Introduction

According to the new World Health Organization (WHO) classification of Gastroenteropancreatic Neuroendocrine Neoplasia (GEP-NEN), [1] Neuroendocrine Carcinoma (NEC) is defined as a distinct disease entity different from neuroendocrine tumor (NET) that constitutes well-differentiated NEN. Specifically, NEC is characterized by poor differentiation, mitotic rates greater than 20 mitoses per 2mm² and a Ki-67 index higher than 20%. While the origin of GEP-NEC remains elusive, [2] the existence of mixed neuroendocrine-non-neuroendocrine neoplasms (MiNEN) suggests that GEP-NEN and non-NEN components develop from a common precursor lesion. [3] A comprehensive molecular characterization by whole-genome sequencing has revealed frequent genetic alterations in TP53 and RB1 in GEPNECs. [2,4,5] Because of the scarcity of patients with NEC, there are no standard treatment flowcharts established. Since NECs are invariably aggressive and frequently metastasize to distant organs, resulting in an extremely poor prognosis, the first-choice treatment is considered to be intense systemic chemotherapy even if radical resection seems feasible. The clinical benefit of the resection of primary colorectal and metastatic lesions for macroscopic eradication of cancer cells has yet to be determined, so the actual management of well-advanced NECs depends on the decisions arrived at through multidisciplinary discussions in each case. Here, we present an extremely rare case of a rectal NEC with a solitary renal metastasis which were macroscopically resected by a laparoscopic approach following intensive systemic chemotherapy with CDDP and CPT11.

Case Presentation

A 56-year-old woman was referred to our hospital for further examination of melena. There were no significant abnormalities including tumor markers (CEA: 1.4ng/ml, CA153: 2.0U/ml). Upon rectal examination, a tethered tumor was felt on the anterior wall of the rectum, 8 cm above the anal verge. Subsequent colonoscopy revealed a type 2 tumor at the upper rectum, and pathological examination of biopsied samples from it showed an infiltrative proliferation of carcinoma cells characterized by a high nuclear-cytoplasmic (N/C) ratio and prominent nucleoli. Immunohistochemical staining was positive for both synaptophysin and chromogranin A, and the Ki-67 index was higher than 90% (Figure 1). These pathological findings confirmed by two experienced pathologists (HK and KN) led to a definitive diagnosis of rectal NEC. Contrast-enhanced Computed Tomographic (CT) scans revealed a tumor of 30 mm in size on the anterior-left wall of the rectum, which was suspected to infiltrate through the rectal wall. Although no significantly swollen lymph nodes were detected, an enhanced mass lesion of 15 mm in size was observed at the upper pole of the right kidney (Figure 2). On renal magnetic resonance (MR) imaging, the tumor was isointense and hyperintense compared to renal parenchyma on T1- and T2weighted images, respectively. In addition, the mass lesion was hyperintense on diffusion-weighted images and clearly enhanced with contrast material (Figure 2). Positron emission tomography with 18-Fluorodeoxyglucose (FDG-PET) showed increased uptake signals in the mass lesions on the anterior-left rectal wall (SUVmax 19.8) and at the upper pole of the right kidney (SUVmax 11.0). There were no other uptake signals detected suggestive of distant metastases. Furthermore, octreotide scintigraphy showed similar mild uptake signals in the primary rectal lesion and right renal tumor (Figure 2). Taken together, the most probable diagnosis for the renal tumor was that it was a solitary metastatic lesion from the rectal NEC. Thus, the patient was diagnosed with rectal NEC cT3N0M1a, cStage IVA. Intensive systemic chemotherapy with cisplatin plus irinotecan was initiated. During the treatment, the tumor response was evaluated regularly by contrast-enhanced CT scans, and at the completion of the fourth course of chemotherapy, both the primary and metastatic tumors were evaluated radiographically to be stable diseases according to the Response Evaluation Criteria in Solid Tumors (RECIST) criteria. Since it would have been difficult to cure the intractable disease by systemic chemotherapy alone and there were no new metastatic lesions detected, we chose to perform a simultaneous radical resection of the two lesions after multidisciplinary, indepth discussions. We proceeded with laparoscopic low anterior resection along with a diverting loop ileostomy and simultaneous partial nephrectomy. On laparoscopic examination, there were no peritoneal disseminations and the tumor at the upper pole of the right kidney was surrounded by Gerota’s fascia, a finding compatible with renal metastasis, which was extirpated laparoscopically with satisfactory circumferential clearance (Figure 3).

