Accelerated instability testing reveals quantitative mass spectrometry overcomes specimen storage limitations associated with PD-L1 immunohistochemistry

Abstract

Immunohistochemistry (IHC) using formalin-fixed, paraffin embedded (FFPE) tissue is limited by epitope masking, posttranslational modification and immunoreactivity loss that occurs in stored tissue by poorly characterized mechanisms. Conformational epitopes recognized by many programmed-death-ligand-1 (PD-L1) IHC assays are particularly susceptible to degradation and provide an ideal model for understanding signal loss in stored FFPE tissue. Here we assessed 1206 tissue sections to evaluate environmental factors impacting immunoreactivity loss. PD-L1 IHC using four antibodies (22C3, 28-8, E1L3N, and SP142), raised against intracellular and extracellular epitopes, was assessed in stored FFPE tissue alongside quantitative mass spectrometry (MS). Global proteome analyses were used to assess proteome-wide oxidation across an inventory of 3041 protein groups (24,737 distinct peptides). PD-L1 quantitation correlated well with IHC expression on unaged sections (R² = 0.744; P < 0.001), with MS demonstrating no loss of PD-L1 protein, even in sections with significant signal loss by IHC impacting diagnostic category. Clones 22C3 and 28-8 were most susceptible to signal loss, with E1L3N demonstrating the most robust signal (56%, 58%, and 33% reduction respectively; p < 0.05). Increased humidity and temperature resulted in significant acceleration of immunoreactivity loss, which was mitigated by storage with desiccant. MS demonstrated only modest oxidation of 274 methionine-containing peptides and aligned with IHC results suggesting peptide oxidation is not a major factor. These data imply immunoreactivity loss driven by humidity and temperature results in structural distortion of epitopes rendering them unsuitable for antibody binding following epitope retrieval. Limitations of IHC biomarker analysis from stored tissue sections may be mitigated by cost-effective use of desiccant when appropriate. In some scenarios, complementary MS is a preferred approach for retrospective analyses of archival FFPE tissue collections.

Fig. 1. PD-L1 expression in aged tissue and tissue under accelerated conditions.

Representative PD-L1 expression assessed by E1L3N IHC in FFPE gastric carcinoma under normal atmospheric conditions (a–c) and in NSCLC under acceleration conditions (d–f). a Day 0, b 4.5 months, c 24 months; d Day 0, e Day 9, f Day 28. PD-L1 programmed-death-ligand-1, IHC immunohistochemistry, FFPE formalin-fixed, paraffin embedded, NSCLC non-small cell lung cancer.

Fig. 2. PD-L1 by clinical cutoffs in FFPE NSCLC sections over time in the acceleration chamber with conditions of 100% oxygen, 80% humidity, and 37 °C for 22C3, 28-8, E1L3N, and SP142 PD-L1 clones.

Bars represent number of cases in series with PD-L1 expression equal or above TPS clinical cutoff thresholds. PD-L1 programmed-death-ligand-1, TPS tumor proportion score, TC tumor cell, NSCLC non-small cell lung cancer, FFPE formalin-fixed, paraffin embedded.

Fig. 3. PD-L1 expression in NSCLC under varying accelerated conditions.

Placenta and tonsil FFPE sections incubated in the acceleration chamber under different environmental conditions at day 28; a–d: 100% oxygen and 80% humidity at either 20 °C or 37 °C, then stained for PD-L1 (E1L3N) or pan-CK (AE1/AE3): a Placenta PD-L1, b Tonsil PD-L1, c Placenta pan-CK, d Tonsil pan-CK. 100% oxygen and 37 °C at either 45% or 80% humidity at day 28, e Placenta PD-L1, f Tonsil PD-L1. Control conditions: 20 °C, atmospheric humidity and oxygen. Bar represents mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. PD-L1 programmed-death-ligand-1, CK cytokeratin, FFPE formalin-fixed, paraffin embedded.

Fig. 4. PD-L1 expression by E1L3N in FFPE placenta sections at days 0, 1, 3, 7, 14, 21, and 28.

The first row shows tissue sections stored under normal atmospheric conditions, the second and third row show tissue sections within an incubator at 100% oxygen, 37 °C, and 80% humidity without (second row) and with (third row) desiccant. PD-L1 programmed-death-ligand-1, FFPE formalin-fixed, paraffin embedded.

Fig. 5. PD-L1 expression by E1L3N in FFPE tonsil sections at days 0, 1, 3, 7, 14, 21, and 28.

The first row shows tissue sections stored under normal ambient conditions, the second and third row shows tissue sections within an incubator at 100% oxygen, 37 °C and 80% humidity without (second row) and with (third row) desiccant. PD-L1 programmed-death-ligand-1, FFPE formalin-fixed, paraffin embedded.

Fig. 6. Correlation of PD-L1 protein expression by immunohistochemistry (by TPS) with PD-L1 abundance measured by MS in FFPE sections prior to incubation in the accelerated loss chamber.

PD-L1 programmed-death-ligand-1, TPS tumor proportion score, MS mass spectrometry, FFPE formalin-fixed, paraffin embedded.

Fig. 7. MS quantitation of PD-L1 peptides LQDAGVYR (LQD) and AEVIWTSSDHQVLSGK (AEV) in FFPE sections incubated in accelerated loss chamber at baseline (0), 9 and 28 days of incubation.

MS mass spectrometry, PD-L1 programmed-death-ligand-1, FFPE formalin-fixed, paraffin embedded.

Fig. 8. Global proteome analyses to assess proteome-wide oxidation under baseline (wet ox baseline) and acceleration conditions (wet ox day 28) compared with samples of naturally aged placenta and tonsil tissue stored under normal ambient conditions.

The plotted values are log2 ratios of numbers of MS/MS spectra corresponding to oxidized and unoxidized methionine-containing peptides. Higher log2 ratios correspond to greater extent of proteome oxidation.