|Year : 2020 | Volume
| Issue : 2 | Page : 221-225
|Loss of ASXL1 expression is associated with lymph node metastasis in colorectal cancer
Jun H Lee1, Ju-Hee Lee2, Byung K Ahn3, Seung S Paik4, Hyunsung Kim4, Kang H Lee3
1 Department of Surgery, Hanyang University Guri Hospital, Guri, Republic of Korea
2 Department of Surgery, Dongguk University Medical Center, Gyeongju, Republic of Korea
3 Department of Surgery, College of Medicine, Hanyang University, Seoul, Republic of Korea
4 Department of Pathology, College of Medicine, Hanyang University, Seoul, Republic of Korea
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|Date of Web Publication||18-Apr-2020|
| Abstract|| |
Context: The function of ASXL1 in colorectal cancer (CRC) has not been investigated yet. Aims: The purpose of this study was to investigate the clinicopathological and prognostic impact of ASXL1 expression on CRC. Settings and Design: The intensity of expression was scored as 0–3, and the extent of staining was scored as 0–4, based on the percentage of positive cells. The immunoreactivity score (IRS) was calculated by multiplying the two scores. Materials and Methods: We performed immunohistochemical staining of ASXL1 using tissue microarrays of 408 CRCs, 46 normal colonic mucosae, 48 adenomas, and 92 metastatic lymph nodes. Statistical Analysis Used: Clinicopathological variables were compared using Fisher's exact test, χ2-test, or unpaired Student's t-test, depending on the nature of the data. Results: A negative expression of ASXL1 was observed in 10.9% of normal mucosae, 27.1% of adenomas, 55.6% of adenocarcinomas, and 71.7% of metastatic lymph nodes (P < 0.001). With respect to the IRS cut-off score, lymph node metastasis and lymphatic invasion were more frequent in the IRS 0–6 group than in the IRS 8–12 group (56.3% vs. 33.3%, P = 0.034; 56.0% vs. 33.3%, P = 0.035). The 5-year disease-free survival rate was significantly lower in patients with IRS 0–6 group than those with IRS 8–12 group (78.7 ± 2.5 vs. 100%, P = 0.034). Conclusion: ASXL1 might act as a tumor suppressor in CRC. The loss of ASXL1 expression might be associated with lymph node metastasis and lymphatic invasion in CRC.
Keywords: ASXL1, colorectal cancer, lymph node, metastasis, prognosis
|How to cite this article:|
Lee JH, Lee JH, Ahn BK, Paik SS, Kim H, Lee KH. Loss of ASXL1 expression is associated with lymph node metastasis in colorectal cancer. Indian J Pathol Microbiol 2020;63:221-5
|How to cite this URL:|
Lee JH, Lee JH, Ahn BK, Paik SS, Kim H, Lee KH. Loss of ASXL1 expression is associated with lymph node metastasis in colorectal cancer. Indian J Pathol Microbiol [serial online] 2020 [cited 2020 May 30];63:221-5. Available from: http://www.ijpmonline.org/text.asp?2020/63/2/221/282718
| Introduction|| |
The human additional sex combs-like(ASXL) 1 gene is located on chromosome 20q11.21 and is a member of the ASXL family (ASXL1, ASXL2, and ASXL3).,, ASXL family members are scaffolding proteins that recruit and assemble various epigenetic regulators and transcription factors to specific genomic loci with histone modifications. These proteins are involved in transcriptional repression through interactions with polycomb repressive complex 2 (PRC2) and transcription activation via interactions with the BRCA1-associated protein 1 (BAP1) and nuclear hormone receptor complexes. Thus, ASXL activates or repress the transcription of their target genes in a context-dependent manner.
Although the association between epigenetic aberrations in ASXL1 and cancer predisposition is unclear, nonsense or frame-shift mutations in ASXL1 are observed in various hematologic malignancies,, and are associated with poor prognosis.,,,,, Truncations of ASXL1 infrequently occur in other types of cancers, for example, castration-resistant prostate cancers (2.0%), head and neck squamous cell carcinomas (1.4%), and breast cancers (1.0%). In contrast, ASXL1 is amplified and overexpressed in 5.1% of cervical cancers.
ASXL1 is frequently mutated in colorectal cancer (CRC) cell lines with microsatellite instability (MSI); however, its functional role has not been investigated in patients with CRC. We previously perform comprehensive mutational analysis by next-generation sequencing (NGS) in advanced CRC with infiltrative growth and peritoneal seeding and find that an ASXL1truncation as common among them. Based on this result, we hypothesized that ASLX1 is associated with metastasis and tumor invasion. Thus, we elucidated the functional roles and clinical effects of ASXL1 protein in CRC.
| Materials and Methods|| |
Patients and enrolment
This study enrolled 408 patients with CRC who underwent surgery between February 2005 and December 2010. Tissue samples of 46 normal colonic mucosae, 48 adenomas, and 92 metastatic lymph nodes from patients with CRC were included. The median follow-up period was 56.1 months (interquartile range, 27.1–79.1 months). This study was approved by the institutional review board (IRB No. 2017-05-026).
