Potential application of cryobiopsy for histo-molecular characterization of mediastinal lymph nodes in patients with thoracic malignancies: a case presentation series and implications for future developments.

BACKGROUND: The management of non-small cell lung cancer (NSCLC) has become increasingly complex due to the evolution of personalized medicine approaches. Such approaches are characterized by the necessity of adequate tumor samples; hence, improved biopsy techniques are needed. Transbronchial lung cryobiopsy is a novel endoscopic procedure designed to collect peripheral pulmonary tissue, and it is currently employed in interstitial lung diseases. The use of this technique in oncology might result in improved mediastinum staging and molecular characterizations; however, available data involving the use of a cryoprobe on mediastinal lymph nodes are still limited. CASE PRESENTATION: Here we present a series of five consecutive patients who underwent endoscopic assessment of mediastinal lymph nodes for oncologic reasons. All patients were subjected both to endobronchial ultrasound-guided transbronchial needle aspiration (EBUS TBNA) and cryobiopsy of mediastinal lymph nodes during the same procedure, and no complications were observed. In three of the reported cases, both cryobiopsy and cell block from EBUS TBNA were positive, while in one case cryobiopsy was not diagnostic and EBUS TBNA was negative; moreover, one case showed discordance between the procedures, as cryobiopsy was negative and cell block obtained from multiple stations was diagnostic for small cell lung cancer. In one case involving a patient treated for lymphoma, cryobiopsy provided more complete histologic characterization, and in another case involving a patient affected by NSCLC cryobiopsy provided more material for molecular analyses. CONCLUSION: This case presentation series suggests that cryobiopsy, which has been generally used on peripheral lung lesions so far, is a feasible and safe approach for diagnosis and staging of mediastinal lymph nodal involvement, especially when station 7 is involved. Compared to EBUS TBNA, cryobiopsy might provide more adequate histological samples, with a possible impact on molecular characterizations and, therefore, therapeutic decisions. However, the learning curve of the procedure has not to be understated and optimal protocols for implementing this technique are needed. In our opinion, further studies designed to integrate the routine use of cryobiopsy in current practice for solid and eventually hematologic tumors with mediastinal lymph node involvement are warranted.

Phenotypic characterization of tumor CTLA-4 expression in melanoma tissues and its possible role in clinical response to Ipilimumab.

The expression of the immune checkpoint molecule CTLA-4 has been almost exclusively studied in the T cell lineage, but increasing evidence has shown its expression on tumors with implications for immunotherapy. To date, the degree of expression of CTLA-4 on tumor cells as a predictive biomarker of response to immune checkpoint inhibitors has not been studied. In this report, we analyzed this issue in melanoma patients treated with CTLA-4 inhibitor Ipilimumab (IPI). We show that the level of CTLA-4 expression on melanoma cells is higher than that on tumor infiltrating lymphocytes (TIL) and it is associated with clinical response to IPI therapy supporting the idea of its possible role as a predictive biomarker.

Response to ipilimumab therapy in metastatic melanoma patients: potential relevance of CTLA-4+ tumor infiltrating lymphocytes and their in situ localization.

Immune checkpoint inhibitors, including ipilimumab (IPI), achieve a clinical benefit in a small proportion of melanoma patients highlighting the need to investigate predictive biomarkers. In this study, we characterized tumor infiltrating lymphocytes (TILs), focusing on the CTLA-4+ subset, and evaluated their possible predictive significance. We characterized TIL density, cell type, and localization in 40 melanoma lesions from 17 patients treated with IPI. Associations of TILs with IPI timing, tissue localization, and response to IPI were estimated using a linear mixed-effects modelling approach. We found that most of TIL subsets increased in situ upon IPI therapy, with particular reference to FoxP3+ cells. TILs and TIL subsets, such as CD3+, CD45RO+, CTLA-4+, CD4+, CD8+ T cells, CD20+ B cells, and NKp46+ NK cells, showed significantly different spatial distributions in the tumor microenvironment being higher at the invasive margin (IM) as compared to the tumor center (TC) (P value < 0.001 for TIL score and P value < 0.05 for all subsets). Remarkably, high TIL score and density of CD3+, CD8+ T cells, and CTLA-4+ immune cells were significantly associated with a better response to IPI (P values = 0.002, 0.023, 0.007, and 0.001, respectively, for responders vs non-responders). In conclusion, we provide a detailed analysis of CTLA-4+ TIL distribution in melanoma tissues taking into account localization, relationship with CD3+/CD8+ TILs, and changes in response to IPI treatment. We identified that CTLA-4+ TILs may represent a marker of IPI response, alone or with CD3+/CD8+ subsets, although this requires confirmation in larger studies.

