Introduction
Neuroendocrine neoplasms (NEN) of the lung represent 20–30% of all NEN and about 20% of all lung malignancies [
1]. According to the recent World Health Organization (WHO) – International Agency for the Research on Cancer proposal for a standardized NEN nomenclature and the current WHO classification, typical and atypical lung carcinoids (TC and AC) belong to the well-differentiated neuroendocrine tumor (NET) family and are graded as G1 and G2, respectively, while the small cell lung cancer (SCLC) and the large cell neuroendocrine carcinoma (LCNEC) are poorly differentiated neuroendocrine carcinomas (NEC) by default of grade 3 [
2,
3]. SCLC is by far the largest fraction of lung NEN accounting for 15% of lung cancer; minor fractions are LCNEC accounting for only 3% of resected lung cancers as well as TC and AC, which account for ≤ 2% of all lung malignancies with prevalent TC vs AC [
4].
Like NETs of other anatomical sites, most lung NETs express somatostatin receptors (SSTRs), mainly the subtype 2 (SSTR2) [
5]. This feature is the basis for the widespread application of diagnostic and therapeutic strategies with “cold” or radiolabeled somatostatin analogs (SSA) in NETs [
6,
7]. SSTR-positron emission tomography/computed tomography (SSTR-PET/CT) with
68Ga-labeled-SSA is currently used in lung TC-AC/NET for in vivo SSTR characterization and staging [
8,
9].
18F-FDG PET/CT, which is the elective functional imaging modality for the highly proliferative SCLC and LCNEC [
10], has also been explored in lung TC-AC/NET and proved to correlate with cancer cell proliferation [
11,
12].
In clinical routine, in vitro SSTR expression can be detected on tissue samples by immunohistochemistry (IHC), a reproducible and sensitive routine procedure, which provides information on cellular/subcellular SSTR subtype distribution in tumor cells [
13]. An alternative method is reverse transcriptase-polymerase chain reaction (RT-PCR), which demonstrates SSTR mRNA [
14]; however, its use is hampered in pathology services by limited access to molecular technology with relative complexity and high costs as compared to the widespread access, robustness, and low cost of IHC. The correlation of SSTR2-IHC expression vs functional imaging data (in vivo expression) has been assessed in mixed series of patients with various NET types by using SSTR-scintigraphy (Octreoscan®) or
68Ga-DOTATOC PET/CT [
13,
15]. Limited data are available for lung NET [
5].
The aim of this study was to assess the somatostatin receptor and proliferative activity profile (SSTR2, SSTR5, Ki-67) at IHC in a retrospective selected series of lung NET, to correlate it with SSTR-PET/CT imaging features, and to specifically assess the potential role of IHC in predicting in vivo SSTR expression. Secondly, we aimed to correlate proliferative activity by Ki-67 with 18F-FDG-PET/CT parameters (when available) in the same cohort.
Discussion
In this study, we found an overall good agreement (75.0%,
p = 0.003) between in vitro SSTR expression and tumor uptake of
68Ga-DOTA-SSA at qualitative assessment in a retrospective series of patients with lung NET. For positive SSTR expression, 100% concordance was found. Similarly, higher SUV
max values corresponded to membranous staining, which indicates positive receptor expression [
13]. These findings may impact on clinical practice in cases where SSTR-PET/CT is not available for preoperative and staging purposes. In these cases, the demonstration of membranous staining in NET biopsy may surrogate SSTR-PET/CT for treatment with “cold” or radiolabeled SSA [
14,
23‐
25]. Indeed,
68Ga-DOTA-SSA-PET/CT, showing high affinity for SSTR2 (higher binding affinity of the analog DOTATATE followed by DOTANOC and then by DOTATOC) and, with some ligands, also for SSTR3 (DOTANOC) and/or SSTR5 (DOTANOC and DOTATOC), is considered the most reliable method for in vivo assessment of somatostatin receptor status [
26]. It provides information on the presence of SSTR and its affinity for the radio-ligand and allows tumor localization with high sensitivity. Moreover, the intensity of tracer uptake is used to tailor treatment with SSA and select patients suitable for peptide receptor radionuclide therapy [
23‐
25].
All patients included in the present cohort had a well-differentiated lung NET, a finding that is a consequence of the retrospective nature of our study as well as the inclusion criteria. Indeed, patients with poorly differentiated/high-grade lung NEN are rarely submitted to PET/CT studies for staging purposes. Among lung NET patients, we found an 81% prevalence of TC/NET-G1, which is in line with literature data [
4].
