Mod Pathol. 2024 Jun;37(6):100491. doi: 10.1016/j.modpat.2024.100491.
AIG에서 adenocarcinoma 발생에 대한 위험성은 아직 불분명합니다. 이번 연구에서는 비록 적은 수이긴 하지만, AIG 환자 중 gastric cancer sample을 이용하여 분자유전학적 특징을 살펴본 연구 입니다.
Introduction
The increased risk of type 1 NETs is well-documented, and, even though AIG and pernicious anemia have been recognized as risk factors for gastric carcinoma (GC) over the years, their actual impact in the absence of H. pylori is still debated.
일관되게, according to the studies looking at the natural history of “pure” AIG the risk of developing GC is virtually absent.
반면에 certain risk of developing GC was noticed when deliberately including patients with corpus-restricted atrophy with and without H. pylori infection. (이러한 의미에서 H. pylori infection rate가 높은 우리나라에서는 <"pure" AIG는 GC 위험성이 없으니까 괜찮아>가 아니라 체부위축이 있는 AIG 환자에서 H. pylori infection은 어떠한 영향을 줄까에 대해 더욱 관심을 갖고 연구가 이루어 져야 하지 않을까 생각됩니다. 최근 2024 UEG의 포스터 section을 살펴보면, AIG 환자에서 GC 발생한 경우 H. pylori 유전자가 있더라 / 혹은 없더라 .. 에 대한 연구 결과가 발표될 예정입니다.)
The histopathological and molecular features, including the status of predictive biomarkers, in a cohort of GCs arising in the context of AIG.
Materials and methods
Study cohort
26 patients diagnosed between 2012 and 2022 with AIG-GC (with clinical, serological, and histological features of AIG according to internationally adopted criteria). H. pylori infection was ruled out by histologic examination.
Clinical and pathologic data on sex, age, primary tumor site, pTNM stage, synchronous and/or metachronous NETs, time of AIG diagnosis, OLGA staging at AIG diagnosis, symptoms at presentation, and previous records of H. pylori infection were collected from the pathology reports and clinical records. (7 patients, only biopsy samples / 19 cases surgical samples)
Establish pTNM stage (according to the 8th edition American Joint Committee on Cancer [AJCC] staging system criteria), histotype and grade (according to the 5th edition of WHO digestive tumors classification), absence of parietal cells, presence and type of intestinal metaplasia (complete vs incomplete), and presence and subtype of precursor lesion.
Immunohistochemical (IHC) analysis
HER2 (4B5, Ventana), PD-L1 (22C3; Dako), p53 (clone DO-7; Dako), synaptophysin (clone 27G12, Leica Biosystems), chromogranin-A (clone DAK A3; Dako), CLDN18, (clone 43-14A; Roche Ventana), MUC1 (clone Ma695; Leica Biosystems), MUC2 (clone CCP58; Leica Biosystem), MUC5AC (clone CLH2, Abcam), MUC6 (clone CLH5; Leica Biosystems Newcastle Ltd, Newcastle Upon Tyne, UK), and CDX2 (clone epr2764y; Cell Marque).
Classification of mucin stains (MUC1, MUC2, MUC5AC, and MUC6) and CDX2
- Gastric immunophenotype: positive for MUC5AC and/or MUC6 and/or MUC1 and negative for MUC2 and CDX2
- Intestinal immunophenotype: negative for MUC5AC, MUC6 and MUC1 and positive for MUC2 and/or CDX2
- Mixed immunophenotype: other combinations of mucins and CDX2 expression than those previously mentioned
(Focal expression in less than 5% of tumor cells was considered negative)
Synaptophysin & chromogranin
Neuroendocrine components/differentiation and to evaluate the presence of linear (chains composed of ≥ 5 continuous ECL cells within a single gland) or micronodular (nodular aggregates of ≥ 5 ECL cells) neuroendocrine hyperplasia
MMR status
MLH1, PMS2, MSH2 and MSH6
MMR deficient (MMRd): when one or both proteins from a functional couple resulted negative in the presence of an adequate internal positive control (intra-tumor inflammatory and stromal cells and non-neoplastic cells)
The evaluation of HER2
Four-tiered Hoffmann scoring criteria
Surgical sample: when complete or basolateral membranous reactivity was observed in ≥10% of tumor cells, scores of 1+, 2+ and 3+ were assigned according to the intensity of membranous reactivity (faint, moderate or intense)
Biopsy sample: when membranous reactivity was observed in at least one cancer cell cluster (≥5 cells), scores of 1+, 2+ and 3+ were assigned according to intensity (faint, moderate or intense)
p53 was considered aberrant in the presence of complete loss or diffuse and strong nuclear immunostaining in neoplastic cells
CLDN18: quantitative (percentage of stained tumor cells method)
Tumors with a 2+/3+ score of CLDN18 intensity in ≥ 75% of tumor cells - positive
PD-L1 expression was evaluated by using the Combined Positive Score (CPS)
EBER(EBV-encoded RNA) in situ hybridization
fluorescein-labelled oligonucleotide probes (EBER probe, Ventana) with enzymatic digestion (ISH protease 3, Ventana)
iViewBlue detection kit (Ventana) with use of the BenchMark ULTRA staining system (K Tello)
Molecular analysis
Only 19 samples had sufficient tissue for molecular analysis
TSO500 analysis allows to detect DNA biomarkers including single nucleotide variants (SNVs), insertions, deletions, copy number variants (CNVs) and multinucleotide variants (MNVs) as well as tumor TMB and MSI in DNA. Fusions and splice variants are detected in RNA.
