Estrogen Receptor Pathway Activity Score to Predict Clinical Response or Resistance to Neoadjuvant Endocrine Therapy in Primary Breast Cancer.

Endocrine therapy is important for management of patients with estrogen receptor (ER)-positive breast cancer; however, positive ER staining does not reliably predict therapy response. We assessed the potential to improve prediction of response to endocrine treatment of a novel test that quantifies functional ER pathway activity from mRNA levels of ER pathway-specific target genes. ER pathway activity was assessed on datasets from three neoadjuvant-treated ER-positive breast cancer patient cohorts: Edinburgh: 3-month letrozole, 55 pre-/2-week/posttreatment matched samples; TEAM IIa: 3- to 6-month exemestane, 49 pre-/28 posttreatment paired samples; and NEWEST: 16-week fulvestrant, 39 pretreatment samples. ER target gene mRNA levels were measured in fresh-frozen tissue (Edinburgh, NEWEST) with Affymetrix microarrays, and in formalin-fixed paraffin-embedded samples (TEAM IIa) with qRT-PCR. Approximately one third of ER-positive patients had a functionally inactive ER pathway activity score (ERPAS), which was associated with a nonresponding status. Quantitative ERPAS decreased significantly upon therapy (P < 0.001 Edinburgh and TEAM IIa). Responders had a higher pretreatment ERPAS and a larger 2-week decrease in activity (P = 0.02 Edinburgh). Progressive disease was associated with low baseline ERPAS (P = 0.03 TEAM IIa; P = 0.02 NEWEST), which did not decrease further during treatment (P = 0.003 TEAM IIa). In contrast, the staining-based ER Allred score was not significantly associated with therapy response (P = 0.2). The ERPAS identified a subgroup of ER-positive patients with a functionally inactive ER pathway associated with primary endocrine resistance. Results confirm the potential of measuring functional ER pathway activity to improve prediction of response and resistance to endocrine therapy.

The Prognostic PDE4D7 Score in a Diagnostic Biopsy Prostate Cancer Patient Cohort with Longitudinal Biological Outcomes.

Purpose. To further validate the prognostic power of the biomarker PDE4D7, we investigated the correlation of PDE4D7 scores adjusted for presurgical clinical variables with longitudinal postsurgical biological outcomes. Methods. RNA was extracted from biopsy punches of resected tumors (550 patients; RP cohort) and diagnostic needle biopsies (168 patients; DB cohort). Cox regression and survival were applied to correlate PDE4D7 scores with patient outcomes. Logistic regression was used to combine the clinical CAPRA score with PDE4D7. Results. In univariate analysis, the PDE4D7 score was significantly associated with PSA recurrence after prostatectomy in both studied patient cohorts' analysis (HR 0.53; 95% CI 0.41-0.67; p<1.0E-04 and HR 0.47; 95% CI 0.33-0.65; p<1.0E-04, respectively). After adjustment for the presurgical clinical variables preoperative PSA, PSA density, biopsy Gleason, clinical stage, percentage tumor in the biopsy (data only available for RP cohort), and percentage of positive biopsies, the HR was 0.49 (95% CI 0.38-0.64; p<1.0E-04) and 0.43 (95% CI 0.29-0.63; p<1.0E-04), respectively. The addition of the PDE4D7 to the clinical CAPRA score increased the AUC by 5% over the CAPRA score alone (0.82 versus 0.77; p=0.004). This combination model stratified 14.6% patients of the DB cohort to no risk of biochemical relapse (NPV 100%) over a follow-up period of up to 15 years. Conclusions. The PDE4D7 score provides independent risk information for pretreatment risk stratification. Combining CAPRA with PDE4D7 scores significantly improved the clinical risk stratification before surgery.

Validation of Cyclic Adenosine Monophosphate Phosphodiesterase-4D7 for its Independent Contribution to Risk Stratification in a Prostate Cancer Patient Cohort with Longitudinal Biological Outcomes.

