Impact of delayed and prolonged fixation on the evaluation of immunohistochemical staining on lung carcinoma resection specimen

Seijen, Maartje van; Brčić, Luka; Gonzales, Atilio Navarro; Sansano, Irene; Béndek, Mátyás; Brčić, Iva; Lissenberg‐Witte, Birgit I.; Korkmaz, Halil İbrahim; Geiger, Thomas; Kammler, Rosita; Stahel, Rolf A.; Thunnissen, Erik

doi:10.1007/s00428-019-02595-9

Cited by 42 publications

(30 citation statements)

References 22 publications

(25 reference statements)

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“…One early report from the Dowsett group working with phospho‐proteins in breast cancer tissues showed that there was an almost complete loss of reactivity for p‐AKT and p‐ERK in breast resection tissues in contrast to strong staining seen in matched core biopsies from the same samples . Moreover, in a NSCLC model, delayed formalin fixation (increases in time before the start of fixation) caused significant loss of immunoreactivity : for PD‐L1, there was a reduction in the proportion of tumour cells showing positive membrane staining and the effect was larger with increase in fixation delay. In addition, (5) methods used to decalcify bony tissues and thus enable the preparation of sections from them can also significantly affect IHC results.…”

Section: Discussionmentioning

confidence: 99%

External quality assessment demonstrates that PD‐L1 22C3 and SP263 assays are systematically different

Dodson¹,

Parry²,

Lissenberg‐Witte

et al. 2019

The Journal of Pathology CR

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PD-L1 inhibitors are part of first line treatment options for patients with advanced non-small cell lung cancer. PD-L1 immunohistochemistry (IHC) assays act as either a companion or a complementary diagnostic. The purpose of this study is to describe the experience of external quality assurance (EQA) provider UK NEQAS ICC and ISH with the comparison of different PD-L1 assays used in daily practice. Three EQA rounds (pilot, run A and run B) were carried out using formalin fixed paraffin embedded samples with sample sets covering a range of epitope concentrations, including 'critical samples' near to clinical threshold cut-offs. An expert panel (n = 4) evaluated all returned slides simultaneously and independently on a multi-header microscope together with the participants own in-house control material. The tonsil sample was evaluated as 'acceptable' or 'unacceptable', and for the other samples the percentage of PD-L1 stained tumour cells were estimated in predetermined categories (<1%, 1 to <5%, 5 to <10%, 10 to <25%, 25 to <50%, 50 to <80%, 80 to 100%). In the pilot and the two subsequent runs the number of participating laboratories was 43, 69 and 76, respectively. The pass rate for the pilot run was 67%; this increased to 81% at run A and 82% at run B. For two 'critical samples', in runs A and B, 22C3 IHC had significantly higher PD-L1 expression than SP263 IHC (p < 0.001), whilst the PD-L1 scores for the other six samples were similar for all assays. In run A the laboratory developed tests (LDTs) using 22C3 scored lower than the commercial 22C3 tests (p = 0.01). After the initial testing, improvement in performance of PD-L1 IHC is shown for approved and LDT PD-L1 assays. Equivalency of approved PD-L1 22C3 and SP263 assays cannot be assumed as the scores cross the clinically relevant thresholds of 1% and 50% PD-L1 expression.

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Section: Discussionmentioning

confidence: 99%

External quality assessment demonstrates that PD‐L1 22C3 and SP263 assays are systematically different

Dodson¹,

Parry²,

Lissenberg‐Witte

et al. 2019

The Journal of Pathology CR

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“…For resection specimens, many different protocols are available, and the time from the interruption of the blood supply to the initiation of fixation is likely longer, since surgery is a more complicated procedure than CNB. It is important to adhere to good laboratory practices regarding tissue handling and fixation [ 15 ]. However, at some centres, breast resection specimens are not immediately sliced and fixed, resulting in poor fixation of the tissue.…”

Section: Introductionmentioning

confidence: 99%

Concordance of the 21-gene assay between core needle biopsy and resection specimens in early breast cancer patients

