Lymphocyte antigen 6G6D-mediated modulation through p38α MAPK and DNA methylation in colorectal cancer.

In addition to being novel biomarkers for poor cancer prognosis, members of Lymphocyte antigen-6 (Ly6) gene family also play a crucial role in avoiding immune responses to tumors. However, it has not been possible to identify the underlying mechanism of how Ly6 gene regulation operates in human cancers. Transcriptome, epigenome and proteomic data from independent cancer databases were analyzed in silico and validated independently in 334 colorectal cancer tissues (CRC). RNA mediated gene silencing of regulatory genes, and treatment with MEK and p38 MAPK inhibitors were also tested in vitro. We report here that the Lymphocyte antigen 6G6D is universally downregulated in mucinous CRC, while its activation progresses through the classical adenoma-carcinoma sequence. The DNA methylation changes in LY6G6D promoter are intimately related to its transcript regulation, epigenomic and histological subtypes. Depletion of DNA methyltransferase 1 (DNMT1), which maintains DNA methylation, results in the derepression of LY6G6D expression. RNA-mediated gene silencing of p38α MAPK or its selective chemical inhibition, however, reduces LY6G6D expression, reducing trametinib's anti-inflammatory effects. Patients treated with FOLFOX-based first-line therapy experienced decreased survival due to hypermethylation of the LY6G6D promoter and decreased p38α MAPK signaling. We found that cancer-specific immunodominant epitopes are controlled by p38α MAPKs signaling and suppressed by DNA methylation in histological variants with Mucinous differentiation. This work provides a promising prospective for clinical application in diagnosis and personalized therapeutic strategies of colorectal cancer.

Combi-TED: a new trial testing Tedopi® with docetaxel or nivolumab in metastatic non-small-cell lung cancer progressing after first line.

Despite the positive results obtained by first-line chemoimmunotherapy in patients with metastatic non-small-cell lung cancer (NSCLC), only a few second-line options are available after disease progression. Combi-TED is a phase II international study that will assess the efficacy of Tedopi®, a cancer vaccine, combined with either docetaxel or nivolumab and compared with docetaxel monotherapy in patients with metastatic NSCLC after chemoimmunotherapy. The study, currently in the recruitment phase, will assess 1-year overall survival (primary end point), patient's progression-free survival and overall response rate, as well as the correlation of efficacy with several tumor or blood biomarkers. The results will hopefully provide more information on Tedopi combinational treatment compared with current standard of care in NSCLC patients who fail first-line chemoimmunotherapy. Clinical Trial Registration: NCT04884282 (ClinicalTrials.gov).

Patients with lung cancer that has spread to other parts of the body are usually treated with a combination of chemotherapy and drugs that stimulate the immune system to kill cancer cells, which is referred to as immunotherapy. If after receiving these drugs the cancer still gets worse, patients have only a few treatment options left and are usually treated with chemotherapy only. Researchers will study if a new medicine called Tedopi^®, a vaccine that specifically attacks cancer cells, used together with chemotherapy or immunotherapy, will work better then chemotherapy alone for these patients. The study will monitor how long patients will live after treatment, for how long they will live without their disease getting worse and how many patients will improve after treatment. Moreover, researchers will study if patients present specific features, such as certain molecules in their tumor cells or blood cells, that may indicate that they respond better to certain treatments.

Evidence-Based Second-Line Treatment in RAS Wild-Type/Mutated Metastatic Colorectal Cancer in the Precision Medicine Era.

Target-oriented agents improve metastatic colorectal cancer (mCRC) survival in combination with chemotherapy. However, the majority of patients experience disease progression after first-line treatment and are eligible for second-line approaches. In such a context, antiangiogenic and anti-Epidermal Growth Factor Receptor (EGFR) agents as well as immune checkpoint inhibitors have been approved as second-line options, and RAS and BRAF mutations and microsatellite status represent the molecular drivers that guide therapeutic choices. Patients harboring K- and N-RAS mutations are not eligible for anti-EGFR treatments, and bevacizumab is the only antiangiogenic agent that improves survival in combination with chemotherapy in first-line, regardless of RAS mutational status. Thus, the choice of an appropriate therapy after the progression to a bevacizumab or an EGFR-based first-line treatment should be evaluated according to the patient and disease characteristics and treatment aims. The continuation of bevacizumab beyond progression or its substitution with another anti-angiogenic agents has been shown to increase survival, whereas anti-EGFR monoclonals represent an option in RAS wild-type patients. In addition, specific molecular subgroups, such as BRAF-mutated and Microsatellite Instability-High (MSI-H) mCRCs represent aggressive malignancies that are poorly responsive to standard therapies and deserve targeted approaches. This review provides a critical overview about the state of the art in mCRC second-line treatment and discusses sequential strategies according to key molecular biomarkers.

