The path to a better biomarker: application of a risk management framework for the implementation of PD-L1 and TILs as immuno-oncology biomarkers in breast cancer clinical trials and daily practice.

Immune checkpoint inhibitor therapies targeting PD-1/PD-L1 are now the standard of care in oncology across several hematologic and solid tumor types, including triple negative breast cancer (TNBC). Patients with metastatic or locally advanced TNBC with PD-L1 expression on immune cells occupying ≥1% of tumor area demonstrated survival benefit with the addition of atezolizumab to nab-paclitaxel. However, concerns regarding variability between immunohistochemical PD-L1 assay performance and inter-reader reproducibility have been raised. High tumor-infiltrating lymphocytes (TILs) have also been associated with response to PD-1/PD-L1 inhibitors in patients with breast cancer (BC). TILs can be easily assessed on hematoxylin and eosin-stained slides and have shown reliable inter-reader reproducibility. As an established prognostic factor in early stage TNBC, TILs are soon anticipated to be reported in daily practice in many pathology laboratories worldwide. Because TILs and PD-L1 are parts of an immunological spectrum in BC, we propose the systematic implementation of combined PD-L1 and TIL analyses as a more comprehensive immuno-oncological biomarker for patient selection for PD-1/PD-L1 inhibition-based therapy in patients with BC. Although practical and regulatory considerations differ by jurisdiction, the pathology community has the responsibility to patients to implement assays that lead to optimal patient selection. We propose herewith a risk-management framework that may help mitigate the risks of suboptimal patient selection for immuno-therapeutic approaches in clinical trials and daily practice based on combined TILs/PD-L1 assessment in BC. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Prognostic and Clinicopathologic Associations of LAG-3 Expression in Triple-negative Breast Cancer.

The immune checkpoint molecule lymphocyte activation gene 3 (LAG-3) is currently being investigated as a possible target for immunotherapy in triple-negative breast cancer (TNBC), frequently as an addition to treatment with programmed cell death protein 1/programmed death ligand 1 (PD-L1) inhibition. However, expression of LAG-3, the frequency of coexpression with PD-L1, and the prognostic significance of this marker have not been studied extensively in TNBC. For this study, tissue microarrays (TMAs) were constructed from surgical specimens of 514 patients with TNBC. TMAs were stained immunohistochemically for LAG-3 and PD-L1 expression. Tumor-infiltrating lymphocytes (TILs) were evaluated on full glass slides. LAG-3 expression was significantly associated with improved overall survival and relapse-free survival. When adjusted for clinicopathologic factors, each increment of 10 LAG-3-positive intratumoral lymphocytes per TMA core was associated with improved overall survival (hazard ratio=0.93, 95% confidence interval: 0.89-0.97, P=0.002), and recurrence-free survival (hazard ratio=0.91, 95% confidence interval: 0.85-0.97, P=0.002). PD-L1 expression on immune cells and PD-L1 expression evaluated with the combined positive score and TILs were also associated with improved survival in both univariate and multivariate analyses. PD-L1 expression on tumor cells was only associated with improved survival in univariate analysis. LAG-3 expression was associated with both TILs and PD-L1 expression. Coexpression of LAG-3 and PD-L1 did not confer additional survival benefits. In conclusion, LAG-3 expression is associated with improved survival in TNBC. LAG-3 is often coexpressed with PD-L1, confirming that TNBC is likely a suitable candidate for cotreatment with LAG-3 and programmed cell death protein 1/PD-L1 inhibitors. However, coexpression does not confer additional survival benefits.

Expression of Secretory Phospholipase A2 Group IIa in Breast Cancer and Correlation to Prognosis in a Cohort of Advanced Breast Cancer Patients.

