Impact of Decalcification, Cold Ischemia, and Deglycosylation on Performance of Programmed Cell Death Ligand-1 Antibodies With Different Binding Epitopes: Comparison of 7 Clones.

Programmed cell death ligand-1 (PD-L1) expression levels in patients' tumors have demonstrated clinical utility across many cancer types and are used to determine treatment eligibility. Several independently developed PD-L1 immunohistochemical (IHC) predictive assays are commercially available and have demonstrated different levels of staining between assays, generating interest in understanding the similarities and differences between assays. Previously, we identified epitopes in the internal and external domains of PD-L1, bound by antibodies in routine clinical use (SP263, SP142, 22C3, and 28-8). Variance in performance of assays utilizing these antibodies, observed following exposure to preanalytical factors such as decalcification, cold ischemia, and duration of fixation, encouraged additional investigation of antibody-binding sites, to understand whether binding site structures/conformations contribute to differential PD-L1 IHC assay staining. We proceeded to further investigate the epitopes on PD-L1 bound by these antibodies, alongside the major clones utilized in laboratory-developed tests (E1L3N, QR1, and 73-10). Characterization of QR1 and 73-10 clones demonstrated that both bind the PD-L1 C-terminal internal domain, similar to SP263/SP142. Our results also demonstrate that under suboptimal decalcification or fixation conditions, the performance of internal domain antibodies is less detrimentally affected than that of external domain antibodies 22C3/28-8. Furthermore, we show that the binding sites of external domain antibodies are susceptible to deglycosylation and conformational structural changes, which directly result in IHC staining reduction or loss. The binding sites of internal domain antibodies were unaffected by deglycosylation or conformational structural change. This study demonstrates that the location and conformation of binding sites, recognized by antibodies employed in PD-L1 diagnostic assays, differ significantly and exhibit differing degrees of robustness. These findings should reinforce the need for vigilance when performing clinical testing with different PD-L1 IHC assays, particularly in the control of cold ischemia and the selection of fixation and decalcification conditions.

Comparison of continuous measures across diagnostic PD-L1 assays in non-small cell lung cancer using automated image analysis.

Tumor programmed cell death ligand-1 (PD-L1) expression is a key biomarker to identify patients with non-small cell lung cancer who may have an enhanced response to anti-programmed cell death-1 (PD-1)/PD-L1 treatment. Such treatments are used in conjunction with PD-L1 diagnostic immunohistochemistry assays. We developed a computer-aided automated image analysis with customized PD-L1 scoring algorithm that was evaluated via correlation with manual pathologist scores and used to determine comparability across PD-L1 immunohistochemistry assays. The image analysis scoring algorithm was developed to quantify the percentage of PD-L1 positive tumor cells on scans of whole-slide images of archival tumor samples from commercially available non-small cell lung cancer cases, stained with four immunohistochemistry PD-L1 assays (Ventana SP263 and SP142 and Dako 22C3 and 28-8). The scans were co-registered and tumor and exclusion annotations aligned to ensure that analysis of each case was restricted to comparable tissue areas. Reference pathologist scores were available from previous studies. F1, a statistical measure of precision and recall, and overall percentage agreement scores were used to assess concordance between pathologist and image analysis scores and between immunohistochemistry assays. In total, 471 PD-L1-evalulable samples were amenable to image analysis scoring. Image analysis and pathologist scores were highly concordant, with F1 scores ranging from 0.8 to 0.9 across varying matched PD-L1 cutoffs. Based on F1 and overall percentage agreement scores (both manual and image analysis scoring), the Ventana SP263 and Dako 28-8 and 22C3 assays were concordant across a broad range of cutoffs; however, the Ventana SP142 assay showed very different characteristics. In summary, a novel automated image analysis scoring algorithm was developed that was highly correlated with pathologist scores. The algorithm permitted quantitative comparison of existing PD-L1 diagnostic assays, confirming previous findings that indicate a high concordance between the Ventana SP263 and Dako 22C3 and 28-8 PD-L1 immunohistochemistry assays.

Mapping the binding sites of antibodies utilized in programmed cell death ligand-1 predictive immunohistochemical assays for use with immuno-oncology therapies.