Combination of the PARPi and ARSi in advanced castration resistant prostate cancer: a review of the recent phase III trials.

Tumors with an impaired ability to repair DNA double-strand breaks by homologous recombination, including those with alterations in breast cancer 1 and 2 (BRCA1 and BRCA2) genes, are very sensitive to blocking DNA single-strand repair by inhibition of the poly (ADP-ribose) polymerase (PARP) enzyme. This provides the basis for a synthetic deadly strategy in the treatment of different types of cancer, such as prostate cancer (PCa). The phase 3 PROfound study was the first to lead to olaparib approval in patients with metastatic castration resistant PCa (mCRPC) and BRCA genes mutations. In recent years, the benefit of combination therapy consisted of a PARP inhibitor (PARPi) plus an androgen receptor signalling inhibitor (ARSi), was evaluated as first-line treatment of mCRPC, regardless of the mutational state of genes, participating in the homologous recombination repair (HRR). This review explores the role of PARPi in PCa and analyses the data of latest clinical trials exploring the PARPi-ARSi combinations, and how these results could change our clinical practice.

Association of HLA-G 3'UTR Polymorphisms with Response to First-Line FOLFIRI Treatment in Metastatic Colorectal Cancer.

Microenvironmental factors such as non-classical human leukocyte antigen-G (HLA-G) have been associated with cancer invasiveness and metastatic progression. HLA-G expression has been associated with specific single-nucleotide polymorphisms (SNP) in HLA-G 3'untranslated region (UTR) in several diseases. The primary aim was to investigate the predictive role of HLA-G polymorphisms on treatment efficacy in metastatic colorectal cancer (mCRC) patients homogeneously treated with first-line FOLFIRI (irinotecan, 5-fluorouracil, and leucovorin) and their association with soluble HLA-G (sHLA-G) plasma concentration. HLA-G 3'UTR was sequenced in 248 patients. A set of eight polymorphisms and related haplotypes were analyzed for their association with best tumor response, overall survival (OS), and progression-free survival (PFS). sHLA-G was measured by immunoassay in 35 available plasma samples and correlated with HLA-G 3'UTR polymorphisms/haplotypes. Our results showed that carriers of rs371194629 (+2960)-Ins allele were at risk for lack of complete response (hazard ratio (HR):0.29, pBH = 0.0336), while carriers of rs1710 (+3010)-G allele (rs1063320 (+3142)-C allele in linkage-disequilibrium), and rs9380142 (+3187)-G allele had a higher chance of complete response according to additive models (HR:4.58, pBH = 0.0245; HR:3.18, pBH = 0.0336, respectively). The combination of rs371194629-Del, rs1710-G, and rs9380142-G alleles forms the UTR1 haplotype. Patients who were carriers of UTR1/UTR-1 diplotype had a greater chance of complete response to therapy (HR:10.59, pBH = 0.0294). The same three beneficial alleles showed a trend toward higher pre-treatment sHLA-G plasma levels, supporting a functional role for polymorphisms in protein secretion. In conclusion, genetic variants of HLA-G are associated with treatment efficacy in mCRC patients treated with first-line FOLFIRI. This finding shed light on the combined effect of this immune system factor and chemotherapy in cancer patients.

Propensity for Early Metastatic Spread in Breast Cancer: Role of Tumor Vascularization Features and Tumor Immune Infiltrate.

Breast cancer is a complex and highly heterogeneous disease consisting of various subtypes. It is classified into human epidermal growth receptor 2 (HER-2)-enriched, luminal A, luminal B and basal-like/triple negative (TNBC) breast cancer, based on histological and molecular features. At present, clinical decision-making in breast cancer is focused only on the assessment of tumor cells; nevertheless, it has been recognized that the tumor microenvironment (TME) plays a critical biologic role in breast cancer. This is constituted by a large group of immune and non-immune cells, but also by non-cellular components, such as several cytokines. TME is deeply involved in angiogenesis, immune-evasion strategies, and propensity for early metastatic spread, impacting on prognosis and prediction of response to specific treatments. In this review, we focused our attention on the early morphological changes of tumor microenvironment (tumor vasculature features, presence of immune and non-immune cells infiltrating the stroma, levels of cytokines) during breast cancer development. At the same time, we correlate these characteristics with early metastatic propensity (defined as synchronous metastasis or early recurrence) with particular attention to breast cancer subtypes.