A non-invasive approach to monitor chronic lymphocytic leukemia engraftment in a xenograft mouse model using ultra-small superparamagnetic iron oxide-magnetic resonance imaging (USPIO-MRI).

Chronic lymphocytic leukemia (CLL) is the most prevalent leukemia among adults. Despite its indolent nature, CLL remains an incurable disease. Herein we aimed to monitor CLL disease engraftment and, progression/regression in a xenograft CLL mouse model using ultra-small superparamagnetic iron oxide-magnetic resonance imaging (USPIO-MRI). Spleen contrast enhancement, quantified as percentage change in signal intensity upon USPIO administration, demonstrated a difference due to a reduced USPIO uptake, in the spleens of mice injected with CLL cells (NSG-CLL, n=71) compared to controls (NSG-CTR, n=17). These differences were statistically significant both after 2 and 4weeks from CLL cells injection. In addition comparison of mice treated with rituximab with untreated controls for changes in spleen iron uptake confirmed that it is possible to monitor treatment efficacy in this mouse model of CLL using USPIO-enhanced MRI. Further applications could include the preclinical in vivo monitoring of new therapies and the clinical evaluation of CLL patients.

CTLA-4 in mesothelioma patients: tissue expression, body fluid levels and possible relevance as a prognostic factor.

CTLA-4 function as a negative regulator of T cell-mediated immune response is well established, whereas much less is known about the immunoregulatory role of its soluble isoform (sCTLA-4). No data are available on CTLA-4 expression and prognostic impact in malignant pleural mesothelioma (MPM). We investigated, by immunohistochemistry, CTLA-4 expression in tumor tissues and, by ELISA, sCTLA-4 levels in sera and matched pleural effusions from 45 MPM patients. Prognostic effect of CTLA-4 expression on overall survival (OS) was assessed through Cox regression and prognostic significance expressed as death rate ratio (HR). We found that 56.0 % of MPM tissues expressed CTLA-4 with variable intensity and percentage of positive cells estimated by the immunoreactive score. sCTLA-4 levels were significantly higher in sera (S-sCTLA-4) than in pleural effusions (PE-sCTLA-4) (geometric mean ratio = 2.70, P value = 0.020). CTLA-4 expression at the tissue level was higher in the epithelioid histological subtype than in the sarcomatoid, whereas at the serum level, it was higher in the sarcomatoid subtype. A homogeneous favorable prognostic effect was found for CTLA-4 overexpression in tissue, serum and pleural effusion. Interestingly, only the PE-sCTLA-4 was found to be a statistically significant positive prognostic factor (HR = 0.37, 95 % CI = 0.18-0.77, P value = 0.007). Indeed, PE-sCTLA-4 correlated with CTLA-4 expression in tissues, whereas this latter expression showed a weak association with OS. To confirm our findings, further experimental evidences obtained from a larger cohort of MPM patients are required. However, our results would indicate a positive correlation of PE-sCTLA-4 levels and OS in MPM patients.

Expression of nuclear matrix proteins binding matrix attachment regions in prostate cancer. PARP-1: New player in tumor progression.

