Characterization of clinicopathological features, treatment practices, and outcomes among Finnish advanced breast cancer patients in real-life clinical practice.

PURPOSE: In recent years, several new targeted therapies have emerged for advanced breast cancer (aBC). However, real-life data specific to aBC and different breast cancer subtypes are scarce. This retrospective cohort study was designed to describe the distribution of aBC subtypes, incidence, treatment patterns, survival, and PIK3CA hotspot mutation frequency. METHODS: The study included all patients in the Hospital District of Southwest Finland diagnosed with aBC between 2004 and 2013 and with a sample available in Auria Biobank. In addition to registry-based data collection, 161 HR+/HER2- aBCs were screened for PIK3CA mutations. RESULTS: Altogether, 54.7% of the 444 patients included in the study had luminal B subtype. The smallest representations were in HR-/HER2+ (4.5%) and triple-negative (5.6%) subgroups. The percentage of aBC among all diagnosed breast cancers increased until 2010, after which it remained stable. The triple-negative cancers were associated with shorter median overall survival (5.5 months) compared to other subgroups (16.5-24.6 months). Most (84%) triple-negative cancers also metastasized during the first two years, whereas this was more evenly distributed over time in other subgroups. Of the HR+/HER2- tumors, 32.3% harbored a PIK3CA hotspot mutation. These patients, however, did not have inferior survival compared to patients with PIK3CA wild-type cancers. CONCLUSION: This study described real-world aBC subgroups and indicated that the clinical outcomes of subgroups vary. Although PIK3CA hotspot mutations did not lead to inferior survival, they are relevant as possible treatment targets. Overall, these data could be utilized to further evaluate the subgroup-specific medical needs in breast cancer.

Overall survival of individuals with metastatic cancer in Sweden: a nationwide study.

AIMS: Consistent improvements for overall survival (OS) have been reported for individuals with metastatic cancer. Swedish population-based registers allow national coverage and long follow-up time. The aim of this study was to estimate and explore long-term OS of individuals diagnosed with metastatic cancer using Swedish nationwide health registers. METHODS: Individuals with metastatic breast (MBC), non-small cell lung (MNSCLC), ovary (MOC) or colorectal cancer (MCRC) or metastatic malignant melanoma (MMM) were identified in the Swedish national cancer register and national patient registers. Survival was estimated and stratified by available variables. Potential cure fractions were estimated using mixture cure models. RESULTS: In total, approximately 69,000 individuals were identified. The most common cancers were MCRC (36.2%) and MNSCLC (29.5%). Men were more frequently diagnosed with MNSCLC, MCRC, and MMM compared to women. Except for MOC, about 50% of individuals were 70 years or older at diagnosis. Throughout the study period survival differed across cancers. The longest median OS was observed for individuals with MOC and MBC. At 10 years of follow-up, the survival curves flatten at a survival rate of approximately 10% for all cancers except MNSCLC. The youngest age groups had the longest median OS. Increased survival was also observed for individuals diagnosed in 2015 and 2018 compared to individuals diagnosed during earlier years. The estimated cure fractions were 4% for MBC, 1.5% for MNSCLC, 6.8% for MCRC, 8.6% for MOC and MMM. CONCLUSIONS: Long-term survival has been assessed across all indications except for NSCLC.. The findings may be relevant for healthcare planning to meet the needs of future patients and potential long-term survivors.

Overall survival of patients with metastatic breast cancer in Sweden: a nationwide study.

BACKGROUND: Breast cancer is the most common cancer among women in Sweden. Whereas survival for the overall breast cancer population is well-documented, survival of patients with metastatic breast cancer (MBC) is harder to quantify due to the lack of reliable data on disease recurrence in national cancer registers. METHODS: This study used machine learning to classify the total MBC population in Sweden diagnosed between 2009 and 2016 using national registers, with the aim to estimate overall survival (OS). RESULTS: The total population consisted of 13,832 patients-2528 (18.3%) had de novo MBC whereas 11,304 (81.7%) were classed as having a recurrent MBC. Median OS for patients with MBC was found to be 29.8 months 95% confidence interval (CI) [28.9, 30.6]. Hormone-receptor (HR)-positive MBC had a median OS of 37.0 months 95% CI [35.9, 38.3] compared to 9.9 months 95% CI [9.1, 11.0] for patients with HR-negative MBC. CONCLUSION: This study covered the entire MBC population in Sweden during the study time and may serve as a baseline for assessing the effect of new treatment strategies in MBC introduced after the study period.

