Formalin fixation for optimal concordance of programmed death-ligand 1 immunostaining between cytologic and histologic specimens from patients with non-small cell lung cancer
Koomen, Bregje; van der Starre-Gaal, Jose; Vonk, Judith; von der Thüsen, Jan; van der Meij, Jacqueline; Monkhorst, Kim; Willems, Stefan; Timens, Wim; 't Hart, Nils
(2021) Cancer Cytopathology, volume 129, issue 4, pp. 304 - 317
(Article)
Abstract
Background: Immunohistochemical staining of programmed death-ligand 1 (PD-L1) is used to determine which patients with non-small cell lung cancer (NSCLC) may benefit most from immunotherapy. Therapeutic management of many patients with NSCLC is based on cytology instead of histology. In this study, concordance of PD-L1 immunostaining between cytology cell blocks
... read more
and their histologic counterparts was analyzed. Furthermore, the effect of various fixatives and fixation times on PD-L1 immunoreactivity was studied. Methods: Paired histologic and cytologic samples from 67 patients with NSCLC were collected by performing fine-needle aspiration on pneumonectomy/lobectomy specimens. Formalin-fixed, agar-based or CytoLyt/PreservCyt-fixed Cellient cell blocks were prepared. Sections from cell blocks and tissue blocks were stained with SP263 (standardized assay) and 22C3 (laboratory-developed test) antibodies. PD-L1 scores were compared between histology and cytology. In addition, immunostaining was compared between PD-L1-expressing human cell lines fixed in various fixatives at increasing increments in fixation duration. Results: Agar cell blocks and tissue blocks showed substantial agreement (κ = 0.70 and κ = 0.67, respectively), whereas fair-to-moderate agreement was found between Cellient cell blocks and histology (κ = 0.28 and κ = 0.49, respectively). Cell lines fixed in various alcohol-based fixatives showed less PD-L1 immunoreactivity compared with those fixed in formalin. In contrast to SP263, additional formalin fixation after alcohol fixation resulted in preserved staining intensity using the 22C3 laboratory-developed test and the 22C3 pharmDx assay. Conclusions: Performing PD-L1 staining on cytologic specimens fixed in alcohol-based fixatives could result in false-negative immunostaining results, whereas fixation in formalin leads to higher and more histology-concordant PD-L1 immunostaining. The deleterious effect of alcohol fixation could be reversed to some degree by postfixation in formalin.
show less
Download/Full Text
Keywords: Non-small cell lung cancer, Programmed death-ligand 1, Immunocytochemistry, Immunohistochemistry, Tissue Fixation, 22C3 antibody, SP263 antibody, programmed cell death-ligand 1, tissue fixation, immunocytochemistry, immunohistochemistry, non–small cell lung carcinoma, Oncology, Cancer Research
ISSN: 1934-6638
Publisher: Wiley-Blackwell
Note: Funding Information: This study was supported by an unrestricted research grant from Roche Diagnostics Nederland BV received by Nils A. ’t Hart. The funding source was not involved in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the article for publication. Funding Information: Bregje M. Koomen reports grants from AstraZeneca, Merck Sharp & Dohme, and Roche Diagnostics, outside the submitted work. Jan H. von der Th?sen reports personal fees from Merck Sharp & Dohme, during the conduct of the study. Kim Monkhorst reports grants and personal fees from AstraZeneca; personal fees from Merck Sharp & Dohme, Benecke, Bristol-Myers Squibb, AbbVie, Diaceutics, Lilly, Bayer, and Boehringer Ingelheim; personal fees and nonfinancial support from Roche Diagnostics; and nonfinancial support from PGDX, Delfi, and Takeda, all outside the submitted work. Stefan M. Willems reports grants from Bristol-Myers Squibb, NextCure, Pfizer Inc, Roche Diagnostics, Merck Sharp & Dohme, Amgen, AstraZeneca, and Bayer, outside the submitted work. Wim Timens reports personal fees from Roche Diagnostics/Ventana, Merck Sharp & Dohme, Bristol-Myers Squibb, and AbbVie, outside the submitted work. The remaining authors made no disclosures. This study was supported by an unrestricted research grant from Roche Diagnostics Nederland BV received by Nils A. ?t Hart. The funding source was not involved in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the article for publication. Sections cut from each cell block and tissue block were stained with the Ventana SP263 antibody and the Dako 22C3 antibody. All staining of patient material was performed within 1 center (University Medical Center Groningen). Staining of slides with the SP263 standardized assay was carried out on a Ventana Benchmark Ultra platform according to the manufacturer's instructions. The Dako 22C3 was used as the LDT, also using the Ventana Benchmark Ultra platform. Previously, this LDT was compared with the Dako 22C3 pharmDx assay on Dako Link 48, for optimization and validation of its use in routine clinical practice. The addition of an amplification step led to the best protocol, which was comparable to a previously published protocol by Adam et al.25 The same SP263 standardized assay and the 22C3 LDT were used to stain sections from the CMA blocks containing the cell line cores. In addition, sections