Impact of Prolonged Ischemia on the Immunohistochemical Expression of Programmed Death Ligand 1 (PD-L1).

Antibodies targeting programmed death receptor 1 or programmed death ligand 1 (PD-L1) have become a standard of care to treat different cancers; for some of these tumors, there is a correlation between tissue expression of PD-L1 and response rates in patients. Although most of the analytical challenges in the evaluation of PD-L1 expression have been standardized, preanalytical issues have been less explored. The objective of this study was to evaluate the impact of time of ischemia on the performance of 2 commonly used antibodies against PD-L1. Sixteen tonsillectomy samples were kept in ischemia for <30 minutes from sample obtention (control) and 1, 3, 6, 12, and 24 hours at room temperature before formalin fixation and paraffin embedding. Selected areas were inserted into TMA paraffin recipient blocks stained with SP142 and SP263 antibodies and evaluated by 2 blind observers. The proportion of suboptimally stained samples was significantly higher for samples with cold ischemia times 6 hours or over ( P <0.0001). False-negative results were 25% in samples exposed to 6 hours of ischemia and raised to 34% for samples remaining in ischemia for 12 or 24 hours. When all observations were pooled, SP142 provided suboptimal results in 24% of observations and SP263 in 12.5%; this is a statistically significant difference ( P =0.042). In conclusion, the quality of staining for PD-L1 in tonsil samples varies with the time of cold ischemia. The SP142 antibody presented a significantly lower tolerance to prolonged cold ischemia than SP263. These results reveal the relevance of controlled preanalytical processing of samples.

The Immunohistochemical Expression of Programmed Death Ligand 1 (PD-L1) Is Affected by Sample Overfixation.

Humanized antibodies targeting programmed death receptor 1 (PD-1) or its ligand (PD-L1) have been approved for the treatment of different cancers. Some of these antibodies show a correlation between the tissue expression of PD-L1 and response. Evaluation of PD-L1 expression presents multiple challenges, but some preanalytical issues such as tissue fixation have been scarcely evaluated. With the hypothesis that immunohistochemical staining of PD-L1 may be impacted by the time of specimen fixation, we evaluated differences in its expression in tonsil samples exposed to predefined fixation times. Random nontumoral tonsillectomy specimens were blindly evaluated in tissue microarray slides after staining with SP142 and SP263 antibodies. With fixation times ranging from 12 to 72 hours, between 2.8% and 6.1% of the samples were considered to be suboptimally stained, with no differences between the 2 antibodies within these fixation times. A significantly higher proportion of samples exposed to a fixation time of 96 hours presented suboptimal immunostaining (15.6%, P<0.0001). In addition, suboptimally stained spots were 20.8% using SP142 and 10.4% using SP263 after 96 hours of fixation (P=0.046). In conclusion, the quality of staining for PD-L1 in tonsil samples decreased with overfixation of the specimen at times >72 hours. Samples exposed to formaldehyde for longer periods presented suboptimal results for both clones, but the SP142 antibody presented a significantly lower tolerance to formalin overexposure than SP263. These results indicate the relevance of a controlled preanalytical processing of samples and particularly the length of fixation of tumor specimens.

Metabolic fingerprinting of fresh lymphoma samples used to discriminate between follicular and diffuse large B-cell lymphomas.

OBJECTIVE: To investigate if proton nuclear magnetic resonance ((1)H NMR) spectroscopy-based metabolic profiling was able to differentiate follicular lymphoma (FL) from diffuse large B-cell lymphoma (DLBCL) and to study which metabolites were responsible for the differences. MATERIALS AND METHODS: High-resolution (1)H NMR spectra was obtained from fresh samples of lymph node biopsies obtained consecutively at one center (14 FL and 17 DLBCL). Spectra were processed using pattern-recognition methods. RESULTS: Discriminant models were able to differentiate between the two tumor types with a 86% sensitivity and a 76% specificity; the metabolites that most contributed to the discrimination were a relative increase of alanine in the case of DLBCL and a relative increase of taurine in FL. Metabolic models had a significant but weak correlation with Ki67 expression (r(2)=0.42; p=0.002) CONCLUSIONS: We have proved that it is possible to differentiate between FL and DLBCL based on their NMR metabolic profiles. This approach may potentially be applicable as a noninvasive tool for diagnostic and treatment follow-up in the clinical setting using conventional magnetic resonance systems.