Prostate cancer (PCa) displays infrequent point mutations, whereas genomic rearrangements are highly prevalent. In eukaryotes, the genome is compartmentalized into chromatin loop domains by the attachment to the nuclear matrix (NM), and it has been demonstrated that several recombination hot spots are situated at the base of loops. Here, we have characterized the binding between NM proteins and matrix attachment regions (MARs) in PCa. Nontumor and 44 PCa tissues were analyzed. More aggressive tumors were characterized by an increase in the complexity of the NM protein patterns that was synchronous with a decrease in the number of proteins binding the MAR sequences. PARP-1 was the protein that showed the most evident changes. The expression of the PARP-1 associated with NM increased and it was dependent on tumor aggressiveness. Immunohistochemical analysis showed that the protein was significantly overexpressed in tumor cells. To explore the role of PARP-1 in PCa progression, PCa cells were treated with the PARP inhibitor, ABT-888. In androgen-independent PC3 cells, PARP inhibition significantly decreased cell viability, migration, invasion, chromatin loop dimensions and histone acetylation. Collectively, our study provides evidence that MAR-binding proteins are involved in the development and progression of PCa. PARP could play a key role in the compartmentalization of chromatin and in the development of the more aggressive phenotype. Thus, PARP can no longer be viewed only as an enzyme involved in DNA repair, but that its role in chromatin modulation could provide the basis for a new therapeutic approach to the treatment of PCa.

Analysis of in vitro ADCC and clinical response to trastuzumab: possible relevance of FcγRIIIA/FcγRIIA gene polymorphisms and HER-2 expression levels on breast cancer cell lines.

BACKGROUND: Trastuzumab is a humanized monoclonal antibody (mAb) currently used for the treatment of breast cancer (BC) patients with HER-2 overexpressing tumor subtype. Previous data reported the involvement of FcγRIIIA/IIA gene polymorphisms and/or antibody-dependent cellular cytotoxicity (ADCC) in the therapeutic efficacy of trastuzumab, although results on these issues are still controversial. This study was aimed to evaluate in vitro the functional relationships among FcγRIIIA/IIA polymorphisms, ADCC intensity and HER-2 expression on tumor target cells and to correlate them with response to trastuzumab. PATIENTS AND METHODS: Twenty-five patients with HER-2 overexpressing BC, receiving trastuzumab in a neoadjuvant (NEO) or metastatic (MTS) setting, were genotyped for the FcγRIIIA 158V>F and FcγRIIA 131H>R polymorphisms by a newly developed pyrosequencing assay and by multiplex Tetra-primer-ARMS PCR, respectively. Trastuzumab-mediated ADCC of patients' peripheral blood mononuclear cells (PBMCs) was evaluated prior to therapy and measured by (51)Chromium release using as targets three human BC cell lines showing different levels of reactivity with trastuzumab. RESULTS: We found that the FcγRIIIA 158F and/or the FcγRIIA 131R variants, commonly reported as unfavorable in BC, may actually behave as ADCC favorable genotypes, in both the NEO (P ranging from 0.009 to 0.039 and from 0.007 to 0.047, respectively) and MTS (P ranging from 0.009 to 0.032 and P = 0.034, respectively) patients. The ADCC intensity was affected by different levels of trastuzumab reactivity with BC target cells. In this context, the MCF-7 cell line, showing the lowest reactivity with trastuzumab, resulted the most suitable cell line for evaluating ADCC and response to trastuzumab. Indeed, we found a statistically significant correlation between an increased frequency of patients showing ADCC of MCF-7 and complete response to trastuzumab in the NEO setting (P = 0.006). CONCLUSIONS: Although this study was performed in a limited number of patients, it would indicate a correlation of FcγR gene polymorphisms to the ADCC extent in combination with the HER-2 expression levels on tumor target cells in BC patients. However, to confirm our findings further experimental evidences obtained from a larger cohort of BC patients are mandatory.

Glyceraldehyde-3-phosphate dehydrogenase gene over expression correlates with poor prognosis in non small cell lung cancer patients.

