Clinico-Morphological Correlations with Ki-67 and p53 Immunohistochemical Expression in High-Grade Gastrointestinal Neuroendocrine Neoplasms

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The paper includes 42 high-grade gastrointestinal (stomach, small intestine, and colorectal) neuroendocrine neoplasms: 26 pure (16 NETG3; 10 NEC:  6 SCNEC, and 4 LCNEC) and 16 LCNEC as components of MiNENs, to study clinico-morphological correlations with Ki-67 and p53 immunohistochemical (IHQ) expression.

The authors conclude that the observed significant associations with key morphological parameters reinforce their utility in differentiating NET G3 from NEC, especially in cases with overlapping histological features. The p53 IHQ profile may be a useful diagnostic adjunct in routine practice.

The study may add data for the management of these rare neoplasms if the authors clarify several issues, including:

1. “Formalin-fixed, paraffin-embedded tumor blocks were sectioned at 4 µm thickness and mounted on slides.” Clarify/state the method to select the blocks (one per case?) for the IHQ evaluation.
2. “Nuclear staining (p53) was considered positive”. Clarify/state and discuss briefly the quantification method (e.g., manual/digital). Also, the intensity of the staining (i.e., weak, intense, variable) and the (putative) impact of heterogeneity observed in NETG3, SCNEC, and LCNEC should be stated and discussed.
3. “…a Ki-67 index >20%...”. Clarify and state whether this study included re-evaluating the Ki-67 index in the same sections used for p53 IHQ. Also, the (putative) impact of heterogeneity observed in NETG3, SCNEC, and LCNEC should be stated and discussed.
4. The report does not address necrosis evaluation. The authors may want to improve the paper by including necrosis evaluation and briefly discussing its putative impact in differentiating NET G3 from NEC.
5. The report does not address the lymphocyte density in the stroma evaluation of high-grade gastrointestinal neuroendocrine neoplasms. The authors may want to improve the paper by including the data on this issue, briefly discussing its putative impact in theranostics of NET G3 and NEC, and its impact on the Ki-67 index evaluation.
6. Clarify/state the meaning of the MiNEN dimension (global? NEC component?); and, the NEC component in lymphovascular invasion, peri-neural infiltration, and lymph-node metastases. In many cases, NE vs non-NE carcinomas are challenging when the NE phenotype is incomplete, the two components are not separated, and/or contain amphicrine cells.
7. Discuss briefly and state the putative meaning of p53 IHQ status being “wild-type” and “non-wild-type, in NEC and NEC component of MiNEN (n=2) and NET G3 (n=2), respectively; i.e., its predictive value on the TP53 mutational status.
8. Briefly discuss the role (e.g., putative theranostic impact) of SSTR IHQ in the NETG3 vs NEC setting.
9. Since the authors recommend continued multicentric collaboration to expand sample cohorts and validate their findings in larger populations, providing a supplementary file with detailed data (patient age at diagnosis, sex, tumor site, maximum tumor size, pT category, lymph node metastasis, pTNM stage, lymphovascular invasion, perineural infiltration, NEC subtype - SCNEC or LCNEC, Ki-67 index, and final diagnosis confirmed through IHC) of each case included in the study would be pretty valuable.
10. Improve the quality of Figure 2 to illustrate wild-type and non-wild-type p53 IHQ patterns better, if available, with the corresponding KI-67 IHQ of the same areas.

Author Response

Thank you very much for taking the time to review this manuscript! Please find the detailed responses attached and the corresponding revisions/corrections in track changes in the re-submitted files

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This manuscript addresses an important topic regarding the differentiation between NET G3 and NEC, based on Ki-67 index and p53 immunohistochemical profiles. Although the study is structured and the topic is interesting, this reviewer has the following concerns.

1. The authors stated that differentiating between NET G3 and NEC remains a diagnostic challenge. However, it appears that the classification in this study was performed by a single local institution without centralized or external pathological review. This limitation should be clearly acknowledged in the “Limitations” section of the Discussion.
2.  In the beginning of the Discussion,  The authors compared patient demographics and tumor characteristics with previous large-scale studies. However, given the small sample size of the current study, such comparisons should be made with caution. It is recommended that this introductory portion of the Discussion be substantially shortened.
3.  Several previous studies have already demonstrated the usefulness of p53 IHC. These prior findings are not adequately discussed in the current manuscript as a comparison. As a result, the novelty and added value of the present study should be more clearly articulated.

Author Response

Thank you very much for taking the time to review this manuscript! Please find the detailed responses attached and the corresponding revisions/corrections in track changes in the re-submitted files

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

I believe the new version of the paper, which acknowledges its limitations, may interest readers.

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript was well revised.