DNA and RNA sequencing passed the quality control criteria if the coding region size in megabases was ≥ 1.20 Mb and ranging between 1.20-1.90, respectively.
* Tumor mutation burden (TMB, 종양세포의 돌연변이 수): TMB-High ≥ 10 mutations/Mb and TMB-Low < 10 mutations/Mb
* Microsatellite instability (MSI, 현미부수체 불안정성): highly unstable (MSI-High) if presenting more than 40% unstable loci, stable (MSS) if presenting less than 20% unstable loci and poorly unstable (MSI-Low) if presenting a percentage of unstable loci comprise between 20% and 40%
Mutations detected were classified according to the TIER classification (암 유전체 분석, 변이 여부 분류: TIER III(unknown), IV(benign))
selecting variants with strong clinical significance (IA, IB, IIC, and IID))
Results
Clinico-pathological features
Table 1. Detailed clinico-pathologic features of 26 autoimmune gastritis-associated gastric carcinomas.
Abbreviations: IM= Intestinal metaplasia, PCC= poorly cohesive carcinoma, SR= signet ring cell, AC= adenocarcinoma, NE= neuroendocrine, NET= neuroendocrine tumor, MiNEN= Mixed neuroendocrine non-neuroendocrine neoplasm, LG= low grade, NEC= neuroendocrine carcinoma, C=complete, IN=incomplete, L=linear, M=micronodular, PGA= Pyloric Gland Adenoma, ID= Intestinal Flat Dysplasia.
* Female-to-male ratio was 1.2:1, with a median age at diagnosis of 74.5 years (range, 52-86 years)
* 11 (42.3%) tumors: antrum, 15 (57.7%) tumors: corpus/fundus
* One patient with a GC located in the corpus had Lynch syndrome
* synchronous or metachronous NETs: 5/26 (19.2%) patients (4: NETs G1, 1: NET G2)
anemia and/or B12 hypovitaminosis (10/21, 47.6%), dyspepsia (6/21, 28.6%), abdominal pain (5/21, 23.8%), and weight loss (4/21, 19.0%). One patient received emergency surgery due to gastric perforation.
* Staging was assessable in 22/26 (84.6%) samples.
: The majority of tumors (13/22, 59.1%) were pT1. When lymph node status was assessed, most cases (16/19, 84.2%) showed no evidence of lymph node metastases.
Histology
Tubular adenocarcinoma (8/26, 30.8%), poorly cohesive carcinomas of the signet ring cell type (3/26, 11.5%), mixed adenocarcinoma (6/26, 23.1%), carcinoma with lymphoid stroma (2/26, 7.6%), mucinous carcinoma with signet ring cells (1/26, 3.9%), high-grade solid adenocarcinomas with focal areas (i.e., accounting for <30% of tumor area) of neuroendocrine differentiation (i.e., a focal NEC component positive for synaptophysin; 2/26, 7.6%; Figure 1A,B), mixed neuroendocrine-non neuroendocrine neoplasm (MiNEN) (3/26, 11.5%;), and adenocarcinoma with an amphicrine component (1/26, 3.9%)
* MiNENs: 3 cases (Figure 1C-F): 2 cases NET component (G1 and G2) & 1 case NEC component.
Figure 1. (A) High-grade solid adenocarcinoma (HE, sample #10) with (B) a synaptophysin-positive area. (C) Mixed Neuroendocrine-non neuroendocrine neoplasm (tubular adenocarcinoma and NET, sample #13) showing (D) positivity for synaptophysin in the NET component. Mixed Neuroendocrine-non neuroendocrine neoplasm (tubular adenocarcinoma and NET, sample #12) at low (E) and high (F) magnification.
* Adenocarcinoma with an amphicrine component:
poorly cohesive signet-ring cell appearance and had both exocrine and neuroendocrine differentiation in a subset of tumor cells (i.e., approximately 20% of cells co-expressed chromogranin A, synaptophysin and PAS/MUC5AC; Figure 2)
Figure 2. Representative images of the mixed amphicrine carcinoma (HE, sample #11) (A) showing poorly cohesive morphology (B) with adjacent ECL-cell hyperplasia. The amphicrine component displays both (C) chromogranin-A (in a double chromogranin [brown]/cytokeratin AE1/A3 [red] stain) and (D) PAS positivity in a subset of tumor cells.
All the cases with neuroendocrine differentiation or a neuroendocrine component (high-grade solid adenocarcinomas, MiNENs, and the adenocarcinoma with an amphicrine component) were located in the corpus/fundus.
Clinico-pathologic features of AIG-GCs as a whole and of corpus/fundus AIG-GCs (excluding the Lynch syndrome patient)
Table 2. Clinico-pathologic features of all autoimmune gastritis-associated gastric carcinomas (AIG-GCs) of our cohort (n=26) and of the AIG-GCs of the corpus/fundus (excluding the Lynch syndrome-associated case, n=14), compared with data from the subset of gastric carcinomas of the previously published cohort by Angerilli et al.
일반적인 Gastric adenocarcinoma의 subset 과 비교한 결과,
AIG-GCs는 corpus/fundus에 주로 위치하고, Lower pT & pN stage가 대부분이었으며, MiNEN/amphicrine carcinomas 와 high-grade solid carcinomas의 비율이 높았다.
