Cjh van de Velde

Severe hepatic dysfunction after adenovirus-mediated transfer of the herpes simplex virus thymidine kinase gene and ganciclovir administration

The use of so-called ‘suicide’ genes to activate prodrugs has been effective in animal models for several solid tumor types and is now in phase I and II clinical trials. We have exploited adenovirus vectors (Ad) for transfer and expression of the herpes simplex virus thymidine kinase (HSVtk) gene to render rat colorectal liver metastases sensitive to the anti-herpetic agent ganciclovir (GCV). The efficacy and toxicity of this enzyme–prodrug combination were tested after in situ transduction of rat colorectal tumor cells and after intraportal administration of the vector Ad.CMV.TK. Our results demonstrate the validity of the approach but reveal that hepatic expression of HSVtk, both in tumor bearing and in tumor-free rats, provokes severe liver dysfunction and mortality upon GCV administration. These data show, that in contrast to the common assumption, normally non-mitotic tissues too, can be affected by adenovirus-mediated HSVtk transfer and subsequent GCV treatment. Given the hepatotropic nature of systemically administered adenovirus type 2- and 5-derived vectors, it will be essential to monitor liver functions of patients included in all gene therapy trials involving adenoviral vectors with the HSVtk gene.

Gastric cancers in young and elderly patients show different genomic profiles.

Although most gastric cancers occur in elderly patients, a substantial number of cases of this common disease occur in young patients. Gastric cancer is a heterogeneous disease at the genomic level and different patterns of DNA copy number alterations are associated with different clinical behaviour. The aim of the present study was to explore differences in DNA copy number alterations in relation to age of onset of gastric cancer. DNA isolated from 46 paraffin‐embedded gastric cancer tissue samples from 17 patients less than 50 years of age [median 43 (21–49) years] and 29 patients greater than or equal to 70 years of age [median 75 (70–83) years] was analysed by genome‐wide microarray comparative genomic hybridization (array CGH) using an array of 5000 BAC clones. Patterns of DNA copy number aberrations were analysed by hierarchical cluster analysis of the mode‐normalized and smoothed log2 ratios of tumour to normal reference fluorescence signal intensities using TMEV software, after which cluster membership was correlated with age group. In addition, supervised analysis was performed using CGH Multi‐array. Hierarchical cluster analysis of the array CGH data revealed three clusters with different genomic profiles that correlated significantly with age (p = 0.006). Cluster 1 mainly contained young patients, while elderly patients were divided over clusters 2 and 3. Chromosome regions 11q23.3 and 19p13.3 contributed most to age‐related differences in tumour profiles. Gastric cancers of young and old patients belong to groups with different genomic profiles, which likely reflect different pathogenic mechanisms of the disease. Copyright

Liver and tumour tissue concentrations of TNF-alpha in cancer patients treated with TNF-alpha and melphalan by isolated liver perfusion

In this study we determined the level of tumour necrosis factor alpha (TNF-alpha) in liver and tumour tissue samples obtained from patients with colorectal metastases confined to the liver, who were treated with isolated liver perfusion with TNF-alpha and melphalan. We adapted a standard enzyme-linked immunosorbent assay kit for the quantification of TNF-alpha in serum to measure the amount of this cytokine in solid tissue. For this purpose, we developed a buffer that lysed the tissues without affecting the TNF-alpha present. The minimum detection level was about 2 pg of TNF-alpha per mg tissue. Using this technique, we found a significant increase in the TNF-alpha level after perfusion in the liver tissue of all evaluable patients, which may explain the transient liver toxicity we observed in all patients. In tumour tissue, a significant TNF-alpha increase was observed in one out of five patients. The level of TNF-alpha in all liver tissue samples and some of the tumours after treatment by isolated liver perfusion was much higher than the peak serum concentrations obtained after systemic administration of the maximum tolerated dose of TNF-alpha. Furthermore, we demonstrated that the level of TNF-alpha in the liver tissue samples was about seven to eight times higher than in tumour tissue. We concluded that regional liver treatment resulted in a relatively high local level of TNF-alpha, but also that this cytokine did not preferentially accumulate in tumour tissue.

