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


Immunophenotypic Characterization of the LCNEC Cohort

Neuroendocrine marker immunoreactivity was variable among the confirmed LCNECs (Supplemental Table 1; all supplemental materials can be found at American Journal of Clinical Pathology online). Eight cases (36%) were positive for all three neuroendocrine markers (synaptophysin, chromogranin, and CD56), 10 cases were positive for two markers (45%), and four cases were positive for one marker (18%). In our series, the most sensitive neuroendocrine marker was synaptophysin, similar to prior reports.[8] This contrasts with small cell carcinoma, in which CD56 is the most commonly expressed marker (90%-100%).[8,9]

In our NSCLC TMA, only 2.7% of NSCLCs displayed variable staining with neuroendocrine markers (Supplemental Table 1). Tumors that were neuroendocrine marker positive included two large cell carcinomas, two squamous cell carcinomas, and one unspecified NSCLC. This is slightly lower than the typical reported range of 6% to 20%.[10–12] Four of these five cases displayed only focal and weak immunoreactivity for neuroendocrine markers. The one case with diffuse moderate staining with chromogranin was also patchy and weakly positive for synaptophysin and was one of the 33 tumors on the TMA with missing histologic classification. Based on our morphologic assessment, however, this case is likely to have been a true LCNEC. Three of the four weakly focally staining cases were positive for only one marker. The remaining one case, which was positive for two markers (synaptophysin and CD56), was classified on the original resection material as large cell carcinoma with focal neuroendocrine differentiation. Morphologically, this case did not have the typical LCNEC features and its average total score by the proposed criteria was 3.25 (Supplemental Table 1), which is below the proposed diagnostic threshold for LCNEC.

There was no correlation between the number, type, or strength of neuroendocrine marker immunoreactivity and the total score (P = .5), as expected by design. When immunohistochemistry for neuroendocrine markers was scored proportionally to the number and strength of marker expression, true LCNEC cases were missed, resulting in decreased sensitivity.

Morphologic Characterization and Scoring Criteria for LCNEC Diagnosis on Biopsy Material

As described in "Materials and Methods," scoring criteria were applied that included Ki-67 LI in addition to defined morphologic and immunophenotypic features (Table 1 and Image 1). Of the three scored morphologic criteria, architectural pattern and necrosis were significantly different between NSCLC and LCNEC Table 2. The presence of two or more architectural criteria (palisading, rosette-like structures, and organoid nesting) was the most specific morphologic feature for diagnosis of LCNEC (98%) but lacked sufficient sensitivity (63%). In contrast to the report by Watanabe et al,[7] nucleolar prominence was neither sensitive nor specific for detecting LCNEC in our cohort (Table 2). It is unclear if the difference was due to utilized criteria for determining nucleolar prominence, as that was not explicitly defined in the previous study. Alternatively, because prominent nucleoli are a common feature of high-grade adenocarcinomas and squamous cell carcinomas of the lung as is also evident from our data (Table 2), it may not provide additional value to this differential diagnosis. Nucleolar prominence was therefore excluded from the proposed scoring scheme.

Image 1.

Morphologic and immune phenotypes of sample large cell neuroendocrine carcinoma (LCNEC) and non-LCNEC tumors (original magnification, ×20). Total score for LCNEC was 7 (architecture = 2, necrosis = 1, neuroendocrine marker positivity = 3, high Ki-67 labeling index [≥40%] = 1). Total score for non-LCNEC was 3 (architecture = 2, necrosis = 0, neuroendocrine marker positivity = 0, high Ki-67 labeling index [≥40%] = 1).

While the vast majority of NSCLCs had total scores less than 2, 100% of LCNECs had total scores of at least 4 Table 3. The average total scores ranged between 0 and 5 for NSCLCs, with a median of 0.25 and a mean of 0.51 (±0.81 SEM). For LCNECs, the range was 4 to 7, with a median of 6 and a mean of 5.63 (±0.73 SEM). The difference between the mean total scores for the two diagnostic categories was statistically significant (P < .0001) Figure 1. While napsin-A positivity among NSCLCs predicted a lower total score (data not shown), its significance on the morphologic score of LCNECs could not be assessed due to a low number of such cases (two of 22; average total scores of 4.25 and 5.25).

Figure 1.

Average total scores in large cell neuroendocrine carcinoma (LCNEC) and non-small cell lung carcinoma (NSCLC). Each dot represents an individual case but cases with overlapping total scores (average from four pathologists) are displayed as a single dot. The mean total scores (long solid lines) with the corresponding values (μ) and the standard deviations (short solid lines) for each phenotypic category are displayed on the graph. The mean total score for LCNEC (5.65) was significantly higher than that for NSCLC (0.51, P < .0001).