from the CMA blocks were stained with the Dako 22C3 pharmDx assay, used on a Dako Autostainer Link 48 platform according to the manufacturer's instructions (performed in Martini Hospital, Groningen, the Netherlands). Staining patterns were analyzed in cores from each cell line and compared visually between the 20 different fixation schemes. Differences in PD-L1 expression between cell lines were quantified by determining PD-L1 H-scores for each cell core, using an application in Visiopharm software (Visiopharm A/S).26 The H-score was calculated by determining staining intensity in each cell (divided into levels 0, 1+, 2+, and 3+), followed by application of the following formula: 1 * (% of cells with staining intensity level 1+) + 2 * (% of cells with staining intensity level 2+) + 3 * (% of cells with staining intensity level 3+).27,28 All stained slides from the paired cell and tissue blocks of included patients were reviewed independently by 2 trained pathologists. Cases of disagreement were resolved through discussion. PD-L1 expression was scored in tumor cells according to the guidelines provided by Roche/Ventana or Dako as part of the PD-L1 IHC pharmDx test. For each slide, the pathologists determined the tumor proportion score (TPS), which is constructed by determining the percentage of viable tumor cells that show membranous PD-L1 immunostaining relative to the total amount of tumor cells. This score was used to categorize the samples into 3 groups: TPS <1% (negative), TPS 1% to 49% (weakly positive), and TPS ?50% (strongly positive). Five Dutch pathology laboratories collaborated to collect material for the comparison of PD-L1 immunostaining in histologic and cytologic specimens from patients with NSCLC (University Medical Center Groningen, Isala Zwolle, Erasmus Medical Center Rotterdam, Netherlands Cancer Institute Amsterdam, and Pathology Friesland). In each center, paired histologic and cytologic samples were collected. To do so, FNAs were performed on pneumonectomy or lobectomy specimens with a palpable or visible tumor to obtain cytologic samples that were as close as possible to routine FNA-derived specimens. The collection of cytologic material was performed before further preparation and fixation of the resection specimen. It has been demonstrated previously that collecting FNA material this way can be done without compromising routine histologic evaluation of the tumor, thus it is a safe method that can be used within the outlines of the code of conduct for responsible use of residual human tissue for research established by the Federation of Dutch Medical Scientific Societies.23 Histology samples were taken after 18 to 72 hours following routine protocols used in clinical practice. The mean estimated fixation time was between 18 hours (overnight fixation) and 72 hours (resection specimens that remained in formalin over the weekend). All patient material was used anonymously and was collected and used in accordance with the Federation of Dutch Medical Scientific Societies code of conduct and with the General Data Protection Regulation. After obtaining the FNA specimen, each center was allowed to use their routine method for fixation and generation of a cell block (see Supporting Table 1). In this way, concordance of PD-L1 immunostaining could be analyzed between histologic tissue and cell blocks that were processed in different ways, reflecting normal, everyday practice. Depending on the locally developed protocols, either a formalin-fixed, agar-based cell block or a CytoLyt/PreservCyt-fixed Cellient cell block was prepared. The Cellient Automated Cell Block System (Hologic Inc) was used for the latter. From each cell block, a slide was cut and stained with hematoxylin and eosin to check for the presence of tumor cells. If enough viable tumor cells were present (?100 tumor cells), sequential, 3-?m-thick slices were cut for staining with 2 separate PD-L1 antibodies, ie, the Ventana SP263 standardized assay (Ventana Medical Systems Inc) and a Dako 22C3 laboratory-developed test (LDT). Formalin-fixed, paraffin-embedded (FFPE) tissue sections were cut from a routinely made histologic tissue block from the same tumor. These sections were also stained for PD-L1 with both antibodies (SP263 and 22C3) and were used for histologic comparison (for a schematic representation of the study design, see Fig. 1). To further evaluate the effects of different fixatives on PD-L1 immunostaining in cytologic samples, commercially available cell lines with high PD-L1 expression were used (T-cell non-Hodgkin lymphoma cell lines from HistoCyte Laboratories Ltd24). The cell lines were fixed in either 5 mL 10% neutral-buffered formalin (NBF) or 25 mL CytoLyt, PreservCyt, CytoRich Red, or Carbowax. CytoLyt and PreservCyt (Hologic Inc) are methanol-based fixatives, while Carbowax (Dow Chemical Company) contains ethanol and polyethylene glycol. CytoRich Red (Thermo Scientific) is alcohol-based as well (methanol, isopropyl alcohol, and ethylene glycol) but also contains a small amount of formaldehyde. Each is used as a (pre-)fixation solution in clinical practice. In addition, cell lines were fixed in CytoLyt, PreservCyt, CytoRich Red, or