Histologic features
Table 3. Histologic features of the gastritis and associated precursor lesions of 26 autoimmune gastritis-associated gastric carcinomas.
Abbreviations: PGA= Pyloric Gland Adenoma
* Most patients (21/26, 80.8%) had stage II OLGA gastritis.
* No patient had evidence of a previously eradicated H. pylori infection
* Parietal cells were absent in 24/26 (92.3%) cases.
* Intestinal metaplasia in the corpus/fundus was observed in 25/26 (96.2%) cases; complete and incomplete intestinal metaplasia was found in 20 and 5 cases, respectively.
* Majority of cases displayed micronodular ECL-cell hyperplasia (23/26, 88.5%)
* Precursor lesions: PGAs (2 cases), and gastric adenocarcinomas of intestinal or mixed immunophenotype (see below) were associated with flat intestinal-type dysplasia (8 cases)
Immunohistochemical analysis
Gastric immunophenotype: 7 (26.9%)
Intestinal immunophenotype: 10 (38.5%)
Mixed immunophenotype: 9 (34.9%)
p53 expression: aberrant in 7 (26.9%) cases
PD-L1 expression: (CPS≥1) in the majority of AIG-GCs (23/26, 88.5%), but CPS≥10 was observed in 10 GCs (38.5%)
MMR deficiency (MMRd): 7 AIG-GCs (26.9%) (5: MLH1/PMS2 loss, 1: MSH2/MSH6 loss (the patient with Lynch syndrome), 1: isolated loss of PMS2)
HER2 overexpressing (3+): 3 AIG-GCs (11.5%)
CLDN18 positive: 6 (23.1%)
All cases were negative for EBV in situ hybridization.
In case #13 and #14 (HER2 overexpression and PMS2 loss): adenocarcinomatous component of the two MiNENs 에서 만 관찰됨.
반대로.. CLDN18 staining은 MiNENs의 NET components에서는 관찰되었지만, NEC component에서는 관찰되지 않음.
<일반적인 Gastric adenocarcinoma의 subset 과 비교한 결과 Table 4.>
Table 4. Molecular features of all autoimmune gastritis-associated gastric carcinomas (AIG-GCs) of our cohort (n=26) and the AIG-GC of the corpus/fundus-restricted (excluding the Lynch syndrome-associated case, n=14), compared with data from the subset of gastric carcinomas of the previously published cohort by Angerilli et al.
Abbreviations: IF= immunophenotype, MMR= Mismatch Repair, CLDN=Claudin, MSI=Microsatellite Instability, TMB=Tumor Mutational Burden, NS= not significant.
HER2, MMR, EBER, and PD-L1: No significant difference
Molecular analysis
Revealed a total of 119 mutations affecting 72 different genes with a range of 0 to 36 variants per sample (Figure 3)
Figure 3. Summary of clinico-pathologic features, biomarker status and genetic alterations of 26 gastric carcinomas in the context of autoimmune gastritis (AIG-GC), of which 19 underwent extensive molecular profiling. AIG-GC of the corpus/fundus, the Lynch-syndrome associated AIG-GC and AIG-GC of the antrum are represented separately.
Abbreviations: PCC= poorly cohesive carcinoma, AC= adenocarcinoma, PGA= pyloric gland adenoma, C= carcinoma, MiNEN= Mixed Neuroendocrine Non-neuroendocrine neoplasm, IF= immunophenotype, MMRp= Mismatch repair proficient, MMRd= Mismatch repair deficient, MSS= microsatellite stable, MSI-L= microsatellite instability-low, MSI-H= microsatellite instability-high, TMB= tumor mutational burden, CNV = copy number variation.
* TP53, RNF43 mutations: The most frequent in the cohort (8/19, 42.1% and 7/19, 36.8% of cases, respectively)
* ARID1A (6/19, 35.3%), ERBB2 (5/19, 26.3%), PIK3CA (4/19, 21.1%), PTEN (4/19, 21.1%), ANKRD26 (3/19, 15.8%), CUX1 (3/19, 15.8%)
* Other pathogenic/likely pathogenic variants
: APC, BCOR, CARD11, CDH1, CSF3R, Journal Pre-proof EP300, FBXW7, KRAS, NBN, NF1, RASA1, ZFHX3 and ALOX12B: 2/19 cases (10.5%)
: ANKRD11, ARID2, ASXL1, ATR, AXIN1, AXIN2, B2M, BAP1, BARD1, CDKN1B, CIC, CTNNB1, CYLD, EED, GATA2, HNF1A, MEN1, MLH1, MRE11A, MSH2, PARK2, PAX5, PPM1D, SMAD4, PTPN11, SMC1A: 1/19 cases (5.2%)
* Frameshift deletion/insertions: The most common variants (74/119, 62.2%)
* non-synonymous coding variants (44/119, 37.0%), in-frame deletion (1/119, 0.8%)
* Analysis of CNVs: 5/19 (26.3%) samples of the cohort displayed a copy number variation (samples #2, #10, #18, #20 and #26), involving primarily EGFR (2/19, 10.5%) and ERBB2 (2/19, 10.5%). ERCC1, CCND3, CCNE1, CDK6, FGF10, FGFR3, MYC, KRAS, and RICTOR (1/19, 5.2%)
* TMB analysis: 7/19 (36.8%) cases were TMB-high (≥ 10 muts/Mb) and 12/19 (63.2%) were TMB-low (< 10 muts/Mb)
* MSI: 5/19 (26.3%) cases. All MSI samples were MMRd and TMB-high.