BACKGROUND: Glycolysis in presence of oxygen with high glucose consumption is known to be the metabolism of choice in many tumors. In lung cancer this phenomenon is routinely exploited in diagnostic PET imaging of fluorodeoxyglucose uptake, but not much is known about the prognostic capabilities of glycolysis level assessment in resected lung tumor samples. METHODS: In this retrospective study, we used real time polymerase chain reaction(RQ-PCR) to assess the expression level of the gene for Glyceraldehyde 3-phosphate dehydrogenase(GAPDH), key enzyme for glucose breakdown, in tumor samples from 82 consecutive early stages resected non small cell lung cancer(NSCLC) patients. We then compared our results in six large publicly available NSCLC microarray datasets collecting data from over 1250 total patients. RESULTS: In our study GAPDH gene over expression was found to be an adverse prognostic factor in early stages NSCLC (n = 82 HR = 1.30 p = 0.050). This result was confirmed in 5 of 6 public datasets analyzed: Shedden et al. 2008: n = 442 HR = 1.54 p < 0.0001; Lee et al. 2008: n = 138 HR = 1.31 p = 0.043; Tomida et al. 2009: n = 117 HR = 1.59 p = 0.004; Roepman et al. 2009: n = 172 (TPI1 gene) HR = 1.51 p = 0.009; Okayama et al. 2012: n = 226 HR = 3.19 p < 0.0001; Botling et al. 2013: n = 196 HR = 1.00 p = 0.97). Furthermore, in the large and clinically well annotated Shedden et al. microarray dataset, GAPDH hazard ratio did not change whether calculated for the whole dataset or for the subgroup of adjuvant naive patients only (n = 330 HR = 1.49 p < 0.0001). CONCLUSION: GAPDH gene over expression in resected tumor samples is an adverse prognostic factor in NSCLC. Our results confirm the prognostic value of glucose metabolism assessment in NSCLC.

Expression of immunoglobulin receptors with distinctive features indicating antigen selection by marginal zone B cells from human spleen.

Marginal zone (MZ) B cells, identified as surface (s)IgM(high)sIgD(low)CD23(low/-)CD21(+)CD38(-) B cells, were purified from human spleens, and the features of their V(D)J gene rearrangements were investigated and compared with those of germinal center (GC), follicular mantle (FM) and switched memory (SM) B cells. Most MZ B cells were CD27(+) and exhibited somatic hypermutations (SHM), although to a lower extent than SM B cells. Moreover, among MZ B-cell rearrangements, recurrent sequences were observed, some of which displayed intraclonal diversification. The same diversifying sequences were detected in very low numbers in GC and FM B cells and only when a highly sensitive, gene-specific polymerase chain reaction was used. This result indicates that MZ B cells could expand and diversify in situ and also suggested the presence of a number of activation-induced cytidine deaminase (AID)-expressing B cells in the MZ. The notion of antigen-driven expansion/selection in situ is further supported by the VH CDR3 features of MZ B cells with highly conserved amino acids at specific positions and by the finding of shared ("stereotyped") sequences in two different spleens. Collectively, the data are consistent with the notion that MZ B cells are a special subset selected by in situ antigenic stimuli.

The engagement of CTLA-4 on primary melanoma cell lines induces antibody-dependent cellular cytotoxicity and TNF-α production.