Finally, and most notably, utilizing the total score cutoff of 4 or higher resulted in the diagnosis of LCNEC with a 90% to 99% specificity and 95% to 100% sensitivity among individual pathologists. When the average total scores from the four participating pathologists were used, LCNEC was correctly diagnosed in all 22 cases (100% sensitivity), and the specificity reached 99%, even when other neuroendocrine tumors (carcinoids and small cell carcinomas) in both TMAs were included in the analysis. The sensitivity and specificity held up in secondary analysis (100% and 98.9%, respectively), which included neuroendocrine marker and Ki-67 LI evaluation by all four participating pathologists rather than a consensus/average from two pathologists in our primary analysis. The specificity was 100% if other neuroendocrine tumors were excluded. This highlights that additional validation with modification of the scoring criteria components could be considered to specifically address the differential diagnosis of LCNEC vs SCLC vs atypical carcinoid.

Proliferative Activity (Ki-67 LI)

Compared to the NSCLC cohort, a significantly greater proportion of LCNEC cases displayed high proliferative activity, defined as Ki-67 LI greater than or equal to 40% (P < .0001) Table 4. Thus, elevated Ki-67 LI was a highly specific (93%), albeit not very sensitive (64%), feature of LCNEC, and thereby provided a significant value to accurate diagnosis of LCNEC.

Additionally, among all examined cases (both NSCLC and LCNEC), those expressing napsin-A by immunohistochemistry, including both weakly napsin-A positive LNCECs, had significantly lower proliferative activity with 96% displaying Ki-67 LI less than 40% (P = .0004; Table 4). It was also noted that the majority of LCNEC lesions that were immunoreactive for chromogranin displayed low Ki-67 LI (90%; P = .01; Table 4). This lower proliferative activity is likely a reflection of a more mature/differentiated phenotype as is implied by the corresponding immunophenotype.

Intratumor Heterogeneity

To examine reproducibility of the proposed scoring system and to assess intratumor heterogeneity, three different parts of the tumor represented on the three LCNEC TMAs were separately scored by all four pathologists. The LCNEC case from the archived NSCLC TMA utilized in all other parts of the study could not be used for this analysis due to the inability to access corresponding tissue blocks, leaving a total of 21 cases for this assessment. The SEM ranged from 0.02 to 0.53, with a median of 0.12, reflecting a good overall reproducibility and minimal intratumor heterogeneity. In addition, of the 21 LCNEC cases, 19 (90%) were consistently scored above the diagnostic threshold for LCNEC diagnosis (total score ≥4) Figure 2. This further highlights the reproducibility of the scoring system and the overall homogeneity of the tumor. The remaining two cases (10%) had mean total scores very close to the cutoff of 4, which made them fall outside the LCNEC diagnostic interpretation when SEMs were considered (score <4; Figure 2). This reflects reduced diagnostic reproducibility of the proposed scoring system for tumors that are on the proposed diagnostic cusp. Independent scoring of neuroendocrine marker immunohistochemistry and Ki-67 LI by four participating pathologists did not significantly alter these findings (data not shown).

Figure 2.

Intratumor heterogeneity and reproducibility of the proposed large cell neuroendocrine carcinoma (LCNEC) scoring system. Each dot represents an individual case and the bars represent standard errors of the mean (SEM). A horizontal dotted line is drawn at a mean score of 4, which is the diagnostic cutoff for LCNEC based on the proposed scoring criteria.

Agreement Among Pathologists

There was moderate agreement among pathologist on the major morphologic criteria Table 5, with greater variability for architecture (κ = 0.42–0.55), which is attributed to its multivariable/ordinal (comprised of three distinct scoring categories adding up to a final score of 0–3) rather than binary nature. Although there was some disagreement for each individual component of the score, the final classification into LCNEC vs non-LCNEC diagnosis demonstrated strong agreement among pathologists (κ = 0.93–0.98; Table 5). The κ values were not altered significantly when Ki-67 LI and neuroendocrine marker immunohistochemistry were scored independently by all four pathologists (Supplemental Table 2). The lower limit of the κ range (0.74) was attributed to one of the four pathologists scoring neuroendocrine markers slightly more liberally than the other three despite the predefined criteria for positivity.

Based on the total score cutoff of 4 or higher, there was complete agreement (100%) among the four pathologists on the final assignment of tumors to LCNEC vs non-LCNEC diagnostic category in 202 of 208 analyzed cases (97%), with an average agreement of 99% ± 5% for all examined tumors. When considered separately, the agreement was significantly better for non-LCNEC than for LCNEC cases Table 6. This latter finding is primarily due to two factors: a small cohort size and a small number of evaluating pathologists. While the former is a function of low LCNEC incidence and a single institution experience, the latter assigns substantial weight to each assigned score. Specifically, in eight of nine LCNEC cases with diagnostic disagreement (89%), there was only one divergent opinion (one of four pathologists assigning a score below the threshold), and only one case which was assigned to the LCNEC diagnostic category by half of the pathologists and to the non-LCNEC category by the other half. These divergent opinions did not correlate with the pathologists' experience. Excluding neuroendocrine tumors other than LCNEC from the analysis resulted in further improvement in interobserver agreement, which was not affected significantly in the secondary analysis with independent neuroendocrine immunohistochemistry and Ki-67 LI scoring by all four participating pathologists (Supplemental Table 2).