Method of pathologic examination
Tissue microarrays (TMAs) were constructed from the samples of 408 patients with CRC. Hematoxylin-and-eosin-stained slides from formalin-fixed, paraffin-embedded blocks were used to define representative tumor areas. Single tissue cores (2 mm in diameter) were sampled from each paraffin block and assembled into a recipient paraffin block using a TMA instrument (AccuMax Array, ISU ABXIS, Seoul, Korea). Tissue sections (4-μm-thick) were cut from TMAs, and immunohistochemical staining for ASXL1 was performed using the Bond Polymer Refine Detection Kit (Leica Microsystems GmbH, Wetzlar, Germany) with an anti-ASXL1 antibody (LS-C156556, polyclonal, 1:100; LSBio, Seattle, WA, USA). ASXL1 expression was evaluated by two independent pathologists (SS Paik and SH Jang) who were blinded to the study. Immunoreactivity was semi-quantitatively assessed based on the intensity and proportion of positive cells. The expression intensity of ASXL1 protein was scored as 0 (negative staining), +1 (weak staining), +2 (moderate staining), or +3 (strong staining). The extent of staining was scored according to the percentage of positive cells: 0 (<10% of the cells), +1 (10%–25%), +2 (26%–50%), +3 (51%–75%), or + 4 (≥76%) [Figure 1]. A final immunoreactivity score (IRS) was calculated by multiplying the above two scores. The IRS was ranged from 0 to 12. We divided negative expression (IRS 0), moderate expression (IRS 1–6), and abundant expression (IRS 8–12) groups by IRS.
|Figure 1: The intensity of ASXL1 protein expression was scored as 0 (negative staining), +1 (weak staining), +2 (moderate staining), or +3 (strong staining)|
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The presence of MSI was examined in 217 patients. The specimens were amplified with 5–10 mono- and dinucleotide microsatellite loci derived from the panel recommended by the NCI, which includes BAT26, BAT25, D5S346, D2S123, and D17S250 markers.
Clinicopathological variables were compared using Fisher's exact test, χ2-test, or unpaired Student's t-test, depending on the nature of the data. Survival graphs were plotted using Kaplan-Meier method and compared using log-rank test. Data were considered statistically significant at P value ≤ 0.05. Statistical analyses were performed using SPSS version 21 (SPSS Inc., Chicago, IL, USA).
| Results|| |
A negative expression (IRS 0) of ASXL1 was observed in 10.9% (5/46) of normal mucosae, 27.1% (13/48) of tubular adenomas, 55.6% (227/408) of adenocarcinomas, and 71.7% (66/92) of metastatic lymph nodes (P < 0.001). Moderate expression (IRS 1–6) of ASXL1 was observed in 87.0% (40/46) of normal mucosae, 68.6% (33/48) of tubular adenomas, 38.5% (157/408) of adenocarcinomas, and 28.3% (26/92) of metastatic lymph nodes. None of the metastatic lymph nodes expressed ASXL1 with IRS 8–12 [Table 1].
|Table 1: IRSs of normal mucosae, adenomas, adenocarcinomas, and metastatic lymph nodes|
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The relationship between ASXL1 expression and clinicopathological features is shown in [Table 2]. The IRS 0–6 and 8–12 groups had 384 and 24 patients, respectively. The mean age of the patients was 63 ± 12 years. In all, 249 patients were men and 159 patients were women. At cut-off IRS of 0–6 versus 8–12, age, sex, tumor location, gross appearance of the tumor, histological grade, vascular invasion, and perineural invasion were not significantly different between groups. The mean tumor size was smaller in the IRS 0–6 group than in the IRS 8–12 group (4.9 ± 2.1 vs. 6.3 ± 2.7 cm, P = 0.002). Lymph node metastasis was more frequent in the IRS 0–6 group than in the IRS 8–12 group (56.3% vs. 33.3%, P = 0.034). Lymphatic invasion was more frequent in the IRS 0–6 group than in the IRS 8–12 group (56.0% vs. 33.3%, P = 0.035). Stage III and IV disease was more frequent in the IRS 0–6 group than in the IRS 8–12 group (56.7% vs. 37.5%, P = 0.018). MSI high was 7.6% and 16.7% in the IRS 0–6 group and 8–12 group, respectively [Table 2].
|Table 2: Clinicopathological features of patients according to IRS (IRS 0-6 vs. 8-12)|
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The 5-year disease-free survival (DFS) rate was significantly different between the two groups (78.7 ± 2.5 vs. 100%, P = 0.034). However, there was no significant difference in the 5-year DFS rate between the two groups at the same stage (Stage II, 83.0 ± 3.9 vs. 100%, P = 0.125; Stage III, 69.2 ± 4.1 vs. 100%, P = 0.140, respectively) [Figure 2].
