Overall survival in the OlympiA phase III trial of adjuvant olaparib in patients with germline pathogenic variants in BRCA1/2 and high-risk, early breast cancer.

BACKGROUND: The randomized, double-blind OlympiA trial compared 1 year of the oral poly(adenosine diphosphate-ribose) polymerase inhibitor, olaparib, to matching placebo as adjuvant therapy for patients with pathogenic or likely pathogenic variants in germline BRCA1 or BRCA2 (gBRCA1/2pv) and high-risk, human epidermal growth factor receptor 2-negative, early breast cancer (EBC). The first pre-specified interim analysis (IA) previously demonstrated statistically significant improvement in invasive disease-free survival (IDFS) and distant disease-free survival (DDFS). The olaparib group had fewer deaths than the placebo group, but the difference did not reach statistical significance for overall survival (OS). We now report the pre-specified second IA of OS with updates of IDFS, DDFS, and safety. PATIENTS AND METHODS: One thousand eight hundred and thirty-six patients were randomly assigned to olaparib or placebo following (neo)adjuvant chemotherapy, surgery, and radiation therapy if indicated. Endocrine therapy was given concurrently with study medication for hormone receptor-positive cancers. Statistical significance for OS at this IA required P < 0.015. RESULTS: With a median follow-up of 3.5 years, the second IA of OS demonstrated significant improvement in the olaparib group relative to the placebo group [hazard ratio 0.68; 98.5% confidence interval (CI) 0.47-0.97; P = 0.009]. Four-year OS was 89.8% in the olaparib group and 86.4% in the placebo group (Δ 3.4%, 95% CI -0.1% to 6.8%). Four-year IDFS for the olaparib group versus placebo group was 82.7% versus 75.4% (Δ 7.3%, 95% CI 3.0% to 11.5%) and 4-year DDFS was 86.5% versus 79.1% (Δ 7.4%, 95% CI 3.6% to 11.3%), respectively. Subset analyses for OS, IDFS, and DDFS demonstrated benefit across major subgroups. No new safety signals were identified including no new cases of acute myeloid leukemia or myelodysplastic syndrome. CONCLUSION: With 3.5 years of median follow-up, OlympiA demonstrates statistically significant improvement in OS with adjuvant olaparib compared with placebo for gBRCA1/2pv-associated EBC and maintained improvements in the previously reported, statistically significant endpoints of IDFS and DDFS with no new safety signals.

The National Lung Matrix Trial: translating the biology of stratification in advanced non-small-cell lung cancer.

BACKGROUND: The management of NSCLC has been transformed by stratified medicine. The National Lung Matrix Trial (NLMT) is a UK-wide study exploring the activity of rationally selected biomarker/targeted therapy combinations. PATIENTS AND METHODS: The Cancer Research UK (CRUK) Stratified Medicine Programme 2 is undertaking the large volume national molecular pre-screening which integrates with the NLMT. At study initiation, there are eight drugs being used to target 18 molecular cohorts. The aim is to determine whether there is sufficient signal of activity in any drug-biomarker combination to warrant further investigation. A Bayesian adaptive design that gives a more realistic approach to decision making and flexibility to make conclusions without fixing the sample size was chosen. The screening platform is an adaptable 28-gene Nextera next-generation sequencing platform designed by Illumina, covering the range of molecular abnormalities being targeted. The adaptive design allows new biomarker-drug combination cohorts to be incorporated by substantial amendment. The pre-clinical justification for each biomarker-drug combination has been rigorously assessed creating molecular exclusion rules and a trumping strategy in patients harbouring concomitant actionable genetic abnormalities. Discrete routes of pathway activation or inactivation determined by cancer genome aberrations are treated as separate cohorts. Key translational analyses include the deep genomic analysis of pre- and post-treatment biopsies, the establishment of patient-derived xenograft models and longitudinal ctDNA collection, in order to define predictive biomarkers, mechanisms of resistance and early markers of response and relapse. CONCLUSION: The SMP2 platform will provide large scale genetic screening to inform entry into the NLMT, a trial explicitly aimed at discovering novel actionable cohorts in NSCLC. CLINICAL TRIAL ISRCTN: 38344105.

The role of cardiopulmonary fitness and its genetic influences on surgical outcomes.