* MMRd: 7 cases (2 cases: MSS, 1 case: TMB-low)
--> AIG-GCs of the corpus/fundus (excluding the Lynch syndrome associated case), the most frequently mutated genes were TP53 (4/11, 36.4%), KRAS, PTEN, RNF43, PIK3CA, and ERBB2 (2/11, 18.2%). 2 cases harbored CNVs, both having ≥3 CNVs. MSI was found in 1 case (1/11, 9%), and TMB-high in 3 cases (3/11, 27.3%).
Statistical integration of IHC and molecular analyses
* MSI and MMRd AIG-GCs were enriched in TMB-high (both p=0.002), RNF43 mutations (p=0.038 and p=0.029), ARID1A mutations (both p<0.001), but not PD-L1 CPS ≥ 10.
* TMB high was also associated with PD-L1 CPS ≥ 10 (p=0.0029), aberrant p53 (p=0.005), RNF43 mutations (p=0.0029), ARID1A mutations (p<0.001), and ERBB2 mutations (p=0.038).
* RNF43 and ERBB2 mutations were associated with PD-L1 CPS ≥ 10 (p=0.0129 and p=0.038, respectively).
No statistically significant difference in the IHC and genomic profile was found between the AIG-GCs of the corpus-fundus (excluding the Lynch syndrome-associated case) and the remaining AIG-GCs.
Discussion
To conclude,
AIG-GCs appear distinct clinco-pathologic features, including low T stage, proximal location, low-risk OLGA stages, but end-stage atrophic-metaplastic background. Our cohort revealed the presence of peculiar phenotypes and a high rate of lesions displaying a neuroendocrine component, suggesting that the landscape of neuroendocrine lesions may be wider than previously stated in the AIG context.
At the molecular level, AIG associated GCs were not associated with a specific molecular signature but appeared to be characterized by relatively high rates of PD-L1 expression, MSI/MMRd, and TMB-high, no association with EBV and low rates of CDH1 mutations.
Although in patients with AIG the risk of developing overt GC seems to be almost non-existent in patients who are endoscopically followed up, this risk may be higher in those patients with longstanding, occult AIG.
Histologic markers of severe AIG may help identify patients at risk for GC. The peculiar phenotypes and molecular features of AIG-GC warrant further research.
일반적인 gastric cancer는 H. pylori infection과 연관성이 높기 때문에 여러 phenotypes이나 molecular features 는 H. pylori infection과 연관하여 발생하는 변화들과는 다소 다른 부분이 있을 수 있겠습니다. AIG에서 gastric cancer의 발생이 있긴 한데... 정말 "pure AIG" 에서는 관련이 없을 수도 있겠다.. 관찰되는 몇몇 phenotypes나 molecular features는 사라진 H. pylori infection, 또는 microbiome 이나 diet 등의 영향이었을까?
어렵네요... 아직 갈길이 먼 것 같긴 합니다. ㅠㅠ
Abstract
Patients with autoimmune gastritis (AIG) have a 13-fold risk of developing type-1 neuroendocrine tumors, whereas the risk for gastric adenocarcinoma is still uncertain. Here we describe the clinicopathological and molecular features of a series of gastric carcinomas (GC) arising in the context of AIG.
A total of 26 AIG-associated GC specimens were collected from four Italian Institutions. Immunohistochemistry for MUC1, MUC2, MUC5AC, MUC6, CDX2, HER2, PD-L1, CLDN18, Mismatch Repair (MMR) proteins, and p53 and EBER in situ hybridization were performed. Histologic features and IHC were jointly reviewed by five expert gastrointestinal pathologists.
Next generation sequencing analysis (TrueSight Oncology 500, Illumina) of 523 cancer-related genes was performed on 19 cases. Most tumors were diagnosed as pT1 (52%), were located in the corpus/fundus (58%) and were associated with OLGA stage II gastritis (80.8%), absence of parietal cells, complete intestinal metaplasia and ECL-cell micronodular hyperplasia. Only 4 (15.4%) GCs were diagnosed during follow-up for AIG. The following histotypes were identified: 20 (77%) adenocarcinomas; 3 (11%) mixed neuroendocrine non-neuroendocrine neoplasms, and 2 (8%) high grade solid adenocarcinomas with focal neuroendocrine component, 1 (4%) adenocarcinoma with an amphicrine component. Overall, 7 cases (27%) showed MMR deficiency, 3 (12%) were positive (score 3+) for HER2, 6 (23%) were CLDN18 positive, and 11 (42%) had PD-L1 Combined Positive Score ≥ 10. EBER was negative in all cases. Molecular analysis revealed 5/19 (26%) MSI cases and 7 (37%) TMB-high. The most frequently altered genes were: TP53 (8/19, 42%), RNF43 (7/19, 37%), ERBB2 (7/19, 37% [two amplified and five mutated cases]), ARID1A (6/19, 32%), and PIK3CA (4/19, 21%).
In summary, AIG-associated GCs are often diagnosed at low stage in patients with long standing misrecognized severe AIG; they often display a neuroendocrine component or differentiation, have relatively higher rates of MMR deficiency, and TMB-high.