Recent trends and future perspectives in isolated hepatic perfusion in the treatment of liver tumors

Isolated hepatic perfusion (IHP) involves a method of complete vascular isolation of the liver to enable treatment of liver tumors with high drug doses without systemic toxicity. Recent clinical studies have mainly employed IHP with melphalan with or without tumor necrosis factor-α and mild hyperthermia. The results of these studies demonstrate that high response and survival rates can be achieved with IHP. The current status, recent developments and future perspectives of IHP are discussed in this review.

Abstract S1-04: Optimal duration of extended letrozole treatment after 5 years of adjuvant endocrine therapy; results of the randomized phase III IDEAL trial (BOOG 2006-05)

Despite the success of adjuvant endocrine therapy in early breast cancer, approximately 50% of all recurrences still occur after the initial 5 years of follow-up. Earlier studies confirmed that endocrine therapy extension after 5 years of adjuvant tamoxifen with either tamoxifen or aromatase inhibitors up to 10 years leads to an improvement in survival. However, aromatase inhibitors are currently standard of care in the initial 5 years of adjuvant therapy, and the benefit of extended use beyond 5 years of AI based therapy is still debated. The randomized phase III IDEAL trial was designed to study the optimal duration of extended adjuvant endocrine therapy after the initial 5 years of any endocrine therapy. Between April 2007 and November 2011, 1824 postmenopausal, HR-positive early breast cancer patients were included by 74 hospitals in the Netherlands. Enrolled patients earlier received 5 years of any endocrine therapy (87.9% AI based),completed this treatment no longer than 2 years before randomization, and did not have any recurrence at the moment of inclusion. The included patients were randomly allocated to either 2.5 or 5 years of extended letrozole (daily 2.5mg). Primary outcome was disease free survival (DFS), secondary endpoints were overall survival (OS), distant disease free survival (DDFS), contralateral breast cancer and safety. Results & Discussion: Results will become available soon. Citation Format: Blok EJ, van de Velde CJH, Meershoek-Klein Kranenbarg EM, Putter H, van den Bosch J, Maartense E, van Leeuwen-Stok AE, Liefers G-J, Nortier JWR, Rutgers EJT, Kroep JR. Optimal duration of extended letrozole treatment after 5 years of adjuvant endocrine therapy; results of the randomized phase III IDEAL trial (BOOG 2006-05) [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr S1-04.

Abstract S2-4: Final Results of a Prospectively Planned Biomarker Analysis: HER1-3 as Predictive Markers of Benefit from Early Treatment with Aromatase Inhibitors Versus Tamoxifen in the TEAM Pathology Sub-Study.

Background: The Tamoxifen and Exemestane Adjuvant Multinational (TEAM) trial included prospectively planned biomarker studies to identify predictive biomarkers for patients receiving endocrine therapy. Quantitative IHC data for ER/PgR, HER1, HER2, HER3 and FISH analysis of HER2 in all cases was available for the current analysis relative to outcome of estrogen receptor-positive (ER+) early breast cancer patients treated with exemestane versus tamoxifen. Patients & Methods: Pathology blocks from 4598 TEAM patients were collected and tissue microarrays constructed. Quantitative analysis of receptors (HER1/2/3) by conventional IHC, and FISH (for HER2 only) were analysed relative to disease-free survival and treatment on an intent to treat basis using survival data for the first 2.75 years of the TEAM trial. Results: Of 4595 eligible cases samples received, 16 were excluded, and 4010 had complete biomarker data for all markers (HER1/HER2 & HER3) for the final biomarker analysis, 3.5% were HER1 positive, 13% HER2 positive & 21% HER3 positive. 1248 (31%) cases were HER1or2or3 positive (HER1-3+ve). HER1-3 positivity was associated with poor outcome (HR=1.6 95%CI=1.3-2.0). In HER1-3 negative patients the hazard ratio (for risk of relapse on exemestane versus tamoxifen in the first 2.75 years) was 0.68 (95% CI = 0.52-0.89), for the HER1-3 positive cases the hazard ratio was 1.14 (95% CI = 0.83-1.56) with a significant treatment by marker interaction (HR=1.68 95%CI=1.1-2.5; p=0.0014 in multivariate analysis). Trends for similar effects were seen for HER1 negative (-ve) vs HER1 positive (+ve) (HRs 0.80, 95%CI=0.65-0.99 vs 1.63 95%CI=0.74-3.59), HER2-ve vs HER2+ve (HRs 0.71, 95%CI=0.57- 0.9 vs 1.69 95%CI=108-2.63) and HER3-ve vs HER3+ve (HRs 0.78 95%CI=0.62-0.99 vs 1.04 95%CI=0.67-1.62) breast cancers. Conclusion: Preferential exemestane versus tamoxifen treatment benefit was seen in HER1/2/3 negative cases, whilst HER1/2/3 positive cases had a poor prognosis in this endocrine treated population (suggesting a degree of resistance to endocrine therapy), and no evidence of additional benefit from AIs versus tamoxifen. These three Type I receptor tyrosine kinases appear to identify breast cancers with relative resistance to all forms of endocrine therapy. This prospectively planned and powered treatment by marker analysis provides high level scientific evidence which may assist clinicians and patients in determining optimal AI schedules for women with early breast cancer. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr S2-4.