BACKGROUND: Outcome after major surgery remains poor in some patients. There is an increasing need to identify this cohort and develop strategies to reduce postsurgical morbidity and mortality. Central to outcome is the ability to mount cardiovascular output in response to the increased oxygen demand associated with major surgery. METHODS: A medline search was performed using keywords to identify factors that affect, and genetic influences in, disease and outcome from surgery, and all relevant English language articles published between 1980 and 2005 were retrieved. Secondary references were obtained from key articles. RESULTS: Preoperative cardiopulmonary exercise testing assesses patient fitness, highlights those at particular risk and, combined with triage to critical care, facilitates significant improvement in surgical outcome. However, genetic factors also influence responses to increased oxygen demand, and some patients are genetically predisposed to mounting increased inflammatory responses, which raise oxygen demand further. Polymorphisms in genes influencing fitness (angiotensin converting enzyme) and immune and inflammatory responses (such as interleukin 6) may associate with surgical outcome. CONCLUSIONS: Development of preoperative screening methods like cardiopulmonary exercise testing and genotype analysis to identify index factors may permit better patient stratification, provide targets for future tailored treatments and so improve surgical outcome.

Intermittent claudication: exercise-increased walking distance is not related to improved cardiopulmonary fitness.

OBJECTIVES: To assess if exercise training improves the symptoms of intermittent claudication by improvement in cardiopulmonary fitness. METHODS: Claudication distance (CD), maximum walking distance (MWD), calf endurance (repetitive heel raises), cardiovascular fitness (VO2 peak), and ankle-brachial pressure index (ABPI) were measured in 16 subjects with intermittent claudication before, and following an 8-week treadmill training programme. RESULTS: Training resulted in a median increase in CD of 65.5 m (p<0.01), MWD of 339.5 m (p<0.001) and HR of 19 (p<0.03). Notably, improvements in MWD correlated with those in HR (p=0.001; R=0.75). There was no training-associated change in VO2 peak (median increase of only 0.35 ml/kg/min; p=0.60) or ABPI (median increase of only 0.01; p=0.64). CONCLUSION: In this study, overall improvement in claudication was not related to an improvement in cardiopulmonary fitness.

Activity (transcription) of the genes for MLH1, MSH2 and p53 in sporadic colorectal tumours with micro-satellite instability.

Micro-satellite instability (MSI) is relevant in the management of colorectal cancers (CRC) and relies on analysis of gene mutations, or production of the proteins involved in DNA mismatch repair (e.g. MLH1, MSH2). p53 mutation is also relevant in MSI, but high-level CRC (MSI-H) demonstrate fewer mutations than low-level (MSI-L) or stable (MSS) cancers. Recently, the importance of gene activity (transcription) in MSI has been identified, where rather than being mutated genes have been downregulated. In this study, 67 sporadic CRC and eight samples of normal bowel were analysed for MSI status (by SSCP) and levels of MLH1, MSH2 and p53 gene transcription (by RT-PCR and scanning densitometry). Micro-satellite instability correlated with gender and site, with more MSI-H CRC in females (P<0.02) and in the right colon (P<0.04). In MSI-H, p53 transcription was markedly reduced (P<0.003). Compared to normal bowel, MLH1 transcription was elevated in all cancers (P<0.01), while MSH2 transcription was elevated only in MSI-H (P<0.04). There was a direct correlation between MLH1 and MSH2 transcription (P<0.001). Although fewer mutations are reported in MSI-H than MSI-L/MSS, these results suggest that reduced p53 transcription might account for decreased tumour suppression in MSI-H. The direct correlation between MLH1 and MSH2 transcription suggests that control of these genes might be coordinated.

Primary varicose veins: altered transcription of VEGF and its receptors (KDR, flt-1, soluble flt-1) with sapheno-femoral junction incompetence.

OBJECTIVE: Loss of regulation of vascular endothelial growth factor (VEGF) production and action disturbs vascular homeostasis leading to pathology. Primary varicose veins (VVs) demonstrate aberrant production/release of VEGF. Our aim was to examine transcription of genes for VEGF (VEGF(121)/VEGF(165)) and its receptors (KDR, flt-1, s.flt-1) in VVs, in relation to underlying venous incompetence. MATERIALS AND METHODS: Samples of varicose (n=83, 18 patients) or normal (n=14, five subjects) great saphenous vein were divided into segments, determined by anatomical position from the sapheno-femoral junction (SFJ). SFJ and segmental incompetence were determined from duplex scans. Gene transcripts were amplified by RT-PCR, analysed by scanning densitometry, and the levels of transcription determined by ratio to control gene GADPH-3 (GAP-3). RESULTS: VEGF(121)/(165), KDR and flt-1 transcription was elevated in VVs overall (p<0.001), and in VVs with an incompetent SFJ (p<0.001), but not when the SFJ was functional; s.flt-1 was unaltered. Notably, gene transcription was unaffected by segmental position, or incompetence. Position below the SFJ correlated with increased transcription of s.flt-1 when the SFJ was incompetent (p<0.04), and s.flt-1 and VEGF(121) when the segment was incompetent (p<0.03). CONCLUSIONS: SFJ incompetence is associated with altered transcription of VEGF and its receptors reflecting an aetiological mechanism or later stage of disease development. Altered VEGF(121) and s.flt-1 transcription may be an early event in varicogenesis.