BACKGROUND: CTLA-4 (Cytotoxic T lymphocyte antigen-4) is traditionally known as a negative regulator of T cell activation. The blocking of CTLA-4 using human monoclonal antibodies, such as Ipilimumab, is currently used to relieve CTLA-4-mediated inhibition of anti-tumor immune response in metastatic melanoma. Herein, we have analyzed CTLA-4 expression and Ipilimumab reactivity on melanoma cell lines and tumor tissues from cutaneous melanoma patients. Then, we investigated whether Ipilimumab can trigger innate immunity in terms of antibody dependent cellular cytotoxicity (ADCC) or Tumor Necrosis Factor (TNF)-α release. Finally, a xenograft murine model was set up to determine in vivo the effects of Ipilimumab and NK cells on melanoma. METHODS: CTLA-4 expression and Ipilimumab reactivity were analyzed on 17 melanoma cell lines (14 primary and 3 long-term cell lines) by cytofluorimetry and on 33 melanoma tissues by immunohistochemistry. CTLA-4 transcripts were analyzed by quantitative RT-PCR. Soluble CTLA-4 and TNF-α were tested by ELISA. Peripheral blood mononuclear cells (PBMC), NK and γδT cells were tested in ADCC assay with Ipilimumab and melanoma cell lines. TNF-α release was analyzed in NK-melanoma cell co-cultures in the presence of ipilimumab. In vivo experiments of xenotransplantation were carried out in NOD/SCID mice. Results were analyzed using unpaired Student's t-test. RESULTS: All melanoma cell lines expressed mRNA and cytoplasmic CTLA-4 but surface reactivity with Ipilimumab was quite heterogeneous. Accordingly, about 2/3 of melanoma specimens expressed CTLA-4 at different level of intensity.Ipilimumab triggered, via FcγReceptorIIIA (CD16), ex vivo NK cells as well as PBMC, IL-2 activated NK and γδT cells to ADCC of CTLA-4+ melanoma cells. No ADCC was detected upon interaction with CTLA-4- FO-1 melanoma cell line. TNF-α was released upon interaction of NK cells with CTLA-4+ melanoma cell lines. Remarkably, Ipilimumab neither affected proliferation and viability nor triggered ADCC of CTLA-4+ T lymphocytes. In a chimeric murine xenograft model, the co-engraftment of Ipilimumab-treated melanoma cells with human allogeneic NK cells delayed and significantly reduced tumor growth, as compared to mice receiving control xenografts. CONCLUSIONS: Our studies demonstrate that Ipilimumab triggers effector lymphocytes to cytotoxicity and TNF-α release. These findings suggest that Ipilimumab, besides blocking CTLA-4, can directly activate the elimination of CTLA-4+ melanomas.

Evaluation of CTLA-4 expression and relevance as a novel prognostic factor in patients with non-small cell lung cancer.

The role of CTLA-4 in negative regulation of T-cell mediated immune response is particularly well established. Much less is known about its expression and function in tumour cells, and to our knowledge, no data are available on its possible impact on prognosis of NSCLC patients. We investigated CTLA-4 expression and prognostic role in 81 patients with radically resected stage I-III NSCLC. The analysis was performed by tissue microarray immunohistochemistry, and the median H-score of 20 was used as a threshold to define CTLA-4 overexpressing tumours. Correlation with standard prognostic factors was performed by using absolute and relative fold change indexes. Hazard ratios (HR) and corresponding 95% confidence limits (95% CL) were computed through the Cox model. A higher frequency of CTLA-4 overexpression (>20) was found in non-squamous than in squamous NSCLC (52.8 vs. 35.7%) and in Ki67 ≤ 15 expressing tumours, as compared to those with Ki67 > 15 (51.5 vs. 38.7%). A reduced death rate was found in CTLA-4 overexpressing tumours (HR = 0.60, 95% CL = 0.28/1.23), and a further decrease was observed when considering tumours with CTLA-4 > 20 and Ki67 ≤ 15, in comparison with tumours with CTLA-4 ≤ 20 and Ki67 > 15 (HR = 0.41; 95% CL = 0.15/1.13). Our observational and exploratory study provides a first and promising indication for an independent prognostic effect of CTLA-4 overexpression in radically resected NSCLC. We presume that this effect relies on modulation of the interaction of microscopic disease with CTLA-4-ligands expressing cells leading to NSCLC cell death.

Prognostic value of stromal and epithelial periostin expression in human prostate cancer: correlation with clinical pathological features and the risk of biochemical relapse or death.