Molecular and Clinical Insights in Malignant Brenner Tumor of the Testis With Liver Metastases:A Case Report.

Malignant Brenner Tumor (mBT) is extremely rare. Although BT are almost exclusive ovarian neoplasms, they may constitute a highly unusual tumor of the testis; in fact, only seven fully documented cases have been reported to date. Because of their rarity, the pathogenesis of these tumors has not been clarified and there is no standard therapeutic approach. We report the first case of epididymal mBT with synchronous, multiple, liver metastases and a very dramatic clinical course. Both primary tumor and metastasis were subjected to mutational analysis of 20 cancer associated genes. Primary tumor showed FGFR3 Tyr375Cys and PIK3CA His1047Arg missense mutations. Both mutations are reported as pathogenic in ClinVar database. The same FGFR3 mutation was present in liver metastasis. Based on these results we believe that the FGFR pathway could be an ideal candidate for personalized treatment, offering hope to a subset of patients with mBT. Personalized approach, including mutational analysis and molecular testing should be required in patients with rare tumors in order to clarify diagnosis and improve therapeutic strategies.

[Target therapies in translocated ALK lung cancer: treatment management in oncology center of Lazio.] / Target therapies nel tumore al polmone ALK-positivo: gestione del trattamento nei centri di oncologia del LazioIncontro di approfondimento e confronto sulla gestione dei pazienti e sulla strategia terapeutica nei carcinomi polmonari positivi al riarrangiamento di ALK.

Lung cancer is a disease extremely heterogeneous in the molecular aspect and knowing the mutational profile of patients is essential in order to initiate the most appropriate treatment. In 2018, alectinib was approved in Italy for the first-line treatment of patients with anaplastic lymphoma kinase (ALK)-positive advanced non-small cell lung cancer (NSCLC), becoming a new therapeutic option for this patient group which constitutes approximately 3-7% of patients with NSCLC. On October 26th a virtual meeting was held in which 10 clinicians from various oncology centers in Lazio took part on the management of therapy of patients with Alk translocation, directed by Dr. Maria Rita Migliorino. The aim of the meeting was to share their clinical experience and to provide a series of practical that can help clinicians during treatment with target therapies in ALK-positive NSCLC.

Urothelial bladder carcinoma metastasizing to the eye: A systematic review and case report.

The eye is a rare site for disseminated malignancies; nevertheless, several tumors may metastasize to ocular structures. Few cases of urothelial and bladder cancer with eye involvement have been described in the literature thus far. The rarity of metastatic ocular localization implies an accurate differential diagnosis among the possible primary tumor sites. However, a specific diagnostic algorithm is not currently available, nor a defined therapeutic approach. Eye metastases are associated with advanced disease and poor prognosis. Physicians should be made aware of the possibility of eye involvement in patients with a past medical history of urothelial bladder cancer associated with ocular symptoms. The present case reports discusses the first documented case, to the best of our knowledge, of an urothelial bladder cancer metastasizing to the retro bulbar region that infiltrates the lacrimal gland. Furthermore, the report provides a systematic qualitative review of the current literature on eye metastases from urothelial bladder cancer using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses.

Colonic Ewing Sarcoma/PNET associated with liver metastases: A systematic review and case report.

Ewing Sarcoma is a highly lethal undifferentiated tumor of bone. ES is a small round cell tumor with etiological and characteristic chromosomal translocations between TET/FET (TLS/FUS, EWSR1, and TAF15) and ETS (E26 transformation-specific) family genes. Generally, therapeutic approach for metastatic Ewing Sarcoma includes both local (surgery and radiotherapy) and systemic (chemotherapy) disease control with an overall cure rate of 20%. For extra-osseous tumors, the most common primary sites of disease are trunk, extremities, head and neck, retroperitoneum. Among other sites, Ewing Sarcoma/PNET may also rarely arise in colon and rectum. Even if colonic Ewing Sarcoma/PNET have been previously reported in 5 cases, none of those reports came from right side of the colon. In this article, we report the first case of right-sided Ewing Sarcoma with synchronous liver metastases completely responding to first line chemotherapy. Furthermore, we provide a systematic qualitative review of the current literature on adult colorectal Ewing Sarcoma using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA).