Primary varicose veins: the sapheno-femoral junction, distribution of varicosities and patterns of incompetence.

OBJECTIVE: to determine the patterns of long saphenous vein (LSV) disease in primary varicose veins (VVs). DESIGN: a retrospective analysis of venous duplex scans performed on patients referred for treatment of primary VVs. METHODS: analysis was made of sapheno-femoral junction (SFJ) incompetence, non-SFJ incompetence, segmental and perforating vein incompetence, distribution of varicosities, deep venous insufficiency, and short saphenous incompetence. RESULTS: four hundred and eighty-one patients were assessed (median age 50 (range 12-98) years; male:female ratio 1:1.95), comprising 706 limbs. Forty-six per cent of limbs had a competent SFJ, 64% of which had no incompetent perforating vessels associated. Disease was more widespread when the SFJ was incompetent. Varicosities were most common in the calf, occurring at or below the level of incompetence within the LSV. Incompetent segments occurred most commonly above-knee. There was no obvious correlation between incompetent perforators and distribution of varicosities, or incompetent segments. Short saphenous incompetence and non-SFJ groin recurrence were associated more with a competent SFJ, the converse being true for the Giacomini vein. CONCLUSION: primary VVs develop in isolated segments of the superficial venous system (without connection to the deep system) at, or distal to, the underlying main trunk incompetence, suggesting a process of "spreading incompetence" from one focal point, producing varicosities (mainly in tributaries).

"Long haul" flight and deep vein thrombosis: a model to help investigate the benefit of aspirin and below-knee compression stockings.

OBJECTIVE: to develop a model simulating factors of "long haul" flight to investigate the relationship with DVT. MATERIALS AND METHODS: volunteers (19 males: 20 females) sat for 6 h in a warm (>25 degrees C), dry environment, limited in movement, consuming alcohol (40 ml of 40% alcohol/hour) and salted foods (300 g). Half of the subjects received 150 mg aspirin and wore especially designed below-knee, compression stockings (Class 1 profile). Changes in full blood counts were recorded, and as an indication of DVT formation, plasma was analysed for D-dimer. Limb swelling was assessed from leg measurements. RESULTS: after 6 h, in controls, there were significant rises in platelet packing (Pct p<0.04), total platelet numbers (p<0.003) and total numbers of white blood cells (WBC's p<0.001). With aspirin plus stockings, there were similar significant rises in total platelet numbers (p<0.002) and total WBC's (p<0.001). In both groups, significant rises were seen in all WBC types (except basophils). Wearing compression stockings prevented calf swelling seen in controls after 6 h (p<0.002). No subject developed a DVT, or a change in levels of D-dimer. CONCLUSION: changes in the cellular components of blood, particularly WBC's, combined with vaso-compression and reduced flow could predispose towards DVT. Aspirin, combined with compression stockings, may provide prophylaxis.

Varicose veins: loss of release of vascular endothelial growth factor and reduced plasma nitric oxide.

OBJECTIVES: To examine the release of vascular endothelial growth factor (VEGF) and nitric oxide (NO) in primary varicose veins (VVs) and normal vein controls following experimentally-induced venous stasis. DESIGN, MATERIALS AND METHODS: Patients with primary VVs (n=21) and control subjects (n=11) were rested supine for 15 min. Blood was collected from both an arm and foot vein. A below-knee cuff was applied and inflated to 90-95 mmHg for 10 min (to prompt venous stasis). Further blood samples were collected from the foot vein. Levels of plasma VEGF and NO were analysed. RESULTS: In control subjects, application of the cuff increased levels of plasma VEGF in the foot (p<0.025). In contrast, in patients with VVs, there was little or no change in these levels of plasma VEGF. Cuff application had little effect on levels of plasma NO in either controls, or those with VVs. When compared to controls however, the levels of plasma NO in all samples with VVs (arm, or foot before, or after, cuff application) were reduced (all, p<0.05). CONCLUSIONS: Loss of VEGF release with experimentally-induced venous stasis, and reduced levels of plasma NO may suggest a mechanism important in the development of primary VVs.

Delivery of low molecular weight heparin for prophylaxis against deep vein thrombosis using a novel, needle-less injection device (J-Tip).

