. Liquid biopsy might allow real-time sampling of patients for PD-L1 through the course of the disease. Twenty-four stage IV NSCLC patients included in the Expanded Access Program with Nivolumab were enrolled. Circulating tumor cells (CTCs) were analyzed by CellSearch with anti-human B7-H1/PD-L1 PE-conjugated antibody. PD-L1 expressing CTCs were assessed at baseline, at 3 and 6 months after starting therapy, and correlated with outcome. At baseline and at 3 months of treatment, the presence of CTCs and the expression of PD-L1 on their surface were found associated to poor patients outcome. Nevertheless, the high frequency of PD-L1 expressing CTCs hampered to discriminate the role of PD-L1 in defining prognosis. Conversely although CTCs were found in all patients 6 months after treatment, at this time patients could be dichotomized into two groups based PD-L1 expression on CTCs. Patients with PD-L1 negative CTCs all obtained a clinical benefit, while patients with PD-L1 (+) CTCs all experienced progressive disease. This suggests that the persistence of PD-L1(+) CTCs might mirror a mechanism of therapy escape.

Chemosensitivity profile assay of circulating cancer cells: prognostic and predictive value in epithelial tumors

The prognostic value associated with the detection of circulating tumor cells (CTCs) in metastatic breast cancer by the CellSearch™ technology raise additional issues regarding the biological value of this information. We postulated that a drug‐resistance profile of CTCs may predict response to chemotherapy in cancer patients and therefore could be used for patient selection. One hundred 5 patients with diagnosis of carcinoma were enrolled in a prospective trial. CTCs were isolated from peripheral blood, and positive samples were evaluated for the expression of a panel of genes involved in anticancer drugs resistance. The drug‐resistance profile was correlated with disease‐free survival (DFS; patients in adjuvant setting) and time to progression (TTP; metastatic patients) in a 24‐months follow‐up. Objective response correlation was a secondary end point. Fifty‐one percent of patients were found positive for CTCs while all blood samples from healthy donors were negative. The drug‐resistance profile correlates with DFS and TTP (p < 0.001 in both). Sensitivity of the test: able to predict treatment response in 98% of patients. Specificity of the test: 100%; no sample from healthy subject was positive for the presence of CTCs. Positive and negative predictive values were found to be 96.5 and 100%, respectively. We identified a drug‐resistance profile of CTCs, which is predictive of response to chemotherapy, independent of tumor type and stage of disease. This approach may represent a first step toward the individualization of chemotherapy in cancer patients.

Prevalence of human papillomavirus, cytomegalovirus, and Epstein-Barr virus in the cervix of healthy women.

The prevalence of some sexually transmitted viruses, possibly involved in cervical carcinogenesis, was studied in the cervix of women with normal cytology. The presence of human papillomaviruses (HPV) type 16 and 18, cytomegalovirus (CMV) and Epstein‐Barr virus (EBV) genomes in cervical cells taken from 143 healthy Italian women was investigated using the polymerase chain reaction (PCR). The study population was divided into four groups with respect to age as follows: group I, 17 to 25 years, n = 48 women; group II, 26 to 35 years, n = 30; group III, 36 to 50 years, n = 32; and group IV, 51 to 70 years, n = 33. In the first age group prevalence rates of HPV 16, CMV and EBV infection of 23%, 21% and 19% were found respectively. The infection rates of HPV 16 and CMV were shown to decrease with age, with prevalences of HPV 16 at 10% in the second group, 6% in the third and 3% in the fourth and of CMV at 13% in the second and third and 6% in the fourth groups. The prevalence of EBV infection did not decrease with increasing age (19% in the first and third groups, 20% in the second and 18% in the fourth). The occurrence of HPV 18 genome was very low (0‐3%) and independent of age. In the first age group a higher percentage of double infections (16.6%) was found than in the three other age groups (6% in the second and third and 3% in the fourth). The finding of multiple infections in younger women requires further study in order to clarify the implications of such viral infections in healthy women and their contribution to the development of genital tract malignancies.