BACKGROUND: The purpose of the present study was to evaluate the prognostic value of POSTN expression following prostatectomy. METHODS: Periostin (POSTN) expression in prostate cancer (PCa) and in normal specimens was evaluated in 90 patients by an immuno-reactive score(IRS) based on the intensity of immunostaining and on the quantity of stained cells. The t-test was applied to compare IRS values in cancer specimens to values in normal specimens. Pearson's test was used to correlate POSTN expression to clinical pathologic features. PSA progression-free and survival curves were constructed by the Kaplan-Meier method and compared using the log-rank test. Multi-parametric models were constructed according to the Cox technique adding all the covariates predicting for either PSA progression or death into the models after univariate analysis. RESULTS: Both stromal and epithelial POSTN expression were significantly increased in tumor tissues. In particular, we found stromal expression to be significantly higher than epithelial expression as compared to normal tissues (p<0.000 and p=0.001).A significant correlation between POSTN epithelial expression and extra-prostatic extension was found (p=0.03). While high stromal expression was significantly associated with shorter survival (p=0.008), a low epithelial score significantly correlated with shorter PSA-free survival (p=0.04), suggesting that POSTN plays an apparently opposing biological role depending on its compartmentalization.Regardless of the mechanism that is involved, patients showing both high stromal and low epithelial expression made up a subgroup with a very bleak prognosis. CONCLUSIONS: Although requiring further validation through larger studies, our findings show that POSTN might represent a novel prognostic marker for PCa.

In vivo imaging of human breast cancer mouse model with high level expression of calcium sensing receptor at 3T.

OBJECTIVES: To demonstrate that manganese can visualise calcium sensing receptor (CaSR)-expressing cells in a human breast cancer murine model, as assessed by clinical 3T magnetic resonance (MR). METHODS: Human MDA-MB-231-Luc or MCF7-Luc breast cancer cells were orthotopically grown in NOD/SCID mice to a minimum mass of 5 mm. Mice were evaluated on T1-weighted sequences before and after intravenous injection of MnCl(2). To block the CaSR-activated Ca(2+) channels, verapamil was injected at the tumour site 5 min before Mn(2+) administration. CaSR expression in vivo was studied by immunohistochemistry. RESULTS: Contrast enhancement was observed at the tumour periphery 10 min after Mn(2+) administration, and further increased up to 40 min. In verapamil-treated mice, no contrast enhancement was observed. CaSR was strongly expressed at the tumour periphery. CONCLUSION: Manganese enhanced magnetic resonance imaging can visualise CaSR-expressing breast cancer cells in vivo, opening up possibilities for a new MR contrast agent. KEY POINTS: • Manganese contrast agents helped demonstrate breast cancer cells in an animal model. • Enhancement was most marked in cells with high calcium sensing receptor expression. • Manganese uptake was related to the distribution of CaSR within the tumour. • Manganese MRI may become useful to investigate human breast cancer.

MiR-146b-5p regulates IL-23 receptor complex expression in chronic lymphocytic leukemia cells.

Chronic lymphocytic leukemia (CLL) cells express the interleukin-23 receptor (IL-23R) chain, but the expression of the complementary IL-12Rß1 chain requires cell stimulation via surface CD40 molecules (and not via the B-cell receptor [BCR]). This stimulation induces the expression of a heterodimeric functional IL-23R complex and the secretion of IL-23, initiating an autocrine loop that drives leukemic cell expansion. Based on the observation in 224 untreated Binet stage A patients that the cases with the lowest miR-146b-5p concentrations had the shortest time to first treatment (TTFT), we hypothesized that miR-146b-5p could negatively regulate IL-12Rß1 side chain expression and clonal expansion. Indeed, miR-146b-5p significantly bound to the 3'-UTR region of the IL-12Rß1 mRNA in an in vitro luciferase assay. Downregulation of miR-146b-5p with specific miRNA inhibitors in vitro led to the upregulation of the IL-12Rß1 side chain and expression of a functional IL-23R complex similar to that observed after stimulation of the CLL cell through the surface CD40 molecules. Expression of miR-146b-5p with miRNA mimics in vitro inhibited the expression of the IL-23R complex after stimulation with CD40L. Administration of a miR-146b-5p mimic to NSG mice, successfully engrafted with CLL cells, caused tumor shrinkage, with a reduction of leukemic nodules and of IL-12Rß1-positive CLL cells in the spleen. Our findings indicate that IL-12Rß1 expression, a crucial checkpoint for the functioning of the IL-23 and IL-23R complex loop, is under the control of miR-146b-5p, which may represent a potential target for therapy since it contributes to the CLL pathogenesis. This trial is registered at www.clinicaltrials.gov as NCT00917540.

