Role of Synaptophysin, Chromogranin and CD56 in adenocarcinoma and squamous cell carcinoma of the lung lacking morphological features of neuroendocrine differentiation: a retrospective large-scale study on 1170 tissue samples

Background Synaptophysin, chromogranin and CD56 are recommended markers to identify pulmonary tumors with neuroendocrine differentiation. Whether the expression of these markers in pulmonary adenocarcinoma and pulmonary squamous cell carcinoma is a prognostic factor has been a matter of debate. Therefore, we investigated retrospectively a large cohort to expand the data on the role of synaptophysin, chromogranin and CD56 in non-small cell lung cancer lacking morphological features of neuroendocrine differentiation. Methods A cohort of 627 pulmonary adenocarcinomas (ADC) and 543 squamous cell carcinomas (SqCC) lacking morphological features of neuroendocrine differentiation was assembled and a tissue microarray was constructed. All cases were stained with synaptophysin, chromogranin and CD56. Positivity was defined as > 1% positive tumor cells. Data was correlated with clinico-pathological features including overall and disease free survival. Results 110 (18%) ADC and 80 (15%) SqCC were positive for either synaptophysin, chromogranin, CD56 or a combination. The most commonly positive single marker was synaptophysin. The least common positive marker was chromogranin. A combination of ≤2 neuroendocrine markers was positive in 2–3% of ADC and 0–1% of SqCC. There was no significant difference in overall survival in tumors with positivity for neuroendocrine markers neither in ADC (univariate: P = 0.4; hazard ratio [HR] = 0.867; multivariate: P = 0.5; HR = 0.876) nor in SqCC (univariate: P = 0.1; HR = 0.694; multivariate: P = 0.1, HR = 0.697). Likewise, there was no significant difference in disease free survival. Conclusions We report on a cohort of 1170 cases that synaptophysin, chromogranin and CD56 are commonly expressed in ADC and SqCC and that their expression has no impact on survival, supporting the current best practice guidelines.


Background
Synaptophysin, chromogranin and CD56 are recommended markers to identify pulmonary tumors with neuroendocrine differentiation [1]. These markers are frequently used to confirm a diagnosis of typical carcinoid, atypical carcinoid, small cell lung cancer and large cell neuroendocrine carcinoma (LCNEC). In the routine diagnostic setting, particularly the differentiation of LCNEC and pulmonary adenocarcinoma (ADC) with solid growth pattern or non-keratinizing squamous cell carcinoma (SqCC) might be challenging. According to current guidelines only non-small cell carcinomas (NSCLC) that exhibit morphological features of neuroendocrine differentiation should be stained with neuroendocrine markers. In case of a negative result these tumors should be labelled NSCLC with neuroendocrine morphology in biopsy specimens with a comment that the tumor is suspected to exhibit neuroendocrine differentiation that could not be confirmed by immunobiological staining. On the other hand, ADC and SqCC may show the expression of neuroendocrine markers despite the lack of neuroendocrine morphology. The clinical significance in this constellation has been investigated in previous studies [2][3][4][5][6][7][8][9][10][11]. While some of the studies suggested an impact of neuroendocrine marker expression on survival [4,7,[12][13][14][15][16] most of the studies reported no prediction of survival [2,10,11]. In this study we investigated over 1000 patient samples to expand the data on the role of synaptophysin, chromogranin and CD56 in NSCLC lacking morphological features of neuroendocrine differentiation.

Patient cohort
Formalin fixed and paraffin embedded NSCLC specimens resected from 2002 to 2010 in the Thoracic Hospital Heidelberg at Heidelberg University were extracted from the archive of the Institute of Pathology, Heidelberg University, with the support of the tissue bank of the National Center for Tumour Diseases. Tissues were used in accordance with the ethical regulations of the NCT tissue bank defined by the local ethics committee (#S315-2020, NCT#2603). Diagnoses were made according to the recommendations of the 2015 world health classification of tumours of the lung, thymus and heart [1]. One thousand one hundred seventy patients with NSCLC including ADC and SqCC were selected. Tissue microarrays were constructed as described previously [17,18].

Immunohistochemistry
Immunohistochemical (IHC) staining was performed as previously described [18,19]. In brief, slides were deparaffinized, pretreated with an antigen retrieval buffer and stained using an automated device. Immunohistochemical stainings were performed on a Ventana Benchmark Ultra (Roche, Switzerland). The antibody and staining conditions are shown in Table 1. The evaluation was carried out by an experienced pathologist (MK). Synaptophysin and chromogranin were considered when located in the cytoplasm, CD56 was evaluated when located on the membrane. Positivity of a marker was defined as > 1% positive tumor cells, as in previous studies [2]. Typical examples of positive and negative staining results of ADC and SqCC are shown in Figs. 1 and 2. The results from the conventional NSCLC markers TTF-1 and p40 were published previously [20,21].

Molecular data
Molecular data included results for KRAS, EGFR, BRAF, ROS1 and ALK testing were available for ADC from a previous investigation [22]. In brief, cases were analyzed  by Sanger sequencing for KRAS (exon 1), EGFR (exons [18][19][20][21] and BRAF (exon 15). Cases tested for ROS1 and ALK were prescreen using IHC, results were subsequently validated by fluorescence in situ hybridization (FISH) using a break-apart probe. Only cases with FISHconfirmation were considered positive.

