Gastroenterology 2024 Sep 3:S0016-5085(24)05415-5. doi: 10.1053/j.gastro.2024.08.032.
"AIG에서는 complete metaplasia, Hp gastritis에서는 incomplete metaplasia를 주로 일으키기 때문에 pure AIG에서 gastric cancer 가능성은 낮다." 라는 기존의 주장에 대한 다른 입장의 논문입니다. Hp infection 및 AIG 모두 다양한 형태의 metaplasia 발생이 가능하며, cancer-related metaplastic cell expression이 일어날 수 있으므로 이 두 질환에 대해서는 gastric cancer의 risk를 줄이기 위해 early detection을 위한 guide가 필요하다는 내용으로 한번 살펴 보겠습니다. (임상에서 이해하기는 매우 어려운 내용이긴 합니다..)
Abbreviations used in this Q4 paper: AIG, autoimmune gastritis; ANPEP, alanyl aminopeptidase N; ECL, enterochromaffin-like; GIF, gastric intrinsic factor; GKN3, gastrokine 3; Hp, Helicobacte rpylori; IM, intestinal metaplasia; MUC6, mucin 6; SPEM, spasmolytic polypeptide expressing metaplasia; TACSTD2, tumor-associated calcium signal transducer 2; Tff2, trefoil factor2.
Introduction
This study was conducted to enhance our understanding of metaplasia development in the context of 2 settings of chronic inflammation: Helicobacter pylori (Hp) infection and autoimmune gastritis (AIG).
Incomplete IM is reported to pose a higher cancer risk over complete IM and SPEM .
미국에서 H. pylori infection에 대한 incidence는 최근 십년간 줄어들고 있으나 <50세의 여성에서 incidence는 증가되고 있고, autoimmunity의 incidence가 높은 분율을 차지 하고 있습니다. AIG는 chronic gastritis, metaplasia를 유도하고, neuroendocrine tumors와 연관성은 있지만 H. pylori 만큼의 adenocarcinoma의 risk를 갖는지는 아직도 불분명 합니다.
이러한 의미에서 이번 연구는, murine models와 human disease에서 H. pylori infection과 AIG에서 유도된 metaplasia에 대해 직접 비교 분석을 수행하였습니다. Mouse models에서 Histologic 및 transcriptional 분석을 실시 하였으며, 임상적인 연관성을 확인 하기 위해 H. pylori infection 된 환자와 AIG 환자의 biopsy sample에서도 함께 분석을 실시 하였습니다. alanyl aminopeptidase N (ANPEP) expression 여부를 확인 하였는데, 이는 metaplastic 및 cancerous gastric tissue에서 확인 된 물질로, H. pylori infection과 AIG에서는 ANPEP를 통해서 cancer associated incomplete IM의 출현을 촉진 시킨다는 것입니다.
Materials and Methods
Murine Tissue Samples
C57BL/6mice (The Jackson Laboratory, BarHarbor,ME) were infected at age 6 to 8 weeks with ~1x108colony forming units of Hp, pre mouse Sydney strain1.
AIG was modeled in transgenic TxA23mice.
To compare with C57BL/6-infected animals, BALB/cTxA23 animals were backcrossed toC57BL/6J animals expressing BALB/ c major histocompatibility complex for11 generations.
Human Study Approval
Healthy stomachs / Hp-infected patients / AIG biopsy specimens
Hp-infected samples,an equal mix of sexes were used, but all AIG-derived samples were from women due to female predominance of disease.
Histopathology
Three blinded, independent investigators scored tissues for pathology following established guidelines.
Immunofluorescence
Epithelial Single-Cell Isolation
Chromium Single-Cell Library Construction: Library construction of mouse tissues, construction of AIG human libraries
Human Data Download: Hp data sets from10 Hp-positive gastric cancer patients
Results
1. Pathologic Comparison of Helicobacter pylori and Autoimmune Gastritis Mouse Models of Disease
<2 well-established mouse>
* Hp infection: C57BL/6 mice (Gastric metaplasia를 유도하는 야생형 인간 분리주(wild-type human isolate) premous Sydney strain 1에 감염 된 mice)
* Model AIG: BALB/c TxA23 mouse model (C57BL/6 infected mice와 비교하기 위해 C57BL/5를 바탕으로 역 교배(backcrossed)하여 인간형 AIG의 조직학적 특징을 갖도록 함)
- 두 모델 모두: Moderate to severe inflammation, atrophy, mucous neck cell expansion, and mucosal hyperplasia (Figure1A).
- Pathologic severity: AIG (corpus-wide pathology) >> Hp (multifocal inflammatory nature) (Figure1C).
- Hp mice: mild inflammation (antrum), No advanced antral pathology or neuroendocrine cell hyperplasia
- Histopathologic characteristics 는 두 models에서 유사하게 나타남
* Immunofluorescent staining (Mucin 6 (MUC6, green), Gastric intrinsic factor (GIF, cyan), Gastrokine 3 (GKN3, red))
- Figure 1B:
: Healthy tissue - MUC6, GIF dysplayed normal staining (healthy neck & chief cells)
: Hp & AIG metaplastic glands - aberrant GKN3 staining & expanded MUC5 staining
- Figure 1D:
: Metaplasia가 진행된 glands의 비율은 Hp-infected mice에서는 ~40%, AIG 에서는 ~80% 관찰됨
* 이러한 결과를 보면, Hp infection과 AIG에서 murine models 사이에는 histopathologic 측면에서는 비율에서 차이는 있지만 거의 비슷하다고 볼 수 있습니다.
Figure 1. Similar gastric pathologies observed between models of Hp and AIG. (A) Representative H&Es of corpora from a healthy (left), a 6-month Hp-infected (center), and a 6-month-old TxA23 (right) mouse. Scalebars:100mm. (B)Representative immunofluorescent images of corpora from a healthy (left), a13-month Hp-infected diseased area (center left) and non diseased area (center right), and a 6-month-old TxA23 (right) mouse. Tissue is stained with Hoechst (blue), GKN3 (red), MUC6 (green), and GIF (cyan). Scalebars: 100mm. (C) Summary stomach pathology scores from 6-to 13-month-old Hp-infected Q10 (red, n=15) and 4-to 6-month-old TxA23 (blue, n=13) mice. Box-and-whisker plot: The boxes indicate the 25th percentile (bottom border), median (center line), and 75th percentile (top border), and the whiskers Q11 show the maximum and minimum ranges. (D) Quantification of GKN3+ metaplastic glands between Hp-infected (n=13) and AIG (n=12) mice.