Abstract S1-5: PIK3CA mutations are linked to PgR expression: a Tamoxifen Exemestane Adjuvant Multinational (TEAM) pathology study.

Background: PIK3CA is mutated in about 26% of breast cancers (BC) and is the most frequently mutated gene in (BC). Almost 95% of mutations occur in exons 9 (E9) or 20 (E20). PIK3CA mutations may be associated with increased survival in endocrine-treated patients; however the impact of mutations in E9 vs E20 is not clear. We assessed 10 common PIK3CA mutations (95% of all mutations), in ER-positive (+ve) samples from the TEAM pathology study (n = ∼4500), and determined the impact of PIK3CA mutations on survival. We report an interim analysis of 1969 TEAM cases. Methods: DNA was extracted from formalin-fixed paraffin embedded sections. Mutational analyses were performed on 25 mutations in 6 genes (PIK3CAx10, Akt1x1, K RAS x5, H RAS x3, N RAS x2 & B RAF x4), using Sequenom MassArray. Results: Mutations were found in PIK3CA: 37.5%; Akt1: 3.3%; K RAS: 0.3%; and BRAF : 0.1% of cases. No mutations were found in H RAS or N RAS (n = 1969). 90% of PIK3CA mutations were located in E9 and E20. Outcome data was available for 1958/1969 patients. Patients whose tumours contained any PIK3CA mutations (n = 739) were at lower risk of distant metastasis, Hazard ratio=0.86 (0.67–1.11), when compared to those without PIK3CA mutations (n = 1219); although this difference was not statistically significant (Cox Regression; p = 0.24). PIK3CA mutations were significantly more frequent in HER2-negative (−ve) (39%) than in HER2+ve samples (25%) (p = 0.001), without evidence that PIK3CA mutations differentially impacted outcome in HER2+ve vs HER2-ve patients. A positive correlation was demonstrated between PIK3CA mutations and PgR Allred score (p = 0.002) but not ER Allred score (p = 0.37). With increasing PgR Allred score increase there is an increased frequency of mutations in E20 but not E9 (Table 1). Discussion: This study indicates a higher percentage of PIK3CA mutations in ER+ve BC samples than previously demonstrated, either for BC as a whole or for ER+ve cases, suggesting that in ER+ve early BC, PIK3CA mutations are more common than previously reported. Furthermore, increased PIK3CA mutation frequency is significantly associated with increasing PgR Allred score and this appears solely due to increased numbers of patients with E20 mutations further complicating the analysis of the impact of PIK3CA mutations in BC. This may explain current uncertainty regarding the impact of PIK3CA mutations in E9 vs E20 with respect to clinical outcome. Whilst we were unable to show a significant impact on outcome in patients whose tumours contained PIK3CA mutations, we believe the complex relationship between PgR expression (good prognosis indicator) and PI3K mutations requires further exploration in the full dataset using interaction techniques adjusting for the impact of PgR on outcome. Mutational analysis and correlation with clinical outcome data for the remaining ∼2500 DNA samples, along with the existing data for 1969 patients, will be presented. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr S1-5.