Use of classifiers to optimise the identification and characterisation of metastatic breast cancer in a nationwide administrative registry.

BAKGROUND: The prognosis for patients with metastatic breast cancer (MBC) is substantially worse when compared with patients with earlier stage disease. Therefore, understanding the differences in epidemiology between these two patient groups is important. Studies using population-based cancer registries to identify MBC are hampered by the quality of reporting. Patients are registered once (at time of initial diagnosis); hence only data for patients with de novo MBC are identifiable, whereas data for patients with recurrent MBC are not. This makes accurate estimation of the epidemiology and healthcare utilisation of MBC challenging. This study aimed to investigate whether machine-learning could improve identification of MBC in national health registries. MATERIAL AND METHODS: Data for patients with confirmed MBC from a regional breast cancer registry were used to train machine-learning algorithms (or 'classifiers'). The best performing classifier (accuracy 97.3%, positive predictive value 85.1%) was applied to Swedish national registries for 2008 to 2016. RESULTS: Mean yearly MBC incidence was estimated at 14 per 100,000 person-years (with 18% diagnosed de novo and 76% of the total with HR-positive MBC). CONCLUSION: To our knowledge, this is the first study to use machine learning to identify MBC regardless of stage at diagnosis in health registries covering the entire population of Sweden.

Socioeconomic cost of AML in Sweden-A population-based study using multiple nation-wide registers.

Acute myeloid leukemia (AML) is associated with a high economic and clinical burden. Recently novel therapies have been added to standard treatment regimens. Here, we evaluated the economic impact of AML up until the introduction of these novel therapies. Individual data on 2954 adult patients diagnosed from 2007 to 2015 from five Swedish national population-based registers were used, enabling analyses from diagnosis to either death or 5-year follow-up for survival, inpatient and outpatient costs, costs of prescribed drugs, sick leave, and early retirement. Costs per patient were stratified by age group, treatment options, and FLT3-ITD status. The expected 5-year costs per patient differed substantially between age groups. Patients aged 18-59 years had an expected mean cost per patient of €170,748, while age groups 60-69 years, 70-79 years, and >80 years incurred an expected mean cost of €92,252, €48,344, and €24,118, respectively, over 5 years. Patients <60 years undergoing stem cell transplantation had the highest costs (€228,525 over 5 years). About 60% of costs for these patients were from hospitalizations and 20% from sick leave and early retirement; cost per day was highest from the first admission to complete remission. This study provides a baseline for socioeconomic evaluations of novel therapies in AML in Sweden.

A systematic literature review of time to return to work and narcotic use after lumbar spinal fusion using minimal invasive and open surgery techniques.

BACKGROUND: Chronic low back pain is a common health problem for adult workers and causes an enormous economic burden. With the improvement of minimally invasive surgical techniques (MIS) in spinal fusion and the development of fusion devices, more lumbar operations are today being performed through a less invasive technique. When compared with open surgeries (OS), MIS has demonstrated better clinical outcomes including operation time, blood loss, complication rates and length of hospital stay. The aim of this review was to identify and summarize evidence on the time to return to work and the duration of post-operation narcotic use for patients who had lumbar spinal fusion operations using MIS and OS techniques. METHODS: A systematic literature review was performed including studies identified from PubMed, EMBASE, the Cochrane Collaboration, and the Centre for Review and Dissemination (CRD) (January 2004­April 2014) for publications reporting on time to return to work and post-operation narcotic use after MIS or OS lumbar spinal fusion surgeries. RESULTS: Out of a total of 36 included studies, 28 reported on the time to return to work and 17 on the narcotic use after MIS or OS. Four studies described the time to return to work directly comparing MIS and OS. Three studies, from the US, directly compared the duration of narcotic use between MIS- transforaminal lumbar interbody fusion (TLIF) and OS-TLIF. In addition to the time to return to work, 23 studies reported on the rate of return to work and the employment rate before and after surgery, and two Swedish studies presented sick leave data. CONCLUSION: There is a gap of good quality data describing the time to return to work and narcotic use after lumbar spinal fusion operations using MIS or OS techniques. However, the current systematic literature review indicates that patients who have lumbar spinal fusion operations, with the MIS procedure, generally return to work after surgery more quickly and require less post-operation narcotics for pain control compared to patients who have OS.