2. Single-Cell Transcriptomic Comparison of Metaplasia Arising in Helicobacter pylori and Autoimmune Gastritis Models
* Gastric cell types (both Hp and AIG data sets): metaplasia, parietal, neck, pit, chief, proliferating epithelial, enterochromaffin like (ECL), and various immune cells (Figure2A–D)
* 17 gastric metaplasia-associated transcripts (such as trefoil factor 2 (Tff2), GKN3, aquaporin 5 (Aqp5), CD44)
* Figure 2C and D: 두 질환에서 metaplasia signature expression은 유사한 pattern을 보였습니다. (대부분의 metaplastic cells은 metaplastic & neck cell clusters에서 isolation 됨)
* 반복적으로 clustering 되어지는 gastric metaplasia의 10가지 subclusters를 분석 하였습니다. (Figure 2E)
* Gastric metaplasia-associated transcripts within each metaplastic subcluster (Figure 2F)
: All subclusters에서 pyloric metaplasia와 일치하는 Tff2, Muc6, GIF, GKN 3 가 함께 발현 되었습니다.
* 이러한 결과는 Hp와 AIG models에서 기원하는 metaplastic cells는 같은 transcriptional diversity에서 originating 한다고 볼 수 있습니다.
Figure 2. Metaplasia is transcriptionally similar across chronic inflammatory models. (A) Experimental design to run single-cell RNA sequencing on corpora of Hp-infected (n=7, 16,000 cells analyzed) and AIG(n=2, 12,000 cells analyzed) mice. (Stomach adapted from Willet SG, Mills JC. Stomach organ and cell lineage differentiation: from embryogenesis to adult homeostasis. Cell Mol Gastroenterol Hepatol 2016;2:546-559.) Feature Uniform Manifold Approximation and Projection (UMAP) visualizing epithelial cell clusters in (B) both, (C) Hp only, and (D) AIG only. UMAP is colored based on enrichment for the Gastric Metaplasia Signature Score. (E) UMAP of reclustered metaplastic cells colored by subcluster. (F) Violin plots measure expression levels of various metaplasia-associated transcripts for each subcluster.
3. Defining Metaplastic Subtypes That Arise Within in Helicobacter pylori and Autoimmune Gastritis Models
* Figure 3A and B: 각 질환 별 10 metaplastic subclusters - 유사한 repertories를 보임
* Cluster-specific gene metaplastic subclusters를 5 cluster-defining transcripts 씩 나눠서 mapping 을 실시함. Figure 3C)
- Cluster 6: Gkn1와 Gkn2가 분명한 foveolar cells에서 풍부함
- Cluster 7: Atp4a와 Atp4b를 포함하는 parietal cells과 유사한 transcriptional profile을 나타냄
- 다른 clusters에서는 pyloric metaplasia와 연관성이 있는 transcripts들이 관찰 되었습니다.
: cluster 1 (Xist), cluster 2 (CD44)
-> Cluster를 정의하는 transcripts 들의 구분되는 발현을 살펴 보면, metaplastic cells내 transcriptional diversity를 갖는다는 것을 확인 할 수 있음
* Phenotypes of the different metaplastic cells (Figure 3D-G)
- Cluster 0, 1: gastric chief cell에서 가장 높게 나타남
- Cluster 1, 3, 4, 7: gastric proliferative cell에서 가장 높게 나타남
- Cluster 5, 8: ECL cell에서 가장 높게 나타남
* 이러한 결과는 Hp와 AIG models 사이에서 metaplastic phenotypes 또한 유사하게 유도되어 진다는 것입니다.
Figure 3. Common and diverse subtypes of metaplasia in mouse models of Hp and AIG. Uniform Manifold Approximation and Projections (UMAPs) representing metaplastic cells found (A) only in Hp (900 cells analyzed) or (B) only in AIG (1900 cells analyzed). The pie charts show metaplasia subcluster frequencies. (C) Heat map of 5 cluster-defining transcripts for each metaplastic subcluster. (D–G) Violin plots visualize signature score for each metaplastic subcluster. The black downward arrows highlight highest scoring subcluster (s) for each signature. Plots are colored by subcluster identity.
4. Pathologic Comparison of Helicobacter pylori Infection and Autoimmune Gastritis in Human Disease
* Pathologic features of disease: similar between patients with Hp and AIG (Figure 4A and B)
- Pyloric and intestinal metaplasia (Figure 4A)
- Pyloric metaplasia containing MUC6, TFF2, CD44v9 (Hp and AIG biopsy specimens, Figure 4B)
--> 이러한 결과는 humans에서 Hp와 AIG 에서 발생한 metaplasia의 경우도 mouse models에서 처럼 차이점을 구분하기 어렵습니다.
* Cellular & molecular levels에서의 metaplasia를 비교하기 위해 3명의 AIG 환자 (Hp: negative, gastric cancer(-))의 체부에서 조직을 얻었고, mouse models과 같은 방식으로 single-cell RNA sequencing을 시행하였고, gastric cancer를 가진 10명의 Hp-positive 환자의 tumor에 인접한 체부에서 조직에 대한 결과를 downloaded 하였습니다. (Figure 4C)
- All expected cell types were identified and highest-scoring cells from Hp and AIG settings mapped to the sam clusters (Figure 4D).
--> 이러한 결과는 Hp-infected gastric cancer 환자와 AIG 환자에서 metaplastic cells은 유사한 tanscrpition을 한다는 것을 보여 줍니다.
Figure 4. Similar gastric pathologies observed in Hp and AIG patient biopsy specimens. (A) Representative H&Es of corpus from a healthy (left), a Hp-infected (center), and AIG (right) patient. Arrows: highlight areas of intestinal (yellow) and pyloric (green) metaplasia. (B) Representative immunofluorescent images of corpus of a healthy (left), an Hp-infected (center), and an AIG (right) patient biopsy specimen. Tissue is stained with Hoechst (blue), MUC6 (green), cluster of differentiation 44 (CD44v9; cyan), and TFF2 (red). (C) Experimental design for conducting single-cell RNA sequencing on Hp-infected (n=10, 30,000cells analyzed) and AIG (n=3, 13,000 cells analyzed) patient biopsy specimens. (D) Feature Uniform Manifold Approximation and Projections (UMAPs) visualizing epithelial cell clusters in Hp only (left) and AIG only (right). UMAP is colored based on enrichment for the Gastric Metaplasia Signature Score.