Programmed cell death ligand-1 (PD-L1) expression levels in patient tumor samples have proven clinical utility across various cancer types. Several independently developed PD-L1 immunohistochemical (IHC) predictive assays are commercially available. Published studies using the VENTANA PD-L1 (SP263) Assay, VENTANA PD-L1 (SP142) Assay, Dako PD-L1 IHC 22C3 pharmDx assay, Dako PD-L1 IHC 28-8 pharmDx assay, and laboratory-developed tests utilizing the E1L3N antibody (Cell Signaling Technology), have demonstrated differing levels of PD-L1 staining between assays, resulting in conjecture as to whether antibody-binding epitopes could be responsible for discordance between assays. Therefore, to understand the performance of different PD-L1 predictive immunohistochemistry assays, we aimed to distinguish the epitopes within the PD-L1 protein responsible for antibody binding. The sites at which antibody clones SP263, SP142, 22C3, 28-8, and E1L3N bind to recombinant PD-L1 were assessed using several methods, including conformational peptide array, surface plasmon resonance, and/or hydrogen/deuterium exchange mass spectrometry. Putative binding sites were confirmed by site-directed mutagenesis of PD-L1, followed by western blotting and immunohistochemical analysis of cell lines expressing mutant constructs. Our results demonstrate that clones SP263 and SP142 bind to an identical epitope in the cytoplasmic domain at the extreme C-terminus of PD-L1, distinct from 22C3 and 28-8. Using mutated PD-L1 constructs, an additional clone, E1L3N, was also found to bind to the cytoplasmic domain of PD-L1. The E1L3N binding epitope overlaps considerably with the SP263/SP142 binding site but is not identical. Clones 22C3 and 28-8 have binding profiles in the extracellular domain of PD-L1, which differ from one another. Despite identifying epitope binding variance among antibodies, evidence indicates that only the SP142 assay generates significantly discordant immunohistochemical staining, which can be resolved by altering the assay protocol. Therefore, inter-assay discordances are more likely attributable to tumor heterogeneity, assay, or platform variables rather than antibody epitope.

Correction: Mapping the binding sites of antibodies utilized in programmed cell death ligand-1 predictive immunohistochemical assays for use with immuno-oncology therapies.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Tumor Mutational Burden as a Predictor of Survival with Durvalumab and/or Tremelimumab Treatment in Recurrent or Metastatic Head and Neck Squamous Cell Carcinoma.

PURPOSE: Biomarkers that predict response to immune checkpoint inhibitors (ICI) in recurrent or metastatic head and neck squamous cell carcinoma (R/M HNSCC) are needed. This retrospective study assessed tumor mutational burden (TMB) and outcomes in the phase II HAWK and CONDOR and phase III EAGLE studies of durvalumab with or without tremelimumab in platinum-resistant R/M HNSCC. PATIENTS AND METHODS: Tumor samples from HAWK/CONDOR (N = 153) and blood samples from EAGLE (N = 247) were analyzed for TMB. Associations with survival were evaluated for tissue TMB (tTMB) at cutoffs from 10 to 20 mutations/megabase (mut/Mb) and for blood plasma TMB (bTMB) at cutoffs from 8 to 24 mut/Mb. RESULTS: In HAWK/CONDOR, overall survival (OS) with durvalumab with or without tremelimumab was longer for high versus low tTMB: statistically significant differences were observed with durvalumab plus tremelimumab at tTMB ≥ 10 mut/Mb [HR, 0.52 (95% confidence interval, CI, 0.28-0.98)] and tTMB ≥ 12 mut/Mb [HR, 0.46 (95% CI, 0.24-0.86)]. In EAGLE, a significant OS benefit versus chemotherapy was observed with durvalumab and durvalumab plus tremelimumab at bTMB≥16 mut/Mb [HR, 0.39 (95% CI, 0.20-0.76) and 0.38 (95% CI, 0.19-0.78), respectively] but not bTMB < 16 mut/Mb [HR, 0.92 (0.61-1.37) and 0.92 (95% CI, 0.62-1.36), respectively]. A significant progression-free survival benefit was also observed in the ICI arms versus chemotherapy at bTMB ≥ 16 mut/Mb. CONCLUSIONS: Findings support TMB as a biomarker for predicting survival in patients with platinum-resistant R/M HNSCC treated with ICIs. The analysis of EAGLE demonstrated that bTMB was predictive of survival with ICI treatment versus chemotherapy in a large, randomized controlled study population.

Domain Adaptation-Based Deep Learning for Automated Tumor Cell (TC) Scoring and Survival Analysis on PD-L1 Stained Tissue Images.