Secreted phospholipase A2 group IIa (sPLA2-IIa) has been shown to promote tumor genesis and cell proliferation. The properties of this group of enzymes are utilized in liposomal drug delivery of chemotherapy. sPLA2-IIa is also under investigation as a possible treatment target in itself, and as a prognostic marker. The expression of sPLA2-IIa in breast cancer has not been examined extensively, and never using immunohistochemistry. We sought to investigate the expression of sPLA2-IIa in a cohort of advanced breast cancer patients with correlation to known clinicopathologic risk factors and survival. Material from 525 breast cancer patients (426 primary tumors and 99 metastases or local recurrences) was examined for sPLA2-IIa expression using immunohistochemistry. Out of these, 262 showed expression of sPLA2-IIa. We found that there was no correlation to clinicopathologic characteristics, and no impact of sPLA2-IIa expression on prognosis. However, we found that a large proportion of patients in our study had high levels of sPLA2-IIa expression, and that sPLA2-IIa was equally expressed in primary tumors and metastases. These findings may be significant in the future development of liposomal drug delivery or targeted sPLA2-IIa treatment.

The immune microenvironment and relation to outcome in patients with advanced breast cancer treated with docetaxel with or without gemcitabine.

Preclinical studies suggest that some effects of conventional chemotherapy, and in particular, gemcitabine, are mediated through enhanced antitumor immune responses. The objective of this study was to use material from a randomized clinical trial to evaluate whether patients with preexisting immune infiltrates responded better to treatment with gemcitabine + docetaxel (GD) compared to docetaxel alone. Formalin fixed, paraffin-embedded breast cancer tissues from SBG0102 phase 3 trial patients randomly assigned to treatment with GD or docetaxel were used. Immunohistochemical staining for CD8, FOXP3, LAG3, PD-1, PD-L1 and CD163 was performed. Tumor infiltrating lymphocytes (TILs) and tumor associated macrophages were evaluated. Prespecified statistical analyses were performed in a formal prospective-retrospective design. Time to progression was primary endpoint and overall survival secondary endpoint. Correlations between biomarker status and endpoints were evaluated using the Kaplan-Meier method and Cox proportional hazards models. Biomarker data was obtained for 237 patients. There was no difference in treatment effect according to biomarker status for the whole cohort. In planned subgroup analysis by PAM50 subtype, in non-luminal (basal-like and HER2E) breast cancers FOXP3 was a significant predictor of treatment effect with GD compared to docetaxel, with a HR of 0.22 (0.09-0.52) for tumors with low FOXP3 compared to HR 0.92 (0.47-1.80) for high FOXP3 TILs (Pinteraction = 0.01). Immune biomarkers were not predictive of added benefit of gemcitabine in a cohort of mixed breast cancer subtypes. However, in non-luminal breast cancers, patients with low FOXP3+ TILs may have significant benefit from added gemcitabine.

Systemic oxidative stress to nucleic acids is unaltered following radioiodine therapy of patients with benign nodular goiter.

BACKGROUND: Little is known about the whole body oxidative stress burden following radioactive iodine ((131)I) therapy of thyroid diseases. METHODS: We studied 17 patients with benign nodular goiter treated with (131)I therapy. The targeted thyroid dose was 50 Gy in 11 patients pretreated with 0.1 mg of recombinant human TSH (rhTSH). In 6 patients, the applied thyroid dose was 100 Gy without rhTSH prestimulation. Well-established biomarkers of oxidative stress to RNA (8-oxo-7,8-dihydroguanosine; 8-oxoGuo) and DNA (8-oxo-7,8-dihydro-2'-deoxyguanosine; 8-oxodG) were measured in freshly voided morning urine (normalized against the creatinine concentration) at baseline, and 7 and 21 days after rhTSH (not followed by (131)I), and 7 and 21 days after (131)I therapy, respectively. RESULTS: The baseline urinary excretions of 8-oxoGuo and 8-oxodG were 2.20 ± 0.84 and 1.63 ± 0.70 nmol/mmol creatinine, respectively. We found no significant changes in the excretion of any of the metabolites, neither after rhTSH stimulation alone nor after (131)I therapy. Also, no significant differences were found between the rhTSH group (low dose, median (131)I: 152 MBq) and the non-rhTSH group (high dose, median (131)I: 419 MBq; 8-oxoGuo: p = 0.66, 8-oxodG: p = 0.71). CONCLUSION: Systemic oxidative stress, as detected by nucleic acids metabolites in the urine, is not increased after thyroid stimulation with 0.1 mg of rhTSH, or after (131)I therapy. Our method cannot quantify the oxidative stress induced locally in the thyroid gland, but the study supports that (131)I therapy of benign nodular goiter carries no or only a minute risk of developing subsequent malignancies. It remains to be explored whether our findings also apply to hyperthyroid disorders.