5. Single-Cell Transcriptomic Comparison of Metaplasia Arising in Helicobacter pylori and Autoimmune Gastritis Patients
* 10th percentile for the human metaplasia signature were isolated and subjected to unbiased reintegration and reclustering (Figure 5A)
* Metaplasia signature-enriched cells의 대부분은 metaplastic and neck cell cllusters에서 isolation 되었습니다.
* 12 metaplastic cell subtypes
- Subclusters 0, 1: ~60% of all metaplastic cells in the Hp // only 20% in AIG specimens (Figure 5B and C)
- Hp보다 AIG에서 보다 다양한 metaplastic subtypes을 보였습니다. (AIG: 12 metaplastic subtypes, Hp: 10 subtypes)
- Subclusters 10, 11: unique to AIG
--> 이러한 현상은 human gastric metaplasia가 보다 heterogeneous 하며, 염증성 손상이 metaplastic cells의 다양성에 영향을 미친다고 볼 수 있습니다. 또한, Hp-infected patients의 모든 metaplastic subtypes는 AIG patients에서 또한 관찰되었다는 것도 주목할 부분 입니다.
* Expression of metaplasia associated transcripts (Figure 5D)
- 대부분의 sublclusters에서 spasmolytic polypeptide, TFF2의 expression이 높게 나타났습니다. (subclusters 2, 4 제외)
- subclusters 9번을 제외한 모든 subclusters에서 neck cell-associated transcript MUC6가 expression 되었습니다.
- Subclusters 8, 9 cells에서는 goblet cell trascript인 TFF3 (intestinal metaplasia (IM) 와 연관)이 높게 expression 되었습니다.
- 몇몇 subclusters에서는 dysplasia와 연관성이 있다고 reported 되어진 Tumor-associated calcium signal transducer 2 (TACSTD2) 또는 TROP2가 높게 발현되었습니다.
* Top 5 cluster-defining genes in a heat map (Figure 5E)
- Gastric epithelial cell과 연관된 transcripts (cluster 2: chief cell, cluster3: foveolar cell)
- Pyloric metaplasia와 연관된 transcripts (cluster 4: cystic fibrosis transmembrane conductance regulator (CFTR) and olfactomedin 4 (OLFM4), cluster 10: WAP four disulfide core domain 2 (WFDC2))
* Metaplastic cells by performing signature analyses (Figure 5F-J)
- Gastric proliferative cell: subclusters 1 and 7 (highest scores) (Figure 5F)
- Chief cell: subcluster 2 and AIG-unique subcluster 11 (Figure 5G)
- Immature enterocyte: Subcluster 5 (Figure 5H)에서 가장 높게 나왔는데, 이 cluster에서 TFF2, MUC6, TFF3, TACSTD2의 co-expression이 관찰 되었으며 (Figure 5D), subcluster 5는 incmplete IM를 의미한다고 볼 수 있습니다.
- ECL cell: Metaplastic subcluster 6가 Hp biopsy에서 보다 AIG에서 2배 높은 빈도로 나타나며, ECL cell 에서 가장 높게 나타났습니다. (Figure 5I)
- Goblet cell: subcluster 9이 가장 높게 나타났는데, 이는 complete IM를 의미합니다.
* 이러한 소견은 Hp infected 및 AIG 환자에서 metaplastic cell 분포에 대한 variability가 있음에도 불구하고, 두 환경에서 disease progression (proliferative metaplasia, comoplete IM, incomplete IM) 과 연관된 metaplastic subtypes이 모두 확인 되었다는 것입니다.
Figure 5. Heterogeneous gastric metaplastic cells identified across patient disease settings. (A) Uniform Manifold Approxi mation and Projections (UMAPs) of reclustered metaplastic cells colored by subclusters. UMAP of metaplastic subclusters in (B) Hp-infected biopsy specimens (1400 cells analyzed) and (C) AIG biopsy specimens (1500 cells analyzed), colored by subcluster. The pie charts show subcluster frequencies. (D) Violin plots measuring expression levels of metaplasia- or dysplasia-associated transcripts for each metaplastic subcluster. (E) Heat map of 5 selected cluster-defining transcripts for each metaplastic subcluster. (F–J) Violin plots visualize signature score for each metaplastic subcluster. The black downward arrows: highlight the highest scoring subcluster (s) for each signature. Plots are colored by subcluster identity.
6. Evaluating Relatedness to Cancer Across Metaplastic Subtypes
* 20 gastric cancer associated transcripts
* Cancer cluster in tumor specimens from the antrum and corpus (Figure 6A)
* Metaplastic subcluster 5 (incomplete IM) / subcluster 9 (completeIM) (Figure 6B and C)
* Tumor derived epithelial cell types을 예측하여 분석하였는데, cluster 5가 양측 diseases setting에서 cancer cells와 가장 연관성을 보이는 metaplastic subcluster로 확인 되었습니다. (Figure 6D)
* subcluster 0, 1, 3, 6, 7, 9, 10도 cancer cell에서 mapped 되었으나, incomplete IM subtype (cancer-related metaplastic cell)인 subcluster 5는 Hp와 AIG, 모두에서 발현되었습니다.
* potential cancer-associated biomarker를 확인 하기 위해 cluster-defining transcripts를 확인 하였으며, subcluster5-defining gene으로 ANPEP를 확인할 수 있었습니다. ANPEP는 Hp-infected tumor biopsy specimens 에서만 unique 하게 up regulation 되었습니다.
* 이러한 결과는, incomplete IM 와 유사한 metaplasia는 Hp-induced 및 AIG induced metaplasia 환자에서 모두 확인할 수 있었고, 하위 cluster에서 보았을 때 gastric cancer와 연관성이 있으며 특히, ANPEP의 level이 unique하게 높게 나타났습니다.
