Gastro-entero-pancreatic neuroendocrine neoplasm are heterogenous tumors with variable disease progression and outcome. To optimize treatment protocol and prognostication, the classification and grading system for gastro-entero-pancreatic neuroendocrine neoplasms has undergone a series of updates over the past several decades. Better understanding of molecular genetics of high grade NEN (Ki-67 > 20%) and outcomes of several studies focusing on patient survival lead to categorization into two distinct subgroups i.e well differentiated Grade 3 NET and NEC[1, 2] among the previously described neuroendocrine carcinoma of WHO 4th edition. It is now understood that G3NET and NEC have distinct molecular pathways and hence recommended to have different management strategy and treatment protocols[3]. Velayoudom-Cephise et al reported that 31% NEC patients had an objective response to platinum–etoposide versus 0% for the NET G3 group[8]. For these reasons ,WHO 5th edition emphasizes that well differentiated G3NETs must be distinguished from NEC. This distinction, however, relies completely on morphology. In a recent study of 33 G3 pancreatic NENs, where 3 experts were required to assign a diagnosis of G3NET, large cell NEC, small cell NEC, or uncertain, a unanimous agreement on a confident diagnosis of G3NET or NEC was reached only in 14 (42%) patients[3]. This highlights that a histomorphological assessment of the degree of differentiation of high grade tumours can be challenging at times and consensus is difficult to obtain even among expert pathologists [2].
The clinical presentation may help to some extent in this distinction between the 2 high grade tumours. Patients with NECs present with nonspecific symptoms of an aggressive systemic illness and normal chromogranin levels while patients with NET G3 are more likely to have a functional tumor[9]. Morphologically NECs are denovo high grade while G3 NETs retains the monotony of neuroendocrine tumours but with increased mitoses(> 20/2mm2).
There are no reliable markers / adjunct tests which can aid the morphological distinction of WD NET Grade 3 and NEC[10]. Several studies have highlighted the presence of p53/ RB1 mutations in NEC and loss of ATRX/ DAXX in NET, which facilitated the morphological subtyping of WD NET Grade 3 and NEC[2, 11]. However there are no established guidelines to use IHC surrogates(p53 and ATRX ) in routine practice to make this distinction between the two categories[2]. Unlike other cancer types, the role of p53 and ATRX as prognostic marker in neuroendocrine neoplasm has not been explicitly described in the literature, as shown in this study.
P53, a tumor suppressor protein, acts as the gate keeper of the cell cycle. Mutation involving p53 gene is seen is several cancers. Mutated protein accumulates into tumor cell nuclei which can be detected by immunohistochemistry[12]. Identification of p53 mutation facilitates grading of several tumors and plays major role in patient prognostication and treatment such as ovarian serous carcinomas[4]. IHC surrogate for TP53 mutation has been used for molecular classification of endometrial carcinomas[5]. Tang LH, proposed that expression of p53 aids in differentiating WDG3NET from NEC[3]. However, unlike the ovarian and endometrial cancers, a cut off value for mutated pattern of p53 in neuroendocrine neoplasms has not been clearly described[2]. Abir Salwa Ali et al, in their study of p53 in High grade gastroenteropancreatic neuroendocrine neoplasms, semi-quantitatively assessed P53 (in percent) by assessing the area of immunopositive tumor cells vs the total tumor area by light-microscopy at a magnification of x40 and a cut off of > 10% was considered as positive[6]. Kirstine Nielsen, in a study of p53 as prognostic marker in high grade net of gastro-entero-pancreas, p53 was scored as negative (0%), heterogeneously positive (1–30%) and strongly positive (> 30%), the latter was considered abnormal and indicated mutations in the TP53 gene. Negative( null type) p53-staining was also considered abnormal[2]. Junjie Li et al found 3 patterns of immunohistochemical staining of p53, viz. absent staining(null- pattern A) which correlated with loss of function mutation, strong staining of > 60% ( pattern C) which correlated with gain of function mutation, whereas heterogenous staining ( pattern B) was considered as wild type p53[11]. Majority of pattern C was associated with NEC and pattern B was associated with NET which was comparable to our study(Table 2) Unlike the other studies, our study assessed p53 in grade 2 NETs apart from NEC and G3 NETs. The NULL pattern of p53 was seen in 3 cases of NEC, none of grade 3 NETs. However 4 of Grade 2 NETs showed null pattern of p53 in our study. Qiong Dai et al in their study of molecular alteration in GI NENs, also found mutated pattern / Null pattern of p53 in two of G2 NETs[12]. Disease free survival among patients of Grade 2 NET with null pattern and grade 3 tumors with null pattern in our study were comparable (42 month and 32 months respectively), though one patient grade 2 NET had a higher overall survival of 114 months with disease and died of disease. Null pattern/ mutated pattern in grade 2 NET could represent transformation of grade 2 NET to higher grade and poor survival[13]. Qiong Dai et al commented that null pattern IHC expression of p53 cannot be used to reliably distinguish NETs and NEC[12]. Like our study Qiong Dai et al found a significant difference in expression of p53 between NET( G2 NET, G3NET) and NEC[12]. Amongst high grade tumors( Ki-67 > 20%)though combined NULL pattern and mutated pattern of p53 were common among NEC compared to Grade 3 NET, the difference was not statistically significant possibly due to the small sample size in our study. Andrew M Bellizzi et al recommended use of panel of IHC markers such as clusterin, SSTR2A, and CXCR4 apart from p53 to distinguish G3 NET from NEC[14].
Loss of ATRX and DAXX has been used as a surrogate to identify WDG3 NET and Grade 2 NET in several studies [3, 15]. On the contrary, our study did not find significant difference in expression pattern of ATRX in Grade 2 and Grade 3 NETs and NEC.
We found that using cut off of p53 staining > 50% ( score > 9) patients has significantly lower DFS and OS compared to patients with P53 staining < 50% ). Like wise in a study by Abir Salwa Ali, et al, (with cut off of > 10% ) mutated p53 staining in patients with colorectal NEC had significantly reduced progression free survival [6]. A study by Shu-Zheng Liu et al revealed that high levels of p53 protein in NENs of gastro enteropancreas showed shorter disease free survival[16]. Molecular profiling of Neuroendocrine malignancies by Namrata vijayvergia et al showed that amongst the various mutations detected in neuroendocrine tumors, TP53 mutation was associated with worse overall survival. Hence p 53 can be used as an independent prognostic marker in NENs regardless of the grades of tumour[17].