Circulating tumor cells (CTCs) in metastatic breast cancer (MBC): prognosis, drug resistance and phenotypic characterization.

BACKGROUND: the expression of ATP-binding cassette transporters on circulating tumor cells (CTCs) is predictive of response to chemotherapy in cancer patients. We tested the hypothesis that drug-resistant CTCs might have predictive value in metastatic breast cancer (MBC) and possibly retain stem-like properties. PATIENTS AND METHODS: CTCs obtained from 42 MBC patients were evaluated for multidrug-resistance-related proteins (MRPs), aldehyde dehydrogenase 1 (ALDH1), estrogen receptor α (ERα) and human epidermal growth factor receptor 2 (HER2/neu). Primary objective was to evaluate the prognostic and predictive value of CTCs profile. Secondary end points were the level of concordance in ERα and HER2/neu status between primary tumors and CTCs and the correlation in CTCs between ALDH1, drug resistance profile and number of MRPs. RESULTS: A difference in progression-free survival (PFS) was found between CTCs-positive and CTCs-negative patients. PFS was shorter in patients with a 'drug resistance' CTCs profile and in patients whose CTCs expressed two or more MRPs. No correlation was found between tumor characteristics and ALDH1. ALDH1 correlated to negative ERα and positive HER2/neu status in CTCs. The correlation between the number of MRPs expressed in CTCs and ALDH1 was statistically significant. CONCLUSION: in MBC, the presence of CTCs expressing MRPs and ALDH1 is predictive of response to chemotherapy.

Circulating tumor cells in cancer therapy: are we off target?

What clinical oncologists learned about metastatic process, is that it is the main cause of cancer-related deaths. What scientists learned about metastatic disease, is that it is due to a highly selective process, which involves a minority of tumor cells that are able to survive within the bloodstream, and to initiate a new growth in distant sites. These cells "in transit" are known as circulating tumor cells (CTCs). Although their nature is not fully understood, what is widely accepted, is that they are drug resistant, and that their presence may represent the main reason for treatment failure. Despite this body of evidence, the pharmacological approach against cancer, with both chemotherapic and biological drugs, is still targeted on the primary tumor, raising the question as to whether we are missing the target. Targeting circulating tumor cells, may represent a new promising approach to indivisualize anticancer therapy.

Overcoming endocrine resistance in breast cancer.

Approximately 70% of breast cancers express the estrogen receptor (ER) and endocrine therapy is the most important component of systemic therapy for hormone-responsive breast cancer. Unfortunately, endocrine-resistant ER-positive disease represents up to one-quarter of all breast cancers and a number of different mechanisms have been implicated in endocrine resistance, either intrinsic, occurring de novo at the initial exposure to endocrine therapies or acquired, occurring after an initial response to therapy. In the present work a number of molecular mechanisms accounting for intrinsic and acquired resistance to hormonal therapies have been reviewed and the most promising strategies to overcome endocrine resistance have been highlighted.

Pancreatic cancer: from molecular signature to target therapy.

Pancreatic adenocarcinoma is a leading cause of cancer death in western countries. The treatment of advanced disease with gemcitabine has only a modest activity on survival with a favourable impact on quality of life. However, recent data support the evidence that the combination of gemcitabine with erlotinib, capecitabine or platinum compounds could be more active than gemcitabine alone in advanced pancreatic cancer. New therapeutic strategies, particularly using molecular target agents, are under evaluation. A number of molecular mechanisms responsible of transformation and progression of pancreatic cancer have been identified, opening the possibility to identify also possible pharmacological targets. A promising approach is the pharmacological inhibition of tumor angiogenesis with anti-vascular endothelial growth factor (VEGF) agents, such as bevacizumab, cyclooxygenase-2 inhibitors (celecoxib), thalidomide and others. Also epidermal growth factor receptor (EGFR) plays an important role in progression of pancreatic cancer. Erlotinib, an oral available anti-EGFR compound, was the first agent capable to significantly improve overall survival in a phase III trial, leading to its approval by Food and Drug Administration (FDA) in combination with gemcitabine as first-line therapy. Ongoing studies are exploring the role of targeted therapy in the adjuvant setting. However, despite these promising results, several questions remain to be resolved, including the rational selection of the patients who are more likely to obtain benefit of target therapy, the choice of the optimal therapeutic schedule of therapy, the clinical setting of choice, and the management of the toxicity.

Weekly vinorelbine and docetaxel as second-line chemotherapy for pretreated non-small cell lung cancer patients: a phase I-II trial.