Figure 1: Microscopic findings of the biopsied specimen from the rectum. Carcinoma cells with a high N/C ratio and prominent nucleoli showed an infiltrative proliferation (hematoxylin and eosin staining) (a). Immunohistochemical staining was positive for synaptophysin (b) and chromogranin A (c), and the Ki-67 labeling index was evaluated as higher than 90% (d). All the figures ((a)- (d)) were taken by 40x objective lens.

Figure 2: Preoperative imaging findings. Abdominal contrast-enhanced CT (a, b) showed a 30-mm tumor on the anterior-left wall of the rectum with a sign of infiltration beyond the wall (a), along with an enhanced mass lesion of 15 mm in size at the upper pole of the right kidney (b). On renal MR images (c-f), the renal tumor was depicted as an isointense and hyperintense mass in comparison to renal parenchyma on T1- (c) and T2- (d) weighted images, respectively. Furthermore, the mass lesion showed a hyperintense signal on diffusion-weighted images (e) and a clear enhancement with contrast material (f). FDG-PET (g) and octreotide scintigraphy (h) showed significant (SUVmax 11.0) and mild uptake in the mass lesion at the upper pole of the right kidney, respectively.

Figure 3: Surgical findings. The tumor at the upper pole of the right kidney was covered by Gerota’s fascia, suggestive of a tumor arising from the kidney. There was no evidence of any metastases in the abdominal cavity.

Macroscopically, the resected specimens of the rectal and renal lesions were a T3 tumor of type 2 (55×50 mm) and a white, solid tumor (20×10 mm), respectively. As in the biopsied samples, pathological findings of the rectal tumor showed that carcinoma cells with a high N/C ratio and prominent nucleoli proliferated extensively in an infiltrative way and were immunopositive for synaptophysin and chromogranin A with a Ki-67 index of 80-90%, all of which were compatible with NEC (Figure 4). There were no components of adenocarcinoma detected and prominent lymphovascular invasion was evident. With regard to the histological assessment of response to preoperative systemic chemotherapy, the treatment effect was minimal: tumor cell necrosis or degeneration was present in less than one-third of the entire lesion. Based on the pathological similarities confirmed by the two pathologists (HK and KN), the renal tumor was diagnosed as a solitary renal metastasis from the rectal NEC (Figure 4), resulting in a final diagnosis of ypT3N0M1a, ypStage IVA. The postoperative course was uneventful and she was discharged on the 13th day after the operation. The patient is currently being monitored carefully, undergoing three-monthly CT scans without any adjuvant chemotherapy. At two years after the operation, there is no evidence of local recurrence or distant metastasis.

Figure 4: Microscopic findings of the resected specimen of the rectum (a-d) and kidney (e,f). In both tumors from the rectum (a) and kidney (e,f), carcinoma cells with a high N/C ratio and prominent nucleoli proliferated in an infiltrative pattern similar to that found in biopsied samples (hematoxylin and eosin staining). Immunostaining was positive for synaptophysin (b) and chromogranin A (c), and the Ki-67 labeling index was 80 to 90% (d) in the rectal tumor. Figures 4a-4d and 4f were taken by 40x objective lens, and figure 4e was taken by 4x objective lens.

Genetic Mutation Profiles

Whole exome and genome sequencing showed 76 and 73 biologically significant exonic mutations including amino acid substitution, frameshift and deletion except synonymous mutations in the primary tumor (1T) and the renal metastatic lesion (3KM), respectively (Figure 5). Of these mutations, 63 were common, accounting for 82.9% and 86.3% in 1T and 3KM, respectively, indicating that the primary and metastatic tumors had very similar mutation profiles. The recurrent mutations common in NEC⁶ were detected in both lesions. These included two different APC mutations (p.K149Rfs*30 (frame-shift) and p.E1277X (stopgain)), a non-synonymous, pathogenic mutation in TP53 (p.C3F) and 4bp deletion in RB1 resulting in splicing alteration. There were no genetic mutations detected in KRAS or BRAF.

Figure 5: Genetic mutation profiles. Whole exome sequencing showed 76 and 73 somatic mutations including nonsynonymous SNV and indel mutations in the primary tumor (1T) and the renal metastatic lesion (3KM), respectively. Of these mutations, 63 were common, indicating that the primary and metastatic tumors had very similar mutation profiles.