A Cost-Effectiveness Analysis of Minimally Invasive versus Open Surgery Techniques for Lumbar Spinal Fusion in Italy and the United Kingdom.

OBJECTIVE: Evaluate the cost-effectiveness of minimally invasive surgery (MIS) compared with open surgery (OS) techniques for one- or two-level lumbar spinal fusion in the treatment of degenerative lumbar spinal conditions in the United Kingdom and Italy. METHODS: A health economic model was developed on the basis of results from a systematic literature review and meta-analysis to determine the cost-effectiveness of MIS compared with OS for lumbar spinal fusion. The analysis was conducted from a health care payer perspective. Parameters included in the model were surgery, blood loss, duration of hospitalization, postoperative complications, and health-related quality of life (HRQOL). Cost-effectiveness was determined by the incremental cost per quality-adjusted life-year gained. RESULTS: MIS was the dominant strategy compared with OS (i.e., yielding both cost savings and improved HRQOL). Cost savings were driven mainly by shorter length of hospital stay, reduced blood loss, and fewer complications such as surgical site infection. The total cost saving per procedure was €973 for Italy and €1666 for the United Kingdom, with an improvement of 0.04 quality-adjusted life-year over 2 years in HRQOL. One-way sensitivity analyses and predefined scenario(s) analyses confirmed the robustness of the model. CONCLUSIONS: MIS is a less expensive and a more effective treatment compared with OS for spinal lumbar fusion in both Italy and the United Kingdom. Lower downstream costs and increased HRQOL in the MIS group compensate for potential higher upfront costs of MIS implants and surgery equipment.

AmotL2 links VE-cadherin to contractile actin fibres necessary for aortic lumen expansion.

The assembly of individual endothelial cells into multicellular tubes is a complex morphogenetic event in vascular development. Extracellular matrix cues and cell-cell junctional communication are fundamental to tube formation. Together they determine the shape of endothelial cells and the tubular structures that they ultimately form. Little is known regarding how mechanical signals are transmitted between cells to control cell shape changes during morphogenesis. Here we provide evidence that the scaffold protein amotL2 is needed for aortic vessel lumen expansion. Using gene inactivation strategies in zebrafish, mouse and endothelial cell culture systems, we show that amotL2 associates to the VE-cadherin adhesion complex where it couples adherens junctions to contractile actin fibres. Inactivation of amotL2 dissociates VE-cadherin from cytoskeletal tensile forces that affect endothelial cell shape. We propose that the VE-cadherin/amotL2 complex is responsible for transmitting mechanical force between endothelial cells for the coordination of cellular morphogenesis consistent with aortic lumen expansion and function.

VE-PTP regulates VEGFR2 activity in stalk cells to establish endothelial cell polarity and lumen formation.

Vascular endothelial growth factor (VEGF) guides the path of new vessel sprouts by inducing VEGF receptor-2 activity in the sprout tip. In the stalk cells of the sprout, VEGF receptor-2 activity is downregulated. Here, we show that VEGF receptor-2 in stalk cells is dephosphorylated by the endothelium-specific vascular endothelial-phosphotyrosine phosphatase (VE-PTP). VE-PTP acts on VEGF receptor-2 located in endothelial junctions indirectly, via the Angiopoietin-1 receptor Tie2. VE-PTP inactivation in mouse embryoid bodies leads to excess VEGF receptor-2 activity in stalk cells, increased tyrosine phosphorylation of VE-cadherin and loss of cell polarity and lumen formation. Vessels in ve-ptp(-/-) teratomas also show increased VEGF receptor-2 activity and loss of endothelial polarization. Moreover, the zebrafish VE-PTP orthologue ptp-rb is essential for polarization and lumen formation in intersomitic vessels. We conclude that the role of Tie2 in maintenance of vascular quiescence involves VE-PTP-dependent dephosphorylation of VEGF receptor-2, and that VEGF receptor-2 activity regulates VE-cadherin tyrosine phosphorylation, endothelial cell polarity and lumen formation.

Vaccination with dendritic cells loaded with tumor apoptotic bodies (Apo-DC) in patients with chronic lymphocytic leukemia: effects of various adjuvants and definition of immune response criteria.