Docetaxel was proven to be effective as second-line therapy for patients with advanced NSCLC after failure of platinum-based front-line chemotherapy. We designed this phase I/II study to define the Maximum Tolerated Dose of weekly docetaxel combined with weekly vinorelbine, and subsequently evaluate tolerability and activity of this schedule in NSCLC patients who were progressive after treatment with either cisplatin and gemcitabine or carboplatin and paclitaxel regimens. To be eligible for the study, patients were required to have a WHO performance status < or =2, failure after at least two cycles of first platinum-based chemotherapy, and no prior treatment with docetaxel and vinorelbine. A total of 27 patients were enrolled in this phase I/II study. A weekly docetaxel dose of 25 mg/m2 was recommended in combination with fixed vinorelbine dose of 20 mg/m2, and 24 patients were treated at this dose level. Severe neutropenia (62%) and febrile neutropenia (29%) were the most frequent toxicities, with 83% of patients requiring dose modification or delay. In the phase II study, 5 (21%) patients obtained a partial response, 8 (33%) patients had stable disease, whereas 10 (42%) patients progressed. After a median follow-up of 18.7 months, median survival was 8 months, with 30% surviving at 1 year. Regardless of the use of weekly docetaxel schedule, this regimen was highly myelosuppressive, and did not seem to improve response rate and survival compared to single-agent docetaxel. No further developments of this schedule are warranted.

Nitric oxide synthases in normal and benign hyperplastic human prostate: immunohistochemistry and molecular biology.

The expression of nitric oxide synthase (NOS) isoforms has been investigated in normal (three subjects) and benign hyperplastic prostate (ten patients) by immunohistochemistry and reverse transcriptase-polymerase chain reaction (RT-PCR). The inducible NOS (iNOS or NOS-2) is not detected in normal prostate, while it is expressed in the prostate of all benign prostatic hyperplasia (BPH) patients, even in the absence of prostatitis or systemic signs of an inflammatory condition. This suggests that sex hormones may be involved in iNOS induction and that there may be a role for NO in the pathogenesis of BPH. Constitutive NOSs (nNOS and eNOS) are expressed in both normal and hyperplastic prostate and are co-expressed in epithelial cells. eNOS, however, is present mainly in the basal layer cells; nNOS seems abundantly expressed in the more superficial cells of the affected prostate. This indicates that the switching between the two constitutive isoforms may be part of the usual process of cell differentiation from the basal to the secretory layer of the epithelium.

Expression of p53 and retinoblastoma gene in high-grade nodal peripheral T-cell lymphomas: immunohistochemical and molecular findings suggesting different pathogenetic pathways and possible clinical implications.

The expression of p53 and the retinoblastoma gene has been investigated by immunohistochemical and molecular analysis in 45 cases of nodal peripheral T-cell lymphoma with high-grade histology. Most cases (73.3 per cent) were primary nodal lymphomas without any extra-nodal site involvement. Most of them (75.6 per cent) were histologically classified as pleomorphic, small, medium, and large cell type. Immunohistochemistry detected p53 in nine cases (20 per cent). In each of these cases, the polymerase chain reaction (PCR)/heteroduplex analysis did not show the presence of mutations, this finding being consistent with an alteration of the p53 functional pathway, in the presence of a wild-type protein. The retinoblastoma gene product was detected by immunohistochemistry in 35 cases (77.8 per cent) and not detected in ten cases (22.2 per cent). In the latter cases, the reverse transcription (RT)-PCR analysis showed the presence of a specific retinoblastoma gene transcript in six cases and was negative in the remaining four cases. The immunohistochemical and molecular findings seem to be consistent with abnormalities of retinoblastoma gene expression at either the transcriptional or the post-transcriptional level. Since all nine p53-positive cases by immunohistochemical analysis were also retinoblastoma gene product-positive, and all ten retinoblastoma gene product-negative cases were also p53-negative, two different and mutually exclusive pathways of possible pathogenetic significance may be suggested, the former involving abnormalities of the functional pathway of p53 in the absence of mutations and the latter abnormalities of retinoblastoma gene expression at the transcriptional and/or post-transcriptional level. Finally, the clinico-pathological correlations showed that p53 immunohistochemical expression is significantly associated with a poorer response to intensive chemotherapy.

Involvement of bcl-2 and bax gene expression in apoptosis and differentiation of the non-tumorigenic murine hematopoietic cell line, 32DC13(G).