JAK/Stat5-mediated subtype-specific lymphocyte antigen 6 complex, locus G6D (LY6G6D) expression drives mismatch repair proficient colorectal cancer.

BACKGROUND: Human microsatellite-stable (MSS) colorectal cancers (CRCs) are immunologically "cold" tumour subtypes characterized by reduced immune cytotoxicity. The molecular linkages between immune-resistance and human MSS CRC is not clear. METHODS: We used transcriptome profiling, in silico analysis, immunohistochemistry, western blot, RT-qPCR and immunofluorescence staining to characterize novel CRC immune biomarkers. The effects of selective antagonists were tested by in vitro assays of long term viability and analysis of kinase active forms using anti-phospho antibodies. RESULTS: We identified the lymphocyte antigen 6 complex, locus G6D (LY6G6D) as significantly overexpressed (around 15-fold) in CRC when compared with its relatively low expression in other human solid tumours. LY6G6D up-regulation was predominant in MSS CRCs characterized by an enrichment of immune suppressive regulatory T-cells and a limited repertoire of PD-1/PD-L1 immune checkpoint receptors. Coexpression of LY6G6D and CD15 increases the risk of metastatic relapse in response to therapy. Both JAK-STAT5 and RAS-MEK-ERK cascades act in concert as key regulators of LY6G6D and Fucosyltransferase 4 (FUT4), which direct CD15-mediated immune-resistance. Momelotinib, an inhibitor of JAK1/JAK2, consistently abrogated the STAT5/LY6G6D axis in vitro, sensitizing MSS cancer cells with an intact JAK-STAT signaling, to efficiently respond to trametinib, a MEK inhibitor used in clinical setting. Notably, colon cancer cells can evade JAK2/JAK1-targeted therapy by a reversible shift of the RAS-MEK-ERK pathway activity, which explains the treatment failure of JAK1/2 inhibitors in refractory CRC. CONCLUSIONS: Combined targeting of STAT5 and MAPK pathways has superior therapeutic effects on immune resistance. In addition, the new identified LY6G6D antigen is a promising molecular target for human MSS CRC.

A New Genetic Risk Score to Predict the Outcome of Locally Advanced or Metastatic Breast Cancer Patients Treated With First-Line Exemestane: Results From a Prospective Study.

INTRODUCTION: Approximately 50% of locally advanced or metastatic breast cancer (MBC) patients treated with first-line exemestane do not show objective response and currently there are no reliable biomarkers to predict the outcome of patients using this therapy. The constitutive genetic background might be responsible for differences in the outcome of exemestane-treated patients. We designed a prospective study to investigate the role of germ line polymorphisms as biomarkers of survival. PATIENTS AND METHODS: Three hundred two locally advanced or MBC patients treated with first-line exemestane were genotyped for 74 germ line polymorphisms in 39 candidate genes involved in drug activity, hormone balance, DNA replication and repair, and cell signaling pathways. Associations with progression-free survival (PFS) and overall survival (OS) were tested with multivariate Cox regression. Bootstrap resampling was used as an internal assessment of results reproducibility. RESULTS: Cytochrome P450 19A1-rs10046TC/CC, solute carrier organic anion transporter 1B1-rs4149056TT, adenosine triphosphate binding cassette subfamily G member 2-rs2046134GG, fibroblast growth factor receptor-4-rs351855TT, and X-ray repair cross complementing 3-rs861539TT were significantly associated with PFS and then combined into a risk score (0-1, 2, 3, or 4-6 risk points). Patients with the highest risk score (4-6 risk points) compared with ones with the lowest score (0-1 risk points) had a median PFS of 10 months versus 26.3 months (adjusted hazard ratio [AdjHR], 3.12 [95% confidence interval (CI), 2.18-4.48]; P < .001) and a median OS of 38.9 months versus 63.0 months (AdjHR, 2.41 [95% CI, 1.22-4.79], P = .012), respectively. CONCLUSION: In this study we defined a score including 5 polymorphisms to stratify patients for PFS and OS. This score, if validated, might be translated to personalize locally advanced or MBC patient treatment and management.

Gemcitabine in advanced biliary tract cancers.