For in vitro studies, we applied the scoring system proposed by Volante and coworkers [
13] which evaluates the subcellular localization (membranous or cytoplasmatic) and the extent of staining. Consistent with previous data obtained in a larger population, SSTR2 was the predominantly expressed receptor subtype with positive findings in 62.5% of samples [
27]. SSTR5 was poorly represented with a positive rate of 19.4%, not improving the overall concordance between in vitro and in vivo results. Based on this, further considerations on our findings are essentially based on SSTR2 expression. Scores 2–3 were predominantly detected in TC/NET-G1 (69% vs 33% of AC/NET-G2), even though the difference was not statistically significant, likely due to the few samples of AC/NET-G2 analyzed. In this histotype, score 3 indicating the highest receptor density was never observed in the present series. Among all patients studied, 6 presented lymph node (2 cases) and/or distant metastases (5 cases). IHC for SSTRs was done on the primary site, and it is known to be consistent in primary and metastatic deposits of lung NEN [
27]. Even though we observed a higher rate of positive SSTR2-IHC in the metastatic group (83.3%) compared to non-metastatic NENs (57.7%), the difference was not statistically significant, probably due to the small number of patients included. In the study by Righi and coworkers, SSTR2A was overexpressed in metastatic TCs when compared to clinically benign TCs, thus suggesting that SSTR2A status is involved in the metastatic propensity of lung carcinoids [
28].
The detection rate of SSTR-PET/CT was 88.5% for TC/NET-G1 and 83.3% for AC/NET-G2, with no significant difference. Therefore, a little amount of SSTR-negative tumors belonged to both histotypes; in other words, SSTR in vivo expression was independent from tumor grade in lung NET. Even when analyzing semi-quantitative data, no significant difference was found between TC/NET-G1 and AC/NET-G2 according to SSTR-PET parameters (tumor SUVmax and SUVT/S). These observations are in line to what found in corresponding tissue samples at IHC.
We did not find any case of positive IHC and negative SSTR-PET/CT, thus confirming the role of a reference method assigned to functional imaging with radio-receptor PET/CT. Conversely, Papotti et al. and Volante et al. reported cases of positive IHC and negative radio-receptor scintigraphy, a technique that is characterized by lower spatial resolution and lower sensitivity than PET/CT imaging [
5,
13]. Moreover, the binding affinity for SSTR2 and SSTR5 of the radiolabeled SSA is significantly affected by structural changes of the octapeptide as well as the choice of chelator (DTPA vs DOTA) or metal used for labeling, with a clear advantage for
68Ga-DOTA-labeled compounds over
111In-DTPA-octreotide in terms of binding affinity and consequently of imaging findings [
29].
As for the IHC negative cases of the present series, only 33.3% concordance was found since SSTR-PET/CT was positive in eight cases, even though with low-grade uptake (Krenning score = 1) in all but one of them. Similar discrepancies have been previously observed, even though in mixed series of NEN patients studied by PET/CT [
15] or in patients with lung NEN submitted to
111In-DTPA-octreotide scintigraphy [
5,
13]. In our series, the visualization (although with low uptake) of a lung lesion with negative SSTR2-IHC could be partly explained by the binding affinity of
68Ga-DOTATOC and
68Ga-DOTANOC to SSTR2, higher than that of Octreoscan® [
29]. In any case, current imaging selection criteria do not consider peptide receptor radionuclide therapy for lesions showing no or low uptake, so most negative cases for SSTR at IHC should remain unsuitable (seven out of eight in this series) for peptide receptor radionuclide therapy despite slightly in vivo positivity.
In addition to neuroendocrine tumor cells, other non-neoplastic cells such as lymphoid and endothelial cells express SSTRs [
30,
31]. As functional radio-receptor imaging cannot define the cellular receptor localization, we quantified the intra-tumoral inflammatory component on tissue samples as potential source of positive SSTR-PET/CT imaging. When present (45.2% of samples), the intra-tumoral inflammatory component was low, making it unlikely to be the determinant of radioligand uptake. Conversely, the discordant finding of SSTR expression in vivo vs in vitro could be explained by SSTR expression levels below the detection limit of IHC. On this line, the low values of SUV
max observed in discrepant cases would support this potential explanation.