Drug Sensitivity Screening Tests

The Patient-Derived Cell Lines (PDCs) were established from the primary and metastatic lesions, named JC-581-TR and JC581-Ren. Using these PDCs, we examined the drug sensitivity to originally established drug inhibitor library consisting of target wellidentified or approved inhibitors.⁷ Neither cytotoxic inhibitors L-OHP (50 μM) nor SN-38 (500 nM) reduced cell viability, while several inhibitors (infigratinib, Bcl family inhibitors, and proteasome inhibitors) potently inhibited the growth of both JC-581-TR and JC-581Ren cells (Figure 6). Infigratinib is an approved FGFR inhibitor for Non-Small-Cell Lung Cancer (NSCLC) and cholangiocarcinoma, but phospho-RTK (receptor tyrosine kinase) array analysis suggested that the FGFR family (FGFR1 to FGFR4) did not seem to be a direct target of infigratinib in JC-581-TR cells (Figure 7). According to a previous report,^[2] infigratinib can inhibit multiple other kinases, and treatment with another FGFR inhibitor, CH5183284, did not inhibit the growth of JC-581-TR or JC-581-Ren cells at all (Figure 7). These results suggested that other targets or multiple targets of infigratinib may play an important role in JC-581-TR and JC-581-Ren cell growth. Obatoclax, a Bcl-2/Bcl-xL/Mcl-1 inhibitor, showed a marked suppression of cell viability in JC-581-TR cells. Moreover, a potent Bcl-2/Bcl-xL dual inhibitor, navitoclax, also inhibited the cell growth of JC-581-TR (Figure 7), indicating that the cell survival of JC-581-TR might be highly dependent on Bcl family proteins.

Figure 6: Drug sensitivity screening tests using PDCs established from the primary and metastatic lesions (JC-581-TR and JC-581Ren, respectively). A heatmap shows that several inhibitors (infigratinib, Bcl family inhibitors, and proteasome inhibitors) exerted a significant growth inhibition of both JC-581-TR and JC-581-Ren cells. In contrast, neither cytotoxic inhibitors L-OHP (50 μM) nor SN-38 (500 nM) reduced cell viability.

Figure 7: Drug sensitivity profiles. The growth of JC-581-TR and JC-581-Ren cells was not inhibited by administration of a different FGFR inhibitor, CH5183284 (a,b). Phospho-RTK array analysis shows that the FGFR family (FGFR1 to FGFR4) was unlikely to be a direct target of infigratinib in JC-581-TR cells (c). Obatoclax, a Bcl-2/Bcl-xL/Mcl-1 inhibitor, and navitoclax, a potent Bcl-2/Bcl-xL dual inhibitor, showed a marked suppression of cell viability in JC-581-TR cells (d).

Methods

The patient provided written informed consent for research use of samples including genetic analyses, establishment of PDCs and drug screening tests. Analyses were performed in accordance with protocols approved by the Institutional Review Board of the Japanese Foundation for Cancer Research.

Whole Exome and Whole Genome Sequencing

The sequence library for whole exome sequencing was prepared using a SureSelect V6 kit (Agilent Technologies). Then 151 bp paired-end libraries were subjected to mass sequencing using a HiSeq 2000 (Illumina). The sequence library for whole genome sequencing was prepared using TruSeq DNA PCR-Free (Illumina) to obtain a final library of 300-400 bp average insert size. The sequence data were processed through the Genomon pipeline (http://genomon.hgc.jp/exome/). The sequence reads were aligned to the NCBI Human Reference Genome Build 37 hg19 with BWA version 0.5.10 using default parameters (http:// bio-bwa.sourceforge.net/). PCR duplicate reads were removed with Picard (http://www.picard.sourceforge.net). Mutation calling was performed using the EBcall algorithm,^[3] and somatic mutations were called by comparing a tumor specimen and its matched normal colon tissue. The mutation was annotated by ANNOVAR (http://www.openbioinformatics.org/annovar/).

Establishment of patient-derived cells (PDCs) and culture condition

JC-581-TR and JC-581-Ren cells were established from surgically resected tumor samples of the primary and metastatic lesions using StemPro-hESC medium (Invitrogen) supplemented with 1.6% BSA (Invitrogen), 8 ng/ml bFGF (Nacalai Tesque), 100 μM 2-Mercapto-ethanol (Invitrogen), 10 μM Y-27632 (LC Laboratories), and 1×antibiotic–antimycotic mixed stock solution (Nacalai Tesqu). In brief, a few pieces of tumors from primary and renal metastasized site were obtained right after surgical resection. Tumor pieces were immediately placed in ice-cold culture medium with antibiotic-antimycotic (Gibco). Tumor tissues were cut into small fragments, and washed twice with ice-cold PBS supplemented with antibiotic–antimycotic. Tumor pellets were enzymatically digested with collagenase/dispase (Roche) and DNase I in StemPro-hESC medium for 30 to 60 minutes. After washing with antibiotic–antimycotic and 0.2% BSA-containing PBS, the cell pellets were cultured in the StemPro-hESC medium to establish the patient-derived JC-581-TR or JC-581-Ren cell lines. After establishing the cells, cell line authentication with 10 locus STR analysis was performed (Supplementary Table S1). The picture of the established cell line was shown in Supplementary Figure S1.