Prevalence of papillomavirus, Epstein-Barr virus, cytomegalovirus, and herpes simplex virus type 2 in urinary bladder cancer

Recent epidemiological studies suggest that the risk for urological malignancies may be related to the exposure to infectious agents. Human Papillomaviruses type 16 and 18 (HPV 16, HPV 18), Epstein‐Barr virus (EBV), cytomegalovirus (CMV) and herpes simplex virus type 2 (HSV‐2) have been suggested previously as cofactors in the pathogenesis of some malignancies in humans. The present paper, the presence of HPV 16, HPV 18, EBV, CMV and HSV‐2 genomes was investigated in a panel of 35 biopsies from urinary bladder carcinomas using the polymerase chain reaction (PCR). Sequences of EBV, HPV, CMV and HSV‐2 genomes were detected in 34%, 31%, 11% and 9% of tissue samples respectively, while in 20% of patients we found more than one viral infection. Absence of viral genomes was found in normal bladder. To our knowledge, this is the first report concerning the association of EBV, CMV and HSV‐2 with bladder cancer. This finding may raise the question whether such viral infection may contribute to development and progression of some types of urological malignancies in humans. J. Med. Virol. 55:262–267, 1998.

Follicle-stimulating hormone, testosterone, and hypoxia differentially regulate UDP-glucuronosyltransferase 1 isoforms expression in rat sertoli and peritubular myoid cells.

Uridine diphosphoglucuronosyltransferases (UGTs) are detoxifying enzymes responsible for the metabolism of endogenous and xenobiotics compounds. UGT isoforms are widely distributed in rat tissues showing a constitutive and inducible gene expression. However, little information is available concerning UGTs expression in testis. The UGT1A1, UGT1A2, and UGT1B1 mRNAs expression in whole rat testis, in Sertoli and peritubular myoid cells in basal conditions, and after hormonal and hypoxic stimulation were investigated by reverse transcriptase-polymerase chain reaction (RT-PCR). Constitutive expression of each UGT1 isoform was present in rat testis with higher levels of UGT1A2. UGT transcripts were also detected in Sertoli and peritubular myoid cells. After FSH stimulation, Sertoli cells showed an increase in UGT1B1 mRNA expression, whereas the levels of UGT1A1 and UGT1A2 resulted unmodified. The main effect induced by testosterone was a decrease of UGT1B1 mRNA expression in peritubular myoid cells, whereas in Sertoli cells an increase in UGT1A1 and UGT1B1 was observed. In hypoxic conditions, a reduction in UGTs mRNA levels was detected in both cell types. These findings suggest that rat UGT1 isoforms are regulated in testis by hormonal and environmental factors. Thus, it was speculated that alterations in UGTs expression and/or activity may be involved in the pathogenesis of testis injury.

Fibronectin and laminin expression in sentinel lymph nodes of patients with malignant melanoma

