Barbara Oliviero

Pilot Study of the Mechanism of Action of Preoperative Trastuzumab in Patients with Primary Operable Breast Tumors Overexpressing HER2

Purpose: To elucidate the mechanism by which trastuzumab, a humanized monoclonal antibody against HER2 with proven survival benefit in women with HER2-positive metastatic breast cancer, mediates its antitumor activity. Experimental Design: A pilot study including 11 patients with HER2-positive tumors treated in a neo-adjuvant setting with trastuzumab was performed. Trastuzumab was administered i.v. at a dose of 4 mg/kg followed by three weekly i.v. doses of 2 mg/kg. The primary tumor was surgically removed 7 days after the last treatment. Surgical samples, tumor biopsies, and lymphocytes from these patients were collected for biological studies. Result: Clinical data indicated one complete pathological remission and four partial remissions using RECIST (Response Evaluation Criteria in Solid Tumors). Trastuzumab was well tolerated and neither serious adverse events nor changes in cardiac function were observed during this short-term treatment and after surgery. The biological data showed that, independent of response, (a) all patients showed high levels of circulating trastuzumab; (b) saturating level of trastuzumab was present in all of the tumors; (c) no down-modulation of HER2 was observed in any tumors; (d) no changes in vessel diameter was observed in any tumors; (e) no changes in proliferation was observed in any tumors; and (f) a strong infiltration by lymphoid cells was observed in all cases. Patients with complete remission or partial remission were found to have a higher in situ infiltration of leukocytes and a higher capability to mediate in vitro antibody-dependent cellular cytotoxicity activity. Conclusions: The results of this pilot study argue against trastuzumab activity in patients through down-modulation of HER2 but in favor of antibody-dependent cellular cytotoxicity guiding efforts to optimize the use of trastuzumab in breast cancer patients.

Natural Killer Cell Functional Dichotomy in Chronic Hepatitis B and Chronic Hepatitis C Virus Infections

BACKGROUND & AIMS The phenotypic and functional characteristics of natural killer (NK) cells in chronic hepatitis B virus (HBV) and hepatitis C virus (HCV) infections are incompletely defined and largely controversial. METHODS We studied NK cell receptor expression, cytotoxic activity, and cytokine production in peripheral blood mononuclear cells from 35 patients with chronic hepatitis C, 22 with chronic hepatitis B, and 30 healthy controls. RESULTS Patients with chronic HBV infection had an increased proportion of NKG2C(+) NK cells with normal inhibitory receptor expression and a lower proportion of activated NK cells compared with HCV(+) patients, which was associated with normal or reduced cytolytic activity and markedly dysfunctional tumor necrosis factor-alpha and interferon-gamma production. Patients with chronic HCV infection showed a predominantly activating phenotype, featuring a decreased percentage of cells expressing the inhibitory receptor KIR3DL1 and a concomitant increase in the proportion of NKG2D(+) NK cells. Expression of the CD69 early activation antigen on NK cells positively correlated with serum alanine aminotransferase and HCV RNA values, suggesting participation of virus-induced effector NK cells in liver necroinflammation. Phenotypic changes in HCV(+) patients were associated with enhanced cytokine-induced cytolytic activity and increased usage of natural cytotoxicity and NKG2D receptor pathways, accompanied by defective cytokine production, although to a lesser extent than patients with chronic HBV infection. CONCLUSIONS These findings provide evidence for a functional dichotomy in patients with chronic HBV and HCV infections, featuring conserved or enhanced cytolytic activity and dysfunctional cytokine production, which may contribute to virus persistence.

Elements Related to Heterogeneity of Antibody-Dependent Cell Cytotoxicity in Patients Under Trastuzumab Therapy for Primary Operable Breast Cancer Overexpressing Her2

Preliminary results from a pilot trial on trastuzumabs mechanism of action against operable breast tumors overexpressing Her2 suggested a role for antibody-dependent cell cytotoxicity (ADCC). To examine factors affecting ADCC intensity and variability, we extended this study to the phenotypic and functional analysis of circulating mononuclear cells in 18 patients. ADCC was induced by trastuzumab therapy in 15 of 18 patients (83%). Inability to develop ADCC in three patients did not depend on inadequate levels of trastuzumab because further increase in its concentration in vitro was ineffective. Rather, susceptibility to develop ADCC was fairly predicted by test with trastuzumab before therapy and was correlated to the number of lymphocytes coexpressing CD16 and CD56. Phenotypic analysis at the end of ADCC evaluating down-regulation of CD16, and up-regulation of CD69 and CD107a, confirmed that natural killer (NK) cells and CD56(+) T cells were involved in productive engagement of trastuzumab. Also, the killing efficiency of CD16(+) lymphocytes was influenced by 158 V/F polymorphism of Fc gamma RIII (CD16), whereas variations of CD247 on NK cells were consistent with trends between ADCC before and after therapy. Complete pathologic response was observed in one patient showing ADCC of outstanding intensity, whereas four cases of partial response showed intermediate ADCC; none of the three patients unable to mount ADCC had significant tumor regression. These data indicate that quantity and lytic efficiency of CD16(+) lymphocytes are major factors for ADCC induction by trastuzumab, and confirm that breast cancer responses to short-term trastuzumab monotherapy may depend on involvement of the ADCC mechanism.