Abstract PD07-06: NEO-ZOTAC: Toxicity data of a phase III randomized trial with NEOadjuvant chemotherapy (TAC) with or without ZOledronic acid (ZA) for patients with HER2-negative large resectable or locally advanced breast cancer (BC)

Background: The role of bisphosphonates (BP) when added to the (neo)adjuvant treatment of BC in enhancing the efficacy of therapy is still unknown. NEOZOTAC investigates the efficacy of ZA added to neoadjuvant chemotherapy in patients with HER2-negative BC. Trial design: NEOZOTAC is a Dutch multicenter study. Patients are 1:1 randomized to 3-weekly TAC (docetaxel 75mg/m 2 , adriamycin 50 mg/m 2 and cyclophosphamide 500 mg/m 2 i.v., day 1) chemotherapy supported by pegfilgrastim (6 mg sc), day 2 with or without ZA (4 mg i.v. within 24 hr after chemotherapy) q3 weeks. Eligibility criteria: Main inclusion criteria: stage II or III, measurable, HER2-negative BC, age ≥18 years, WHO 0–2, adequate bone marrow-, renal-, and liver function, absence of prior BP usage and absence of active dental problems. Study endpoint: The primary endpoint is the pathologic complete response (pCR) rate. Secondary endpoints are toxicity, clinical response, tumor heterogeneity in core biopsy vs. operation specimen, and (disease free) survival. Optional side studies include fluorescent imaging (SoftScan®), changes in bone markers, single nucleotide polymorphisms and the insulin-like growth factor pathway, circulating tumor and endothelial cells and the false-negative rate of the sentinel node biopsy after neoadjuvant chemotherapy. Statistical Methods: Using a 5% significance level based on the two-sided Fishers exact test with a power of 80%, 250 patients (125/arm) are needed to show an improvement of the pCR-rate from 17% to 34% in the experimental arm. Randomization was done according to the Pococks minimisation technique stratified by cT, cN, and estrogen receptor status. Toxicity is analyzed using the Exact (2-sided) Chi-Square test. Results: From July 2010 to April 2012, 250 patients from 25 participating sites were randomized. Toxicity data of 173 patients are currently available and data of all 250 patients will be presented at SABCS. Patient characteristics are presented in table 1. Hematological and non-hematological toxicities were not significantly different between both treatment arms. Main grade 3/4 NCI-CTCv4 toxicities were neutropenia (8%), followed by febrile neutropenia (7%), fatigue (6%), diarrhea, hypertension, nausea (3%) and vomiting (1.2%). Bone pain, myalgia, and hypocalcemia occurred in one patient in the TAC-ZA arm (0.6%). Osteonecrosis of the jaw was not observed. Conclusions: Neoadjuvant TAC supported by pegfilgrastim plus ZA is feasible. No significant difference in toxicity are reported compared with the control arm. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr PD07-06.

Abstract P2-09-08: Safety assessment of extended adjuvant endocrine therapy with letrozole; results of the randomized phase III IDEAL trial (BOOG 2006-05)

The implementation of adjuvant aromatase inhibitors (AI) has contributed significantly to an ongoing improvement in survival of early breast cancer patients, but the impact of extended AI on toxicity after use in the initial 5 years has not yet been studied. Earlier, it was established that even after 5 years of adjuvant tamoxifen, extended letrozole for 5 years was comparable to the earlier reported toxicities, without an effect on overall quality of life. The aim of the current study is to assess the reported toxicity in the IDEAL trial, investigating extended letrozole after 5 years of endocrine therapy. The Dutch phase III IDEAL trial was designed to study the optimal duration of extended adjuvant letrozole, by randomizing to either 2.5 or 5 years of letrozole after the completion of 5 years of any endocrine therapy. All patients were disease-free at the time of randomization. The primary outcome of this study, disease free survival after extended endocrine therapy, will be reported separately. Toxicity data were collected every 6 months in the first year, and annually thereafter until study completion. 1824 patients were included in the trial, of which 26 patients never started with therapy (n=1798). In total, 3434 adverse events were reported by 1283 patients, causing 360 (20%) patients to stop therapy due to these events. In total, 643 patients randomized to 5 years of therapy reported 1834 AEs (71.2%), whereas 640 patients on 2.5 years of therapy reported 1587 events (71.5%). In the 5 year AI group, 1456 (80%) of the AEs were reported within the initial 2.5 years causing 177 patients to stop therapy in this period, versus 29 patients who stopped due to toxicity after the therapy extension beyond 2.5 years. There was no significant difference in the proportion of grade 3/4 events between both groups (2.5yr: 9.8%, 5yr: 9.7%, X 2 p=0.52). Most reported AEs were arthralgia (n=248, 13.8% of all patients , 8.9% grade 3/4), hot flushes (n=215, 12%, 6.5% grade 3/4), osteoporosis (n=181, 10%, 2.2% grade 3/4), fatigue (n=156, 8.7%, 5.1% grade 3/4) and a decrease in joint function (n=114, 6.3%, 3.5% grade 3/4). In total, only one grade 5 AE was reported, which was unrelated to the treatment (bleeding and sepsis after cholecystectomy). Although the toxicity pattern is comparable to regular AI based adjuvant therapy, the percentage of specific adverse events like arthralgia and hot flushes is lower than expected. This observation is possibly a selection bias, since patients who encountered side effects during regular adjuvant therapy could have been less willing to participate in this trial. Remarkably, low numbers of AEs and therapy refusal due to toxicity were reported after therapy extension beyond 2.5 years. In conclusion, extended adjuvant endocrine therapy shows an acceptable toxicity pattern, and therapy extension beyond 2.5 years up to 5 years is well tolerated and safe. Citation Format: Blok EJ, Kroep JR, Meershoek-Klein Kranenbarg EM, Duym-de Carpentier M, Putter H, van den Bosch J, Maartense E, van Leeuwen-Stok AE, Liefers G-J, Nortier JWR, Rutgers EJT, van de Velde CJH. Safety assessment of extended adjuvant endocrine therapy with letrozole; results of the randomized phase III IDEAL trial (BOOG 2006-05) [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P2-09-08.