Supplementary Figure S1: Photos of the established patient derived cancer cells. JC-581-TR cells, established from primary tumor in rectum and JC-581-Ren cells from the renal metastasized tumor was cultured in the collagen coated culture dishes and taken photos with the phase contrast microscope. Scale bars are shown in the picture.

Supplementary Table S1: The information of the STR analysis of tumor tissues and the established cells

Reagents

Infigratinib was purchased from Shanghai Biochempartner. CH5183284 was purchased from ActiveBiochem. Obatoclax and Navitoclax were purchased from AdooQ BioScience (Irvine, CA, USA). All inhibitors were dissolved in Dimethyl Sulfoxide (DMSO) for the experiments. Detailed information of the other inhibitors used for the originally established drug inhibitor library is shown in Table 1.

Table 1: The information of the inhibitors using for the originally established drug inhibitor library.

Drug screening

3,000 cells/well were seeded in triplicate in 96-Well Collagen-Coated Plates (IWAKI) and cultured for 24 hours. Cells were then treated with the originally established drug inhibitor library at a low or high concentration. After 72 hours of incubation, cell viability was assessed using CellTiter-Glo assay reagent (Promega) by measuring luminescence with a Tristar LB941 microplate luminometer (Berthold Technologies). The relative cell viability was calculated as a ratio to the DMSO control. The average relative cell viability was shown as a heat map using the ComplexHeatmap package (version 2.4.3, R version 4.0.2). Original data are shown in Table 2.

Table 2: Drug sensitivity of JC-581-TR and Ren.

Cell viability assay

For evaluating cell viability to each inhibitor, cells were seeded in triplicate at 3,000 cells/well in 96-well collagen-coated plates. After culture for 24 hours, the cells were treated with the indicated concentration of drugs for 72 hours. The cell viability was measured using CellTiter-Glo assay reagent as indicated in the previous section. GraphPad Prism version 9.1.2 (GraphPad Software) was used to analyze the data.

Phospho-RTK array assay

Cells were seeded at 3×10⁶ cells in a collagen-coated dish and cultured for 24 hours. The cells were treated with DMSO control or 1 μM of infigratinib. After 6 hours of drug treatment, the cell lysates were collected and applied to a phospho-RTK array assay according to the manufacturer’s instructions for the Proteome Profiler Human Phospho-Kinase Array Kit (R&D Systems). The signal was detected using an Amersham ImageQuant 800 (GE Healthcare).

Discussion

As metastatic malignancies in the kidney are rare and asymptomatic in most cases, their exact incidences are difficult to evaluate. In autopsy-based analysis, metastatic renal tumors were incidentally found in 7.2 to 18.8%, of which 2.9 to 4.4% were metastatic lesions derived from primary colon malignancies. [10,11] Although there are some case reports describing renal metastasis from colorectal cancers, these patients had overt multiple organ metastases besides renal tumors, [12-15] so a solitary renal metastasis from colorectal cancers is thought to be quite rare. Among published reports of colorectal NENs, only one patient with rectal NET G1 has been demonstrated to develop renal metastasis. [14] As far as we know, there have been no previous reports on a solitary metastasis from rectal NEC to the kidney.

In a retrospective study involving 100 patients with highgrade neuroendocrine colorectal carcinomas, 60% had a primary tumor located from the sigmoid colon to rectum, and 64% had already developed distant metastases at the time of the first consultation. The median Overall Survival (OS) was 14.7 months, and the 2- and 5-year OS rates were 23% and 8%, respectively, indicating a very poor prognosis. [16] According to the National Comprehensive Cancer Network (NCCN) Guidelines 2020, because of the severity of biological malignancy, multidisciplinary treatment is recommended even for resectable NEC primary lesions, and chemotherapy-based treatment such as irinotecan/ cisplatin or carboplatin/etoposide is indicated for locally advanced tumors or distant metastases. [17,18] With intensive treatment, however, the Overall Response Rate (ORR) was estimated to be 42% at most [16].