Given daily, low molecular weight (LMW) heparins are established for prophylaxis against deep vein thrombosis (DVT). We describe delivery by a novel, needle-less device that is virtually painless in action. Its use could provide benefits for patients in terms of comfort both psychologically and physically, and for healthcare workers in terms of safety from needle-stick injury. Patients undergoing elective surgery received LMW heparin delivered subcutaneously by either a standard needle and syringe or by the needle-less injection device, J-Tip. Pain was scored at the time of injection and plasma anti-factor Xa levels compared between the two methods of drug delivery 4 h later: 29 patients received LMW heparin delivered by the J-Tip and 31 patients by standard needle and syringe. The J-Tip was significantly more comfortable for the patient as the method of drug delivery (P < 0.001). When delivered by the J-Tip, LMW heparin was equally as efficacious, as plasma anti-factor Xa levels were similar for both methods of delivery (P < 0.42). In summary, delivery of LMW heparin by the J-Tip device was both comfortable and effective. These findings, taken in conjunction with its ease of use and complete freedom from risk of needle-stick injury might encourage further examination and use of this type of product.

Antigen gene transfer to cultured human dendritic cells using recombinant avipoxvirus vectors.

Advances in understanding the role of dendritic cells (DCs) as the major antigen (Ag)-presenting cell type of the immune system combined with the recent development of methods for the ex vivo expansion of human DCs have opened the possibility for the transfer of tumor Ags to DCs with a view toward tumor immunotherapy. In this study, we examined the feasibility of Ag transfer to cultured human DCs using the host range-restricted avipoxvirus, fowlpoxvirus (FWPV). FWPV was found to infect and express a lacZ marker gene in a number of mammalian cell lines of fibroblastic, epithelial, and hemopoietic lineage origins. LacZ recombinant FWPV (rFWPV) was found subsequently to infect human DCs that had been cultured ex vivo from peripheral blood monocytes. Using rFWPV containing lacZ under the control of a vaccinia virus (VV) early/late promoter (p7.5K) and a 10 plaque-forming units per cell multiplicity of infection, >80% of cells expressed the lacZ marker gene. Quantitative analysis showed that the level of expression continued to rise for 5 days postinfection, at which point the experiments were terminated. Replication-competent recombinant VV (rVV) was also shown to be capable of transferring the marker gene to primary DC cultures. However, neither rFWPV nor rVV were able to express transgenes under the control of late viral promoters, indicating that both rFWPV and rVV infections are arrested at an early stage in human DCs. Infection of CD83 + DCs by rFWPV was confirmed by double-staining cytochemistry. We conclude that host range-restricted FWPV can be used efficiently to transfer Ag genes to human DCs ex vivo and may have a role in the development of tumor immunotherapy protocols.

Irradiated NC adenocarcinoma cells transduced with both B7.1 and interleukin-2 induce CD4+-mediated rejection of established tumors.

Previous studies have shown that expression of the immune co-stimulator B7.1 reduces the tumorigenicity of some, but not all, malignant cell lines. However, B7.1-expressing tumor cells are not very effective in inducing the rejection of established tumors. This may in part be due to induction of anergy in the potentially reactive T cells. Previous studies have shown that IL-2 can reverse the anergic state both in vitro and in vivo. Therefore, we have examined the effect of retrovirus-mediated delivery and expression of murine B7.1 and interleukin-2 on tumor formation and rejection of established MHC class I+/II- NC adenocarcinomas. Neither the expression of B7.1 nor IL-2 alone had a significant effect on NC tumorigenicity. In contrast, combined expression of B7.1 and IL-2 substantially decreased the tumorigenicity of these cells in the immunecompetent syngeneic hosts. T-cell depletion studies show this to be dependent primarily on the activation of CD4+ cells. Furthermore, distant subcutaneous injection of irradiated NC/IL-2/B7.1 can induce, much more effectively than NC/B7.1 or NC/IL-2, the rejection of small NC tumors, and prevent the recurrence of large surgically resected tumors. Together, these results suggest that tumor cells genetically modified to express B7.1 and IL-2 can induce the immune-mediated rejection of established class II- tumors by a mechanism involving CD4+ cells.

The effect of combined expression of interleukin 2 and interleukin 4 on the tumorigenicity and treatment of B16F10 melanoma.

