A Semiquantitative Scoring System May Allow Biopsy Diagnosis of Pulmonary Large Cell Neuroendocrine Carcinoma

Experience With Tissue Microarrays

Marina K Baine, MD, PhD; John H. Sinard, MD, PhD; Guoping Cai, MD; Robert J. Homer, MD, PhD


Am J Clin Pathol. 2020;153(2):165-174. 

In This Article

Materials and Methods

Tissue Microarray Construction

A natural language search of the Yale pathology information system was performed for all lung resection cases from 1983 to 2014 containing "large cell" and "neuroendocrine" in the final diagnosis field. All cases were re-reviewed by two of the authors (R.J.H. and M.K.B.). Twenty-seven cases that were either diagnosed and signed out as LCNEC or were morphologically compatible with LCNEC on re-review were identified. For the LCNEC tissue microarrays (TMAs), of the 27 selected resection cases, six were negative for neuroendocrine markers. Two of these neuroendocrine marker-negative cases showed diffuse nuclear positivity for p40, consistent with squamous cell carcinoma. Another two neuroendocrine marker-negative cases were strongly and diffusely napsin-A positive, consistent with adenocarcinoma, while the remaining two were negative for napsin-A or p40, and were morphologically most consistent with large cell carcinoma, not otherwise specified (NOS). These six cases were reclassified in all further analyses. The existing NSCLC TMA included one LCNEC case, which was included in our analysis, resulting in a total of 22 confirmed LCNECs. This last case, however, could not be used in the analysis of intratumor heterogeneity (see below for details) due to inability to retrieve the corresponding tissue block for additional sampling. Thus only 21 cases on the LCNEC TMA were used for that part of the study.

Three replicate TMAs were constructed, each of which included the 27 initially selected putative LCNECs (21 confirmed as LCNEC and six reclassified as other types of NSCLC) and four additional definite non-LCNEC cases (to serve as internal controls and for normalization between the three blocks) comprising one adenocarcinoma, one squamous cell carcinoma, one small cell carcinoma, and one atypical carcinoid, for a total of 31 cases. H&E-stained sections from corresponding representative paraffin-embedded, formalin-fixed blocks containing tumor material were used to define diagnostic areas (ie, composed of viable tumor). To determine diagnostic reproducibility and to account for intratumor heterogeneity, three random representative 1-mm cores were obtained from different areas of the tumor for each case and then inserted in a grid pattern into three corresponding recipient paraffin blocks using a tissue arrayer (Beecher Instruments).

The final LCNEC cohort included eight males and 14 females, ranging in ages from 53 to 81 years old, with a median age at diagnosis of 64.5 years. For this study, the patients were deidentified making it exempt from institutional review board oversight. As a result, no additional clinical information was able to be collected at the time of the study, including treatment or survival data.

A second TMA of primary lung cancer resection specimens was analyzed for comparison. This TMA had been previously constructed from institutional paraffin archives, including 238 cases of NSCLC (adenocarcinoma, squamous cell carcinoma, adenosquamous carcinoma, large cell carcinoma, and NSCLC, NOS), eight carcinoid tumors including one atypical carcinoid, one small cell carcinoma, and 33 tumors without histologic classification.


Sections (4 μm) were cut from each TMA block, mounted onto glass slides, and deparaffinized. Epitope retrieval was performed using Leica Bond ER 1 for 40 minutes on the stainer. Consecutive sections were incubated with napsin-A (Novus, clone TMU-A), p40 (Biocare, clone BC28), Ki-67 (Dako, clone MIB-1), chromogranin (Dako, clone A), synaptophysin (Leica, clone 27G12), and CD56 (Dako, clone NCAM) antibodies. Detection was carried out using Leica Bond Polymer Refine DAB detection kit or Ventana Ultraview DAB detection kit, depending on the antibody. Napsin-A and p40 immunostains were used for confirmation of tumor histotype as adenocarcinoma or squamous cell carcinoma, respectively. Any strength of neuroendocrine marker staining was considered positive, which was determined by consensus between two of the authors (R.J.H. and M.K.B.).

Ki-67 LI in the tumors was assessed independently by two pathologists, and the average was used for primary data analysis. Secondary analysis was performed to include neuroendocrine marker staining and Ki-67 LI interpretation from all four participating pathologists.

Morphologic Assessment and Scoring

Previously described morphologic criteria[3] were independently assessed on H&E-stained sections of the TMA by four pathologists, including three experienced pathologists and one trainee (PGY2 resident) using the scoring scheme outlined in Table 1. Architectural features included peripheral palisading, organoid nesting, and rosette-like structures, each of which received a score of 1 if present, with a maximum score of 3 if all three features were identified. An additional one point was assigned for the presence of extensive necrosis. Three points were assigned for one or more neuroendocrine marker positivity, the presence of which was required for LCNEC diagnosis, and a Ki-67 LI 40% or greater earned an additional point.

Nucleolar prominence was considered in the original scoring scheme, but it appeared to contribute little to differentiating LCNEC from NSCLC. This may not be surprising as this is a commonly shared feature with NSCLC in general. We therefore excluded nucleolar prominence from the final proposed scoring scheme.

Statistical Analysis

JMP version 14.0 software (SAS Institute) was used for statistical evaluation of the data. Receiver operating characteristic curve analysis was performed to determine the cutoff total score (≥4) for assigning tumors into the LCNEC diagnostic category (Table 1). Sensitivity and specificity of the total score for LCNEC diagnosis were manually calculated. Continuous average total scores (among four participating pathologists) for LCNEC and NSCLC were compared by one-way analysis of variance. The mean (among triplicate TMAs) of the LCNEC average total scores and standard error of the mean (SEM) was used to represent the range of all possible scores for the corresponding tumor and to define intratumor heterogeneity. Ki-67 LI was dichotomized into low and high, based on a 40% cutoff, and its sensitivity and specificity with regards to LCNEC diagnosis were manually calculated. The significance of the scored morphologic characteristics, and the associations of tumor histotype, neuroendocrine markers, and napsin-A immunoreactivities with Ki-67 LI were determined using Fisher exact test and χ 2 analysis. Pairwise Cohen κ was used to assess agreement on morphology between participating pathologists, and the range for all pathologist pairs was reported along with the standard deviation.