Mod Pathol. 2024 Jun;37(6):100491. doi: 10.1016/j.modpat.2024.100491.
AIG에서 adenocarcinoma 발생에 대한 위험성은 아직 불분명합니다. 이번 연구에서는 비록 적은 수이긴 하지만, AIG 환자 중 gastric cancer sample을 이용하여 분자유전학적 특징을 살펴본 연구 입니다.
Introduction
The increased risk of type 1 NETs is well-documented, and, even though AIG and pernicious anemia have been recognized as risk factors for gastric carcinoma (GC) over the years, their actual impact in the absence of H. pylori is still debated.
일관되게, according to the studies looking at the natural history of “pure” AIG the risk of developing GC is virtually absent.
반면에 certain risk of developing GC was noticed when deliberately including patients with corpus-restricted atrophy with and without H. pylori infection. (이러한 의미에서 H. pylori infection rate가 높은 우리나라에서는 <"pure" AIG는 GC 위험성이 없으니까 괜찮아>가 아니라 체부위축이 있는 AIG 환자에서 H. pylori infection은 어떠한 영향을 줄까에 대해 더욱 관심을 갖고 연구가 이루어 져야 하지 않을까 생각됩니다. 최근 2024 UEG의 포스터 section을 살펴보면, AIG 환자에서 GC 발생한 경우 H. pylori 유전자가 있더라 / 혹은 없더라 .. 에 대한 연구 결과가 발표될 예정입니다.)
The histopathological and molecular features, including the status of predictive biomarkers, in a cohort of GCs arising in the context of AIG.
Materials and methods
Study cohort
26 patients diagnosed between 2012 and 2022 with AIG-GC (with clinical, serological, and histological features of AIG according to internationally adopted criteria). H. pylori infection was ruled out by histologic examination.
Clinical and pathologic data on sex, age, primary tumor site, pTNM stage, synchronous and/or metachronous NETs, time of AIG diagnosis, OLGA staging at AIG diagnosis, symptoms at presentation, and previous records of H. pylori infection were collected from the pathology reports and clinical records. (7 patients, only biopsy samples / 19 cases surgical samples)
Establish pTNM stage (according to the 8th edition American Joint Committee on Cancer [AJCC] staging system criteria), histotype and grade (according to the 5th edition of WHO digestive tumors classification), absence of parietal cells, presence and type of intestinal metaplasia (complete vs incomplete), and presence and subtype of precursor lesion.
Immunohistochemical (IHC) analysis
HER2 (4B5, Ventana), PD-L1 (22C3; Dako), p53 (clone DO-7; Dako), synaptophysin (clone 27G12, Leica Biosystems), chromogranin-A (clone DAK A3; Dako), CLDN18, (clone 43-14A; Roche Ventana), MUC1 (clone Ma695; Leica Biosystems), MUC2 (clone CCP58; Leica Biosystem), MUC5AC (clone CLH2, Abcam), MUC6 (clone CLH5; Leica Biosystems Newcastle Ltd, Newcastle Upon Tyne, UK), and CDX2 (clone epr2764y; Cell Marque).
Classification of mucin stains (MUC1, MUC2, MUC5AC, and MUC6) and CDX2
- Gastric immunophenotype: positive for MUC5AC and/or MUC6 and/or MUC1 and negative for MUC2 and CDX2
- Intestinal immunophenotype: negative for MUC5AC, MUC6 and MUC1 and positive for MUC2 and/or CDX2
- Mixed immunophenotype: other combinations of mucins and CDX2 expression than those previously mentioned
(Focal expression in less than 5% of tumor cells was considered negative)
Synaptophysin & chromogranin
Neuroendocrine components/differentiation and to evaluate the presence of linear (chains composed of ≥ 5 continuous ECL cells within a single gland) or micronodular (nodular aggregates of ≥ 5 ECL cells) neuroendocrine hyperplasia
MMR status
MLH1, PMS2, MSH2 and MSH6
MMR deficient (MMRd): when one or both proteins from a functional couple resulted negative in the presence of an adequate internal positive control (intra-tumor inflammatory and stromal cells and non-neoplastic cells)
The evaluation of HER2
Four-tiered Hoffmann scoring criteria
Surgical sample: when complete or basolateral membranous reactivity was observed in ≥10% of tumor cells, scores of 1+, 2+ and 3+ were assigned according to the intensity of membranous reactivity (faint, moderate or intense)
Biopsy sample: when membranous reactivity was observed in at least one cancer cell cluster (≥5 cells), scores of 1+, 2+ and 3+ were assigned according to intensity (faint, moderate or intense)
p53 was considered aberrant in the presence of complete loss or diffuse and strong nuclear immunostaining in neoplastic cells
CLDN18: quantitative (percentage of stained tumor cells method)
Tumors with a 2+/3+ score of CLDN18 intensity in ≥ 75% of tumor cells - positive
PD-L1 expression was evaluated by using the Combined Positive Score (CPS)
EBER(EBV-encoded RNA) in situ hybridization
fluorescein-labelled oligonucleotide probes (EBER probe, Ventana) with enzymatic digestion (ISH protease 3, Ventana)
iViewBlue detection kit (Ventana) with use of the BenchMark ULTRA staining system (K Tello)
Molecular analysis
Only 19 samples had sufficient tissue for molecular analysis
TSO500 analysis allows to detect DNA biomarkers including single nucleotide variants (SNVs), insertions, deletions, copy number variants (CNVs) and multinucleotide variants (MNVs) as well as tumor TMB and MSI in DNA. Fusions and splice variants are detected in RNA.