The recent use of interleukin 2 (IL-2) and interleukin 4 (IL-4) single cytokine modified tumour cells in rodent models has demonstrated a potential use of these cytokines to produce autologous cancer cell vaccines. Here we compare the potential therapeutic benefit of transduction with IL-2 or IL-4 alone, and combined IL-2 + IL-4 in B16F10 cells, a murine malignant melanoma of poor immunogenicity. Transduction of B16F10 cells (MHC class I and II negative) to express either IL-2 or IL-4 alone delays the formation of tumours, IL-4 being more effective than IL-2. However, combined expression of IL-2 + IL-4 reduces tumorigenicity more than either cytokine alone. The eventual formation of tumours may result from loss of gene expression, and preliminary results suggest methylation of the retroviral long terminal repeat (LTR), rather than loss of the transduced DNA sequences. Histological examination of tumours expressing either IL-2 or IL-4 alone shows a non-specific inflammatory reaction with an increased tissue infiltrate of immune effectors (monocytes/macrophages, lymphocytes, granulocytes) localised around the tumour. In comparison, when cells expressing combined IL-2 + IL-4 were injected there were more granulocytes present, and perhaps more importantly, these were mainly localised within the tumour. The benefit of combined IL-2 + IL-4 expression results from a local rather than systemic effect as the growth of tumours from cells expressing IL-2 or IL-4 alone injected at distant sites was comparable with a single inoculation of cells expressing either cytokine alone. However, when cells expressing single cytokines IL-2 or IL-4 were mixed and injected at the same site, in comparison with the clonal population of cells expressing combined IL-2 + IL-4, tumour growth was characteristic of IL-4 alone rather than IL-2 + IL-4. Treatment of established tumours with a single injection of lethally irradiated tumour cells expressing IL-2 + IL-4 was sufficient to either reject tumours, or at least delay further tumour development. Furthermore, treatment stimulated an initial non-specific immune reaction that lead to a systemic immunity. Lethally irradiated wild-type cells were also successful in treating some established tumours, although this did not induce any systemic immunity. However, although successful in treatment studies, neither wild-type nor combined IL-2 + IL-4 expressing cells were able to vaccinate animals against a subsequent challenge with live wild-type tumour. These results indicate a potential therapeutic benefit with the use of combination IL-2 + IL-4 transduction of autologous cancer cells.

The effect of folate on the methotrexate/indomethacin interaction in a murine cancer cell line.

1. The effect of folate on the interaction between methotrexate (a folate analogue) and indomethacin has been examined in murine NC carcinoma cells. 2. Conditioning of NC cells to a physiological (20 nM) folate concentration after culture in a high folate concentration increased the response to methotrexate. The sensitivity of these conditioned cells to methotrexate related inversely to the folate concentration. 3. At 20 nM and 2 microM folate, indomethacin 1 micrograms ml-1 potentiated the cytotoxicity of methotrexate 4 and 8 ng ml-1 (both P < 0.03). 4. When NC cells were incubated with [3H]-methotrexate at 20 nM and 2 microM folate, there was a trend for increased tritium accumulation with indomethacin 0.36 micrograms ml-1 (1 microM; P < 0.01). 5. We conclude that the folate concentration can affect the sensitivity of NC cells to methotrexate, although the degree of potentiation of cytotoxicity by indomethacin remains similar.

Interaction between methotrexate and indomethacin on a human normal haemopoietic cell line.

1. The interaction between methotrexate and indomethacin has been examined, at a physiological folate concentration (20 nM), on a human normal lymphoblast-like cell line (RPMI 1788) in vitro. 2. Indomethacin (1 microgram ml-1) increased the reduction of lymphoblast growth caused by methotrexate (10-80 ng ml-1). 3. Indomethacin (0.1 and 1 microgram ml-1) potentiated the cytotoxicity of methotrexate (20 and 40 ng ml-1) after 4 days in culture. 4. Indomethacin (0.4 micrograms ml-1) reduced the accumulation of tritium in lymphoblasts incubated with [3H]-methotrexate after 30 min; therefore initial drug accumulation was not responsible for the potentiation seen after 4 days. 5. If indomethacin increases the killing of human cancer cells by methotrexate in vivo, with a smaller potentiation on lymphoblasts, this combination may be beneficial in treating human malignancy.

Methotrexate alters the fatty acid composition of NC adenocarcinoma cells in culture.

The effects of methotrexate and indomethacin alone and in combination have been examined on the fatty acid (FA) composition of total cellular lipids in cultured NC adenocarcinoma cells. These studies show that methotrexate can alter the lipid content of cancer cells. Methotrexate 16 ng/ml incubated with NC cells for 2 days increased the content of various FAs. When used alone, indomethacin 1 microgram/ml or methotrexate 8 ng/ml had no significant effect, but in combination caused FA increases, usually to about the same extent as with the higher concentration of methotrexate alone. No FA changes were seen up to 3 h with these drug concentrations or with methotrexate up to 10 micrograms/ml alone or with INDO 1 microgram/ml. These effects may explain previous findings that indomethacin potentiates methotrexate, an interaction which may be important in cancer therapy.