Neuroendocrine differentiation in breast carcinoma: clinicopathological features and outcome.

AIMS: Primary neuroendocrine (NE) breast carcinoma (BC) is an entity with a wide range of prevalence and poorly defined clinical behaviour. We evaluated the prevalence, clinicopathological features and clinical outcome of NEBC. METHODS AND RESULTS: Immunohistochemical staining for synaptophysin and chromogranin A was performed on whole sections from 1232 consecutive cases of invasive BC. We divided NEBC into focal (10-49% positive cells) and diffuse (≥50% positive cells) and compared the outcome of patients with NEBC with strictly matched non-NEBC. A total of 128 BC showed NE differentiation (10.4%): 84 diffuse (6.8%) and 44 focal (3.6%). NE differentiation showed a significant association with T4 stage (P = 0.001), solid-papillary and mucinous histotype (P < 0.0001), G2 grading (P = 0.002), positive oestrogen receptor (ER) (P = 0.003) and progesterone receptor (PR) (P = 0.002). Almost 90% of NEBC were ER(+) /HER2(-) and more than half ER(+) /HER2(-) /Ki67≥14%. Kaplan-Meier analysis revealed that patients with NEBC showed worse disease-free survival (DFS) (P = 0.04) compared to matched non-NEBC. We did not find significant differences regarding clinicopathological features, DFS and CSS between diffuse and focal neuroendocrine BC. CONCLUSIONS: This study demonstrates that NEBC represents 7-10% of invasive BC and that NE differentiation does not affect the prognosis of BC in terms of CSS.

Comparison of three validated PD-L1 immunohistochemical assays in urothelial carcinoma of the bladder: interchangeability and issues related to patient selection.

Different programmed cell death-ligand 1 (PD-L1) assays and scoring algorithms are being used in the evaluation of PD-L1 expression for the selection of patients for immunotherapy in specific settings of advanced urothelial carcinoma (UC). In this paper, we sought to investigate three approved assays (Ventana SP142 and SP263, and Dako 22C3) in UC with emphasis on implications for patient selection for atezolizumab/pembrolizumab as the first line of treatment. Tumors from 124 patients with invasive UC of the bladder were analyzed using tissue microarrays (TMA). Serial sections were stained with SP263 and SP142 on Ventana Benchmark Ultra and with 22C3 on Dako Autostainer Link 48. Stains were evaluated independently by two observers and scored using the combined positive score (CPS) and tumor infiltrating immune cells (IC) algorithms. Differences in proportions (DP), overall percent agreement (OPA), positive percent agreement (PPA), negative percent agreement (NPA), and Cohen κ were calculated for all comparable cases. Good overall concordance in analytic performance was observed for 22C3 and SP263 with both scoring algorithms; specifically, the highest OPA was observed between 22C3 and SP263 (89.6%) when using CPS. On the other hand, SP142 consistently showed lower positivity rates with high differences in proportions (DP) compared with 22C3 and SP263 with both CPS and IC, and with a low PPA, especially when using the CPS algorithm. In conclusion, 22C3 and SP263 assays show comparable analytical performance while SP142 shows divergent staining results, with important implications for the selection of patients for both pembrolizumab and atezolizumab.

Expression of programmed cell death ligand 1 in non-small cell lung cancer: Comparison between cytologic smears, core biopsies, and whole sections using the SP263 assay.

BACKGROUND: Evaluation of programmed cell death ligand 1 (PD-L1) expression can be made on both resection specimens and diagnostic biopsies; however, more than 30% of patients with advanced non-small cell lung cancer (NSCLC) do not have adequate histologic material to perform PD-L1 assays and require additional biopsies. In addition, in our practice, more than 16% of cases have cytological smears as the only available material. Our aim was to validate the PD-L1 immunocytochemistry assay on cytological smears and compare its accuracy with the results obtained from tissue cores and whole tumor sections using the clinically relevant cutoff of 50%. METHOD: We compared the PD-L1 staining results of cytological smears to those from tissue cores or whole sections in 50 and 53 NSCLC cases, respectively, using the SP263 assay after scanning hematoxylin and eosin slides. RESULTS: We found an overall agreement of 90.6% between cytological smears and whole sections; specifically, we found absolute concordance between smears with PD-L1 expressed in <10% and ≥50% of cells and whole sections with PD-L1 expressed in <50% and ≥50% of cells, respectively. In addition, slightly lower diagnostic accuracy was found for the cytological smears in comparison with the tissue cores, but the difference was not statistically significant. We found excellent intraobserver and good interobserver agreement in the evaluation of PD-L1 on smears. CONCLUSION: Immunocytochemistry on cytological smears is a reliable method for determination of PD-L1 at the 50% cutoff when positive cells are <10% or ≥50%; for cases showing PD-L1 expression in 10% to 49% of cells, additional tissue sampling may be necessary.

PD-L1 expression comparison between primary and relapsed non-small cell lung carcinoma using whole sections and clone SP263.

We assessed the concordance, in terms of PD-L1 expression, between primary and metastatic non-small cell lung carcinoma (NSCLC) of different histotypes using validated SP263 clone. A few samples of local recurrences have also been analyzed. Whole sections of consecutive cases of primary NSCLC and paired relapses undergone surgical resection have been stained with PD-L1 clone SP263; for scoring purposes, a three-tiered system was applied using the following thresholds: <1%, 1-49% and ≥50%. Eighty-four cases of paired primary and relapsed tumors from 83 patients were analyzed, including 75 metastases and 9 local recurrences. Regarding metastases, when considering a cutoff of 1%, discrepancy in PD-L1 expression occurred in 9/75 (12%) paired samples (kappa value = 0.75); at 50% cutoff, discrepancy in PD-L1 expression was detected in 7/75 (9.3%) of paired samples (kappa value = 0.61). Regarding recurrences, at 1% cutoff, the discrepancy in PD-L1 expression was seen in 3/9 (33%) paired samples and in all cases there was a gained PD-L1 expression; at 50% cutoff, 1/9 (11%) paired samples showed gained PD-L1 expression. Our data provide important information regarding the concordance between primary and relapsed NSCLC and the degree of reliability of metastatic sites in terms of PD-L1 expression evaluation.