We previously demonstrated that autologous dendritic cells that have endocytosed apoptotic bodies of chronic lymphocytic leukemia (CLL) cells (Apo-DC) can stimulate antileukemic T cell responses in vitro. In this phase I study, we vaccinated 15 asymptomatic CLL patients at five time points with Apo-DC administered intradermally either alone (cohort I), or in combination with subcutaneous granulocyte-macrophage-colony-stimulating-factor (GM-CSF) (cohort II) or with GM-CSF and intravenous low-dose cyclophosphamide (cohort III). Aim of the study was to evaluate the safety and immunogenicity of Apo-DC alone or in combination with GM-CSF and low-dose cyclophosphamide in CLL patients. All patients completed the vaccination schedule without dose-limiting toxicity. No objective clinical responses were seen. Vaccine-induced leukemia-specific immune responses were evaluated by IFN-γ ELISpot and proliferation assays over a 52 weeks observation period and immune response criteria were defined. According to these criteria, 10/15 patients were defined as immune responders. The frequency of immune-responding patients was higher in cohorts II (3/5) and III (5/5) than in cohort I (2/5). In order to further characterize the induced immune response, estimation of secreted cytokines and CD107-degranulation assay were performed. Clustering of T and CLL cells was observed in CD107-degranulation assay and visualized by confocal microscopy. Additionally, assessment of regulatory T cells (T(regs)) revealed their significantly lower frequencies in immune responders versus non-responders (P < 0.0001). Cyclophosphamide did not reduce T(regs) frequency. In conclusion, vaccination with Apo-DC + GM-CSF and cyclophosphamide was safe and elicited anti-CLL immune responses that correlated inversely with T(regs) levels. Lack of clinical responses highlights the necessity to develop more potent vaccine strategies in B cell malignancies.

Antibody-dependent natural killer cell-mediated cytotoxicity engendered by a kinase-inactive human HER2 adenovirus-based vaccination mediates resistance to breast tumors.

Cancer vaccines may have applications in the therapy and prevention of mammary carcinoma. To investigate such applications, we constructed a recombinant adenoviral vaccine expressing a kinase-inactive mutant form of human HER2 and introduced this into BALB/c wild-type (WT) or HER2 transgenic mice. Here, we report contributions by antibody responses and natural killer (NK) cells in tumor protection in this model. One i.p. vaccination protected WT mice from the HER2-expressing mouse carcinoma D2F2/E2. Half of the HER2 transgenic mice were protected fully and long term after preventive vaccination. Tumor growth in mice that eventually developed neoplastic lesions was delayed. Protection in WT and HER2 transgenic mice was associated with high or low levels of IgG2a antibodies, respectively, whereas CTLs were observed in WT but not in HER2 transgenic mice. Depleting CD4(+) or CD8(+) cells in vaccinated WT mice had limited effects, suggesting that protection was largely independent of CD4(+) or CD8(+) T cells. In contrast, antibody-mediated tumor rejection seemed to contribute significantly based on a loss of protection in mice deficient for Fc-γ RI/III or B cells. Further, a role for antibody-dependent cellular cytotoxicity (ADCC) mediated by NK cells was indicated by evidence that vaccine protection could be abolished by in vivo depletion of NK cells. Lastly, NK cells and immune sera purified from WT or HER2 transgenic mice exhibited efficient ADCC of HER2-expressing tumor cells in vitro. Our findings define a critical requirement for NK cells in vaccine-induced protection against HER2-expressing tumors.

Angiomotin-like protein 1 controls endothelial polarity and junction stability during sprouting angiogenesis.

RATIONALE: We have previously shown that angiomotin (Amot) is essential for endothelial cell migration during mouse embryogenesis. However, approximately 5% of Amot knockout mice survived without any detectable vascular defects. Angiomotin-like protein 1 (AmotL1) potentially compensates for the absence of Amot as it is 62% homologous to Amot and exhibits similar expression pattern in endothelial cells. OBJECTIVE: Here, we report the identification of a novel isoform of AmotL1 that controls endothelial cell polarization and directional migration. METHODS AND RESULTS: Small interfering RNA-mediated silencing of AmotL1 in mouse aortic endothelial cells caused a significant reduction in migration. In confluent mouse pancreatic islet endothelial cells (MS-1), AmotL1 colocalized with Amot to tight junctions. Small interfering RNA knockdown of both Amot and AmotL1 in MS-1 cells exhibited an additive effect on increasing paracellular permeability compared to that of knocking down either Amot or AmotL1, indicating both proteins were required for proper tight junction activity. Moreover, as visualized using high-resolution 2-photon microscopy, the morpholino-mediated knockdown of amotl1 during zebrafish embryogenesis resulted in vascular migratory defect of intersegmental vessels with strikingly decreased junction stability between the stalk cells and the aorta. However, the phenotype was quite distinct from that of amot knockdown which affected polarization of the tip cells of intersegmental vessels. Double knockdown resulted in an additive phenotype of depolarized tip cells with no or decreased connection of the stalk cells to the dorsal aorta. CONCLUSIONS: These results cumulatively validate that Amot and AmotL1 have similar effects on endothelial migration and tight junction formation in vitro. However, in vivo Amot appears to control the polarity of vascular tip cells whereas AmotL1 mainly affects the stability of cell-cell junctions of the stalk cells.