DNA and RNA sequencing passed the quality control criteria if the coding region size in megabases was ≥ 1.20 Mb and ranging between 1.20-1.90, respectively.
* Tumor mutation burden (TMB, 종양세포의 돌연변이 수): TMB-High ≥ 10 mutations/Mb and TMB-Low < 10 mutations/Mb
* Microsatellite instability (MSI, 현미부수체 불안정성): highly unstable (MSI-High) if presenting more than 40% unstable loci, stable (MSS) if presenting less than 20% unstable loci and poorly unstable (MSI-Low) if presenting a percentage of unstable loci comprise between 20% and 40%
Mutations detected were classified according to the TIER classification (암 유전체 분석, 변이 여부 분류: TIER III(unknown), IV(benign))
selecting variants with strong clinical significance (IA, IB, IIC, and IID))
Results
Clinico-pathological features
Table 1. Detailed clinico-pathologic features of 26 autoimmune gastritis-associated gastric carcinomas.
Abbreviations: IM= Intestinal metaplasia, PCC= poorly cohesive carcinoma, SR= signet ring cell, AC= adenocarcinoma, NE= neuroendocrine, NET= neuroendocrine tumor, MiNEN= Mixed neuroendocrine non-neuroendocrine neoplasm, LG= low grade, NEC= neuroendocrine carcinoma, C=complete, IN=incomplete, L=linear, M=micronodular, PGA= Pyloric Gland Adenoma, ID= Intestinal Flat Dysplasia.
* Female-to-male ratio was 1.2:1, with a median age at diagnosis of 74.5 years (range, 52-86 years)
* 11 (42.3%) tumors: antrum, 15 (57.7%) tumors: corpus/fundus
* One patient with a GC located in the corpus had Lynch syndrome
* synchronous or metachronous NETs: 5/26 (19.2%) patients (4: NETs G1, 1: NET G2)
anemia and/or B12 hypovitaminosis (10/21, 47.6%), dyspepsia (6/21, 28.6%), abdominal pain (5/21, 23.8%), and weight loss (4/21, 19.0%). One patient received emergency surgery due to gastric perforation.
* Staging was assessable in 22/26 (84.6%) samples.
: The majority of tumors (13/22, 59.1%) were pT1. When lymph node status was assessed, most cases (16/19, 84.2%) showed no evidence of lymph node metastases.
Histology
Tubular adenocarcinoma (8/26, 30.8%), poorly cohesive carcinomas of the signet ring cell type (3/26, 11.5%), mixed adenocarcinoma (6/26, 23.1%), carcinoma with lymphoid stroma (2/26, 7.6%), mucinous carcinoma with signet ring cells (1/26, 3.9%), high-grade solid adenocarcinomas with focal areas (i.e., accounting for <30% of tumor area) of neuroendocrine differentiation (i.e., a focal NEC component positive for synaptophysin; 2/26, 7.6%; Figure 1A,B), mixed neuroendocrine-non neuroendocrine neoplasm (MiNEN) (3/26, 11.5%;), and adenocarcinoma with an amphicrine component (1/26, 3.9%)
* MiNENs: 3 cases (Figure 1C-F): 2 cases NET component (G1 and G2) & 1 case NEC component.
Figure 1. (A) High-grade solid adenocarcinoma (HE, sample #10) with (B) a synaptophysin-positive area. (C) Mixed Neuroendocrine-non neuroendocrine neoplasm (tubular adenocarcinoma and NET, sample #13) showing (D) positivity for synaptophysin in the NET component. Mixed Neuroendocrine-non neuroendocrine neoplasm (tubular adenocarcinoma and NET, sample #12) at low (E) and high (F) magnification.
* Adenocarcinoma with an amphicrine component:
poorly cohesive signet-ring cell appearance and had both exocrine and neuroendocrine differentiation in a subset of tumor cells (i.e., approximately 20% of cells co-expressed chromogranin A, synaptophysin and PAS/MUC5AC; Figure 2)
Figure 2. Representative images of the mixed amphicrine carcinoma (HE, sample #11) (A) showing poorly cohesive morphology (B) with adjacent ECL-cell hyperplasia. The amphicrine component displays both (C) chromogranin-A (in a double chromogranin [brown]/cytokeratin AE1/A3 [red] stain) and (D) PAS positivity in a subset of tumor cells.
All the cases with neuroendocrine differentiation or a neuroendocrine component (high-grade solid adenocarcinomas, MiNENs, and the adenocarcinoma with an amphicrine component) were located in the corpus/fundus.
Clinico-pathologic features of AIG-GCs as a whole and of corpus/fundus AIG-GCs (excluding the Lynch syndrome patient)
Table 2. Clinico-pathologic features of all autoimmune gastritis-associated gastric carcinomas (AIG-GCs) of our cohort (n=26) and of the AIG-GCs of the corpus/fundus (excluding the Lynch syndrome-associated case, n=14), compared with data from the subset of gastric carcinomas of the previously published cohort by Angerilli et al.
일반적인 Gastric adenocarcinoma의 subset 과 비교한 결과,
AIG-GCs는 corpus/fundus에 주로 위치하고, Lower pT & pN stage가 대부분이었으며, MiNEN/amphicrine carcinomas 와 high-grade solid carcinomas의 비율이 높았다.