Correction: PD-L1 expression heterogeneity in non-small cell lung cancer: evaluation of small biopsies reliability.

[This corrects the article DOI: 10.18632/oncotarget.21485.].

PD-L1 Expression Heterogeneity in Non-Small Cell Lung Cancer: Defining Criteria for Harmonization between Biopsy Specimens and Whole Sections.

INTRODUCTION: Determination of programmed death ligand 1 (PD-L1) expression defines eligibility for treatment with pembrolizumab in patients with advanced NSCLC. This study was designed to better define which value across core biopsy specimens from the same case more closely reflects the PD-L1 expression status on whole sections and how many core biopsy specimens are needed for confident classification of tumors in terms of PD-L1 expression. METHODS: We built tissue microarrays as surrogates of biopsies collecting five cores per case from 268 cases and compared PD-L1 staining results obtained by using the validated clone SP263 with the results obtained by using whole tumor sections. RESULTS: We found an overall positivity in 39% of cases at a cutoff of 1% and in 10% of cases at a cutoff of 50%. The maximum value across cores was associated with high concordance between cores and whole sections and the lowest number of false-negative cases overall. To reach high concordance with whole sections, four and three cores are necessary at cutoffs of 1% and 50%, respectively. Importantly, with 20% as the cutoff for core biopsy specimens, fewer than three cores showed high sensitivity and specificity in identifying cases with 50% or more of tumor cells positive for PD-L1 on whole sections. Specifically, for PD-L1 expression values of 20% to 49% on cores, the probabilities of a tumor specimen expressing PD-L1 in at least 50% of cells on a whole section were 46% and 24% with one and two biopsy specimens, respectively. CONCLUSIONS: An accurate definition of the criteria to determine the PD-L1 status of a given tumor may greatly help in selecting those patients who could benefit from anti-programmed cell death 1/PD-L1 treatment.

PD-L1 Assays 22C3 and SP263 are Not Interchangeable in Non-Small Cell Lung Cancer When Considering Clinically Relevant Cutoffs: An Interclone Evaluation by Differently Trained Pathologists.

Pembrolizumab is the only programmed cell death 1/programmed death-ligand 1 inhibitor for treatment of patients with non-small cell lung cancer, with a companion diagnostic assay, the 22C3 PharmDx. Although in many studies 22C3 and Ventana's SP263 appear to yield overlapping results, they show discrepancies at clinically relevant cutoffs (1% and 50%). We provide a solid comparison between 22C3 and SP263 assays in a large cohort of non-small cell lung cancer cases taking into account interobserver variability between trained pathologists who are used to either clone in their clinical practice. Serial sections of tissue microarrays, built from 198 cases of resected lung cancer, were stained for 22C3 on the Dako Link-48 platform and for SP263 on the Ventana Benchmark Ultra, following manufacturer's instructions. A protocol was also developed to run the 22C3 antibody on the Ventana platform. The pathologist used to 22C3 scored consistently higher than the pathologist used to SP263 at both 1% and 50% cutoff for all assays. For 22C3 and SP263 on respective platforms, we found statistically significant differences in terms of proportion of positive cases at both cutoffs; at 50% cutoff, around half of the cases positive with SP263 would have been defined negative with 22C3 by both pathologists. Important differences were also observed, when comparing clone 22C3 and SP263, both run on the Ventana platform. The lowest differences were seen with 22C3 run on both platforms. Assays 22C3 and SP263 show important discrepancies in identifying programmed death-ligand 1-positive cases at clinically relevant cutoffs, with possible underestimation of patients suitable for pembrolizumab therapy.

PD-L1 expression heterogeneity in non-small cell lung cancer: evaluation of small biopsies reliability.

Immunotherapy with checkpoint inhibitors, allowing recovery of effector cells function, has demonstrated to be highly effective in many tumor types and represents a true revolution in oncology. Recently, the anti-PD1 agent pembrolizumab was granted FDA approval for the first line treatment of patients with advanced non-small cell lung cancer (NSCLC) whose tumors show PD-L1 expression in ≥ 50% of neoplastic cells and as a second line treatment for patients with NSCLC expressing PD-L1 in ≥1% of neoplastic cells, evaluated with a validated assay. For the large majority of patients such evaluation is made on small biopsies. However, small tissue samples such as core biopsies might not be representative of tumors and may show divergent results given the possible heterogeneous immunoexpression of the biomarker. We therefore sought to evaluate PD-L1 expression concordance in a cohort of 239 patients using tissue microarrays (TMA) as surrogates of biopsies stained with a validated PD-L1 immunohistochemical assay (SP263) and report the degree of discordance among tissue cores in order to understand how such heterogeneity could affect decisions regarding therapy. We observed a discordance rate of 20% and 7.9% and a Cohen's κ value of 0.53 (moderate) and 0,48 (moderate) for ≥ 1% and ≥ 50% cutoffs, respectively. Our results suggest that caution must be taken when evaluating single biopsies from patients with advanced NSCLC eligible for immunotherapy; moreover, at least 4 biopsies are necessary in order to minimize the risk of tumor misclassification.