The annual incidence of hepatobiliary cancer has been steadily increasing in the USA from 15,000 in 1993 to 22,200 in 2000. Despite this increase, it still continues to be a rare neoplasm. Surgical resection is the treatment of choice, but a high percentage of patients are unsuitable for resection. These patients have a very poor prognosis because of the lack of efficacious therapy options. Thus, overall survival in these patients ranges from 3 to 12 months, depending on the extent of disease and its site of origin. For some time, mitomycin C, doxorubicin and 5-fluorouracil have been considered among the most active chemotherapeutic agents, with a response rate ranging from 10 to 20%. More recently, gemcitabine has become the reference agent for these neoplasias because of the histologically common origin of biliary cancer and exocrine pancreatic cancer. However, its role has yet to be well-defined. Here we examine clinical trials designed for locally advanced and metastatic biliary tract cancer and review the existing data supporting palliative therapy with gemcitabine alone or in association with other drugs.

Nano albumin bound-paclitaxel in pancreatic cancer: Current evidences and future directions.

Pancreatic cancer (PDAC) is an aggressive and chemoresistant disease, representing the fourth cause of cancer related deaths in western countries. Majority of patients have unresectable, locally advanced or metastatic disease at time of diagnosis and the 5-year survival rate in these conditions is extremely low. For more than a decade gemcitabine has been the cornerstone of metastatic PDAC treatment, although survival benefit was very poor. PDAC cells are surrounded by an intense desmoplastic reaction that may create a barrier to the drugs penetration within the tumor. Recently PDAC stroma has been addressed as a potential therapeutic target. Nano albumin bound (Nab)-paclitaxel is an innovative molecule depleting tumor stroma, through interaction between albumin and secreted protein acidic and rich in cysteine. Addition of nab-paclitaxel to gemcitabine has showed activity and efficacy in metastatic PDAC first-line treatment improving survival and overall response rate vs gemcitabine alone in the MPACT phase III study. This combination represents one of the standards of care in advanced PDAC therapy and is suitable to a broader spectrum of patients compared to other schedules. Nab-paclitaxel is under investigation as a backbone of chemotherapy in novel combinations with target agents or immunotherapy in locally advanced or metastatic PDAC. In this article, we provide an updated and critical overview about the role of nab-paclitaxel in PDAC treatment based on the latest advances in preclinical and clinical research. Furthermore, we focus on the use of nab-paclitaxel within the context of metastatic PDAC treatment landscape and we discuss about future implications in the light of current clinical ongoing trials.

Oxaliplatin-related neurotoxicity: how and why?

In early clinical trials, oxaliplatin has demonstrated significant activity against colorectal cancer in combination with 5-fluorouracil (5-FU) and folinic acid (FA), both in metastatic as in radically resected disease. The drug differs from the other two most important platinum compounds (cisplatin and carboplatin) for the absence of nephrotoxicity or for the reduced drug-induced ototoxicity. During its administration, two different types of neurological symptoms can be experienced: the first one occurs during or immediately after the end of the infusion and it appears as a transient peripheral sensory neuropathy manifesting as paresthesias and dysesthesia in the extremities sometimes accompanied by muscular contractions of the extremities or the jaw (triggered or enhanced by exposure to cold). The second one occurs after long-term administration of oxaliplatin presenting with deep sensory loss, sensory ataxia and functional impairment (similar to those observed with cisplatin). This type of neurotoxicity is usually late-onset and correlated with the cumulative-dose of oxaliplatin. The aim of this review is to analyse the mechanism underlying induction of neurotoxicity and the possible treatments to prevent and to treat it.

The development of platinum compounds and their possible combination.

Cisplatin plays a central role in cancer chemotherapy in spite of its toxicity. To circumvent this toxicity and to enhance its therapeutic index a lot of preclinical and clinical studies have been conducted and several thousand analogues have been synthesized. Much more analysis remains to be done, but nowadays, the absence of any definitive, biologically interpretable molecular predictor of activity is consistent with the idea that platinum compounds have multiple intracellular targets and that cells can have multiple mechanisms of resistance. This review analyses a part of these platinum compounds analyzed to date, their mechanism of action, resistance and the future trends in this sector.

Stable disease in advanced colorectal cancer: therapeutic implications.

Colorectal cancer is one of the most common neoplasms in Western Countries and ranks second as a cause of death due to cancer. The overall mortality at 5 years is about 40%. Patients with resectable metastatic disease can be cured, but for those who cannot, treatment is purely palliative, and overall survival (OS) is from approximately 7 to 24 months. Infusional regimen with modulated 5Fluorouracil (5FU) gives an objective response rate (RR) of up to 30-40%. The addition of CPT11 or oxaliplatin to 5FU improves RR, time to progression (TTP) and OS with a stabilization of disease (SD) in 40-70% of cases and 20-40%, respectively. The concurrent utilization of selective biological agents as growth factor receptors acting at a molecular level and influencing the processes of tumor formation and growth, increases tumor cell apoptosis and inhibits tumor growth; as a result, the tumor regresses or is inhibited, with consequently prolonged OS and TTP. This paper examines the problem related to the treatment of metastatic colorectal cancer with SD. Current doubts regarding the continuation of one treatment until disease progression (PD) with a risk of toxicity, whether or not to use a less toxic "maintenance" therapy after a fairly aggressive "induction" therapy in "stabilized" responders, or whether to stop the treatment in the presence of a SD confirmed after at least two consecutive evaluations, are present.