Pan

Bai

et al. 2021

Breast Cancer Res Treat

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Background Adjuvant therapy decisions may be partly based on the results of a multigene quantitative reverse transcription-polymerase chain reaction (RT-PCR)-based assay: the 21-gene recurrence score (RS) test of resection specimens. When necessary, core needle biopsy (CNB) may be considered as a surrogate. Here, we evaluated the concordance in gene expression according to results from RT-PCR-based RS testing between paired CNBs and resection specimens. Methods CNBs and resection specimens from 50 breast cancer (BC) patients were tested to calculate RSs. First, we examined the concordance of the ER, PR and HER-2 status of tissue samples indicated by immunohistochemical (IHC) and RT-PCR analyses. Then, we compared the IHC findings of ER, PR, HER-2 and Ki-67 staining across paired samples. Ultimately, the RS and single-gene results for ER, PR, HER-2 and Ki-67 were explored between paired samples. Results The concordance between IHC and RT-PCR was 100%, 80.0% and 100% for ER, PR and HER-2, respectively, in both resection specimens and CNBs. The concordance for IHC ER, PR, HER-2 and Ki-67 status was 100%, 94.0%, 52.0% and 82.0%, respectively, between paired samples. RS results from paired samples showed a strong correlation. The overall concordance in RS group classification between samples was 74%, 72% and 78% based on traditional cutoffs, TAILORx cutoffs and ASCO guidelines, respectively. ER, PR, HER-2 and Ki-67 were modestly- to- strongly correlated between paired samples according to the RT-PCR results. Conclusion A modest- to- strong correlation of ER, PR, HER-2 and Ki-67 gene expression and RS between CNBs and resection specimens was observed in the present study. The 21-gene RS test could be reliably performed on CNBs. ER, PR and HER-2 status showed remarkable concordance between the IHC and RT-PCR analyses. The concordance between paired samples was high for the IHC ER, PR and Ki-67 results and low for HER-2.

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“…Accordingly, careful assessment of pre‐analytical factors is key to avoiding false negative results when assessing 1% TPS. Investigation on the effects of pre‐analytical factors on PD‐L1 expression has been largely carried out on histological samples 20 . In fact, the only pre‐analytical data available on cytology concern the type of fixation.…”

Section: Introductionmentioning

confidence: 99%

PD‐L1 expression in cell‐blocks of non‐small cell lung cancer: The impact of prolonged fixation

Vigliar

Iaccarino

Campione

et al. 2020

Diagnostic Cytopathology

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Introduction In the selection of non‐small cell lung cancer (NSCLC) patients for immunotherapy, specimen processed as cell blocks (CBs) may be the only available material to assess PD‐L1 expression. Therefore, optimal CB preparation becomes paramount. In this context, here we assessed whether inadequate fixation time might be one of the pre‐analytical factors affecting PD‐L1 expression. Methods Ex vivo CBs from placental (n = 3) and NSCLC (n = 8) resection specimens were obtained. PD‐L1 staining was performed on CBs prepared at increasing fixation times (12 hours, 48 hours, 72 hours, 96 hours, 168 hours and 504 hours) using the companion diagnostic SP263 Assay and a validated 22C3 laboratory developed test (LDT). Staining intensity and percentage of positive cells were evaluated. Results All placental CBs showed moderate to strong PD‐L1 positivity in most cells, regardless of the fixation time. Likewise, the percentage of SP263‐stained NSCLC cells was similar at all fixation times except for one case, which showed less intense SP263 staining at 168 hours. Conversely, in 5/8 cases, the 22C3 LDT percentage of positive cells and staining intensity decreased at 168 hours and 504 hours. Conclusions Our results show that fixation time influences the performance of 22C3 LDT on CBs. Thus, we recommend that the fixation time of cytological materials be carefully checked, especially when PD‐L1 testing is delayed until the oncology request. Indeed, delays in tissue processing and paraffin embedding may lead to sub‐optimal performance of PD‐L1 staining on CBs.

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Impact of delayed and prolonged fixation on the evaluation of immunohistochemical staining on lung carcinoma resection specimen

Cited by 42 publications

References 22 publications

External quality assessment demonstrates that PD‐L1 22C3 and SP263 assays are systematically different

External quality assessment demonstrates that PD‐L1 22C3 and SP263 assays are systematically different

Concordance of the 21-gene assay between core needle biopsy and resection specimens in early breast cancer patients

PD‐L1 expression in cell‐blocks of non‐small cell lung cancer: The impact of prolonged fixation

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