Assays for urinary biomarkers of oxidatively damaged nucleic acids.

The analysis of oxidized nucleic acid metabolites can be performed by a variety of methodologies: liquid chromatography coupled with electrochemical or mass-spectrometry detection, gas chromatography coupled with mass spectrometry, capillary electrophoresis and ELISA (Enzyme-linked immunosorbent assay). The major analytical challenge is specificity. The best combination of selectivity and speed of analysis can be obtained by liquid chromatography coupled with tandem mass spectrometric detection. This, however, is also the most demanding technique with regard to price, complexity and skills requirement. The available ELISA methods present considerable specificity problems and cannot be recommended at present. The oxidized nucleic acid metabolites in urine are assumed to originate from the DNA and RNA. However, direct evidence is not available. A possible contribution from the nucleotide pools is most probably minimal, if existing. Recent investigation on RNA oxidation has shown conditions where RNA oxidation but not DNA oxidation is prominent, and while investigation on DNA is of huge interest, RNA oxidation may be overlooked. The methods for analyzing oxidized deoxynucleosides can easily be expanded to analyze the oxidized ribonucleosides. The urinary measurement of oxidized nucleic acid metabolites provides a non-invasive measurement of oxidative stress to DNA and RNA.

Urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine as a biomarker in type 2 diabetes.

The increasing prevalence of diabetes together with the associated morbidity and mortality calls for additional preventive and therapeutic strategies. New biomarkers that can be used in therapy control and risk stratification as alternatives to current methods are needed and can facilitate a more individualized and sufficient treatment of diabetes. Evidence derived from both epidemiological and mechanistic studies suggests that oxidative stress has an important role in mediating the pathologies of diabetic complications. A marker of intracellular oxidative stress that potentially could be used as a valuable biomarker in diabetes is the DNA oxidation marker 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), which can be assessed noninvasively in the urine, with minimal discomfort for the patient. In this review the analytical validity of 8-oxodG is addressed by highlighting important methodological issues. The available epidemiological evidence regarding urinary 8-oxodG and type 2 diabetes is presented. A possible role for DNA oxidation in cancer development in type 2 diabetes patients is discussed, followed by an evaluation of the potential of urinary 8-oxodG as a clinical biomarker in type 2 diabetes.

Urinary markers of nucleic acid oxidation and long-term mortality of newly diagnosed type 2 diabetic patients.

OBJECTIVE: We analyzed data from a cohort of 1,381 newly diagnosed type 2 diabetic patients to test the hypothesis that urinary markers of nucleic acid oxidation are independent predictors of mortality. RESEARCH DESIGN AND METHODS: We examined the relationship between urinary excretion of markers of DNA oxidation (8-oxo-7,8-dihydro-2'-deoxyguanosine [8-oxodG]) and RNA oxidation (8-oxo-7,8-dihydroguanosine [8-oxoGuo]) and long-term mortality using Cox proportional hazards regression. RESULTS: After multivariate adjustment, the hazard ratios for all-cause and diabetes-related mortality of patients with 8-oxoGuo levels in the highest quartile compared with those in the lowest quartile were 1.44 (1.12-1.85) and 1.54 (1.13-2.10), respectively. Conversely, no significant associations between 8-oxodG and mortality were found in the adjusted analyses. CONCLUSIONS: Urinary excretion of the RNA oxidation marker 8-oxoGuo measured shortly after diagnosis of type 2 diabetes predicts long-term mortality independently of conventional risk factors. This finding suggests that 8-oxoGuo could serve as a new clinical biomarker in diabetes.