Figure 6. Cancer-related metaplastic cells present in patients with Hp and AIG. (A) Feature Uniform Manifold Approximation and Projection (UMAP) visualizing epithelial cell clusters in Hp tumor corpus biopsy specimens (n=10, 30,000 cells analyzed). UMAP is colored based on enrichment for the Gastric Cancer Signature Score. (B) Gastric Cancer Signature feature UMAP for Hp metaplastic cells. (C) Gastric Cancer Signature feature UMAP for AIG metaplastic cells. (D) UMAPs of gastric metaplastic subclusters in all settings (left), Hp only (center), or AIG only (right) labeled with epithelial cell clusters from Hp corpus tumor biopsy specimens. Violin plots demonstrate ANPEP/CD13 transcript E) across metaplastic subclusters and (F) across cell clusters in the Hp corpus tumor biopsy specimens.
7. Alanyl Aminopeptidase N/CD13 as a Biomarker for Cancer-Related Metaplasia in Helicobacter pylori and Autoimmune Gastritis patients
* Expression of ANPEP of patients with Hp or AIG (Figure7)
* Healthy gastric tissue: No detectable expression of intestinal metaplastic proteins TFF3 and MUC2 or ANPEP
* H&E: Hp 와 AIG biopsy specimens에서 IM glands 를 모두 확인 할 수 있었습니다. (Figure 7Aand B)
- 이는 TFF3와 MUC2에 대한 positive staining으로 confirm 가능 하였습니다. (Figure 7C and D)
* IM regions에서 ANPEP expression 또한, 관찰할 수 있었습니다. (Figure 7E and F)
- IM 가진 5명의 Hp patients와 IM 가진 6명의 AIG patients, 모두의 IM glands subset에서 ANPEP expression을 관찰 할 수 있었습니다.
* 이러한 결과는, Hp- 및 AIG-induced metaplasia를 가진 precancerous 환자의 조직에서 metaplastic cells의 subset에 ANPEP이 존재한다는 것을 확인 할 수 있었으며, ANPEP가 cancer progression의 위험성을 가진 환자의 early biomarker 로써 사용할 수 있다는 근거를 제공하는 것입니다.
Figure 7. ANPEP/CD13 identifies cancer-related metaplastic cells in Hp and AIG metaplastic biopsy specimens. (A and B) Representative H&E images of Hp-infected and AIG patient biopsy specimens highlight areas of metaplasia. (C–F) Immunofluorescent images of Hp-infected (left) and AIG patient biopsy specimens (right). (C and D) Tissue is stained with Hoechst (blue), MUC6 (cyan), MUC2 (green), and TFF3 (red). (E and F) Tissue is stained with Hoechst (blue), MUC6 (cyan), MUC2 (green), and ANPEP (red). Scale bars: 100 mm. Sequential imaging of the same section was conducted between A, C, and E (Hp-infected) and B, D, and F (AIG).
Discussion
* Metaplasia는 acute injury에 대한 tissue healing 으로 나타나는 조직변화 이지만, chronic inflammation과 같은 long standing injury는 dysplasia로의 변화를 유도하게 됩니다. 하지만, 적은 일부에서만 나타나기 때문에 적절한 예방과 치료 전략을 위해서 cancer progression의 high risk를 갖는 보다 특징적인 metaplastic lineage를 찾는 것이 필요합니다.
* 이번 연구에서 주목할 점은, 보다 높은 adenocarcinoma risk와 연관된 H. pylori infection이 AIG에서 독특한 subtype의 metaplasia를 유도하지 않았다는 것입니다.
* Mouse models과 human disease에서 chief cell과 proliferative cell과 유사한 일반적인 metaplastic subtypes이 확인 되었고, ECL 과 intestinal lineage의 subtypes 도 함께 확인 할 수 있었습니다. (Paligenosis: chief-like metaplastic cells = chief-drived metaplastic cells)
* 이번 연구에서는 proliferative metaplastic cells를 확인 하였는데, 이들은 human에서 dysplasia-associated TACSTD2 또는, TROP2를 발현시키며 transcriptionally cancer와 연관성이 있습니다. Cancer progression potential을 가진 proliferative metaplastic cells이 있긴 하지만, 이번 연구에서는 cancer-associated IM subtypes에 대해 보다 focusing 하였습니다.
*
Conclusion
This study underscores the significance of comparing the features of metaplasia induced by Hp infection and AIG, revealing that both conditions induce cancer-associated metaplasia. As such, it emphasizes the importance of closely monitoring patients with either disease for early biomarkers, such as ANPEP/CD13, to mitigate adenocarcinoma risk and improve patient outcomes.
Abstract
Background & aims: Gastric metaplasia may arise as a consequence of chronic inflammation and is associated with an increased risk of gastric cancer development. Although Helicobacter pylori (Hp) infection and autoimmune gastritis (AIG) both induce gastric metaplasia, possible distinctions in resulting metaplastic cells and their respective cancer risks requires further investigation.
Methods: Using both mouse models and human participants, we scrutinized the metaplasia originating from Hp infection and AIG. Gastric pathology and metaplasia were examined through histopathologic assessment. Molecular features of metaplastic cells were defined using single-cell transcriptomics in murine models of Hp infection and AIG, as well as in human biopsy specimens from patients with Hp infection and AIG. Expression of a newly defined cancer-related metaplastic biomarker was confirmed through immunofluorescence.
Results: Metaplasia in Hp infection and AIG displayed comparable histopathologic and transcriptional features. Diverse metaplastic subtypes were identified across both disease settings, with subtle differences in the prevalence of certain subtypes between inflammatory contexts. Notably, Hp infection did not drive a unique metaplastic cell phenotype. One metaplastic subtype, which resembled incomplete intestinal metaplasia and shared transcriptional features with gastric cancer, was identified in both diseases. This cancer-like metaplastic subtype was characterized by expression of the cancer-associated biomarker alanyl aminopeptidase N/CD13.
Conclusion: Both Hp infection and AIG trigger a diverse array of metaplastic cell types. Identification of a cancer-related metaplastic cell uniquely expressing alanyl aminopeptidase N/CD13, present in both Hp- and AIG-induced gastritis, indicates the carcinogenic capacity of both diseases. This discovery can guide early detection and risk stratification for patients with chronic gastritis.
Keywords: Autoimmune Gastritis; Gastric Cancer; Gastric Inflammation; Gastric Metaplasia; Helicobacter pylori Infection.