We report the ability of two deep learning-based decision systems to stratify non-small cell lung cancer (NSCLC) patients treated with checkpoint inhibitor therapy into two distinct survival groups. Both systems analyze functional and morphological properties of epithelial regions in digital histopathology whole slide images stained with the SP263 PD-L1 antibody. The first system learns to replicate the pathologist assessment of the Tumor Cell (TC) score with a cut-point for positivity at 25% for patient stratification. The second system is free from assumptions related to TC scoring and directly learns patient stratification from the overall survival time and event information. Both systems are built on a novel unpaired domain adaptation deep learning solution for epithelial region segmentation. This approach significantly reduces the need for large pixel-precise manually annotated datasets while superseding serial sectioning or re-staining of slides to obtain ground truth by cytokeratin staining. The capacity of the first system to replicate the TC scoring by pathologists is evaluated on 703 unseen cases, with an addition of 97 cases from an independent cohort. Our results show Lin's concordance values of 0.93 and 0.96 against pathologist scoring, respectively. The ability of the first and second system to stratify anti-PD-L1 treated patients is evaluated on 151 clinical samples. Both systems show similar stratification powers (first system: HR = 0.539, p = 0.004 and second system: HR = 0.525, p = 0.003) compared to TC scoring by pathologists (HR = 0.574, p = 0.01).

Evaluation of an online training tool for scoring programmed cell death ligand-1 (PD-L1) diagnostic tests for lung cancer.

BACKGROUND: Numerous studies indicate that higher tumour programmed cell death ligand-1 (PD-L1) expression is associated with greater response to anti-programmed cell death-1 (PD-1)/PD-L1 immunotherapy in non-small cell lung cancer (NSCLC). In the era of precision medicine, there is a need to provide reliable, standardised training for pathologists to improve their accuracy of interpretation and scoring, as the results are used directly to inform clinical decisions. Here we present findings regarding reader reproducibility of PD-L1 tumour cell (TC) staining scoring for NSCLC using a PD-L1 e-trainer tool as part of a PD-L1 immunohistochemistry reader training course. METHODS: The PD-L1 training course was developed based on the use of VENTANA PD-L1 (SP263) and Dako PD-L1 IHC PharmDx 22C3 stained NSCLC samples in combination with a PD-L1 e-trainer tool. Five-hundred formalin-fixed, paraffin-embedded archival samples were obtained from commercial sources and stained for PD-L1. Slides were scored by two expert pathologists, then scanned to produce digital images and re-scored. Thirty-three cases were selected and sorted into three sets: a training set and two self-assessment tests (pre-test and 'competence' test). Participants (all selected board-certified pathologists) received face-to-face training including use of an e-trainer tool. Statistical analyses were performed using the competence test set. Overall percentage agreement (OPA) was assessed between the participant pathologists' registered scores and the reference scores assigned by expert pathologists at clinically relevant PD-L1 cut-offs (≥1%, ≥25% and ≥ 50%). RESULTS: Seven sessions were held and 69 participant pathologists completed the training. Inter-reader concordance indicated high OPA (85-95%) for PD-L1 TC scoring at clinically relevant cut-offs, with Fleiss' Kappa > 0.5. CONCLUSIONS: Use of this web-based training tool incorporated into classroom-style training was associated with an overall moderately good level of inter-reader reproducibility at key cut-offs for TC PD-L1 expression testing in NSCLC. Overall, the online training tool offers a means of standardised training for practising pathologists in a clinical setting.

Programmed Cell Death Ligand 1 Expression in Untreated EGFR Mutated Advanced NSCLC and Response to Osimertinib Versus Comparator in FLAURA.