Impaired intrahepatic natural killer cell cytotoxic function in chronic hepatitis C virus infection

Hepatitis C virus (HCV) persistence in the host results from inefficiencies of innate and adaptive immune responses. Most studies addressing the role of innate immunity concentrated on peripheral blood (PB) natural killer (NK) cells, whereas only limited information is available on intrahepatic (IH) NK cells. We therefore examined phenotypic and functional features of IH and PB NK cells in paired liver biopsy and venous blood samples from 70 patients with chronic HCV infection and 26 control persons subjected to cholecystectomy for gallstones as controls. Ex vivo isolated IH NK cells from HCV‐infected patients displayed unique phenotypic features, including increased expression of NKp46‐activating receptor in the face of reduced tumor necrosis factor–related apoptosis‐inducing ligand (TRAIL) and cluster of differentiation (CD) 107a expression, which resulted in impaired degranulation compared with controls. To gain insights into the effect of HCV on NK cells, we exposed peripheral blood mononuclear cells (PBMCs) from patients and healthy donors to cell‐culture–derived HCV (HCVcc) and measured NK cell degranulation, TRAIL, and phosphorylated extracellular signal‐regulated kinase 1/2 (pERK1/2) expression. Exposure of PBMCs to HCVcc significantly boosted NK degranulation, pERK1/2, and TRAIL expression in healthy donors, but not in patients with chronic HCV infection, a defect that was completely reversed by interferon‐alpha. Purified NK cells showed a minimal, though significant, increase in degranulation and TRAIL expression, both in patients and controls, after exposure to HCVcc. Conclusions: These findings indicate dysfunctional IH NK cell cytotoxicity associated with TRAIL down‐regulation in chronic HCV infection, which may contribute to virus persistence. PB NK cell impairment upon exposure to HCVcc suggests the existence of an accessory cell‐dependent NK cell lytic defect in chronic HCV infection predominantly involving the TRAIL pathway. (HEPATOLOGY 2012;56:841–849)

Natural killer cells in viral hepatitis: facts and controversies

Eur J Clin Invest 2010; 40 (9): 851–863

Natural killer cell dynamic profile is associated with treatment outcome in patients with chronic HCV infection

BACKGROUND & AIMS A substantial proportion of patients with chronic hepatitis C virus infection treated with pegylated interferon α/ribavirin fail to achieve sustained virological response (SVR). Since growing evidence suggests that innate immunity may influence treatment responses, we examined natural killer (NK) cell phenotypic and functional changes during standard antiviral therapy. METHODS Expression of several NK-cell regulatory molecules was evaluated by flow cytometry in 37 consecutive patients with chronic HCV infection at baseline and at different time points during and after discontinuation of treatment. Cytokine production was evaluated by intracellular staining. Cytolytic potential was assessed as degranulation and as antibody-dependent cytotoxicity. RESULTS Baseline frequencies of CD56(dim) NK cells and perforin content were significantly higher, whereas CD16 expression was lower in SVR vs. non-responder subjects. Analysis by linear regression for repeated measures during the first 12 weeks showed significantly increased frequencies of activated (CD69(+)) NK cells in rapid virological responders (RVR) and identified a typical NK cell profile associated with SVR, featuring higher NK perforin content, lower CD16 expression, and higher proportion of CD56(dim)/CD16(-) cells. Moreover, SVR patients displayed higher natural and antibody-dependent NK cell cytotoxicity. IL28B rs12979860 CC homozygosis was significantly associated with SVR, independently of NK-cell phenotype and function. CONCLUSIONS Different NK-cell phenotypic and functional features, in patients with chronic hepatitis C treated with standard therapy, were observed between non-responder vs. SVR patients, suggesting a potential role of NK cells in the response to treatment.

Mitomycin C as an alternative to irradiation to inhibit the feeder layer growth in long-term culture assays

BACKGROUND Mitomycin C (MMC), an antitumoral antibiotic, has been described inhibiting the proliferation of different cell types in vitro. Since irradiation is commonly used to stop the cell growth of adherent cells in several experimental models, we aimed to define the optimal dose and incubation time of MMC capable of inhibiting the growth of murine fibroblasts, used as an adherent feeder layer in long-term hematopoietic culture assay. METHODS M2 10B4 (both parental and engineered to produce human IL-3 and G-CSF) and Sl/Sl (engineered to produce human IL-3 and steel factor) murine fibroblast cell-lines, frequently used in LTC-IC assay, were incubated with increasing doses of MMC for either a short (3 h) or a long (16 h) period. The efficiency of MMC in stopping the cell growth was evaluated for 5 days following MMC removal. The effects of MMC treatment on human hematopoietic cells were studied using both LTC-IC and limiting dilution (CAFC) assays. RESULTS The growth of M2 10B4 cells was stopped at 3 and 16 h in the presence of 20 microg/mL and 2 microg/mL of MMC, respectively while Sl/Sl fibroblasts required a lower dose of drug (2 and 0.2 microg/mL, respectively). No significant difference was found between the number of LTC-IC or CAFC obtained from cultures containing irradiated or MMC-treated feeder cells. DISCUSSION MMC inhibits the growth of murine fibroblasts used as adherent feeder cells in long-term culture assays, without interfering with the subsequent growth of co-cultured hemopoietic cells. Different cell types might present a different sensitivity to MMC and therefore a dose-response curve to MMC has to be obtained for each cell type of interest.