BACKGROUND: The clinical metrics used to date to assess the progression risk of newly diagnosed prostate cancer patients only partly represent the true biological aggressiveness of the underlying disease. OBJECTIVE: Validation of the prognostic biomarker phosphodiesterase-4D7 (PDE4D7) in predicting longitudinal biological outcomes in a historical surgery cohort to improve postsurgical risk stratification. DESIGN, PATIENTS, AND METHODS: RNA was extracted from biopsy punches of resected tumors from 550 patients. PDE4D7 was quantified using one-step quantitative reverse transcription-polymerase chain reaction. PDE4D7 scores were calculated by normalization of PDE4D7 to reference genes. Multivariate analyses were adjusted for clinical prognostic variables. Outcomes tested were: prostate-specific antigen relapse, start of salvage treatment, progression to metastases, overall mortality, and prostate cancer-specific mortality. The PDE4D7 score was combined with the clinical risk model Cancer of the Prostate Risk Assessment Postsurgical Score (CAPRA-S) using multivariate regression modeling; the combined score was tested in post-treatment progression free survival prediction. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Correlations with outcomes were analyzed using multivariate Cox regression and logistic regression statistics. RESULTS AND LIMITATIONS: The PDE4D7 score was significantly associated with time-to-prostate specific antigen failure after prostatectomy (hazard ratio [HR]: 0.53, 95% confidence interval [CI]: 0.41-0.67 for each unit increase, p<0.0001). After adjustment for postsurgical prognostic variables the HR was 0.56 (95% CI: 0.43-0.73, p<0.0001). The PDE4D7 score remained significant after adjusting the multi-variate analysis for the CAPRA-S model categories (HR=0.54, 95% CI=0.42-0.69, p<0.0001). Combination of the PDE4D7 score with the CAPRA-S demonstrated a significant incremental value of 4-6% in 2-yr (p=0.004) or 5-yr (p=0.003) prediction of progression free survival after surgery. The combined model of PDE4D7 and CAPRA-S improves patient selection with very high risk of fast disease relapse after primary intervention. CONCLUSIONS: The PDE4D7 score has the potential to provide independent risk information and to restratify patients with clinical intermediate- to high-risk characteristics to a very low-risk profile. PATIENT SUMMARY: In this report, we studied the potential of a novel biomarker to predict outcomes of a cohort of prostate cancer patients who underwent surgery more than 10 yr ago. We found that a gene called phosphodiesterase-4D7 added extra information to the available clinical data. We conclude that the measurement of this gene in tumor tissue may contribute to more effective treatment decisions.

Effects of apricitabine and other nucleoside reverse transcriptase inhibitors on replication of mitochondrial DNA in HepG2 cells.

Several nucleoside reverse transcriptase inhibitors are associated with mitochondrial toxicity resulting from inhibition of DNA polymerase-gamma. This study compared the effects on mitochondrial DNA of apricitabine (previously referred to as AVX754 or SPD754), a novel cytidine analogue under development for the treatment of human immunodeficiency virus (HIV)-1 infection, and other reverse transcriptase inhibitors. Human HepG2 hepatoblastoma were cultured for up to 16 days with test compounds at concentrations of 0.3-300 microM. Mitochondrial DNA replication was assessed by means of a duplex nucleic acid sequence-based amplification technique, which measures the ratio of the number of mitochondrial DNA copies to the number of genomic DNA copies. Apricitabine and tenofovir had no effect on the mitochondrial DNA content. In contrast, alovudine, zalcitabine, didanosine and stavudine markedly reduced mitochondrial DNA content, whereas abacavir, emtricitabine, lamivudine and zidovudine produced slight increases in mitochondrial DNA, which may reflect an adaptive cellular response to mitochondrial dysfunction. These results suggest that apricitabine shows a favorable mitochondrial toxicity profile, which is important for long-term clinical use. Further studies are warranted to define the clinical implications of these findings.

Real-time nucleic acid sequence-based amplification assay to quantify changes in mitochondrial DNA concentrations in cell cultures and blood cells from HIV-infected patients receiving antiviral therapy.

BACKGROUND: To study the clinical relevance of changes in mitochondrial DNA (mtDNA) in peripheral blood mononuclear cells (PBMCs) attributable to HIV infection and/or combination antiretroviral therapy (cART), a high-throughput molecular assay to quantify mtDNA is required. METHODS: We developed a quantitative real-time duplex nucleic acid sequence-based amplification assay in which both mtDNA and nuclear DNA are simultaneously amplified in 1 tube. The assay could accurately quantify mtDNA in a range of 15-1500 copies of mtDNA per 2 genomic copies with an intrarun variation of 11% and an interrun variation of 16%. We compared this real-time assay with the lactate/pyruvate ratios in fibroblasts incubated with glucose and exposed to zalcitabine. Additionally, we studied the effects of platelet contamination and the in vivo effects of cART on mtDNA in PBMCs from a small group of patients. RESULTS: Decreases in mtDNA preceded the increase in lactate/pyruvate ratios and vice versa when zalcitabine was eliminated from the culture. Platelets affected the mtDNA in PBMCs if >5 platelets per PBMC were present. Within 12 weeks, mtDNA increased and remained increased in PBMCs from patients on continuous treatment with zidovudine/lamivudine/indinavir therapy (P = 0.03), but increased if patients were switched to stavudine/didanosine therapy (P = 0.008). CONCLUSION: After drug exposure, the mtDNA assay can detect changes in mtDNA concentrations in cell lines and PBMCs, when properly controlled for platelet effects, earlier than traditional assays.