|Figure 2: The 5-year disease-free survival rate. (a) Stage I–III, (b) Stage II, and (c) Stage III|
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| Discussion|| |
In this study, we evaluated expression and clinicopathological features of ASLX1 in patients with CRC. The negative expression (IRS 0) of ASXL1 was detected more frequently in adenocarcinomas and metastatic lymph nodes than in normal mucosae and adenomas. Furthermore, patients with negative and moderate expression (IRS 0–6) of ASXL1 showed frequent lymph node metastasis. Based on these results, we can assume that ASXL1 might act as a tumor suppressor in CRC. To the best of our knowledge, this is the first study on ASXL1 expression in large clinical samples of CRC. In previous studies, ASXL1 has been speculated to exhibit a tumor-suppressive or oncogenic role in a context-dependent manner. Most mutations detected in ASXL1 were truncations that are observed in various types of human cancers.,,,,, In addition, there is a report that ASXL1 expression upregulated by circular RNA-ITGA7 inhibits CRC cell proliferation. Therefore, similar to the case in most other cancers, ASXL1 appears to exhibit a tumor-suppressive function in CRC.
In a previous study, ASXL1truncation was common in CRC with infiltrative growth and peritoneal seeding. In this study, lymph node metastasis and lymphatic invasion were more frequent in the IRS 0–6 group than the IRS 8–12 group. Furthermore, the negative expression (IRS 0) of ASXL1 was detected more frequently in metastatic lymph nodes than in adenocarcinomas. Although much more evidence is required, our result suggests the loss of ASXL1 expression in CRC might be associated with metastasis and tumor invasion.
The results of immunohistochemical analysis showed that ASLX1 is closely associated with lymph node metastasis and lymphatic invasion, which is a known risk factor for recurrence. In this study, the 5-year DFS rate was significantly lower in patients in the IRS 0–6 group than those in the IRS 8–12 group. In addition, there was no recurrence in the IRS 8–12 group. Although the 5-year DFS rates of Stage II and III cancers did not differ significantly, this result is due to the small number of the IRS 8–12 group. As ASXL1 mutations are associated with a poor prognosis in myeloid malignancies,,,,,, this result suggested that ASXL1 may be a potential prognostic marker in CRC. The relationship between ASXL1 expression and clinical outcomes should be confirmed with a larger cohort to understand the role of ASXL1 in CRC better.
ASXL1 is frequently mutated in CRC cell lines with MSI. The truncation of ASXL1 occurs in 5 of 11 CRC cell lines (45.5%) with MSI. In advanced CRC with infiltrative growth and peritoneal seeding group, ASXL1truncation was detected in 8 of 10 patients (80%) by NGS. Although the mechanism of epigenetic aberrations in the ASXL1 is unclear, a negative expression (IRS 0) of ASXL1 was 55.6% of adenocarcinomas in this study. This prevalence is consistent with previous studies. However, not negative expression (IRS 0) group but moderate expression (IRS 1–6) group showed similar clinicopathological features. In this study, only the abundant expression (IRS 8–12) group exhibited distinctive features, such as larger tumor, less lymph node metastasis, and less lymphatic invasion than the negative and moderate expression (IRS 0–6) groups. A possible explanation for this similarity in terms of the clinicopathological characteristics between the negative expression (IRS 0) group and the moderate expression (IRS 1–6) group could be the dominant-negative effect of truncated ASXL1 protein in the moderate expression (IRS 1–6) group. ASXL1 truncation causes premature protein with loss of the C-terminal plant home domain. The C-terminal truncated form of ASXL1 may interfere with the function of the normal ASXL1., The dominant-negative effect could explain the similarity in terms of the clinicopathological characteristics between the negative expression (IRS 0) group and the moderate expression (IRS 1–6) group.
The limitations of this study include the lack of cytogenetic analysis and difficulty in determining a clear cut-off score for ASXL1 expression because of the dominant-negative effect of the mutant protein. In addition, the results of MSI status were not evaluated for the entire study period. Despite these limitations, we believe that this is the first study on ASXL1 expression in clinical CRC samples. Although much more evidence is required, our work partly depicts a potential way of how ASXL1 acts in CRC, which expands our understanding of the cancer-associated role of ASXL1.
| Conclusion|| |
In conclusion, ASXL1 might act as a tumor suppressor in CRC. The loss of ASXL1 expression might be associated with lymph node metastasis and lymphatic invasion in CRC. A large-scale study on the role of ASXL1 as a prognostic factor is warranted.
Financial support and sponsorship
This work was supported by the research fund of Hanyang University (HY-2015).
Conflicts of interest
There are no conflicts of interest.
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Kang H Lee
222-1 Wangsimni-ro, Seongdong-gu, Seoul - 04763
Republic of Korea
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2]
[Table 1], [Table 2]
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