The NET and NEC are believed to represent two distinct entities with differing biological, histological, and genetic characteristics [34, 37]. The aforementioned concept has recently been challenged by observations that suggest a transformation of G3NET to NEC [30]. In this longitudinal retrospective investigation of G3NET patients with a sudden worsening of the clinical condition, we examined the progression of metastasized NETs under systemic therapy with follow-up biopsies. As sudden and severe deterioration in condition, we called a rather short (1–2 months) phase of illness, in which the patients showed a remarkable enlargement of liver metastases and/or increase in number of tumor lesions along with an increase in transaminases and circulating tumor markers such as chromograninA and/or NSE. We found that a subset of these patients exhibited G3NETs, most of which originated from the pancreas, that had developed NEC-like features, including TP53 mutations. However, no transformation to a typical NEC was observed.

In a cohort of 62 NET patients who underwent a tissue examination at the time of diagnosis and subsequently throughout the course of the disease, approximately two-thirds of patients were found to have a G3NET at the last biopsy (40/62, 65%). The G3NETs were observed to emerge from G1NETs (n = 4) or G2NETs (n = 24), and only 12 patients exhibited a G3NET at the initial examination. Among the G3NET patients, a sudden and severe deterioration in condition was observed in nine patients. In all of these patients, the metastatic tumor tissue exhibited histological changes and immunohistochemical overexpression of p53, accompanied by a corresponding mutation in the TP53 gene. In eight out of nine NEC-like G3NETs, the allele frequency was high, suggesting monoclonality of the mutation. This somewhat unexpected finding may be explained by the fact that most of the debulking sample contained NEC-like G3NET tissue and was probably the only tissue available for molecular examination. The other G3NETs exhibited no alterations in the TP53 gene. The NEC-like changes were characterized by high Ki67 values (mean 65, ranging from 50 to 85), a confluent growth pattern, nuclear pleomorphism, and necrosis, which were consistent with large cell NEC features in all but one case, which exhibited characteristics more akin to small cell NEC. The presence of intratumoral heterogeneity [38], with the presence of NET tissue in proximity to NEC-like changes, was a common observation, particularly evident in larger samples. The TP53 alterations were represented by seven missense mutations with disparate mutation patterns, in addition to a single deletion. RB1 alterations were observed infrequently, manifesting as mutation and loss of expression in a single case. It is noteworthy that the molecular analysis also identified MEN1 and DAXX mutations in four and two pancreatic NEC-like G3NETs, respectively. As these mutations are characteristic of PanNETs but not NECs, their identification provides compelling evidence that the pancreatic NEC-like G3NETs originated from NETs [3, 5, 39]. Another finding that supports the NET origin of NEC-like G3NETs is the frequent expression of SST2. This has been described in many G3NETs, but rarely in NECs [3]. These findings collectively indicate that despite the presence of advanced histological alterations in conjunction with TP53 mutations, there is no complete transformation to NECs. We thus designated these neoplasms as “NEC-like G3NET” (Fig. 5). Seven of our nine NEC-like G3NETs originated from the pancreas (mainly from the tail), and two from the rectum. While the pancreas is already known to be the most common site of G3NET origin [1, 8, 30, 40], the rectum is an uncommon site for G3NET [1]. Consequently, detailed data on high-grade rectal NETs are not available. A review of the literature reveals that the only extrapancreatic NETs that have been observed to progress from G1/G2 to G3NET and acquire a TP53 mutation have been reported in the lung and thymus [28, 29, 41].

The graph illustrates longitudinal trajectories of progressing metastasized G3 neuroendocrine tumors (G3NETs) under systemic therapies. The majority of G3NETs (28/40, 70%) evolved from G1/G2NETs, while only 30% were primary G3NETs (blue trajectories). After a median interval of 24 months, the delta Ki67 reached a value of 19%. Nine of the 40 G3NETs (22%) exhibited an abrupt deterioration after a median interval time of 60 months, accompanied by NEC-like transformation and a TP53 mutation (red trajectories). No NEC transformation occurred

Two recent studies conducted on the pancreas have yielded comparable results regarding the development of high-grade pancreatic neoplasms, aligning with the findings of our investigation [26, 30]. Umetsu et al. employed an integrated molecular and immunohistochemical approach to examine 47 high-grade pancreatic neoplasms, identifying 34 high-grade PanNENs, either as G3NETs or NECs, after excluding 17 mixed acinar NECs [26]. As expected, PanNECs frequently exhibited alterations in TP53, RB1, and CDKN2A, but also in KRAS and SMAD4. In contrast, G3PanNETs harbored NET-typical gene alterations, including MEN1, DAXX, ATRX, TSC2, SETD2, and CDKN2A. Furthermore, 35% (6/17) of these NETs exhibited TP53 alterations, which are comparable to our NEC-like G3NETs [26]. The resemblance of Umetsu’s TP53-mutated G3PanNETs to our NEC-like G3NETs is further substantiated by the observation that their Ki67 values were markedly elevated in comparison to those observed in G3PanNETs lacking TP53 mutation.