Mitochondrial DNA and RNA increase in peripheral blood mononuclear cells from HIV-1-infected patients randomized to receive stavudine-containing or stavudine-sparing combination therapy.

BACKGROUND: Mitochondrial DNA (mtDNA) in peripheral blood mononuclear cells (PBMCs) has been suggested as a potential marker of mitochondrial toxicity associated with nucleoside analogue reverse-transcriptase inhibitor-containing therapy. METHODS: We quantified mtDNA and mitochondrial RNA (mtRNA) in PBMCs over the course of 48 weeks in 78 patients infected with human immunodeficiency virus type 1 (HIV-1) who were randomly assigned to receive ritonavir-boosted indinavir and efavirenz with or without stavudine. Furthermore, we analyzed the association of mtDNA and mtRNA with clinical signs and symptoms and/or abnormalities in laboratory markers attributed to mitochondrial toxicity. RESULTS: No statistically significant difference was found in mtDNA and mtRNA content over time between the 2 treatment arms. When arms were combined, both median mtDNA and mtRNA content showed statistically significant increases over the course of 48 weeks, from 206 to 278 copies/cell (P < .001) and from 154 to 288 copies/cell (P = .003), respectively. No statistically significant difference in mtDNA and mtRNA content was found between patients with and those without adverse events attributed to mitochondrial toxicity. CONCLUSIONS: The observed increases in mtDNA and mtRNA content during the first year of treatment may represent a restorative trend resulting from suppression of HIV-1 infection, independent of the treatment used. Future studies should focus on well-defined mitochondrial toxicities and changes in these markers within the corresponding affected tissues simultaneously with those in PBMCs. Furthermore, with respect to studies of peripheral blood, mtDNA and mtRNA content in individual cell subtypes rather than in PBMCs may be better markers of toxicity and deserve further investigation.

Mitochondrial DNA depletion in HIV-infected patients is more pronounced with chronic hepatitis C and enhanced following treatment with pegylated interferon plus ribavirin.

BACKGROUND: Mitochondrial DNA (mtDNA) damage seems to be responsible for many of the toxicities associated with the long-term use of nucleoside analogues in HIV-infected patients. These adverse effects, mainly lipoatrophy, seem to be even more pronounced in subjects with hepatitis C virus (HCV) co-infection. However, there is no information about a possible additive effect of HCV on mtDNA depletion nor about the impact of ribavirin use in HIV/HCV-coinfected individuals. PATIENTS AND METHODS: mtDNA was measured in peripheral blood mononuclear cells (PBMC) collected from 192 individuals classified into 4 groups: HIV-neg/HCV-neg (control group, n = 11), HIV-pos/HCV-neg (56), HIV-neg/HCV-pos (18) and HIV-pos/HCV-pos (107). A duplex real-time NASBA assay was used to quantify mtDNA on maximal platelet-depleted specimens and all experiments were run in duplicate. The mtDNA copy number per cell was estimated taking as reference the nuclear DNA copy number. RESULTS: The mean mtDNA values in the control group was 757 copies/cell, while it was 428, 349 and 296 for HIV-pos, HCV-pos and HIV/HCV-coinfected individuals, respectively (P < 0.001 for all groups relative to the control group). No significant differences were observed when comparing patients with HIV or HCV infections alone, but coinfected individuals showed a lower mtDNA copy number than patients infected with HIV (P < 0.001) or with HCV (P = 0.089). In a subset of 18 patients with HIV/HCV-coinfection, treatment with pegylated interferon plus ribavirin produced a further reduction in mtDNA (mean value, 189 copies/cell; P = 0.009). CONCLUSIONS: HIV and HCV may independently cause mtDNA depletion in PBMC. Coinfection may result in more pronounced mtDNA depletion. The administration of interferon plus ribavirin may further enhance mtDNA depletion. These findings may explain the greater risk of lipoatrophy of antiretroviral therapy in HIV-infected patients with HCV coinfection and why anti-HCV therapy may aggravate this effect.