DOI: https://doi.org/10.21203/rs.3.rs-2046012/v1
Background
We used percutaneous computed tomography-guided core needle biopsy (PCT-CNB) of liver metastases from (gastroenteropancreatic neuroendocrine tumors) GEP NETs to identify inter-tumor grading classification heterogeneity.
Methods
We retrospectively investigated 92 patients with liver metastases in GEP NETs using PCT-CNB; 76 patients had tissue from liver and primary sites while 16 had tissue from liver and secondary liver sites. For tissue sampling, Ki-67 immunohistochemistry was performed and grading classifications was determined. Inter-tumor grading classification heterogeneity and associated changes on patient survival were also evaluated.
Results
No procedure-related mortality was recorded during and after biopsy. In 37/92 patients (40.2%), grading classifications changed: 13 patients increased from G1 to G2, 2 increased from G1 to G3, 5 decreased from G2 to G1, 14 increased from G2 to G3, 1 decreased from G3 to G1, and 2 decreased from G3 to G2. Patients with NET G1 or NET G2 grades had better progression-free survival (PFS) and overall survival (OS) when compared with NET G3 grade patients (P=0.001 and Pā<ā0.001, respectively). OS rates at 5 and 10 years were 67.5% and 26.0% for stable G2 patients, but decreased to 46.4% and 23.2% for increased G2 patients (P=0.016).
Conclusion
The PCT-CNB of liver metastases from GEP NETs showed grade differences between the liver tumor and primary site/secondary liver metastases. Also, when G2 increased, OS significantly decreased.
Neuroendocrine tumors (NETs) are generated in the neuroendocrine cell system; they form organoid cell aggregations or are composed of disseminated cells in different organs (1, 2). Gastroenteropancreatic NETs (GEP NETs) are the main NET subtypes and account for approximately 66% of all NETs (3). In recent years, GEP NET incidence rates have increased. From 1973–2012, according to National Cancer Institute data (4), GEP NET incidence rates increased 6.4-fold. To combat GEP NET, Ki-67 is used as a cell proliferation nuclear marker, it is an important prognostic disease variable (5, 6), and Ki-67 indices are included in World Health Organization (2010) grading classifications for GEP NETs (7).
Liver metastases are frequently observed in patients with GEP NETs; approximately 65–95% of patients will manifest these symptoms, even when primary tumors are small (8, 9). Also, liver metastases generate a strong prognostic impact in these patients (10, 11), while tumor heterogeneity may significantly impact clinical disease management. Previous studies have considered Ki-67 staining heterogeneity between primaries and metastases (12–15). Therefore, liver biopsies are required for accurate evaluations, and primary and metastatic tumor biopsies are recommended by a National Expert Canadian Group (16). However, some studies have indicated that primary tumor biopsies are not necessary if the liver metastasis biopsy provides positive and comprehensive tumor information (17).
Percutaneous computed tomography (CT) or ultrasound (US)-guided core needle biopsies (PCT-CNB or US-CNB) are widely used to assess diagnostic and prognostic markers and potential therapeutic targets in focal liver lesions (18, 19). While US is predominantly used for biopsies, CT is used for lesions that are not well-visualized by US; this may be due to small lesion size, altered liver morphology, isoechogenicity, body shape/build, or lesions contained at difficult anatomical locations such as the liver dome (20, 21). PCT-CNB is a safe procedure and generates adequate sample quality to study tumor heterogeneity. We used this technique to assess inter-tumor grading classification heterogeneity in GEP NETs. Tumor heterogeneity was investigated: (a) between the liver tumor and primary sites and (b) between the liver tumor and secondary liver metastases. Also, the effects of such changes on patient survival were investigated.
Our institutional review board approved this study and waived informed consent. Between August 2014 and December 2021, 92 patients with liver metastases in GEP NET underwent PCT-CNB; 76 had tissue from liver and primary sites and 16 had tissue from liver and secondary liver sites.