Gastroenterology 2024 Sep 3:S0016-5085(24)05415-5. doi: 10.1053/j.gastro.2024.08.032.
"AIG에서는 complete metaplasia, Hp gastritis에서는 incomplete metaplasia를 주로 일으키기 때문에 pure AIG에서 gastric cancer 가능성은 낮다." 라는 기존의 주장에 대한 다른 입장의 논문입니다. Hp infection 및 AIG 모두 다양한 형태의 metaplasia 발생이 가능하며, cancer-related metaplastic cell expression이 일어날 수 있으므로 이 두 질환에 대해서는 gastric cancer의 risk를 줄이기 위해 early detection을 위한 guide가 필요하다는 내용으로 한번 살펴 보겠습니다. (임상에서 이해하기는 매우 어려운 내용이긴 합니다..)
Abbreviations used in this Q4 paper: AIG, autoimmune gastritis; ANPEP, alanyl aminopeptidase N; ECL, enterochromaffin-like; GIF, gastric intrinsic factor; GKN3, gastrokine 3; Hp, Helicobacte rpylori; IM, intestinal metaplasia; MUC6, mucin 6; SPEM, spasmolytic polypeptide expressing metaplasia; TACSTD2, tumor-associated calcium signal transducer 2; Tff2, trefoil factor2.
Introduction
This study was conducted to enhance our understanding of metaplasia development in the context of 2 settings of chronic inflammation: Helicobacter pylori (Hp) infection and autoimmune gastritis (AIG).
Incomplete IM is reported to pose a higher cancer risk over complete IM and SPEM .
미국에서 H. pylori infection에 대한 incidence는 최근 십년간 줄어들고 있으나 <50세의 여성에서 incidence는 증가되고 있고, autoimmunity의 incidence가 높은 분율을 차지 하고 있습니다. AIG는 chronic gastritis, metaplasia를 유도하고, neuroendocrine tumors와 연관성은 있지만 H. pylori 만큼의 adenocarcinoma의 risk를 갖는지는 아직도 불분명 합니다.
이러한 의미에서 이번 연구는, murine models와 human disease에서 H. pylori infection과 AIG에서 유도된 metaplasia에 대해 직접 비교 분석을 수행하였습니다. Mouse models에서 Histologic 및 transcriptional 분석을 실시 하였으며, 임상적인 연관성을 확인 하기 위해 H. pylori infection 된 환자와 AIG 환자의 biopsy sample에서도 함께 분석을 실시 하였습니다. alanyl aminopeptidase N (ANPEP) expression 여부를 확인 하였는데, 이는 metaplastic 및 cancerous gastric tissue에서 확인 된 물질로, H. pylori infection과 AIG에서는 ANPEP를 통해서 cancer associated incomplete IM의 출현을 촉진 시킨다는 것입니다.
Materials and Methods
Murine Tissue Samples
C57BL/6mice (The Jackson Laboratory, BarHarbor,ME) were infected at age 6 to 8 weeks with ~1x108colony forming units of Hp, pre mouse Sydney strain1.
AIG was modeled in transgenic TxA23mice.
To compare with C57BL/6-infected animals, BALB/cTxA23 animals were backcrossed toC57BL/6J animals expressing BALB/ c major histocompatibility complex for11 generations.
Human Study Approval
Healthy stomachs / Hp-infected patients / AIG biopsy specimens
Hp-infected samples,an equal mix of sexes were used, but all AIG-derived samples were from women due to female predominance of disease.
Histopathology
Three blinded, independent investigators scored tissues for pathology following established guidelines.
Immunofluorescence
Epithelial Single-Cell Isolation
Chromium Single-Cell Library Construction: Library construction of mouse tissues, construction of AIG human libraries
Human Data Download: Hp data sets from10 Hp-positive gastric cancer patients
Results
1. Pathologic Comparison of Helicobacter pylori and Autoimmune Gastritis Mouse Models of Disease
<2 well-established mouse>
* Hp infection: C57BL/6 mice (Gastric metaplasia를 유도하는 야생형 인간 분리주(wild-type human isolate) premous Sydney strain 1에 감염 된 mice)
* Model AIG: BALB/c TxA23 mouse model (C57BL/6 infected mice와 비교하기 위해 C57BL/5를 바탕으로 역 교배(backcrossed)하여 인간형 AIG의 조직학적 특징을 갖도록 함)
- 두 모델 모두: Moderate to severe inflammation, atrophy, mucous neck cell expansion, and mucosal hyperplasia (Figure1A).
- Pathologic severity: AIG (corpus-wide pathology) >> Hp (multifocal inflammatory nature) (Figure1C).
- Hp mice: mild inflammation (antrum), No advanced antral pathology or neuroendocrine cell hyperplasia
- Histopathologic characteristics 는 두 models에서 유사하게 나타남
* Immunofluorescent staining (Mucin 6 (MUC6, green), Gastric intrinsic factor (GIF, cyan), Gastrokine 3 (GKN3, red))
- Figure 1B:
: Healthy tissue - MUC6, GIF dysplayed normal staining (healthy neck & chief cells)
: Hp & AIG metaplastic glands - aberrant GKN3 staining & expanded MUC5 staining
- Figure 1D:
: Metaplasia가 진행된 glands의 비율은 Hp-infected mice에서는 ~40%, AIG 에서는 ~80% 관찰됨
* 이러한 결과를 보면, Hp infection과 AIG에서 murine models 사이에는 histopathologic 측면에서는 비율에서 차이는 있지만 거의 비슷하다고 볼 수 있습니다.
Figure 1. Similar gastric pathologies observed between models of Hp and AIG. (A) Representative H&Es of corpora from a healthy (left), a 6-month Hp-infected (center), and a 6-month-old TxA23 (right) mouse. Scalebars:100mm. (B)Representative immunofluorescent images of corpora from a healthy (left), a13-month Hp-infected diseased area (center left) and non diseased area (center right), and a 6-month-old TxA23 (right) mouse. Tissue is stained with Hoechst (blue), GKN3 (red), MUC6 (green), and GIF (cyan). Scalebars: 100mm. (C) Summary stomach pathology scores from 6-to 13-month-old Hp-infected Q10 (red, n=15) and 4-to 6-month-old TxA23 (blue, n=13) mice. Box-and-whisker plot: The boxes indicate the 25th percentile (bottom border), median (center line), and 75th percentile (top border), and the whiskers Q11 show the maximum and minimum ranges. (D) Quantification of GKN3+ metaplastic glands between Hp-infected (n=13) and AIG (n=12) mice.