HER-2/neu mediated down-regulation of MHC class I antigen processing prevents CTL-mediated tumor recognition upon DNA vaccination in HLA-A2 transgenic mice.

To study DNA vaccination directed against human HER-2 in the HHD mouse Tg strain, we created a novel HER-2-expressing syngeneic tumor transplantation model. We found that a DNA vaccine encoding the full length HER-2 DNA protected HHD mice from HER-2(+) tumor challenge by a CTL independent mechanism. A more efficient approach to induce HLA-A2 restricted CTLs, through immunization with a multi-epitope DNA vaccine expressing the HLA-A2 restricted HER-2 369-377, 435-443 and 689-697 epitopes, resulted in high numbers of peptide specific T cells but failed to induce tumor protection. Subsequently we discovered that HER-2 transfected tumor cells down-regulated MHC class I antigen expression and exhibited a series of defects in the antigen processing pathway which impaired the capacity to produce and display MHC class I peptide-ligands to specific CTLs. Our data demonstrate that HER-2 transfection is associated with defects in the MHC class I presentation pathway, which may be the underlying mechanism behind the inability of CTLs to recognize tumors in this HLA-A2 transgenic model. As defective MHC class I presentation may be a common characteristic of HER-2 expressing tumors, vaccines targeting HER-2 should aim at inducing an integrated immune response where also CD4(+) T cells and antibodies are important components.

Retinoic acid elicits cytostatic, cytotoxic and immunomodulatory effects on uveal melanoma cells.

The current therapy of uveal melanoma (UM) metastases remains inefficient, which warrants the development of new treatment modalities. For the first time we investigated the effects of retinoic acid (RA) on a panel of UM cell lines and found that RA induces morphological changes compatible with differentiation, suppresses proliferation and causes apoptosis in these cells. RA treatment resulted in an increase of p21, p27 and p53 protein levels and G1 arrest in UM cells, which correlated with significant down-modulation of surface Her2/neu proto-oncogene expression. In addition, RA-treated UM cells exhibited increased sensitivity to both MHC class I-restricted killing by cytotoxic T lymphocytes and NK cell-mediated lysis that were accompanied by more efficient conjugate formation between UM cells and killer lymphocytes. Taken together, our results implicate UM as a new target for treatment with retinoids and suggest that retinoids and T- or NK-cell based immunotherapy can have mutually enhancing effects in UM patients.

Improved immunogenicity of an immunodominant epitope of the HER-2/neu protooncogene by alterations of MHC contact residues.

The HER-2/neu (HER-2) oncogene is expressed in normal epithelial surfaces at low levels and overexpressed in several types of tumors. The low immunogenicity against this self tumor Ag can be improved by developing epitopes with amino acid replacements in their sequences. In this study, three HER-2/neu.369 (HER-2.369) analogue peptides, produced by modifying both anchor positions by introducing L, V, or T at position 2 and V at the C terminus, were analyzed for their capacity to induce CTLs in vitro from human PBMC and in vivo in HLA-A2.1/Kb transgenic mice. One of the analogues (HER-2.369 V2V9) sensitized target cells for HER-2-specific recognition by human CTLs and induced specific CTLs in vitro at 100-fold lower concentrations than the HER-2.369 wild-type epitope. These CTLs were also able to recognize the wild-type epitope and HER-2-expressing tumors in an MHC-restricted manner. Furthermore, a 100-fold lower amount of the HER-2.369 V2V9 analogue compared with the wild-type epitope was required to induce CTLs in HLA-A2.1/Kb transgenic mice. However, the V2V9 analogue demonstrated only marginally better binding to the MHC class I A2 allele compared with wild type. To establish thermodynamic parameters, we developed radiolabeled F3*Y analogues from both the HER-2.369 epitope and the V2V9 analogue. Our results indicate that the high biological activity of the HER-2.369 V2V9 epitope is associated with a slower dissociation kinetic profile, resulting in an epitope with greater HLA-A2 stability.