An enhanced CT or magnetic resonance imaging (MRI) scan was required within 30 days before PCT-CNB. The protocol also outlined PCT-CNB procedures, postoperative complication assessments, Ki-67 immunohistochemistry, Ki-67 assessments, follow-up procedures, and overall survival (OS) and progression-free survival (PFS) assessments.
Biopsies were performed using a 64-slice spiral CT scanner (250 mA, 120 kV, and 3 mm thick; Philips Healthcare, Andover, MA, USA). Biopsies were performed by one of five radiologists with > 5 years’ experience. First, when the patient was in an appropriate position, an abdominal CT scan (planning procedure) was performed. During procedures, unenhanced CT images were reviewed with previous diagnostic contrast-enhanced CT or MRI images to locate isoattenuating target lesions, identify cross-sectional levels, and ascertain lesional relationships with internal liver structures (landmarks). After marking the skin, sterile-draping, local anesthetic, and incision, radiologists performed a coaxial technique. For this, 18-gauge biopsy and 17-gauge introducer needles (SuperCore; Argon Medical Devices; Plano, TX, USA) were advanced to the biopsy target edge.
For the avoidance of damage to gallbladder, duodenum, pancreas, stomach, spleen, portal vein, etc., the previous diagnostic contrast-images and immediate CT images were compared using a side-by-side approach (Fig. 1). Tissue samplings numbered 2–3. CT images were immediately gathered after biopsy to identify any procedural issues. An abdominal belt was worn to reduce hepatic hemorrhage. Afterwards, patients were admitted and observed for 4 h, and those without complications were discharged.
Using Ki-67 antibodies, the tumor proliferation index was assessed in liver and primary tumor paraffin block sections. A two-step immunohistochemistry protocol was implemented; from paraffin blocks, 4 µm sections were sectioned and stained using a Ki-67 antibody (MIB-1, Dako Corporation, CA, USA). As positive and negative controls, normal tonsil tissue and a mouse IgG antibody were respectively used. Antigen retrieval was performed in citrate buffer (pH 6) for 3 min under pressure. For detection, an Envision + Dual Link Kit (Dako) was used. Diaminobenzidine was used as a highly sensitive chromogenic substrate and hematoxylin as a counterstain. Brown nuclear staining indicated Ki-67 positivity.
Ki-67 index was also assessed by manually counting camera-captured images, based on assessment of 2000 cells. For this method, each tumor section was manually scanned with a standard Olympus BX41 microscope (Olympus, Center Valley, PA), and the areas of greatest Ki-67 positivity (hot spots) were selected and captured using a camera snapshot and printed. Ki-67-negative and -positive cells were identified, counted, and crossed off once enumerated by two independent pathologists. Dark brown tumor nuclei indicated Ki-67-positivity, whereas light brown nuclei and cytoplasmic staining were ignored. The Ki-67 index indicates the ratio of positive cells to total counted cells (2000 cells) in highlighted areas. Then, using WHO (2010) digestive system tumor classification criteria (Ki-67: G1 = 0–2%; G2 > 2–20%, and G3 > 20%) (22), tumor samples were graded.
Follow-up intervals were dependent on surgery, systemic treatment, or nonsurgical liver-directed therapy. Apart from overseeing physical symptoms, follow-up programs comprised physical examinations, radiological imaging, and laboratory tests. As part of follow-up assessments, some patients underwent somatostatin receptor scintigraphy (Octreoscan) or Ga68 PET-CT. T All assessment results were recorded, including patient survival status, disease recurrence in patients, and progression follow-up times (the last imaging time). The follow-up deadline was March 2022.
All qualitative data were expressed as percentages and quantitative data as the median (range). Kaplan-Meier curves were generated to evaluate OS and PFS and were stratified by tumor grade at initial diagnosis. Statistical analyses were performed using commercially available software (SPSS 20, IBM, Chicago, IL, USA). A P < 0.05 value indicated statistical significance.