Carbowax followed by 30-minute fixation in 10% NBF. Various fixation times were used, which allowed us to evaluate the effect of different fixation periods on PD-L1 immunostaining. The fixation periods used were 2 hours and 24 hours for all fixatives and 48 hours for the cell lines fixed in NBF and CytoLyt only. This design allowed for 20 different fixation schemes (see Supporting Table 2). Subsequently, agarose pellets were created for each cell line and were then processed into paraffin blocks. Cores from each block were assembled in paraffin-embedded cell microarray (CMA) blocks. These were sent to the University Medical Center in Groningen, where slides were cut (3-?m thickness) for PD-L1 immunostaining. Sections cut from each cell block and tissue block were stained with the Ventana SP263 antibody and the Dako 22C3 antibody. All staining of patient material was performed within 1 center (University Medical Center Groningen). Staining of slides with the SP263 standardized assay was carried out on a Ventana Benchmark Ultra platform according to the manufacturer's instructions. The Dako 22C3 was used as the LDT, also using the Ventana Benchmark Ultra platform. Previously, this LDT was compared with the Dako 22C3 pharmDx assay on Dako Link 48, for optimization and validation of its use in routine clinical practice. The addition of an amplification step led to the best protocol, which was comparable to a previously published protocol by Adam et al.25 The same SP263 standardized assay and the 22C3 LDT were used to stain sections from the CMA blocks containing the cell line cores. In addition, sections from the CMA blocks were stained with the Dako 22C3 pharmDx assay, used on a Dako Autostainer Link 48 platform according to the manufacturer's instructions (performed in Martini Hospital, Groningen, the Netherlands). Staining patterns were analyzed in cores from each cell line and compared visually between the 20 different fixation schemes. Differences in PD-L1 expression between cell lines were quantified by determining PD-L1 H-scores for each cell core, using an application in Visiopharm software (Visiopharm A/S).26 The H-score was calculated by determining staining intensity in each cell (divided into levels 0, 1+, 2+, and 3+), followed by application of the following formula: 1 * (% of cells with staining intensity level 1+) + 2 * (% of cells with staining intensity level 2+) + 3 * (% of cells with staining intensity level 3+).27,28 All stained slides from the paired cell and tissue blocks of included patients were reviewed independently by 2 trained pathologists. Cases of disagreement were resolved through discussion. PD-L1 expression was scored in tumor cells according to the guidelines provided by Roche/Ventana or Dako as part of the PD-L1 IHC pharmDx test. For each slide, the pathologists determined the tumor proportion score (TPS), which is constructed by determining the percentage of viable tumor cells that show membranous PD-L1 immunostaining relative to the total amount of tumor cells. This score was used to categorize the samples into 3 groups: TPS <1% (negative), TPS 1% to 49% (weakly positive), and TPS ?50% (strongly positive). To assess agreement of the PD-L1 TPS between histologic and cytologic samples, weighted ? values (linear weights) were calculated. Furthermore, the Cohen ? was calculated using data dichotomized according to the 1% and 50% cutoffs. Overall percent agreement (OPA), positive percent agreement, and negative percent agreement were determined for both cutoffs using histology as the reference standard. In addition, the McNemar-Bowker test of symmetry was applied to assess whether the categorization of PD-L1 expression differed significantly between histologic and cytologic samples. P values <.05 were considered statistically significant. Both interobserver and interassay agreement were assessed using weighted ? and Cohen ? values for the 1% and 50% cutoffs. Statistical analysis was performed using RStudio version 1.1.456 (R Foundation for Statistical Computing) and IBM SPSS Statistics version 25 (IBM Corporation). Funding Information: Bregje M. Koomen reports grants from AstraZeneca, Merck Sharp & Dohme, and Roche Diagnostics, outside the submitted work. Jan H. von der Thüsen reports personal fees from Merck Sharp & Dohme, during the conduct of the study. Kim Monkhorst reports grants and personal fees from AstraZeneca; personal fees from Merck Sharp & Dohme, Benecke, Bristol‐Myers Squibb, AbbVie, Diaceutics, Lilly, Bayer, and Boehringer Ingelheim; personal fees and nonfinancial support from Roche Diagnostics; and nonfinancial support from PGDX, Delfi, and Takeda, all outside the submitted work. Stefan M. Willems reports grants from Bristol‐Myers Squibb, NextCure, Pfizer Inc, Roche Diagnostics, Merck Sharp & Dohme, Amgen, AstraZeneca, and Bayer, outside the submitted work. Wim Timens reports personal fees from Roche Diagnostics/Ventana, Merck Sharp & Dohme, Bristol‐Myers Squibb, and AbbVie, outside the submitted work. The remaining authors made no disclosures. Publisher Copyright: © 2020 The Authors. Cancer Cytopathology published by Wiley Periodicals LLC on behalf of American Cancer Society
(Peer reviewed)