In our case, four courses of systemic intensive chemotherapy

with irinotecan/cisplatin were administered, resulting in the best response of Stable Disease (SD). In a multidisciplinary team meeting, we discussed the pros and cons of the continuation of systemic chemotherapy or possible radical resection of the target lesions. Considering that there are no second-line regimens established for rectal NECs and that no new metastatic lesions had appeared at the completion of four courses of chemotherapy, we concluded that radical resection of the primary and metastatic tumors could cure the disease. Furthermore, minimally invasive surgery for simultaneous resection of the two lesions was considered to be feasible by laparoscopic low anterior resection and by laparoscopic partial resection of the kidney as demonstrated by a previous report. [19] Thus, we chose to perform the laparoscopic radical resection of the target lesions.

In terms of radical resection for NECs with distant metastases, a satisfactory OS rate was achieved by upfront surgical resection of pancreatic NEC along with simultaneous liver metastases in some cases. [20] Furthermore, in 32 NEC cases of the pancreas (14), CRC (12) and others (6), liver metastases were well controlled by RFA (radiofrequency ablation) or surgery, resulting in a satisfactory OS rate. [21] Although these findings that surgical resection might yield a better OS in even some Stage IV NEC cases are encouraging, there are two limitations to be considered. As these reports were based on the previous definitions of NET rather than the new, global classification of WHO 2019, NET G3 and NEC were treated as the same disease entity. The other is that the number of cases involved is very small with various patterns of primary and metastatic sites. Although intensive systemic chemotherapy is primarily recommended for primary NECs with multi-organ metastases or recurrent NECs, there is a possibility that the prognosis can be improved by interposing radical surgical resection during systemic chemotherapy. The true benefit of surgical intervention for an improved prognosis remains to be determined through an accumulation of colorectal NEC cases according to the new version of WHO classification.

Regarding genomic in-depth analysis, genetic mutations in TP53 and RB1 are considered to be characteristic of NEC, and TP53 p.C3F is already registered as a pathogenic mutation. Although a pathogenic effect of 4bp deletion in RB1 is undetermined, this mutation is probably pathogenic, as the deleted nucleotides contained a splicing acceptor site (GTGA), thereby leading to truncation by frame shift. Consistent with this notion, it is not registered in the Human Genetic Variation Database (HGVD) (https://www.hgvd.genome.med.kyoto-u.ac.jp/), a reference database of genetic variations in the Japanese population. These observations may indicate that the RB1 mutation found in this patient is a novel pathogenic mutation.

According to our drug sensitivity screening tests, Bcl family inhibitors and proteasome inhibitors besides infigratinib can also be effective in the treatment of NECs. Previous reports showed that NECs arising in different organs including small-cell lung cancer (SCLC) and Small-Cell Neuroendocrine Prostate Cancer (SCNPC) were sensitive to Bcl-2 inhibition. [22,23] In addition, given that Bcl-2 expression was found to be high in 64% of 25 colorectal NECs, [24] inhibitors to the Bcl family including Bcl-2 might be a therapeutic option for colorectal NECs. In a previous phase II study of a proteasome inhibitor, bortezomib, for metastatic NETs, [25] single-agent bortezomib did not induce any objective responses in the 16 enrolled, chemotherapy-naive patients. However, there is an encouraging recent article describing the possible effectiveness of inhibitors of NAE (NEDD8 (neural precursor cell expressed, developmentally downregulated 8) activating enzyme) in patients with small intestinal NETs (SI-NETs), which can exert a therapeutic effect through cell cycle regulation by inhibiting the ubiquitin-proteasome pathways resulting in p27 stabilization. [26] Considering that NAE inhibitors have been approved by the FDA (Food and Drug Administration) as therapeutic agents for high-risk MDS (myelodysplastic syndrome), and a clinical trial designed to investigate the efficacy of NAE inhibitors for AML (acute myeloid leukemia) and NSCLC patients is currently ongoing, NAE could be a promising candidate for molecular targeted therapy in colorectal NEC patients. Our comprehensive drug screening system using PDCs can serve as a platform for assessing novel therapeutics with the potential to be translated into clinical practice.

Conclusions

We performed laparoscopic radical resection for rectal NEC and its solitary renal metastasis. Since the long-term prognosis of curative resection for stage IV rectal NEC is still undetermined, post-operative careful monitoring of the patient is required. According to the findings of a comprehensive drug-sensitivity screening test, infigratinib, obatoclax and navitoclax were shown to be effective to the cancer cells established from the resected tumor samples.

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