INTRODUCTION: EGFR mutated (EGFRm) NSCLC tumors occasionally express programmed cell death ligand 1 (PD-L1), although frequency and clinical relevance are not fully characterized. We report PD-L1 expression in patients with EGFRm advanced NSCLC and association with clinical outcomes following treatment with osimertinib or comparator EGFR tyrosine kinase inhibitors in the FLAURA trial (phase III, NCT02296125). METHODS: Of 231 tissue blocks available from the screened population (including EGFRm-positive and -negative samples), 197 had sufficient tissue for PD-L1 testing using the SP263 (Ventana, Tucson, Arizona) immunohistochemical assay. Tumor cell (TC) staining thresholds of PD-L1 TC greater than or equal to 1%, TC greater than or equal to 25%, and TC greater than or equal to 50% were applied. Progression-free survival (PFS) was investigator-assessed, per Response Evaluation Criteria in Solid Tumor, version 1.1, according to PD-L1 expressors (TC ≥ 1%) or negatives (TC < 1%) in randomized patients. RESULTS: PD-L1 staining was successful in 193 of 197 patient formalin-fixed paraffin-embedded blocks; of these, 128 of 193 were EGFRm-positive and 106 of 128 patients were randomized to treatment (osimertinib: 54; comparator: 52). At the PD-L1 TC greater than or equal to 25% threshold, 8% (10 of 128) of EGFRm-positive tumors expressed PD-L1 versus 35% (23 of 65) of EGFRm-negative tumors. With the TC greater than or equal to 1% threshold, 51% (65 of 128) versus 68% (44 of 65) were mutation-positive and -negative, respectively, and with the TC greater than or equal to 50% threshold, 5% (7 of 128) versus 28% (18 of 65), were mutation-positive and -negative, respectively. For PD-L1 expressors (TC ≥ 1%), median PFS was 18.4 months with osimertinib and 6.9 months with comparator (hazard ratio = 0.30; 95% confidence interval: 0.15-0.60). For PD-L1-negative patients (TC < 1%), median PFS was 18.9 months with osimertinib and 10.9 months with comparator (hazard ratio = 0.37; 95% confidence interval: 0.17-0.74). CONCLUSIONS: Clinical benefit with osimertinib was unaffected by PD-L1 expression status.

Interobserver Reliability of Programmed Cell Death Ligand-1 Scoring Using the VENTANA PD-L1 (SP263) Assay in NSCLC.

INTRODUCTION: The VENTANA PD-L1 (SP263) Assay is approved for use with anti-programmed cell death-1/programmed cell death ligand-1 (PD-1/PD-L1) therapies in NSCLC and urothelial carcinoma. Here, we investigate interobserver reliability of the SP263 assay, applied to PD-L1 scoring of tumor cells (TCs) in NSCLC. METHODS: Six practicing European pulmonary pathologists independently scored the proportion of TCs expressing PD-L1 (TC score) from 200 archival, commercially sourced, formalin-fixed paraffin-embedded NSCLC resections stained using the SP263 assay. Agreement in scores was analyzed using the intraclass correlation coefficient and concordance in patient's classification using Fleiss' kappa. RESULTS: Results from 172 samples showed strong pair-wise correlations between pathologists (R2 >0.89) for TC scoring with an intraclass correlation coefficient of 0.96. Overall agreement was greater than 90% for TC of 1% and above, and greater than 94% for TCs of at least 25% and at least 50%. Fleiss' kappa showed substantial agreement for TC of 1% and above, and almost perfect agreement for TCs of at least 25% and at least 50%. CONCLUSIONS: Assessment of TC score in NSCLC was highly reproducible using the SP263 assay, building confidence in the accuracy of this assay in selection of patients for anti-PD-1/PD-L1 therapy.

Assessment of PD-L1 mRNA and protein expression in non-small cell lung cancer, head and neck squamous cell carcinoma and urothelial carcinoma tissue specimens using RNAScope and immunohistochemistry.