Data analysis
Statistical analyses were performed using R-Statistical Software (www.r-project.org, v.
These cases also exhibited positivity for TTF-1 in the same tumor cells and showed a typical growth pattern of adenocarcinoma. The vast majority of ADC were negative for p40 (99%). 511 (94%) SqCC were positive with antibodies against p40. Only 6 (1%) SqCC exhibited focal weak TTF-1 positivity. These tumors showed keratinization and intercellular bridges and were therefore classified as SqCC. The majority of SqCC were negative for TTF-1 (99%). None of the ADC and SqCC  Overall, 110 (18%) ADC and 80 (15%) SqCC were positive for either synaptophysin, chromogranin, CD56 or a combination of these. The most commonly positive single marker was synaptophysin in ADC (13%) and SqCC (4%). The least common positive marker was chromogranin in ADC (3%) and CD56 in SqCC (1%). A combination of either two or three neuroendocrine markers was positive in 2-3% of ADC and 0-1% of SqCC. A summary of the expression of p40, TTF-1 and the neuroendocrine markers is provided in Table 2 and Fig. 3. No significant difference of gender, age, T-and N-categories as well as clinical stage were observed between ADC and SqCC with and without expression of neuroendocrine markers (Tables 3 and 4).   (Tables 5 and 6).

Discussion
In the present study we investigated the impact of the expression of synaptophysin, chromogranin and CD56 in ADC and SqCC without neuroendocrine morphology on overall survival in a large study including more than  1000 patients. This is the largest cohort reported on this topic to date. We found that neuroendocrine marker expression is common and is not associated with OS and DFS. Staining a combination of synaptophysin, chromogranin and CD56 is currently advised to establish evidence of neuroendocrine differentiation in thoracic tumors [23]. However, staining should be restricted to NSCLC exhibiting neuroendocrine differentiation, as it has been shown that ADC and SqCC may exhibit positive staining in 10-30% in most studies [2,6]. Studies reporting a higher positivity rate were commonly done on whole slides [3] and not on tissue micro-arrays [2,4,11], with one exception reporting neuroendocrine marker expression in up to 90% of tumors [5]. Thus, our results are in line with the literature [3,6,10]. The differences of the reported positivity rates might also be explained by different cut-offs for the definition of positivity and the application of different antibody clones [5,10,11]. We investigated only one cut-off value for positivity and choose a cut-off of > 1% positive tumor cells. This cut-off has been used in other previous studies but is somewhat arbitrary [2,3]. We decided to use this cut-off as single cell positivity is a physiologic finding in lung tissue and single neuroendocrine cells overgrown by tumor cells and unspecific background staining might not be reliably distinguished from positive tumor cells [24]. Moreover, cut-off values above 1% are rarely helpful in the routine diagnostic setting. Ionescu et al. reported CD56 to be most commonly expressed closely followed by synaptophysin [2], while Sterlacci et al. reported synaptophysin to be the most commonly detected positive marker in ADC and SqCC, as in our study [11]. In line with these large-scale investigations, chromogranin was least commonly expressed in our study.
The impact of neuroendocrine marker expression on survival of patients with ADC and SqCC is controversially discussed. While most investigations found no impact on prognosis, some more recent studies challenged this finding [4,25,26]. Feng et al. investigated the impact of neuroendocrine marker expression on OS and DFS in one of the largest cohorts including a total of 451 patients and found a significantly worse survival in patients with tumors expressing neuroendocrine markers [4]. However, another large study including more than 200 ADC and SqCC did not find any prognostic impact neither on OS nor DFS, in line with the findings of our study [2].
Another marker of neuroendocrine differentiation, Insulinoma-associated Protein 1 (INSM1), has been reported to support the diagnosis of neuroendocrine differentiation in thoracic tumors and has the potential to complement the currently recommended neuroendocrine markers [27,28]. Interestingly, INSM1 has been reported to be more sensitive and specific as compared to the single markers Synaptophysin, Chromogranin and CD56 and was therefore advocated as a first-line stand alone marker or in combination with CD56 to detect neuroendocrine differentiation [28][29][30][31]. INSM1 marker expression  has been suggested to be prognostic in high-grade neuroendocrine neoplasms, but if INSM1 expression has a prognostic impact in ADC or SqCC remains to be investigated [32]. Moreover, we could not detect any differences in the rate of common genetic aberrations in pulmonary ADC when we compared tumors with and without expression of neuroendocrine markers. Although we analyzed a large cohort, these data must be interpreted with caution, because the respective patient subsets were small.
Our study has several limitations: first, the retrospective design of the investigation. Prospective large-scale studies are not available to the best of our knowledge. Second, we used a tissue microarray as a surrogate for the biopsy situation. As only two cores from the whole tumor were investigated, it is not entirely clear if other parts of tumors that were judged negative in our study exhibit neuroendocrine immunoreactivity. This problem is also highlighted by the fact that previous studies on whole slides reported higher rates of neuroendocrine positivity [3]. On the other hand numerous studies comparing the results of tissue microarray studies with the findings from conventional large sections using other biomarkers have shown that all well-established associations between molecular markers and tumor phenotype or patient prognosis can be reproduced with tissue microarrays [33].

Conclusion
In summary, we show that synaptophysin, chromogranin and CD56 are commonly expressed in ADC and SqCC and that their expression as no impact on OS and DFS supporting the current best practice guidelines.