Retinoids act as multistep modulators of the major histocompatibility class I presentation pathway and sensitize neuroblastomas to cytotoxic lymphocytes.

The current therapeutic modalities achieve low response rates in human neuroblastoma, a frequent extracranial malignancy of the early childhood. We have assessed the effect of retinoids, used presently for the treatment of neuroblastoma, on the discrete steps of the MHC class I processing machinery and susceptibility of neuroblastoma cells to CTL-mediated killing. We demonstrate that retinoic acid derivatives induce the expression of proteolytic and regulatory subunits of the immunoproteasome, increase the half-life of MHC class I complexes, and enhance the sensitivity of neuroblastoma cells to both MHC class I-restricted peptide-specific and HLA nonrestricted lysis by CTLs. Importantly, effects of retinoids on the MHC class I pathway appear to be independent of IFN-gamma and/or TNF-alpha as intermediate messengers. To our knowledge, this is the first demonstration of inflammation-unrelated biological molecules that induce systemic modulation of antigen presentation in nonprofessional antigen presenting cells. Our findings suggest that the application of retinoids and T cell-based immunotherapy may be an effective combination for the treatment of neuroblastoma.

Proteasome inhibitors reconstitute the presentation of cytotoxic T-cell epitopes in Epstein-Barr virus-associated tumors.

EBV-infected cells and EBV-associated tumors may evade CTL recognition by defective antigen processing, resulting in poor presentation of CTL epitopes. Since the proteasome is the major source of MHC class I-presented peptides, we analyzed the effect of proteasome inhibitors on the expression of surface HLA class I and the generation of EBV-derived CTL epitopes presented by the HLA-A2 and HLA-A11 alleles. Treatment with covalent and reversible inhibitors of the proteasome partially reduced the total and allele-specific expression of surface HLA class I in EBV-carrying LCLs. HLA-A2 expression was also decreased by treatment with leupeptin and bestatin, while HLA-A11 expression was affected by treatment with phenanthroline. Despite their general inhibitory effect on HLA class I expression, all proteasome inhibitors tested enhanced the presentation of 2 subdominant HLA-A2 epitopes from EBV LMP1 and LMP2, while the presentation of the immunodominant HLA-A11-restricted epitope from EBNA4 was inhibited by MG132 and lactacystin and increased by ZL(3)VS. Treatment with ZL(3)VS restored the presentation of endogenously expressed EBNA4 in 1 HLA-A11-positive BL cell line. These findings suggest that specific inhibitors of the proteasome may be used to increase the antigenicity of virus-infected and malignant cells that are per se inefficient at generating particular CTL target epitopes.

Effect of interferon-alpha therapy on epitope-specific cytotoxic T lymphocyte responses in hepatitis C virus-infected individuals.

The majority of hepatitis C virus (HCV)-infected individuals fail to resolve the infection and become chronically infected despite the presence of HCV-specific CTL responses directed to different HCV-derived peptide antigens. Only a minority of individuals is able to clear the virus by mounting efficient CTL responses early after acute infection, but at present it is not clear whether viral clearance is associated with CTL responses of defined specificity. To elucidate those responses associated with improvement of the disease, we analyzed CTL responses to 16 different HLA-A2-presented, HCV-derived epitopes in 12 chronically infected patients, 14 chronically infected patients treated with interferon-alpha, and in one patient with acute symptomatic disease. We show here that the majority of chronically infected individuals present CTL responses directed to an NS4-derived peptide antigen (amino acids 1789-1797). Treated patients presented stronger HCV-specific CTL responses and therapy-induced changes in CTL target choice. In particular, 13 out of 14 individuals responded to an NS3-derived epitope (amino acids 1073-1081). By longitudinal analysis we show that five individuals responding to IFN-alpha therapy with decreases in alanine aminotransferase levels presented a strong CTL activity directed to the NS3-derived epitope. One patient that spontaneously resolved the infection presented a generally strong CTL activity specific for HCV-derived epitopes with a dominant response to the NS3-derived peptide antigen. This suggests that CTL responses directed to this NS3-derived antigen may be beneficial for the control of HCV infection. Improvement of these responses may represent a therapeutic intervention in chronic HCV infection.