T-cell dynamics after high-dose chemotherapy in adults: elucidation of the elusive CD8+ subset reveals multiple homeostatic T-cell compartments with distinct implications for immune competence.

Recovery of total T cell numbers after in vivo T‐cell depletion in humans is accompanied by complex perturbation within the CD8+ subset. We aimed to elucidate the reconstitution of CD8+ T cells by separate analysis of putative naïve CD95− CD28+, memory CD95+ CD28+ and CD28− T cell compartments after acute maximal depletion by high‐dose chemotherapy (HD‐ChT) in women with high‐risk breast cancer. We found that recovery of putative naïve CD8+ CD95− CD28+ and CD4+ CD95− CD28+ T cells, was compatible with a thymus‐dependent regenerative pathway since their recovery was slow and time‐dependent, their values were tightly related to each other, and their reconstitution patterns were inversely related to age. By analysing non‐naïve T cells, a striking diversion between putative memory T cells and CD28− T cells was found. These latter increased early well beyond normal values, thus playing a pivotal role in total T‐cell homeostasis, and contributed to reduce the CD4 : CD8 ratio. In contrast, putative memory T cells returned to values not significantly different from those seen in patients at diagnosis, indicating that this compartment may recover after HD‐ChT. At 3–5 years after treatment, naïve T cells persisted at low levels, with expansion of CD28− T cells, suggesting that such alterations may extend further. These findings indicate that CD28− T cells were responsible for ‘blind’ T‐cell homeostasis, but support the notion that memory and naïve T cells are regulated separately. Given their distinct dynamics, quantitative evaluation of T‐cell pools in patients undergoing chemotherapy should take into account separate analysis of naïve, memory and CD28− T cells.

Impaired thrombin-induced platelet activation and thrombus formation in mice lacking the Ca2+-dependent tyrosine kinase Pyk2

In the present study, we used a knockout murine model to analyze the contribution of the Ca(2+)-dependent focal adhesion kinase Pyk2 in platelet activation and thrombus formation in vivo. We found that Pyk2-knockout mice had a tail bleeding time that was slightly increased compared with their wild-type littermates. Moreover, in an in vivo model of femoral artery thrombosis, the time to arterial occlusion was significantly prolonged in mice lacking Pyk2. Pyk2-deficient mice were also significantly protected from collagen plus epinephrine-induced pulmonary thromboembolism. Ex vivo aggregation of Pyk2-deficient platelets was normal on stimulation of glycoprotein VI, but was significantly reduced in response to PAR4-activating peptide, low doses of thrombin, or U46619. Defective platelet aggregation was accompanied by impaired inside-out activation of integrin α(IIb)β(3) and fibrinogen binding. Granule secretion was only slightly reduced in the absence of Pyk2, whereas a marked inhibition of thrombin-induced thromboxane A(2) production was observed, which was found to be responsible for the defective aggregation. Moreover, we have demonstrated that Pyk2 is implicated in the signaling pathway for cPLA(2) phosphorylation through p38 MAPK. The results of the present study show the importance of the focal adhesion kinase Pyk2 downstream of G-protein-coupled receptors in supporting platelet aggregation and thrombus formation.

Natural killer cell functional dichotomy: a feature of chronic viral hepatitis?

Natural killer (NK) cells are involved in innate immune responses to viral infections either via direct cytotoxicity which destroys virus-infected cells or production of immunoregulatory cytokines which modulate adaptive immunity and directly inhibit virus replication. These functions are mediated by different NK subpopulations, with cytotoxicity being generally performed by CD56dim NK cells, whereas CD56bright NK cells are mainly involved in cytokine secretion. NK functional defects are usually combined so that impaired degranulation is often associated with deficient cytokine production. Innate immunity is thought to be relevant in the control of hepatitis virus infections such as hepatitis B virus (HBV) and hepatitis C virus (HCV), and recent findings reproducibly indicate that NK cells in chronic viral hepatitis are characterized by a functional dichotomy, featuring a conserved or enhanced cytotoxicity and a reduced production of interferon (IFN)-γ and tumor necrosis factor-α. In chronic HCV infection this appears to be caused by altered IFN-α signaling resulting from increased signal transducer and activator of transcription 1 (STAT1) phosphorylation, which polarizes NK cells toward cytotoxicity, and a concomitantly reduced IFN-α induced STAT4 phosphorylation yielding reduced IFN-γ mRNA levels. These previously unappreciated findings are compatible on the one hand with the inability to clear HCV and HBV from the liver and on the other they may contribute to understand why these patients are often resistant to IFN-α-based therapies.