The other investigation is a longitudinal study that reported a transformation from NET to NEC in seven out of 152 patients with gastroenteropancreatic or pulmonary NETs after PRRT treatment [30]. Molecular analysis of the five high-grade NENs, all of which originated from the pancreas, revealed the presence of TP53 mutations in three of the five cases [30]. Given that the TP53 mutations were partially accompanied by other gene alterations, including ATRX, TSC2, TSC1, and PIK3CA, which have been previously reported in PanNETs [42], it can be inferred that these high-grade PanNENs also belong to the group designated as NEC-like G3NETs. Cordero-Hernandez designated the tumors as NECs, yet a detailed description is absent, thereby rendering it conceivable that the tumors’ histology aligns with ours. Additionally, high-grade transformation with TP53 mutation has been documented in two other PanNETs during tumor progression [27, 43]. The aforementioned studies, including our own, collectively indicate that TP53 mutations that emerge following multimodal treatment may facilitate the evolution of NETs into high-grade NENs. It appears that RB1 does not exert a significant influence on this phenomenon, as evidenced by the observation of a single RB1 alteration in our series of NEC-like G3NETs.

All studies of NET progression, with the exception of one whose study design and complex results are challenging to interpret [31] and compare with other investigations, concur that the majority of tumors evolve from low-grade to high-grade NETs. However, there is a paucity of knowledge regarding the time frame for NET progression, as this is difficult to ascertain due to the definition of the endpoint. Additionally, the endpoint of the interval time could not be precisely defined in our study, as it was dependent on the decision of the patient’s physician regarding the timing and necessity of a biopsy. Nevertheless, the presence of analogous alterations in all endpoint biopsies led to the conclusion that, at the time of biopsy, the transformation process was still ongoing in all patients. Accepting this somewhat imprecise endpoint determination, the interval time between diagnosis and rapidclinical deterioration with concomitant NEC-like histology was unexpectedly much longer with a median interval time of 60 months (range 30–168, with the longest interval time in the two rectal NEC-like G3NETs) than the interval time of 24 months (range 7–180) in G3NET patients without abrupt disease deterioration (Fig. 1). A notable distinction was observed in the Ki67 delta values at the endpoint of the interval time, with a median delta Ki67 value of 53% (range 37 to 68) in NEC-like G3NETs, in contrast to a median delta Ki67 value of 19% in G3NETs without NEC-like features (Fig. 1). Our findings are generally consistent with those of Cordero-Hernandez, who reported a median interval time between diagnosis and transformation to high-grade NEN of 83 months (range 17–210) and Ki67 values up to 90% (range 51 to > 90%) at the endpoint of the interval time [30]. These observations indicate that the TP53 mutation, which is likely to be a primary driver of the accelerated growth observed in one or more of the metastases, is frequently a relatively late event in G3NETs undergoing systemic treatment.

The current standard of care for G3NETs is not yet established and is currently being investigated in clinical trials. These include the use of PRRT, chemotherapy with either CAPTEM (capecitabine and temozolomide) or FOLFOX (folinic acid fluorouracil and oxaliplatin), and everolimus [44]. As demonstrated in the aforementioned study by Cordero-Hernandez, seven patients who exhibited abrupt progression demonstrated a high-grade transformation subsequent to PRRT following temozolomide chemotherapy [30]. In the present study, three of the nine patients with NEC-like G3NETs who received PRRT prior to the final examination were examined for tumor mutational burden, microsatellite instability, and PD-L1 status. No differences were identified compared to the findings in G3NETs without NEC-like transformation. Although the question regarding a potential correlation between treatment and accelerated disease progression is of significant interest, it is beyond the scope of this investigation due to the limited number of NEC-like G3NET patients, which is insufficient to yield meaningful insights.

In conclusion, a retrospective longitudinal study comprising baseline and follow-up biopsies identified the development of G3NETs with NEC-like changes in a cohort of 40 G3NET patients. Of these, nine patients exhibited rapid progression in liver metastases from seven pancreatic NETs and two rectal NETs. It is reasonable to conclude that this phenomenon is attributable to the novel TP53 mutation, which was detected in all nine cases at the last evaluation, and which likely accelerated tumor growth and altered tumor structure. Additionally, our findings indicated that a considerable number of pancreatic NEC-like G3NETs exhibited mutation characteristic of PanNETs, including MEN1 and DAXX. This observation lends support to the hypothesis that no morphomolecular transformation into a typical NEC had occurred. Future studies with spatial transcriptomics may be helpful, to determine the area in the G3NET and NEC-like G3NET where the genetic transformation occurs.