Interleukin (IL)-18, a biomarker of human ovarian carcinoma, is predominantly released as biologically inactive precursor.

Interleukin (IL)-18 is a proinflammatory and immune-enhancing cytokine, which exerts antitumor effects in vivo, mediated by the induction of interferon (IFN)γ. We previously reported that IL-18 processing is defective in epithelial ovarian carcinoma (EOC) cells, which secrete an inactive precursor (pro-IL-18) in vitro. In addition, IL-18 was reported as a potential biomarker of EOC. Here, we further investigated its role as a serological marker in human EOC and addressed its possible biological activity in vivo. Our data indicate that immunoreactive IL-18 is increased in EOC patients' sera at diagnosis as compared with age-matched healthy women. IL-18 levels were higher in the ascitic fluids than in sera, suggesting a local production in the peritoneal cavity. Indeed, immunohistochemical analysis of tumors showed IL-18 expression in cytokeratine-positive neoplastic cells, although also scattered histiocytes and some lymphoid cells stained for IL-18. The detection of human IL-18 in sera and ascitic fluids of immunodeficient mice, orthotopically implanted with human EOC cells, further suggested that circulating IL-18 is tumor-derived. However, IL-18 is not an EOC specific biomarker, as increased serum levels were found also in some endometrial cancer patients. By means of a new monoclonal antibody, we characterized IL-18 present in the ascitic fluid as pro-IL-18, which is biologically inactive. Accordingly, IFNγ was not increased in EOC patients' sera and ascitic fluids and showed no correlation with IL-18 levels. Altogether these data indicate that IL-18 in EOC fluids is predominantly tumor-derived and that its lack of biological activity may represent a mechanism of tumor-escape.

Establishment and Characterization of a Novel Fibroblastic Cell Line (SCI13D) Derived from the Broncho-Alveolar Lavage of a Patient with Fibrotic Hypersensitivity Pneumonitis.

Hypersensitivity pneumonitis (HP) is a diffuse interstitial lung disease (ILD) caused by the inhalation of a variety of antigens in susceptible individuals. Patients with fibrotic HP (fHP) may show histopathological and radiological manifestations similar to patients with idiopathic pulmonary fibrosis (usual interstitial pneumonia-like pattern of fibrosis) that are associated with a worse prognosis. We describe here the establishment and characterization of a fibroblastic cell line derived from the broncho-alveolar lavage (BAL) of a patient with fHP, a 53 year old man who presented at our Pneumology Unit with cough and dyspnea. The fHP diagnosis was based on international criteria and multidisciplinary discussion. Primary fibroblasts were expanded in vitro until passage 36. These fibroblasts displayed morpho/phenotypical features of myofibroblasts, showing high positivity for α-smooth muscle actin, type I collagen, and fibronectin as determined by quantitative RT-PCR and cyto-fluorographic analysis. Cytogenetic analyses further evidenced trisomy of chromosome 10, which interestingly harbors the FGF2R gene. To our knowledge, this is the first fibroblastic cell line derived from an fHP patient and might, therefore, represent a suitable tool to model the disease in vitro. We preliminarily assessed here the activity of pirfenidone, further demonstrating a consistent inhibition of cells growth by this antifibrotic drug.

Radiation-Related Deregulation of TUBB3 and BRCA1/2 and Risk of Secondary Lung Cancer in Women With Breast Cancer.