Four immunohistochemistry (IHC) diagnostic assays have been approved for tumour PD-L1 protein assessment in the clinic. However, mRNA detection by in situ hybridisation (ISH) could be utilised as an alternative to protein detection. Detecting spatial changes in gene expression provides vital prognostic and diagnostic information, particularly in immune oncology where the phenotype, cellular infiltration and immune activity status may be associated with patient survival. Translation of mRNA expression to a clinically relevant cut off or threshold is challenging due to variability between assays and the detection of different analytes. These studies aim to confirm the suitability of formalin fixed paraffin embedded (FFPE) tissue sections for use with RNA ISH. A comparison of mRNA expression and protein expression may inform the suitability of mRNA as a patient selection biomarker in a similar manner to IHC and provide evidence of a suitable scoring algorithm. Ninety patient samples, thirty for each indication of non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC) and urothelial carcinoma (UC), previously assessed using the VENTANA PD-L1 (SP263) Assay were chosen to represent a wide dynamic range of percentage tumour cell staining (TCIHC). Expression of mRNA was assessed by ISH using the RNAScope 2.5 assay and probe CD274/PD-L1 (Advanced Cell Diagnostics) including kit provided positive and negative control probes. Brightfield whole slide images of tissues were captured. The percentage of tumour cells with PD-L1 mRNA expression (%TCmRNA) and mean punctate dots/tumour cell were determined using image analysis. Differences in RNA expression between the IHC derived TCIHC≥25% and <25% groups were assessed using t-tests. For each indication, a receiver-operating characteristic (ROC) analysis identified thresholds for patient classification using %TCmRNA and dots/tumour cell, with reference to TCIHC≥25%. Eighty-six samples were successfully tested; 3 failed due to insufficient control probe staining, 1 due to lack of tumour. Percent TCmRNA staining using RNAScope demonstrated statistical significance (at α = 0.05) in the PD-L1 high (TCIHC ≥25%) vs the PD-L1 low (TCIHC <25%) groups for NSCLC, HNSCC, and UC. The number of punctate dots/tumour cell was significantly higher in the PD-L1 high vs the PD-L1 low groups for NSCLC and HNSCC but not UC. For %TCmRNA; ROC analysis identified thresholds of: NSCLC 18.0%, HNSCC 31.8%, UC 25.8%. For dots/tumour cell, thresholds were: NSCLC 0.26, HNSCC 0.53, UC 0.45. Routine tissue fixation and processing is suitable for RNA detection using RNAScope. PD-L1 mRNA extent and level is associated with PD-L1 status determined by IHC. Threshold optimisation for %TCmRNA and mean dots/tumour cell results in high specificity to IHC PD-L1 classification, but only moderate sensitivity.

Fast fluorescence in situ hybridisation for the enhanced detection of MET in non-small cell lung cancer.

The c-Met/hepatocyte growth factor receptor pathway is frequently dysregulated in multiple cancer types, including non-small cell lung cancer (NSCLC). MET amplification has been shown to develop as a resistance mechanism to treatment in NSCLC. The identification of increased MET copy number within tumour cells is increasingly important to stratify those tumours and patients which are susceptible to treatment targetting MET kinase inhibition. Fluorescence in situ hybridisation (FISH) has been successfully employed to identify patients with abnormal MET gene copy number with numerous probes available for use. Here we report a FISH protocol that reduces probe hybridisation time in NSCLC tissue to 1 hour and compare the results with other protocols. MET gene copy number was determined in 20 NSCLC cases using 3 FISH probes: 1. Kreatech FISH, MET (7q31) SE 7 ready to use probes, hybridised using an overnight protocol; 2. Dako MET IQFISH probe with CEP7 ready to use probe, hybridised for 2 hours; 3. Kreatech MET (7q31) SE 7 XL FISH probe, prepared in SwiftFISH buffer and hybridised for 1 hour. The MET gene copy number and MET: centromere 7 gene ratio were determined for each tissue and cases categorised as having MET high or MET low status. All three FISH probes were shown to demonstrate good agreement with each other. Overall percentage agreement between probes was ≥90%. Intraclass correlation showed good agreement (ICC ≥0.80) between all three assays for MET gene copy number and MET: centromere 7 gene ratio. These FISH protocols provide evidence that rapid laboratory developed FISH assays with short turnaround time perform consistently with standard protocols, potentially enabling faster treatment decisions.

Concordance among four commercially available, validated programmed cell death ligand-1 assays in urothelial carcinoma.

BACKGROUND: Antibodies targeting the programmed cell death-1 (PD-1)/PD-ligand 1 (PD-1/PD-L1) checkpoint have shown promising clinical activity in patients with advanced urothelial carcinoma (UC). Expression of PD-L1 in UC tumors has been investigated using different antibody clones, staining protocols, and scoring algorithms. The aim was to establish the extent of concordance among PD-L1 immunohistochemistry (IHC) assays. METHODS: Tumor biopsy samples (N = 335) were assessed using four commercially available PD-L1 assays: VENTANA SP263, VENTANA SP142, PD-L1 IHC 28-8 pharmDx, and PD-L1 IHC 22C3 pharmDx. PD-L1 analytical staining and classification concordance, including agreement between clinically relevant scoring algorithms, were investigated using overall/positive/negative percentage agreement (OPA/PPA/NPA). RESULTS: Good analytical correlation was observed among the VENTANA SP263, PD-L1 IHC 22C3 pharmDx, and PD-L1 IHC 28-8 pharmDx assays for tumor cell (TC) and immune cell (IC) PD-L1 staining with Spearman rank coefficients of 0.92-0.93 for TCs and 0.88-0.91 for ICs. However, concordance (preset criterion: ≥85%) between patient PD-L1 status when applying the TC or ICICArea ≥ 25% (VENTANA SP263) cutoff was only achieved for PD-L1 IHC 22C3 pharmDx versus VENTANA SP263 (OPA 92.2%, PPA 86.4%, NPA 95.4%). Differences were observed between patient populations with UC tumors classified as PD-L1 high versus PD-L1 low/negative using combined positive score (CPS) ≥1, CPS ≥10, IC ≥5%, and TC/IC ≥25%. CONCLUSIONS: The VENTANA SP263 and PD-L1 IHC 22C3 pharmDx assays are analytically similar in UC. When the different PD-L1 assays were combined with their specified clinical scoring algorithms, differences were seen in patient classification driven by substantial differences in scoring approaches.