2. Single-Cell Transcriptomic Comparison of Metaplasia Arising in Helicobacter pylori and Autoimmune Gastritis Models
* Gastric cell types (both Hp and AIG data sets): metaplasia, parietal, neck, pit, chief, proliferating epithelial, enterochromaffin like (ECL), and various immune cells (Figure2A–D)
* 17 gastric metaplasia-associated transcripts (such as trefoil factor 2 (Tff2), GKN3, aquaporin 5 (Aqp5), CD44)
* Figure 2C and D: 두 질환에서 metaplasia signature expression은 유사한 pattern을 보였습니다. (대부분의 metaplastic cells은 metaplastic & neck cell clusters에서 isolation 됨)
* 반복적으로 clustering 되어지는 gastric metaplasia의 10가지 subclusters를 분석 하였습니다. (Figure 2E)
* Gastric metaplasia-associated transcripts within each metaplastic subcluster (Figure 2F)
: All subclusters에서 pyloric metaplasia와 일치하는 Tff2, Muc6, GIF, GKN 3 가 함께 발현 되었습니다.
* 이러한 결과는 Hp와 AIG models에서 기원하는 metaplastic cells는 같은 transcriptional diversity에서 originating 한다고 볼 수 있습니다.
Figure 2. Metaplasia is transcriptionally similar across chronic inflammatory models. (A) Experimental design to run single-cell RNA sequencing on corpora of Hp-infected (n=7, 16,000 cells analyzed) and AIG(n=2, 12,000 cells analyzed) mice. (Stomach adapted from Willet SG, Mills JC. Stomach organ and cell lineage differentiation: from embryogenesis to adult homeostasis. Cell Mol Gastroenterol Hepatol 2016;2:546-559.) Feature Uniform Manifold Approximation and Projection (UMAP) visualizing epithelial cell clusters in (B) both, (C) Hp only, and (D) AIG only. UMAP is colored based on enrichment for the Gastric Metaplasia Signature Score. (E) UMAP of reclustered metaplastic cells colored by subcluster. (F) Violin plots measure expression levels of various metaplasia-associated transcripts for each subcluster.
3. Defining Metaplastic Subtypes That Arise Within in Helicobacter pylori and Autoimmune Gastritis Models
* Figure 3A and B: 각 질환 별 10 metaplastic subclusters - 유사한 repertories를 보임
* Cluster-specific gene metaplastic subclusters를 5 cluster-defining transcripts 씩 나눠서 mapping 을 실시함. Figure 3C)
- Cluster 6: Gkn1와 Gkn2가 분명한 foveolar cells에서 풍부함
- Cluster 7: Atp4a와 Atp4b를 포함하는 parietal cells과 유사한 transcriptional profile을 나타냄
- 다른 clusters에서는 pyloric metaplasia와 연관성이 있는 transcripts들이 관찰 되었습니다.
: cluster 1 (Xist), cluster 2 (CD44)
-> Cluster를 정의하는 transcripts 들의 구분되는 발현을 살펴 보면, metaplastic cells내 transcriptional diversity를 갖는다는 것을 확인 할 수 있음
* Phenotypes of the different metaplastic cells (Figure 3D-G)
- Cluster 0, 1: gastric chief cell에서 가장 높게 나타남
- Cluster 1, 3, 4, 7: gastric proliferative cell에서 가장 높게 나타남
- Cluster 5, 8: ECL cell에서 가장 높게 나타남
* 이러한 결과는 Hp와 AIG models 사이에서 metaplastic phenotypes 또한 유사하게 유도되어 진다는 것입니다.
Figure 3. Common and diverse subtypes of metaplasia in mouse models of Hp and AIG. Uniform Manifold Approximation and Projections (UMAPs) representing metaplastic cells found (A) only in Hp (900 cells analyzed) or (B) only in AIG (1900 cells analyzed). The pie charts show metaplasia subcluster frequencies. (C) Heat map of 5 cluster-defining transcripts for each metaplastic subcluster. (D–G) Violin plots visualize signature score for each metaplastic subcluster. The black downward arrows highlight highest scoring subcluster (s) for each signature. Plots are colored by subcluster identity.
4. Pathologic Comparison of Helicobacter pylori Infection and Autoimmune Gastritis in Human Disease
* Pathologic features of disease: similar between patients with Hp and AIG (Figure 4A and B)
- Pyloric and intestinal metaplasia (Figure 4A)
- Pyloric metaplasia containing MUC6, TFF2, CD44v9 (Hp and AIG biopsy specimens, Figure 4B)
--> 이러한 결과는 humans에서 Hp와 AIG 에서 발생한 metaplasia의 경우도 mouse models에서 처럼 차이점을 구분하기 어렵습니다.
* Cellular & molecular levels에서의 metaplasia를 비교하기 위해 3명의 AIG 환자 (Hp: negative, gastric cancer(-))의 체부에서 조직을 얻었고, mouse models과 같은 방식으로 single-cell RNA sequencing을 시행하였고, gastric cancer를 가진 10명의 Hp-positive 환자의 tumor에 인접한 체부에서 조직에 대한 결과를 downloaded 하였습니다. (Figure 4C)
- All expected cell types were identified and highest-scoring cells from Hp and AIG settings mapped to the sam clusters (Figure 4D).
--> 이러한 결과는 Hp-infected gastric cancer 환자와 AIG 환자에서 metaplastic cells은 유사한 tanscrpition을 한다는 것을 보여 줍니다.
Figure 4. Similar gastric pathologies observed in Hp and AIG patient biopsy specimens. (A) Representative H&Es of corpus from a healthy (left), a Hp-infected (center), and AIG (right) patient. Arrows: highlight areas of intestinal (yellow) and pyloric (green) metaplasia. (B) Representative immunofluorescent images of corpus of a healthy (left), an Hp-infected (center), and an AIG (right) patient biopsy specimen. Tissue is stained with Hoechst (blue), MUC6 (green), cluster of differentiation 44 (CD44v9; cyan), and TFF2 (red). (C) Experimental design for conducting single-cell RNA sequencing on Hp-infected (n=10, 30,000cells analyzed) and AIG (n=3, 13,000 cells analyzed) patient biopsy specimens. (D) Feature Uniform Manifold Approximation and Projections (UMAPs) visualizing epithelial cell clusters in Hp only (left) and AIG only (right). UMAP is colored based on enrichment for the Gastric Metaplasia Signature Score.