32DCl3(G) is an interleukin-3 (IL-3) dependent, non-tumorigenic murine hematopoietic cell line which undergoes terminal differentiation into granulocytes when exposed to granulocyte-colony stimulating factor (G-CSF). This line therefore offers a convenient system to study the expression of genes involved in apoptosis and differentiation. In our experiments we have acquired evidence that during the differentiation pathway, likewise in apoptosis induced by IL-3 deprivation, detectable levels of bax mRNA appear, while bcl-2 expression decreases. These events are under the control of the p53 tumor-suppressor gene. In these cells, an overexpression of exogenous wild-type p53 leads to a decrease in bcl-2 mRNA and to the appearance of box mRNA, which instead is absent in the parental cells growing in IL-3 conditioned medium. Furthermore, results from experiments on p53 transfected cells demonstrate that excess wild-type p53 activity, on its own, fails to elicit apoptosis as long as IL-3 is present and does not induce differentiation if G-CSF is not added to the culture medium. We conclude that in apoptosis and differentiation of 32DCl3(G) the alterate ratio of bcl-2 and box gene expression, modulated by p53, is an early event dependent on IL-3 withdrawal and that the appearance of bax and the decrease of bcl-2 expression are necessary, but not sufficient for the acquisition of a completely mature granulocytic phenotype.

Detection of cellular heterogeneity by DNA ploidy, 17 chromosome, and p53 gene in primary carcinoma and metastasis in a case of ovarian cancer.

An unusual case of a patient with ovarian carcinoma carrying the p53 point mutation in both metastases (omentum and lymph node), but not in the primary tumor, is described. The presence of a p53 single mutation (G:A) at the second base of codon 248 was examined by polymerase chain reaction-amplification refractory mutation system (PCR-ARMS) analysis. This case was examined also by fluorescent in situ hybrization (FISH) analysis and flow cytometry (FCM) to obtain further information at the single cell level and to detect heterogeneity within a population of cells. FCM analysis evidenced the same multiple aneuploid cell subpopulations in primary and in metastatic samples showing the presence of a cellular heterogeneity. FISH analysis showed a disomic condition for the 17 chromosome in the primary and in one metastasis, while in the other metastasis a monosomic together with a disomic subpopulation was revealed. Our results confirm the independent clonal evolution of the metastasis. The late mutation event observed only in metastatic specimens suggests the hypothesis that in the primary tumor the wild-type gene either does not perform its control role for unknown genetic structural events or the p53 gene in this case does not play a critical role in carcinogenesis.

High frequency of human papillomavirus detection in urinary bladder cancer.

We investigated the presence of human papillomaviruses (HPVs) types 16 and 18 DNA in formalin-fixed, paraffin-embedded tissues from the urinary bladder (46 transitional carcinomas and 10 non-neoplastic normal urinary samples) to find a possible role for HPV types in urinary tract cancerogenesis. The analysis was performed using polymerase chain reaction followed by filter hybridization with oligonucleotide-specific probes. The HPV16 and/or HPV18 genomes were detected in 23 of 46 (50%) bladder carcinomas and in none of 10 (0%) non-neoplastic urinary samples. These results suggest that HPV16 and 18 may carry a risk for the development of malignancy in the urinary tract as it occurs in the anogenital regions.

The hemopoietic progenitor 32DCl3(G) cells synthesize C3 molecules and acquire C3b acceptor sites upon differentiation or neoplastic transformation.

32DCl3(G) cells are a diploid, nontumorigenic, IL-3-dependent hemopoietic progenitor cell line, which undergoes terminal differentiation into neutrophilic granulocytes when cultured in presence of G-CSF. The infection with BALB-Moloney murine sarcoma virus, containing a v-HA-ras oncogene, renders this cell line IL-3 independent and continuously growing in the presence of G-CSF, nontumorigenic and with an apparent block at the level of promyelocyte/myelocyte (32D-Ha-ras). After infection with Abelson murine leukemia virus containing a v-abl oncogene, the cell line originates (32D-abl) that is also IL-3 independent but is tumorigenic and unable to differentiate in the presence of G-CSF. This cellular model allowed us to study the relationship between distinct steps of cell differentiation, neoplastic transformation, and C3 synthesis, activation, and characteristics of binding. We demonstrated that C3 synthesis, release, and cleavage are properties already present in the progenitor 32DCl3(G) cells. The more differentiated 32D-Ha-ras cells acquired C3 acceptor sites, apparently completely saturated by the constitutively released molecules. The transformation with Abelson murine leukemia virus, although it did not affect all these properties, let the cells bind considerable amounts of C3-related fragments activated in normal murine serum through the alternative pathway. This last event was a result of the acquisition of PMSF-sensitive serine proteases associated with the plasma membrane.

Phenotypic and functional modifications of thymocytes during tumor growth.