Abstract PD2-07: 10-year follow-up and biomarker discovery for adjuvant endocrine therapy; results of the TEAM trial

Optimal endocrine therapy for postmenopausal, hormone-receptor positive (HR+) early breast cancer remains a point of discussion. The Tamoxifen Exemestane Adjuvant Multinational (TEAM) phase III trial showed no significant differences for disease free survival (DFS) and overall survival (OS) at 5 years between exemestane monotherapy and sequential treatment (tamoxifen followed by exemestane). We now report disease related outcomes at 10 years of follow-up (FU), and an explorative analysis to assess the predictive value of clinicopathological and immune-related biomarkers. In the TEAM trial, postmenopausal women with HR+ positive early breast cancer were randomly assigned to exemestane alone or sequential therapy. For this analysis, TEAM patients from countries that completed 10 years of FU were included. The primary endpoint was DFS at ten years, analyzed by intention to treat. Secondary outcomes were OS and cumulative incidence of relapse. An explorative per protocol analysis for relapse free survival (RFS) was performed to identify predictive pathological and immunological biomarkers, including centrally determined ER (ER-poor 0-6 vs ER-rich 7-8) and PR (0-4 vs 5-8) Allred scores, and the immunological markers CD8, FoxP3, classical HLA class 1 and HLA-G which were described earlier (Engels et al, Breast Cancer Treat Res, 2015). In total, 6120 patients were eligible for the current analysis, 3075 patients with exemestane monotherapy and 3045 patients randomized to sequential treatment. Median follow up was 9.83 years. DFS was 66.8% in the exemestane group and 66.8% in the sequential group (hazard ratio (HR) 0.96, 95% CI 0.88-1.05, p=0.389). OS was 74% in the exemestane, and 73% in the sequential group, respectively (HR 0.98, 95% CI 0.89-1.09, p=0.737). The cumulative incidence of relapse was 20% and 22% in the exemestane and sequential groups, respectively (HR 0.88, 95% CI 0.79-0.99, p=0.031). In the explorative per protocol analysis (n=4041), Allred score were available for 2996 patients; immunological markers for 1754 patients. Patients with above median numbers of FoxP3-positive T-cells showed a benefit of exemestane monotherapy for RFS (HR 0.56, 95% CI 0.42-0.75, p After ten years of follow up, both exemestane monotherapy and sequential therapy remain appropriate options for postmenopausal HR+ early breast cancer patients. Interestingly, the number of regulatory T-cells was a predictive factor for the benefit of exemestane monotherapy, which implies a role of the local immune system in endocrine therapy. Furthermore, data suggested that patients with a higher differentiation grade or ER-rich tumor derive more benefit from exemestane monotherapy. Citation Format: Blok EJ, Derks MGM, Kuppen PJK, Meershoek-Klein Kranenbarg EM, Engels CC, Liefers G-J, Putter H, Seynaeve CM, Kroep JR, Nortier JWR, Rea DW, Hasenburg A, Markopoulos CJ, Paridaens R, Bartlett JMS, van de Velde CJH. 10-year follow-up and biomarker discovery for adjuvant endocrine therapy; results of the TEAM trial [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr PD2-07.