5. Single-Cell Transcriptomic Comparison of Metaplasia Arising in Helicobacter pylori and Autoimmune Gastritis Patients
* 10th percentile for the human metaplasia signature were isolated and subjected to unbiased reintegration and reclustering (Figure 5A)
* Metaplasia signature-enriched cells의 대부분은 metaplastic and neck cell cllusters에서 isolation 되었습니다.
* 12 metaplastic cell subtypes
- Subclusters 0, 1: ~60% of all metaplastic cells in the Hp // only 20% in AIG specimens (Figure 5B and C)
- Hp보다 AIG에서 보다 다양한 metaplastic subtypes을 보였습니다. (AIG: 12 metaplastic subtypes, Hp: 10 subtypes)
- Subclusters 10, 11: unique to AIG
--> 이러한 현상은 human gastric metaplasia가 보다 heterogeneous 하며, 염증성 손상이 metaplastic cells의 다양성에 영향을 미친다고 볼 수 있습니다. 또한, Hp-infected patients의 모든 metaplastic subtypes는 AIG patients에서 또한 관찰되었다는 것도 주목할 부분 입니다.
* Expression of metaplasia associated transcripts (Figure 5D)
- 대부분의 sublclusters에서 spasmolytic polypeptide, TFF2의 expression이 높게 나타났습니다. (subclusters 2, 4 제외)
- subclusters 9번을 제외한 모든 subclusters에서 neck cell-associated transcript MUC6가 expression 되었습니다.
- Subclusters 8, 9 cells에서는 goblet cell trascript인 TFF3 (intestinal metaplasia (IM) 와 연관)이 높게 expression 되었습니다.
- 몇몇 subclusters에서는 dysplasia와 연관성이 있다고 reported 되어진 Tumor-associated calcium signal transducer 2 (TACSTD2) 또는 TROP2가 높게 발현되었습니다.
* Top 5 cluster-defining genes in a heat map (Figure 5E)
- Gastric epithelial cell과 연관된 transcripts (cluster 2: chief cell, cluster3: foveolar cell)
- Pyloric metaplasia와 연관된 transcripts (cluster 4: cystic fibrosis transmembrane conductance regulator (CFTR) and olfactomedin 4 (OLFM4), cluster 10: WAP four disulfide core domain 2 (WFDC2))
* Metaplastic cells by performing signature analyses (Figure 5F-J)
- Gastric proliferative cell: subclusters 1 and 7 (highest scores) (Figure 5F)
- Chief cell: subcluster 2 and AIG-unique subcluster 11 (Figure 5G)
- Immature enterocyte: Subcluster 5 (Figure 5H)에서 가장 높게 나왔는데, 이 cluster에서 TFF2, MUC6, TFF3, TACSTD2의 co-expression이 관찰 되었으며 (Figure 5D), subcluster 5는 incmplete IM를 의미한다고 볼 수 있습니다.
- ECL cell: Metaplastic subcluster 6가 Hp biopsy에서 보다 AIG에서 2배 높은 빈도로 나타나며, ECL cell 에서 가장 높게 나타났습니다. (Figure 5I)
- Goblet cell: subcluster 9이 가장 높게 나타났는데, 이는 complete IM를 의미합니다.
* 이러한 소견은 Hp infected 및 AIG 환자에서 metaplastic cell 분포에 대한 variability가 있음에도 불구하고, 두 환경에서 disease progression (proliferative metaplasia, comoplete IM, incomplete IM) 과 연관된 metaplastic subtypes이 모두 확인 되었다는 것입니다.
Figure 5. Heterogeneous gastric metaplastic cells identified across patient disease settings. (A) Uniform Manifold Approxi mation and Projections (UMAPs) of reclustered metaplastic cells colored by subclusters. UMAP of metaplastic subclusters in (B) Hp-infected biopsy specimens (1400 cells analyzed) and (C) AIG biopsy specimens (1500 cells analyzed), colored by subcluster. The pie charts show subcluster frequencies. (D) Violin plots measuring expression levels of metaplasia- or dysplasia-associated transcripts for each metaplastic subcluster. (E) Heat map of 5 selected cluster-defining transcripts for each metaplastic subcluster. (F–J) Violin plots visualize signature score for each metaplastic subcluster. The black downward arrows: highlight the highest scoring subcluster (s) for each signature. Plots are colored by subcluster identity.
6. Evaluating Relatedness to Cancer Across Metaplastic Subtypes
* 20 gastric cancer associated transcripts
* Cancer cluster in tumor specimens from the antrum and corpus (Figure 6A)
* Metaplastic subcluster 5 (incomplete IM) / subcluster 9 (completeIM) (Figure 6B and C)
* Tumor derived epithelial cell types을 예측하여 분석하였는데, cluster 5가 양측 diseases setting에서 cancer cells와 가장 연관성을 보이는 metaplastic subcluster로 확인 되었습니다. (Figure 6D)
* subcluster 0, 1, 3, 6, 7, 9, 10도 cancer cell에서 mapped 되었으나, incomplete IM subtype (cancer-related metaplastic cell)인 subcluster 5는 Hp와 AIG, 모두에서 발현되었습니다.
* potential cancer-associated biomarker를 확인 하기 위해 cluster-defining transcripts를 확인 하였으며, subcluster5-defining gene으로 ANPEP를 확인할 수 있었습니다. ANPEP는 Hp-infected tumor biopsy specimens 에서만 unique 하게 up regulation 되었습니다.
* 이러한 결과는, incomplete IM 와 유사한 metaplasia는 Hp-induced 및 AIG induced metaplasia 환자에서 모두 확인할 수 있었고, 하위 cluster에서 보았을 때 gastric cancer와 연관성이 있으며 특히, ANPEP의 level이 unique하게 높게 나타났습니다.