Optimizing single agent panitumumab therapy in pre-treated advanced colorectal cancer.

PURPOSE: To improve the selection of advanced colorectal cancer patients to panitumumab by optimizing the assessment of RAS (KRAS-NRAS) mutations. EXPERIMENTAL DESIGN: Using a centralized pyrosequencing RAS assay, we analyzed the tumors of 94 patients, wild-type for KRAS mutations (codons 12 to 13) by Sanger sequencing (SS), treated with panitumumab. RESULTS: By SS analysis, 94 (62%) of 152 patients were wild-type and their objective response rate to panitumumab was 17%. We first optimized the KRAS test, by performing an accurate tissue-dissection step followed by pyrosequencing, a more sensitive method, and found further mutations in 12 (12.8%) cases. Secondly, tumors were subjected to RAS extension analysis (KRAS, exons 3 to 4; NRAS exons 2 to 4) by pyrosequencing that allowed to identify several rare mutations: KRAS codon 61, 5.3%; codon 146, 5.3%; NRAS, 9.5%. Overall, RAS mutation rate was 32.9%. All patients with additional RAS mutations had progressive or stable disease, except 3 patients with mutations at codon 61 of KRAS or NRAS who experienced partial (2 cases) or complete response. By excluding from the analysis 11 cases with mutations at codons 61, no patient was responsive to treatment (P=.021). RAS wild-type versus RAS mutated cases had a significantly better time to progression (P=.044), that resulted improved (p=.004) by excluding codon 61 mutations. CONCLUSION: This study shows that by optimizing the RAS test it is possible to significantly improve the identification of patients who do not gain benefit of panitumumab. Prospective studies are warranted to determine the clinical significance of rare mutations.

Antiangiogenic therapies in breast cancer.

Angiogenesis is a complex, multistep process that is involved in the progression and prognosis of several types of cancer. In particular, it has been demonstrated that angiogenesis plays a central role in the progression of breast cancer. This review evaluates results from clinical trials in which the efficacy of conventional chemotherapeutic agents was compared with that of novel antiangiogenic compounds for the treatment of breast cancer, and provides a summary of trials that were ongoing at the time of publication in the neoadjuvant and adjuvant settings.

Human fibroblasts from normal and malignant breast tissue grown in vitro show a distinct senescence profile and telomerase activity.

The telomerase activity and the senescence profile of cultured breast fibroblasts from normal human interstitial and malignant stromal tissue were studied in comparison with their proliferation and differentiation pattern. Fibroblasts were grown either in the presence or absence of a conditioned medium (CM) obtained from cultures of the oestrogen receptor-positive breast cancer MCF-7 cell line. At different passages (from the 2nd up to the 48th), fibroblasts were examined for the telomerase activity by the Telomerase Repeats Amplification Protocol (TRAP) assay, for proliferation profile by Ki-67 antigen expression, and the myofibroblast or smooth muscle cell-like differentiation pattern by immunofluorescence with monoclonal antibodies specific for smooth muscle markers. Serial passages of fibroblasts from normal or tumour breast reveal that the relationship between the levels of telomerase activity and phenotypic/proliferation profile changes with cell subcultivation in a different manner in the two cell populations. The fibroblasts from normal tissue completed 12 passages in a CM-independent way prior to senescence whereas fibroblasts from tumour stroma senescence were attained after 48 passages. These cells showed a marked decrease of telomerase activity, growth rate and smooth muscle alpha-actin expressing myofibroblasts after the 32nd passage. CM treatment of this fibroblast population induces a decline in the myofibroblast content, which precedes the changes in telomerase activity. Passaged fibroblasts from normal breast tissue can be converted to myofibroblasts upon CM treatment whereas those from tumour stroma were CM-insensitive. Taken together our data suggest that a heterogeneous fibroblast population with different life span is activated/recruited in the breast interstitium and poses the problem of a unique activation/recruitment of fibroblasts in neoplastic conditions.