Percutaneous biopsy procedures were successfully performed in 92 patients with 97 liver tumors, with no treatment-related deaths and severe complications. Five patients experienced self-limited peri-hepatic hemorrhages, but interventions were not required. Also, no tumor seeding or diaphragmatic injury was observed. Patient clinical characteristics and pathological features are shown (Tables 1 and 2).
Characteristics | Number (Median or mean ± SD) | Percent |
---|---|---|
Patients | 92 | |
Median age (range), years | 48 (27–77) | |
Male/Female | 48/44 | |
Liver tumors (PCT-CNB) | 97 | |
Tumors size (cm, mean ± SD) | 3.9 ± 2.7 | |
Liver segments | ||
S1/2/3/4/5/6/7/8 | 0/3/6/5/12/29/20/22 | 0/3.3/6.5/5.4/12.0/30.4/20.7/21.7 |
PCT-CNB: Percutaneous CT-guided core needle biopsy |
Features | Number | Percent | |
---|---|---|---|
Grading classifications (PCT-CNB) | |||
G1 | 14 | 14.4 | |
G2 | 61 | 62.9 | |
G3 | 22 | 22.7 | |
Grading classifications (Initial diagnosis) | |||
G1 | 20 | 21.7 | |
G2 | 59 | 64.1 | |
G3 | 13 | 14.1 | |
Primary tumor Site | |||
Stomach | 3 | 3.3 | |
Duodenum | 3 | 3.3 | |
Pancreas | 75 | 81.5 | |
Jejunum/ileum | 4 | 4.3 | |
Rectum | 7 | 7.6 | |
Tumor samples Method (liver and primary sites) | |||
PCT-CNB | 97 | 52.7 | |
PUS-CNB | 10 | 5.4 | |
EUS-FNA | 12 | 6.5 | |
Gastroscopic/Colonoscopic biopsy | 4 | 2.2 | |
Surgery | 61 | 33.2 | |
PCT-CNB: Percutaneous CT-guided core needle biopsy; PUS-CNB: Percutaneous US-guided core needle biopsy; EUS-FNA: Endoscopic ultrasonography guided fine needle aspiration |
Tumor grading (in 92 patients) revealed: 14 G1, 61 G2, and 22 G3 grades. At initial diagnosis, 20 G1, 59 G2, and 13 G3 grades were recorded. Primary tumors were located in the duodenum (n = 3), jejunum/ileum (n = 4), stomach (n = 3), rectum (n = 7), and pancreas (n = 75). In addition to 97 PCT-CNB biopsies, other tumor sample methods at liver or primary sites included percutaneous ultrasound-guided core needle biopsy (PUS-CNB) (n = 10), endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) (n = 12), gastroscopic/colonoscopic biopsy (n = 4), and surgery (n = 61).
In 37/92 patients (40.2%), tumor classifications changed: 13 patients increased from G1 to G2, 2 from G1 to G3, and 14 from G2 to G3, while 5 decreased from G2 to G1, 1 from G3 to G1, and 2 from G3 to G2.
In 76 patients with Ki-67 staining of liver metastases and primary tumors, secondary diagnostic tumor grades were compared with initial diagnoses. We identified 25 patients (32.9%) with increased grades; 13 increased from G1 to G2, 2 from G1 to G3, and 10 from G2 to G3. We identified 6 (7.9%) patients with decreased grades; 4 decreased from G2 to G1, 1 from G3 to G1, and 1 from G3 to G2. Also, 45 patients (59.2%) had stable grades: 5 with G1, 32 with G2, and 8 with G3 tumors (Table 3).