Figure 6. Cancer-related metaplastic cells present in patients with Hp and AIG. (A) Feature Uniform Manifold Approximation and Projection (UMAP) visualizing epithelial cell clusters in Hp tumor corpus biopsy specimens (n=10, 30,000 cells analyzed). UMAP is colored based on enrichment for the Gastric Cancer Signature Score. (B) Gastric Cancer Signature feature UMAP for Hp metaplastic cells. (C) Gastric Cancer Signature feature UMAP for AIG metaplastic cells. (D) UMAPs of gastric metaplastic subclusters in all settings (left), Hp only (center), or AIG only (right) labeled with epithelial cell clusters from Hp corpus tumor biopsy specimens. Violin plots demonstrate ANPEP/CD13 transcript E) across metaplastic subclusters and (F) across cell clusters in the Hp corpus tumor biopsy specimens.
7. Alanyl Aminopeptidase N/CD13 as a Biomarker for Cancer-Related Metaplasia in Helicobacter pylori and Autoimmune Gastritis patients
* Expression of ANPEP of patients with Hp or AIG (Figure7)
* Healthy gastric tissue: No detectable expression of intestinal metaplastic proteins TFF3 and MUC2 or ANPEP
* H&E: Hp 와 AIG biopsy specimens에서 IM glands 를 모두 확인 할 수 있었습니다. (Figure 7Aand B)
- 이는 TFF3와 MUC2에 대한 positive staining으로 confirm 가능 하였습니다. (Figure 7C and D)
* IM regions에서 ANPEP expression 또한, 관찰할 수 있었습니다. (Figure 7E and F)
- IM 가진 5명의 Hp patients와 IM 가진 6명의 AIG patients, 모두의 IM glands subset에서 ANPEP expression을 관찰 할 수 있었습니다.
* 이러한 결과는, Hp- 및 AIG-induced metaplasia를 가진 precancerous 환자의 조직에서 metaplastic cells의 subset에 ANPEP이 존재한다는 것을 확인 할 수 있었으며, ANPEP가 cancer progression의 위험성을 가진 환자의 early biomarker 로써 사용할 수 있다는 근거를 제공하는 것입니다.
Figure 7. ANPEP/CD13 identifies cancer-related metaplastic cells in Hp and AIG metaplastic biopsy specimens. (A and B) Representative H&E images of Hp-infected and AIG patient biopsy specimens highlight areas of metaplasia. (C–F) Immunofluorescent images of Hp-infected (left) and AIG patient biopsy specimens (right). (C and D) Tissue is stained with Hoechst (blue), MUC6 (cyan), MUC2 (green), and TFF3 (red). (E and F) Tissue is stained with Hoechst (blue), MUC6 (cyan), MUC2 (green), and ANPEP (red). Scale bars: 100 mm. Sequential imaging of the same section was conducted between A, C, and E (Hp-infected) and B, D, and F (AIG).
Discussion
* Metaplasia는 acute injury에 대한 tissue healing 으로 나타나는 조직변화 이지만, chronic inflammation과 같은 long standing injury는 dysplasia로의 변화를 유도하게 됩니다. 하지만, 적은 일부에서만 나타나기 때문에 적절한 예방과 치료 전략을 위해서 cancer progression의 high risk를 갖는 보다 특징적인 metaplastic lineage를 찾는 것이 필요합니다.
* 이번 연구에서 주목할 점은, 보다 높은 adenocarcinoma risk와 연관된 H. pylori infection이 AIG에서 독특한 subtype의 metaplasia를 유도하지 않았다는 것입니다.
* Mouse models과 human disease에서 chief cell과 proliferative cell과 유사한 일반적인 metaplastic subtypes이 확인 되었고, ECL 과 intestinal lineage의 subtypes 도 함께 확인 할 수 있었습니다. (Paligenosis: chief-like metaplastic cells = chief-drived metaplastic cells)
* 이번 연구에서는 proliferative metaplastic cells를 확인 하였는데, 이들은 human에서 dysplasia-associated TACSTD2 또는, TROP2를 발현시키며 transcriptionally cancer와 연관성이 있습니다. Cancer progression potential을 가진 proliferative metaplastic cells이 있긴 하지만, 이번 연구에서는 cancer-associated IM subtypes에 대해 보다 focusing 하였습니다.
*
Conclusion
This study underscores the significance of comparing the features of metaplasia induced by Hp infection and AIG, revealing that both conditions induce cancer-associated metaplasia. As such, it emphasizes the importance of closely monitoring patients with either disease for early biomarkers, such as ANPEP/CD13, to mitigate adenocarcinoma risk and improve patient outcomes.
Abstract
Background & aims: Gastric metaplasia may arise as a consequence of chronic inflammation and is associated with an increased risk of gastric cancer development. Although Helicobacter pylori (Hp) infection and autoimmune gastritis (AIG) both induce gastric metaplasia, possible distinctions in resulting metaplastic cells and their respective cancer risks requires further investigation.
Methods: Using both mouse models and human participants, we scrutinized the metaplasia originating from Hp infection and AIG. Gastric pathology and metaplasia were examined through histopathologic assessment. Molecular features of metaplastic cells were defined using single-cell transcriptomics in murine models of Hp infection and AIG, as well as in human biopsy specimens from patients with Hp infection and AIG. Expression of a newly defined cancer-related metaplastic biomarker was confirmed through immunofluorescence.
Results: Metaplasia in Hp infection and AIG displayed comparable histopathologic and transcriptional features. Diverse metaplastic subtypes were identified across both disease settings, with subtle differences in the prevalence of certain subtypes between inflammatory contexts. Notably, Hp infection did not drive a unique metaplastic cell phenotype. One metaplastic subtype, which resembled incomplete intestinal metaplasia and shared transcriptional features with gastric cancer, was identified in both diseases. This cancer-like metaplastic subtype was characterized by expression of the cancer-associated biomarker alanyl aminopeptidase N/CD13.
Conclusion: Both Hp infection and AIG trigger a diverse array of metaplastic cell types. Identification of a cancer-related metaplastic cell uniquely expressing alanyl aminopeptidase N/CD13, present in both Hp- and AIG-induced gastritis, indicates the carcinogenic capacity of both diseases. This discovery can guide early detection and risk stratification for patients with chronic gastritis.
Keywords: Autoimmune Gastritis; Gastric Cancer; Gastric Inflammation; Gastric Metaplasia; Helicobacter pylori Infection.