Analytical Validation and Clinical Utility of an Immunohistochemical Programmed Death Ligand-1 Diagnostic Assay and Combined Tumor and Immune Cell Scoring Algorithm for Durvalumab in Urothelial Carcinoma.

CONTEXT.­: Clinical responses to anti-programmed death receptor-1 and anti-programmed death ligand-1 (PD-L1) agents are generally improved in patients with high PD-L1 expression compared with those with low/negative expression across several tumor types, including urothelial carcinoma. OBJECTIVE.­: To validate a PD-L1 immunohistochemical diagnostic test in urothelial carcinoma patients treated with the anti-PD-L1 monoclonal antibody durvalumab. DESIGN.­: The Ventana PD-L1 (SP263) assay was validated for intended use in urothelial carcinoma formalin-fixed, paraffin-embedded samples in studies addressing sensitivity, specificity, robustness, and precision, and implemented in study CD-ON-MEDI4736-1108 (NCT01693562). Efficacy was analyzed in patients classified according to prespecified PD-L1 expression cutoffs: PD-L1 high (if >1% of the tumor area contained tumor-associated immune cells, ≥25% of tumor cells or ≥25% of immune cells stained for PD-L1; if ≤1% of the tumor area contained immune cells, ≥25% of tumor cells or 100% of immune cells stained for PD-L1) and PD-L1 low/negative (did not meet criteria for PD-L1 high). RESULTS.­: The assay met all predefined acceptance criteria for sensitivity, specificity, and precision. Interreader and intrareader precision overall agreement were 93.0% and 92.4%, respectively. For intraday reproducibility and interday precision, overall agreement was 99.2% and 100%, respectively. Interlaboratory overall agreement was 92.6%. In study CD-ON-MEDI4736-1108, durvalumab demonstrated clinical activity and durable responses in both PD-L1-high and PD-L1-low/negative subgroups, although objective response rates tended to be higher in the PD-L1-high subgroup than in the PD-L1-low/negative subgroup. CONCLUSIONS.­: Determination of PD-L1 expression in urothelial carcinoma patients using the Ventana PD-L1 (SP263) assay was precise, highly reproducible, and clinically relevant.

Consistency of tumor and immune cell programmed cell death ligand-1 expression within and between tumor blocks using the VENTANA SP263 assay.

BACKGROUND: Several anti-programmed cell death-1 (PD-1) and anti-programmed cell death ligand-1 (PD-L1) therapies have shown encouraging safety and clinical activity in a variety of tumor types. A potential role for PD-L1 testing in identifying patients that are more likely to respond to treatment is emerging. PD-L1 expression in clinical practice is determined by testing one tumor section per patient. Therefore, it is critical to understand the impact of tissue sampling variability on patients' PD-L1 classification. METHODS: Resected non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC) and urothelial carcinoma (UC) tissue samples (five samples per tumor type) were obtained from commercial sources and two tumor blocks were taken from each. Three sections from each block (~ 100 µm apart) were stained using the VENTANA PD-L1 (SP263) assay, and scored based on the percentage of PD-L1-staining tumor cells (TCs) or tumor-infiltrating immune cells (ICs) present. Each section was categorized as PD-L1 high or low/negative using a variety of cut-off values, and intra-block and intra-case (between blocks of the same tumor) concordance (overall percentage agreement [OPA]) were evaluated. An additional 200 commercial NSCLC samples were also analyzed, and intra-block concordance determined by scoring two sections per sample (≥70 µm apart). RESULTS: Concordance in TC PD-L1 classification was high at all applied cut-offs. Intra-block and intra-case OPA for the 15 NSCLC, HNSCC or UC samples were 100% and 80-100%, respectively, across all cut-offs; intra-block OPA for the 200 NSCLC samples was 91.0-98.5% across all cut-offs. IC PD-L1 classification was less consistent; intra-block and intra-case OPA for the 15 NSCLC, HNSCC or UC samples ranged between 70 and 100% and between 60 and 100%, respectively, with similar observations in the intra-block analysis of the 200 NSCLC samples. CONCLUSIONS: These results show the reproducibility of TC PD-L1 classification across the depth of the tumor using the VENTANA PD-L1 (SP263) assay. Practically, this means that treatment decisions based on TC PD-L1 classification can be made confidently, following analysis of one tumor section. Although more variable than TC staining, consistent IC PD-L1 classification was also observed within and between blocks and across cut-offs.