Histologic features
Table 3. Histologic features of the gastritis and associated precursor lesions of 26 autoimmune gastritis-associated gastric carcinomas.
Abbreviations: PGA= Pyloric Gland Adenoma
* Most patients (21/26, 80.8%) had stage II OLGA gastritis.
* No patient had evidence of a previously eradicated H. pylori infection
* Parietal cells were absent in 24/26 (92.3%) cases.
* Intestinal metaplasia in the corpus/fundus was observed in 25/26 (96.2%) cases; complete and incomplete intestinal metaplasia was found in 20 and 5 cases, respectively.
* Majority of cases displayed micronodular ECL-cell hyperplasia (23/26, 88.5%)
* Precursor lesions: PGAs (2 cases), and gastric adenocarcinomas of intestinal or mixed immunophenotype (see below) were associated with flat intestinal-type dysplasia (8 cases)
Immunohistochemical analysis
Gastric immunophenotype: 7 (26.9%)
Intestinal immunophenotype: 10 (38.5%)
Mixed immunophenotype: 9 (34.9%)
p53 expression: aberrant in 7 (26.9%) cases
PD-L1 expression: (CPS≥1) in the majority of AIG-GCs (23/26, 88.5%), but CPS≥10 was observed in 10 GCs (38.5%)
MMR deficiency (MMRd): 7 AIG-GCs (26.9%) (5: MLH1/PMS2 loss, 1: MSH2/MSH6 loss (the patient with Lynch syndrome), 1: isolated loss of PMS2)
HER2 overexpressing (3+): 3 AIG-GCs (11.5%)
CLDN18 positive: 6 (23.1%)
All cases were negative for EBV in situ hybridization.
In case #13 and #14 (HER2 overexpression and PMS2 loss): adenocarcinomatous component of the two MiNENs 에서 만 관찰됨.
반대로.. CLDN18 staining은 MiNENs의 NET components에서는 관찰되었지만, NEC component에서는 관찰되지 않음.
<일반적인 Gastric adenocarcinoma의 subset 과 비교한 결과 Table 4.>
Table 4. Molecular features of all autoimmune gastritis-associated gastric carcinomas (AIG-GCs) of our cohort (n=26) and the AIG-GC of the corpus/fundus-restricted (excluding the Lynch syndrome-associated case, n=14), compared with data from the subset of gastric carcinomas of the previously published cohort by Angerilli et al.
Abbreviations: IF= immunophenotype, MMR= Mismatch Repair, CLDN=Claudin, MSI=Microsatellite Instability, TMB=Tumor Mutational Burden, NS= not significant.
HER2, MMR, EBER, and PD-L1: No significant difference
Molecular analysis
Revealed a total of 119 mutations affecting 72 different genes with a range of 0 to 36 variants per sample (Figure 3)
Figure 3. Summary of clinico-pathologic features, biomarker status and genetic alterations of 26 gastric carcinomas in the context of autoimmune gastritis (AIG-GC), of which 19 underwent extensive molecular profiling. AIG-GC of the corpus/fundus, the Lynch-syndrome associated AIG-GC and AIG-GC of the antrum are represented separately.
Abbreviations: PCC= poorly cohesive carcinoma, AC= adenocarcinoma, PGA= pyloric gland adenoma, C= carcinoma, MiNEN= Mixed Neuroendocrine Non-neuroendocrine neoplasm, IF= immunophenotype, MMRp= Mismatch repair proficient, MMRd= Mismatch repair deficient, MSS= microsatellite stable, MSI-L= microsatellite instability-low, MSI-H= microsatellite instability-high, TMB= tumor mutational burden, CNV = copy number variation.
* TP53, RNF43 mutations: The most frequent in the cohort (8/19, 42.1% and 7/19, 36.8% of cases, respectively)
* ARID1A (6/19, 35.3%), ERBB2 (5/19, 26.3%), PIK3CA (4/19, 21.1%), PTEN (4/19, 21.1%), ANKRD26 (3/19, 15.8%), CUX1 (3/19, 15.8%)
* Other pathogenic/likely pathogenic variants
: APC, BCOR, CARD11, CDH1, CSF3R, Journal Pre-proof EP300, FBXW7, KRAS, NBN, NF1, RASA1, ZFHX3 and ALOX12B: 2/19 cases (10.5%)
: ANKRD11, ARID2, ASXL1, ATR, AXIN1, AXIN2, B2M, BAP1, BARD1, CDKN1B, CIC, CTNNB1, CYLD, EED, GATA2, HNF1A, MEN1, MLH1, MRE11A, MSH2, PARK2, PAX5, PPM1D, SMAD4, PTPN11, SMC1A: 1/19 cases (5.2%)
* Frameshift deletion/insertions: The most common variants (74/119, 62.2%)
* non-synonymous coding variants (44/119, 37.0%), in-frame deletion (1/119, 0.8%)
* Analysis of CNVs: 5/19 (26.3%) samples of the cohort displayed a copy number variation (samples #2, #10, #18, #20 and #26), involving primarily EGFR (2/19, 10.5%) and ERBB2 (2/19, 10.5%). ERCC1, CCND3, CCNE1, CDK6, FGF10, FGFR3, MYC, KRAS, and RICTOR (1/19, 5.2%)
* TMB analysis: 7/19 (36.8%) cases were TMB-high (≥ 10 muts/Mb) and 12/19 (63.2%) were TMB-low (< 10 muts/Mb)
* MSI: 5/19 (26.3%) cases. All MSI samples were MMRd and TMB-high.