SIR, Malignant melanoma (MM) has shown an alarming increase in incidence over the last few decades; currently it represents roughly 5% of skin cancers and 1% of all malignant tumours, with an annual increase in incidence and mortality of 2–3% during the last 30 years. Sentinel lymph node (SLN) biopsy has greatly improved the staging and prognostic evaluation of primary cutaneous MM; in particular, the Reverse Transcriptase–Polymerase Chain Reaction (RT-PCR), which is able to detect melanoma cells expressing tyrosinase mRNA, offers an additional technique to the histopathological examination. The remodelling of cell adhesion molecules during the process of tumour progression is known to play a key functional role in cancer progression and metastasis. To date, although there have been several attempts to identify molecular markers among cell adhesion molecules in melanoma lesions and in peripheral blood from patients with MM, few studies have focused on the involvement of cell adhesion molecules in SLNs of such patients. Fibronectin and laminin are two adhesion molecules that have been shown to be involved in melanoma progression even though their role in SLNs has been poorly investigated. Recently, our group investigated the molecular profile of SLNs, suggesting a role as prognostic indicators for survivin, tyrosinase, MIA and Mart-1 expression. In this study we investigated the role of fibronectin and laminin mRNA expression as prognostic indicators in SLNs of patients with MM and correlated the results to progression of disease in a median follow-up of 51Æ5 months. The presence of capsular naevus cells in SLNs was tested and the positive samples were excluded from the study. We analysed 72 SLNs from 48 patients (mean age 58Æ3 years) with MM; informed consent was obtained from each patient. There were 22, 20 and six patients with stage I, II and III disease, respectively. Sections of each lymph node were stained with haematoxylin and eosin, and immunohistochemistry was performed by using antibodies to HMB-45 antigen and S100 protein that were detected with the avidinbiotin-peroxidase technique. Negative controls were obtained by using normal animal serum instead of specific primary antibodies. The results are shown in Table 1. After surgical excision the SLNs were frozen in liquid nitrogen and stored at –80 C until use in the RT-PCR assay. One microgram of total RNA extracted from the frozen tissues was reverse transcribed in a final volume of 20 lL, then 5 lL of cDNA was amplified in PCR buffer containing 25 pmol each of upstream and downstream specific primers. All the recommended precautions were taken to avoid the possibility of false-positive results. Each RT-PCR experiment included a sample without RNA as negative control and RNA extracted from the SK-MEL 5 melanoma cell line as positive control. The amplification products were separated by electrophoresis on 2% agarose gel: those showing specific amplification products were considered positive. The suitability of all samples was investigated by an RT-PCR with b-actin-specific primers. Laminin expression was found to be positive in 45 of 72 and negative in 27 of 72 samples examined. Positive expression was associated with death or disease progression in 20 of 45 cases, whereas negativity was associated with disease progression in 16 of 27 cases; 11 of 27 remain disease free. Fibronectin gene expression was found in 61 of 72 specimens: positivity was associated with disease progression in 26 of 61 positive samples, while 35 of 61 were disease free. Eleven of 72 samples did not have fibronectin gene expression: negativity was associated with death or disease progression in 10 of 11 cases, whereas one of 11 is currently disease free. The features of patients and the results obtained by the RT-PCR assay are listed in Table 1. Agarose gel electrophoresis of RT-PCR products is shown in Figure 1. In order to verify the statistical significance of our results we first determined the correlation between overall survival, Clark level and fibronectin and laminin expression by the Kaplan– Meier method. We failed to find a statistically significant correlation for the expression of laminin (P = 0Æ57); in contrast, a significant correlation was found between the Clark level and overall survival (P = 0Æ0036) and between negativity for fibronectin and overall survival (P = 0Æ0024). The log-rank test showed a strong correlation between absence of fibronectin expression and Clark level (Spearman correlation P = 0Æ35). The Cox proportional hazards model was applied to the multivariate survival analysis and demonstrated that absence of fibronectin expression and Clark level are statistically significant negative prognostic factors (P = 0Æ012 and P = 0Æ030, respectively).

Integrin Pattern and Effect on Contraction in Cultured Testicular Peritubular Myoid Cells

PROBLEM: Neither the integrin pattern nor the biological functions of integrins have been extensively documented in human cultured testicular peritubular myoid cells (TPMC). The integrin pattern and the presence of some proteins of the immunoglobulin superfamily on human TPMC as well as the role of integrins in TPMC contraction were examined.
 METHOD OF STUDY: Integrin expression was evaluated by immunofluorescence and FACS analysis. To assess the role of integrin in TPMC contraction, human and rat cells were added to a collagen gel system and exposed to contractile stimuli.
 RESULTS: The immunofluorescence and cytofluorimetric analysis showed that human cultured TPMC express α1, α2, α3, α5, α6, αv, β1, β3, and β4 integrin subunits, and significant amounts of intercellular adhesion molecule‐1 (ICAM‐1), whereas they do not present α4, β2, β7 subunits, nor intercellular adhesion molecule‐2 (ICAM‐2) and neural cell adhesion molecule (NCAM). The preincubation of human cells with an anti‐β1 mAb and of rat cells with a polyclonal anti‐β1 antibody inhibited TPMC contraction induced by different contractile stimuli.
 CONCLUSION: Our investigation documented a broad integrin pattern on human cultured TPMC as well as a role for integrins in human and rat TPMC contraction.