Patients | Grade at Initial diagnosis | Grade at second diagnosis |
---|---|---|
5 | G1 | G1 |
13 | G1 | G2 |
2 | G1 | G3 |
4 | G2 | G1 |
32 | G2 | G2 |
10 | G2 | G3 |
1 | G3 | G1 |
1 | G3 | G2 |
8 | G3 | G3 |
PCT-CNB: Percutaneous CT-guided core needle biopsy |
In 16 patients with Ki-67 staining of liver and secondary liver sites, secondary diagnostic tumor grades were compared with initial diagnoses. We identified 6 patients (37.5%) with changed classifications: 4 patients increased from G2 to G3, 1 decreased from G2 to G1, and 1 decreased from G3 to G2. Ten patients (62.5%) had stable grades: 8 with G2 and 2 with G3 tumors (Table 4).
Patients | Grade at initial diagnosis | Grade at second diagnosis |
---|---|---|
0 | G1 | G1 |
0 | G1 | G2 |
0 | G1 | G3 |
1 | G2 | G1 |
8 | G2 | G2 |
4 | G2 | G3 |
0 | G3 | G1 |
1 | G3 | G2 |
2 | G3 | G3 |
PCT-CNB: Percutaneous CT-guided core needle biopsy; PUS-CNB: Percutaneous US-guided core needle biopsy |
For all patients, the median follow-up time was 44.0 months (range = 4.3–240.0). OS rates at 5 and 10 years were 89.2% and 64.5% for G1 grades, 59.4% and 24.0% for G2 grades, and 10.1% and 0.0% for G3 grades (P < 0.001) (Fig. 2), respectively.
For stable G2 grades, OS rates at 5 and 10 years were 67.5% and 26.0%, but this decreased to 46.4% and 23.2% for increased G2 grades (P = 0.016) (Fig. 3).
PFS rates at 5 and 10 years were 62.4% and 41.6% for G1 grades, 34.3% and 0% for G2 grades, and 0% and 0% for G3 grades (P = 0.001) (Fig. 4).
We used PCT-CNB for liver metastases in GEP NETs to identify inter-tumor grading classification heterogeneity. From this research, we made three important observations. Firstly, PCT-CNB was safely performed with sufficient tumor sample for tumor grading. Secondly, PCT-CNB showed frequent grade differences between liver tumor and primary site/secondary liver metastases. Thirdly, when G2 grades increased, the OS decreased significantly.
PCT-CNB for liver tumors is a minimally invasive tools that supplements clinical disease management, including diagnostics, prognostic assessments (disease staging), and/or making therapeutic decisions (20, 23). The biopsy technique may be performed in a coaxial or non-coaxial manner; however, it appears the former technique is safer when compared with other biopsy techniques as multiple needle passes may be made with a single pleural puncture, thereby reducing pneumothorax incidence rates. The coaxial approach is particularly advantageous as a single hepatic puncture is made to permit multiple cuttings, while a single needle technique may generate two or more hepatic punctures per procedure (24). For NETs, EUS-FNA sampling cannot generate specimens for histology while FNA-yield may be insufficient to determine Ki-67 indices. Therefore, the EUS-FNA appears suboptimal for the pretreatment grading of NETs (25, 26).
We used the coaxial technique in this study; 2–3 specimens were acquired and reflected standard practices at our institution, and importantly, provided sufficient tumor sampling material for liver metastasis classification grading of GEP NETs. Moreover, percutaneous biopsy procedures were technically successful with no treatment-related deaths and severe complications recorded.
Inter-tumor Ki-67 heterogeneity values were previously reported (12, 14, 15). Dhall et al. (12) identified differences between different tumor sites in ileal NET, where 3/60 (5%) patients showed Ki-67 heterogeneity between different primary tumor sections. Grillo et al.(14) identified increased Ki-67 labeling indices between primary tumor and metastatic sites in 19/49 (38.8%) patients with GEP NETs, while a recent study (15) identified increased grading classifications in 35/103 (34.0%) cases. Similar to these data, we observed similar rates of change between liver tumors and primary site/secondary liver metastases in 37/92 (40.2%) patients.