A relevant observation of our study was that in vitro SSTR scores and SUV values at SSTR-PET/CT proceeded at the same rate. Cytoplasmatic staining, indicating negative receptor expression, matched low SUV values, and membranous staining, indicating positive receptor expression, matched higher SUV values (
p = 0.002) [
13]. Therefore, SUV values can be used as a parameter of SSTR2 density. Miederer and coworkers in a mixed series of NEN [
15] and Kaemmerer and coworkers in gastro-entero-pancreatic NETs reported a similar highly significant correlation [
32]. However, some discrepant results were also observed in our series, as one of 12 tumors with IHC scores 0–1 showed high SUV values (9.7 in one AC/NET-G2) and 3 of 20 tumors with IHC scored 2–3 showed low SUV values (< 4.0, all TC/NET-G1). These discrepancies on the tissue side may reflect non-standard tissue handling for histology processing with resulting poor SSTR preservation, and on the in vivo side, undetermined reduced SSTR expression status or SSTR blockade.
Besides analyzing the receptor profile, we also studied proliferative activity by Ki-67 staining, which, as expected, proved to be an important discriminant factor between TC/NET-G1 and AC/NET-G2, with higher values of positivity in AC/NET-G2 (
p = 0.0001). Parallel to this observation, a correlation of strong statistical significance between
18F-FDG-SUV
max (available in 14 patients only) and Ki-67 was found in lung AC/NET-G2 (
p < 0.0001), even though the calculation could be affected by the small number of patients studied with
18F-FDG-PET/CT. Conversely, we did not find any correlation between SUV
max at SSTR-PET/CT and Ki-67 values, both in the overall series and when analyzing separately TC/NET-G1 and AC/NET-G2. These findings are in agreement with those reported by Campana and coworkers in a mixed series of NET patients studied with
68Ga-DOTANOC [
33].
In line with our previous study, the use of SUV
max ratio between
68Ga-DOTA-peptides and
18F-FDG-PET/CT allowed the distinction between TC/NET-G1 and AC/NET-G2 in those lung lesions that are visualized by both tracers. This could be a valid help in surgical management, potentially influencing the extension of parenchymal resection and lymph node dissection, considering that the differentiation between the two histotypes is rarely feasible through pre-operative biopsies [
20].
This study has some limitations. First is its retrospective nature. Second, two different SSA were used as radio-ligand, with different affinities to SSTR. To assess the influence on the pharmacokinetics of the two different peptides, we calculated possible variations in splenic uptake; no significant differences were observed. As in a previous multicenter study, we chose the spleen as reference, because it is a healthy organ showing high physiologic uptake of radio-peptides that is homogeneous throughout the splenic parenchyma, and is due to high expression of SSTR [
20]. Third, tissue handling though following rigid standard procedures may slightly vary between cases, with improper receptor antigen preservation and poor resulting IHC. Finally, we did not investigate the prognostic value of SSTR2-IHC and SSTR-PET/CT findings since the goal of the original study was to assess the potential role of IHC in predicting in vivo SSTR expression. A long-term analysis of clinical outcome is needed to clarify whether SSTR2-IHC and SSTR-PET/CT findings are predictors of patient outcome, able to stratify lung NET patients with poor prognosis. Nonetheless, this study was performed in a highly selected and well-annotated series of lung NET, while available literature data are relatively old and not specifically targeting lung NET [
5,
13,
15,
32].
In conclusion, information on comparative SSTR expression in vitro and in vivo in patients with lung NET is limited. This retrospective study showed an overall good agreement between SSTR2-IHC on tissue and tumor uptake at 68Ga-SSA-PET/CT in patients with lung NET. PET/CT with 68Ga-SSA was confirmed to be the most reliable method for in vivo assessment of receptor status. In clinical practice, SSTR-PET/CT SUVmax values can be used as a parameter of SSTR2 density. Membranous staining in tissue samples is feasible and informative for follow-up studies with SSTR-PET/CT and potentially for treatment with “cold” or radiolabeled SSA analogs. Within the limits imposed by the relatively small cohort here reported, our data suggest that SSTR2-IHC may surrogate SSTR-PET/CT in selected lung NET patients for effective clinical decision making when SSTR-PET/CT is not available or where financial constraints limits the access to in vivo SSTR assessment.
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