* MMRd: 7 cases (2 cases: MSS, 1 case: TMB-low)
--> AIG-GCs of the corpus/fundus (excluding the Lynch syndrome associated case), the most frequently mutated genes were TP53 (4/11, 36.4%), KRAS, PTEN, RNF43, PIK3CA, and ERBB2 (2/11, 18.2%). 2 cases harbored CNVs, both having ≥3 CNVs. MSI was found in 1 case (1/11, 9%), and TMB-high in 3 cases (3/11, 27.3%).
Statistical integration of IHC and molecular analyses
* MSI and MMRd AIG-GCs were enriched in TMB-high (both p=0.002), RNF43 mutations (p=0.038 and p=0.029), ARID1A mutations (both p<0.001), but not PD-L1 CPS ≥ 10.
* TMB high was also associated with PD-L1 CPS ≥ 10 (p=0.0029), aberrant p53 (p=0.005), RNF43 mutations (p=0.0029), ARID1A mutations (p<0.001), and ERBB2 mutations (p=0.038).
* RNF43 and ERBB2 mutations were associated with PD-L1 CPS ≥ 10 (p=0.0129 and p=0.038, respectively).
No statistically significant difference in the IHC and genomic profile was found between the AIG-GCs of the corpus-fundus (excluding the Lynch syndrome-associated case) and the remaining AIG-GCs.
Discussion
To conclude,
AIG-GCs appear distinct clinco-pathologic features, including low T stage, proximal location, low-risk OLGA stages, but end-stage atrophic-metaplastic background. Our cohort revealed the presence of peculiar phenotypes and a high rate of lesions displaying a neuroendocrine component, suggesting that the landscape of neuroendocrine lesions may be wider than previously stated in the AIG context.
At the molecular level, AIG associated GCs were not associated with a specific molecular signature but appeared to be characterized by relatively high rates of PD-L1 expression, MSI/MMRd, and TMB-high, no association with EBV and low rates of CDH1 mutations.
Although in patients with AIG the risk of developing overt GC seems to be almost non-existent in patients who are endoscopically followed up, this risk may be higher in those patients with longstanding, occult AIG.
Histologic markers of severe AIG may help identify patients at risk for GC. The peculiar phenotypes and molecular features of AIG-GC warrant further research.
일반적인 gastric cancer는 H. pylori infection과 연관성이 높기 때문에 여러 phenotypes이나 molecular features 는 H. pylori infection과 연관하여 발생하는 변화들과는 다소 다른 부분이 있을 수 있겠습니다. AIG에서 gastric cancer의 발생이 있긴 한데... 정말 "pure AIG" 에서는 관련이 없을 수도 있겠다.. 관찰되는 몇몇 phenotypes나 molecular features는 사라진 H. pylori infection, 또는 microbiome 이나 diet 등의 영향이었을까?
어렵네요... 아직 갈길이 먼 것 같긴 합니다. ㅠㅠ
Abstract
Patients with autoimmune gastritis (AIG) have a 13-fold risk of developing type-1 neuroendocrine tumors, whereas the risk for gastric adenocarcinoma is still uncertain. Here we describe the clinicopathological and molecular features of a series of gastric carcinomas (GC) arising in the context of AIG.
A total of 26 AIG-associated GC specimens were collected from four Italian Institutions. Immunohistochemistry for MUC1, MUC2, MUC5AC, MUC6, CDX2, HER2, PD-L1, CLDN18, Mismatch Repair (MMR) proteins, and p53 and EBER in situ hybridization were performed. Histologic features and IHC were jointly reviewed by five expert gastrointestinal pathologists.
Next generation sequencing analysis (TrueSight Oncology 500, Illumina) of 523 cancer-related genes was performed on 19 cases. Most tumors were diagnosed as pT1 (52%), were located in the corpus/fundus (58%) and were associated with OLGA stage II gastritis (80.8%), absence of parietal cells, complete intestinal metaplasia and ECL-cell micronodular hyperplasia. Only 4 (15.4%) GCs were diagnosed during follow-up for AIG. The following histotypes were identified: 20 (77%) adenocarcinomas; 3 (11%) mixed neuroendocrine non-neuroendocrine neoplasms, and 2 (8%) high grade solid adenocarcinomas with focal neuroendocrine component, 1 (4%) adenocarcinoma with an amphicrine component. Overall, 7 cases (27%) showed MMR deficiency, 3 (12%) were positive (score 3+) for HER2, 6 (23%) were CLDN18 positive, and 11 (42%) had PD-L1 Combined Positive Score ≥ 10. EBER was negative in all cases. Molecular analysis revealed 5/19 (26%) MSI cases and 7 (37%) TMB-high. The most frequently altered genes were: TP53 (8/19, 42%), RNF43 (7/19, 37%), ERBB2 (7/19, 37% [two amplified and five mutated cases]), ARID1A (6/19, 32%), and PIK3CA (4/19, 21%).
In summary, AIG-associated GCs are often diagnosed at low stage in patients with long standing misrecognized severe AIG; they often display a neuroendocrine component or differentiation, have relatively higher rates of MMR deficiency, and TMB-high.