Thymocytes (T) from mice bearing the syngeneic tumor 3LL inhibit the immune response to SRBC by syngeneic splenocytes derived from control mice and cultured in Mishell and Dutton's antibody forming systems. T from control mice are devoid of this capacity but acquire it after in vitro incubation with tumor cells. It has been shown that metabolites of arachidonic acid (PGE-2, LTB-4 and LTC-4) synthesized in excess by tumor cells act as mediators for the acquisition of the above capacity by thymic cells in vitro. Surface phenotypical analysis by flow-cytometry demonstrated that in the thymus of tumor bearing mice the proportion of both single positive thymocytes (L3T4+/LY2- and L3T4-/LY2+) and of double negative thymocytes (L3T4-/LY2-) increased with a parallel decrease in the proportion of double positive cells (L3T4+/LY2+). In the mean time the total number of thymocytes markedly decreased. An attempt was done to mimic in vitro what happens in vivo. For this purpose T from control mice were incubated with conditioned culture medium in which 3LL cells had grown or with arachidonic acid metabolites. After one or the other of these treatments T from control mice did not modify their phenotypic antigenic pattern but acquired the capacity to negatively interfere with the in vitro immune response of normal spleen cells to SRBC. Since serum from tumor bearing mice contains large amounts of PGE-2, LTB-4 and LTC-4 we tested its effects on normal syngeneic T. Serum from tumor bearing mice, but not serum from normal mice, induces T from control syngeneic animals to acquire both immunosuppressive capacity and differentiation pattern clusters similar to those that characterize T derived from the thymus of tumor bearing mice. We demonstrated that metabolites of arachidonic acid are active as inducers of immunosuppressive capacity in T. On the other hand, a yet unknown factor present in serum of tumor bearing mice plays a role as inducer of differentiation of these cells.

Alteration of growth and differentiation factors response by Kirsten and Harvey sarcoma viruses in the IL-3-dependent murine hematopoietic cell line 32D C13(G).

32D C13(G) is an interleukin 3(IL3)-dependent non-tumorigenic murine hematopoietic cell line which undergoes terminal differentiation into granulocytes when exposed to granulocytic colony stimulating factor (G-CSF). Infections of 32D C13(G) cells with either Kirsten rat sarcoma virus or Balb murine sarcoma virus, both containing a v-ras oncogene, generates clones that can permanently grow in G-CSF without differentiation. 32D-Ki-ras cells show a heterogeneous morphology ranging from the promyelocytic to the myelocytic stage of differentiation, and express high levels of both myeloperoxidase (MPO) and lactoferrin (LF) mRNA. 32D-Ha-ras cells show a more immature phenotype and express MPO but no LF mRNA. The apparent differentiation block of both 32D Ki-ras and 32D Ha ras can be reversed by treatment with the chemical inducers retinoic acid, sodium butyrate or dimethylsulphoxide, which leads to terminal differentiation into granulocytes. When 32D-Ki-ras and 32D-Ha-ras cells are cultured in medium containing IL-3 they become adherent and express some monocyte-macrophage markers. Upon prolonged exposure to IL3, 32D-Ki-ras, but not 32D-Ha-ras, resume suspension growth. Both 32D-Ki-ras and 32D-Ha-ras rapidly die if grown in chemically defined medium in the absence of any growth factor and are non-tumorigenic in immunosuppressed mice. These findings indicate that ras activation may interfere with the normal response to growth and differentiation factors in cells of the granulocytic lineage. These alterations may represent a critical, although non-sufficient, step in leukemogenesis.

Immunosubversive role of PGE2 in tumor bearing mice.

The immunosuppressive activity of tumor cells was studied in vivo and in vitro using C57BL/6 mice and Lewis lung carcinoma (3LL) cells. The SRBC immunization of tumor-bearing mice in vivo gave a lower number of PFC than the control mice. In vitro, employing the Mishell and Dutton technique, the primary immune response of splenocytes from tumor-bearing mice was significantly reduced. The in vitro primary immune response of normal splenocytes was also reduced when the tumor cells or supernatants of tumor cell cultures were present during SRBC immunization. 3LL cells synthesize a large quantity of PGE2 which was also demonstrated in the supernatants of 3LL cell cultures. Nevertheless, as the addition of indomethacin, a potent inhibitor of the prostaglandin synthesis, only partially reduces the tumor cell immunosuppressive action, prostaglandins are conceivably only one of the factors responsible for the immunodepression exerted by the tumor cells.

[The dopaminergic system and aldosterone secretion. I. Suppressive effects of L-dopa on secondary hyperaldosteronism]. / Sistema dopaminergico e secrezione di aldosterone. Nota I. Effetti soppressivi della L-dopa sull'ipera dosteronismo secondario.

The authors studied the blood aldosterone, cortisol and kalium levels, the plasma renin activity and the aldosterone urinary excretion following L-DOPA (0.50 g per os) administration in seven patients affected by hypertension and secondary hyperaldosteronism. The results suggest that L-DOPA administration, by stimulating the dopaminergic biosynthesis. Nevertheless such a conclusion needs further investigations.