Relevance of deep learning to facilitate the diagnosis of HER2 status in breast cancer.

Tissue biomarker scoring by pathologists is central to defining the appropriate therapy for patients with cancer. Yet, inter-pathologist variability in the interpretation of ambiguous cases can affect diagnostic accuracy. Modern artificial intelligence methods such as deep learning have the potential to supplement pathologist expertise to ensure constant diagnostic accuracy. We developed a computational approach based on deep learning that automatically scores HER2, a biomarker that defines patient eligibility for anti-HER2 targeted therapies in breast cancer. In a cohort of 71 breast tumour resection samples, automated scoring showed a concordance of 83% with a pathologist. The twelve discordant cases were then independently reviewed, leading to a modification of diagnosis from initial pathologist assessment for eight cases. Diagnostic discordance was found to be largely caused by perceptual differences in assessing HER2 expression due to high HER2 staining heterogeneity. This study provides evidence that deep learning aided diagnosis can facilitate clinical decision making in breast cancer by identifying cases at high risk of misdiagnosis.

PD-L1 expression in advanced NSCLC: Insights into risk stratification and treatment selection from a systematic literature review.

Tumors can evade immune detection by exploiting inhibitory immune checkpoints such as the programmed cell death-1 (PD-1)/programmed cell death ligand-1 (PD-L1) pathway. Antibodies that block this pathway offer a promising new approach to treatment in advanced/metastatic non-small cell lung cancer (NSCLC). A systematic review of the literature was conducted to assess the association of PD-L1 with important patient and disease characteristics, the prognostic significance of PD-L1 expressing NSCLC tumors, and the value of PD-L1 as a predictive biomarker of response to anti-PD-1/PD-L1 treatments in advanced/metastatic NSCLC. A total of 35 eligible studies were selected for analysis. Methods used to determine PD-L1 in NSCLC tissue varied considerably; with different PD-L1 antibodies, antibody detection methods, and staining cut-offs. Immunohistochemistry was the most frequent type of PD-L1 assay. Overall, study evidence did not support an association between PD-L1 expression and gender, age, smoking history, tumor histology (adenocarcinoma vs. squamous cell carcinoma), performance status, pathologic tumor grade or EGFR/KRAS/ALK mutational status. In several studies, high PD-L1 expression was associated with shorter survival compared with low expression. Most evidence indicated that patients with high vs. low PD-L1 expression were more likely to experience treatment benefit with anti-PD-1/PD-L1 agents (nivolumab, pembrolizumab, durvalumab, atezolizumab, and avelumab) in advanced NSCLC. Variability in the methods used to determine PD-L1 expression in NSCLC tissue suggests a need for standardized use of well-validated PD-L1 diagnostic assays. Although considerable research links PD-L1 expression in tumors to shorter survival in advanced/metastatic NSCLC, its use as a prognostic factor requires more study. As studies of anti-PD-1/PD-L1 agents continue, PD-L1 is likely to play an important role as a predictive biomarker for selecting patients deriving most benefit from anti-PD-1/PD-L1 monotherapy and directing patients with lower levels of tumor PD-L1 expression (with a high unmet medical need), to alternative treatments, such as combination immunotherapies.

Minimum lesion detectability as a measure of PET system performance.