INTRODUCTION: Breast cancer survivors are at increased risk of developing unrelated primary cancers, particularly lung cancer. Evidence indicates that sex hormones as well as a deregulation of DNA-repair pathways may contribute to lung cancer onset. We investigated whether the hormone status and expression of markers involved in DNA repair (BRCA1/2, ERCC1, and P53R2), synthesis (TS and RRM1), and cell division (TUBB3) might be linked to lung cancer risk. PATIENTS AND METHODS: Thirty-seven breast cancer survivors with unrelated lung cancer and 84 control subjects comprising women with breast cancer (42/84) or lung cancer (42/84) were enrolled. Immunohistochemistry on tumor tissue was performed. Geometric mean ratio was used to assess the association of marker levels with patient groups. RESULTS: Estrogen receptor was expressed in approximately 90% of the breast cancer group but was negative in the majority of the lung cancer group, a result similar to the lung cancer control group. Likewise, ER isoform ß was weakly expressed in the lung cancer group. Protein analysis of breast cancer versus control had a significantly lower expression of BRCA1, P53R2, and TUBB3. Likewise, a BRCA1 reduction was observed in the lung cancer group concomitant with a BRCA2 increase. Furthermore, BRCA2 and TUBB3 increased in ipsilateral lung cancer in women who had previously received radiotherapy for breast cancer. CONCLUSION: The decrease of DNA-repair proteins in breast cancer could make these women more susceptible to therapy-related cancer. The increase of BRCA2 and TUBB3 in lung cancer from patients who previously received radiotherapy for breast cancer might reflect a tissue response to exposure to ionizing radiation.

Insights from a Long-Term Follow-Up Evaluation of Early Breast Cancer Outcomes by Tumor Subtype.

INTRODUCTION: Our understanding of the granularity of breast cancer (BC) clinical outcomes by biologic subtype may be impaired by limited study follow-up times. OBJECTIVE: We evaluated the impact of modern immunohistochemistry (IHC)-based BC subtypes on long-term mortality. METHOD: We used a cohort of 200 women diagnosed with stage I-III BC in the period 1985-1990. Surgical samples underwent centralized pathology review. Multivariate models assessed associations of subtype with overall survival (OS) and BC-related survival (BCRS). RESULT: 42.0% women had luminal A-like, 32.5% luminal B-like/human epidermal growth factor receptor (HER)2-negative, 8.5% had HER2-positive, and 17.0% had triple-negative BC. 53.0% had tumor size (T) >2 cm and 47.5% had a positive nodal status (N). Over 18.7 years of median follow-up (range 0.3-32.0 years),140 deaths were recorded (75 BC-related). Median OS was longest for patients with luminal A-like tumors (21.2 years; 95% confidence interval [CI] 17.4-24.9]). The luminal B-like/HER2-negative subtype was significantly associated with worse BCRS (adjusted hazard ratio [HR] = 1.86; 95% CI 1.09-3.16). After multivariable analysis, T >2 cm (HR [vs. ≤2 cm] = 1.71 [95% CI 1.03-2.84]) and positive N (HR [vs. negative] = 2.19 [95% CI 1.03-4.65]) impacted BCRS. CONCLUSION: IHC-defined subtype will continue informing treatment algorithms for BC, until more precise tools like molecular profiling become widely available. Although confirmation in larger and adequately powered studies is warranted, modern surrogate subtype definitions produced a valid long-term prognostic stratification in this mature cohort.

Manganese-enhanced MRI (MEMRI) in breast and prostate cancers: Preliminary results exploring the potential role of calcium receptors.

PROCEDURES: To preliminary assess the relationship between Manganese Enhanced Magnetic Resonance Imaging (MEMRI) and the expression of calcium receptors in human prostate and breast cancer animal models. METHODS: NOD/SCID mice were inoculated with MDA-MB-231 breast cancer cells and prostate PC3 cancer cells to develop orthotopic or pseudometastatic cancer animal models. Mice were studied on a clinical 3T scanner by using a prototype birdcage coil before and after intravenous injection of MnCl2. Assessment of receptor's status was carried out after the MR images acquisition by immunohistochemistry on excised tumours. RESULTS: Manganese contrast enhancement in breast or prostate cancer animal models well correlated with CaSR expression (p<0.01), whereas TRPV6 expression levels appeared not relevant to the Mn uptake. CONCLUSION: Our preliminary results suggest that MEMRI appears an efficient tool to characterize human breast and prostate cancer animal models in the presence of different expression level of calcium receptors.