Previously, associations between increased grades and patient survival were evaluated (2, 14, 15), with shifts to next grades mainly occurring from G1 to G2 (14, 15). Keck et al. (15) reported that patients with increased metastasis grades performed poorly; however, no significant differences were reported for PFS (p = 0.55) or OS (p = 0.32). This observation concurred with our data where grades in 13 patients increased from G1 to G2. However, comparisons were not made for patient survival between the increased G1 group and the stable G1 group as this latter group was had low numbers (n = 5). While academically interesting, these data exert no impact on NET patients and their clinical management, as patients with G1 and G2 tumors receive the same treatment (27). Additionally, in 10 of our patients with GEP-NET, the Ki-67 index changed from G2 to G3 during the disease course. Twenty-three patients had stable grade G2 tumors. OS rates at 5 and 10 years were 67.5% and 26.0% for the stable G2 group, but this decreased to 46.4% and 23.2% for the increased G2 group (P = 0.016). This finding agreed with previous reports (2, 15, 28) showing that increased grades were associated with decreased OS.
Currently, it is unclear why Ki-67 indices are often heterogeneous in GEP NETs. It was previously hypothesized [1] that this heterogeneity was attributed to genetic variations in NETs, with similar explanations for other solid cancers, whereas another study [2] theorized that Ki-67 index changes were possibly due to therapy resistance and treatment effects. Further studies are warranted to explore these underlying mechanisms.
G1 to G2 tumor changes in patients would be considered well-differentiated, therefore treatments would not necessarily change under existing clinical management approaches (15). However, well-differentiated G2 GEP NET changes may advance to G3 tumors which are morphologically differentiated and may require other therapeutic strategies (29). Several strategies are available for the clinical management of liver metastases in GEP NET (17) and range from surgery to ablation with various interventional radiology procedures (30, 31), including regional and systemic therapy with varied cytotoxic, biological, or targeted agents (32–35). Therefore, it is plausible that PCT-CNB analysis of liver metastases provides comprehensive insights for therapeutic GEP NET decision-making.
Our study had several limitations. Firstly, the study was retrospective in nature with a low number of stable G1 patients (n = 5), therefore, no survival comparisons were made between stable G1 and increased G1 patients. Secondly, while CT provides excellent anatomical information and improved deep lesion visualization when compared with US, repeated CT scans deliver more ionizing radiation. Thirdly, real-time guidance is not provided by CT as biopsies are taken. Finally, “beam hardening” from biopsy needle metal artifacts can visually obscure target lesions.
PCT-CNB was safely performed with sufficient tumor sampling for reliable Ki-67-indexing and grading classifications of liver metastases from GEP NETs. Our data identified grade differences between liver tumor and primary site/secondary liver metastases, and critically, when the G2 grade increased, OS was significantly decreased.
percutaneous CT-guided core needle biopsy
Gastroenteropancreatic neuroendocrine tumors
endoscopic ultrasound-guided fine needle aspiration
percutaneous ultrasound-guided core needle biopsy
overall survival
progression-free survival
Ethics approval and consent to participate
This study was approved by the ethics committee of Fudan University Shanghai Cancer Center (Decision/protocol number of ethics committee approval: 2011226-4.), and performed in accordance with the Declaration of Helsinki. As it was a retrospective study, all data were collected from a medical records system. Therefore, the study was exempt from the requirement to obtain individual informed consent, based on the Ethical Guidelines of the Ethics Committee of Fudan University Shanghai Cancer Center.
Consent to publish
Not applicable.
Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.
Competing interests
The authors declare that they have no competing interests.
Funding
This work was supported by National Natural Science Foundation of China (Grant No. 82072034).
Authors' Contributions
Wentao Li, Chao Chen, and Ying Wang designed/performed most of the investigation, data analysis and wrote the manuscript; Xinhong He and Chao Chen contributed to interpretation of the data and analyses. All of the authors have read and approved the manuscript.
Acknowledgements
None.