BACKGROUND: A phantom in combination with an imaging protocol was developed to measure the limit of small lesion detection on different PET systems. Seven small spheres with inner diameters ranging from 3.95 up to 15.43 mm were imaged in a Jaszczak ECT Phantom, in air, in a cold background, and with sphere to background contrast ratios of 15:1 down to 1.88:1. The imaging times varied from 1 to 16 min. The imaging protocol was performed on the Gemini TF and Vereos by Philips, the mCT and HRRT by Siemens, and the Discovery 710 by General Electric. For each scanning condition, the images were reconstructed with image voxel sizes of 1 to 4 mm cubic voxels. The reconstruction method used for each system was the one recommended by the manufacture to achieve best small image lesion detection results. A human observer study was performed to determine the smallest observable sphere for each scanning condition. RESULTS: All systems were able to image the smallest sphere of 3.95 mm inner diameter at the 15 to 1 signal to background ratio when imaged for 16 min. For a typical whole body per bed position scan time of 2 to 4 min, the smallest imaged sphere varied between 4.95 and 6.23 mm at the 15:1 contrast ratio and 12.43 and 15.43 mm at a contrast ratio of 1.88:1. In general, all systems were consistent with the Rose criteria when determining lesion detectability. CONCLUSIONS: Besides demonstrating that the current state of the art clinical PET/CT systems have the same lesion detection ability, the study demonstrates how sensitive scan time can be to detecting small lesions which have a relatively small contrast uptake in the range of just 2:1. This should help guide imaging protocols to use longer scan times over regions of the subject in which small lesions are suspect.

Agreement between Programmed Cell Death Ligand-1 Diagnostic Assays across Multiple Protein Expression Cutoffs in Non-Small Cell Lung Cancer.

Purpose: Immunotherapies targeting programmed cell death-1 (PD-1) and programmed cell death ligand-1 (PD-L1) demonstrate encouraging antitumor activity and manageable tolerability in non-small cell lung cancer (NSCLC), especially in patients with high tumor PD-L1 expression, as detected by companion or complementary diagnostic assays developed for individual agents. A laboratory is unlikely to use multiple assay platforms. Furthermore, commercially available diagnostic assays are not standardized, and different assay methods could lead to inappropriate treatment selection. This study establishes the extent of concordance between three validated, commercially available PD-L1 IHC diagnostic assays for NSCLC patients [Ventana SP263 (durvalumab), Dako 22C3 (pembrolizumab), and Dako 28-8 (nivolumab)].Experimental Design: Five hundred formalin-fixed, paraffin-embedded archival NSCLC samples were obtained from commercial sources. Stained slides were read in batches on an assay-by-assay basis by a single pathologist trained in all methods, in a Clinical Laboratory Improvements Amendments program-certified laboratory. An additional pathologist performed an independent review of 200 stained samples for each assay.Results: PD-L1 expression was evaluable with all assays in 493 samples. The three assays showed similar patterns of tumor membrane staining, with high correlation between percent PD-L1 staining. An overall percentage agreement of >90% was achieved between assays at multiple expression cutoffs, including 1%, 10%, 25%, and 50% tumor membrane staining.Conclusions: This study builds optimism that harmonization between assays may be possible, and that the three assays studied could potentially be used interchangeably to identify patients most likely to respond to anti-PD-1/PD-L1 immunotherapies, provided the appropriate clinically defined algorithm and agent are always linked. Clin Cancer Res; 23(14); 3585-91. ©2017 AACR.

Nondestructive quality control of HER2 control cell line sections: the use of interferometry for measuring section thickness and implications for HER2 interpretation on breast tissue.

The field of companion diagnostics is an expanding area in which industry can make significant contributions to the quality and reliability of prognostic and predictive assays. HER2 testing by immunohistochemistry and in situ hybridization, by virtue of its semi-quantitative nature demands the highest level of reagent quality and consistency. We describe a unique manufacturing practice that is used to guarantee the quality of control cell line slides provided for controlling the staining procedure and ensuring the validity of the reagent performance. An IHC control that could reflect variations in fixation, processing, section thickness, section storage and staining protocols would provide a useful tool for improved standardization. One way to ensure such accuracy and reproducibility of HER2 testing is by using robust internal controls. We intend to present for the first time, details of a unique manufacturing process, that of interferometry, the technique of using the pattern of interference created by the superposition of 2 or more waves to diagnose the properties of the aforementioned waves, that is, "section thickness," to guarantee the quality and consistency of internal cell line controls that can be used in a HER2 testing assay.