Abstract
Claudin 18.2 (CLDN18.2) immunohistochemical expression can be used to select patients with gastric/gastroesophageal junction adenocarcinomas for zolbetuximab (IMAB362) therapy, a monoclonal antibody targeting CLDN18.2. The aim of this study was to correlate immunohistochemical expression of CLDN18.2 with clinicopathologic and molecular features in a large series of digestive tract cancers. Immunohistochemistry (IHC) for CLDN18.2 was performed on tissue microarrays from 1404 adenocarcinomas including 155 gastric/gastroesophageal, 74 pancreatic ductal, 1175 colorectal (576 in initial test cohort; 599 in subsequent validation cohort), and correlated with HER2 and mismatch repair (MMR) status. Cases were scored as CLDN18.2 positive or negative, with positivity defined as moderate to strong membranous staining in ≥75% of tumor cells. CLDN18.2 expression was correlated with clinicopathologic and molecular features for all colorectal adenocarcinomas. CLDN18.2 was positive in 39% (61/155) of gastric/gastroesophageal adenocarcinomas, 38% (28/74) of pancreatic ductal adenocarcinomas, and 3.4% (40/1175) of colorectal adenocarcinomas (p<0.001). For gastric/gastroesophageal and pancreatic ductal adenocarcinoma, there was no correlation between CLDN18.2 expression and either HER2 or MMR status. In contrast, CLDN18.2-positive colorectal adenocarcinomas had distinct clinicopathologic and molecular features. CLDN18.2-positive colorectal adenocarcinomas were more frequently proximally located and were more often MMR deficient and BRAF V600E positive (all with p<0.05). Quantitative pathologic analysis using the digital pathology biomarker QuantCRC demonstrated marked differences in histologic features between CLDN18.2-positive and negative colorectal adenocarcinomas, with CLDN18.2-positive tumors having increased tumor:stroma ratio and %mucin but decreased %immature stroma in both the test and validation cohorts (all with p<0.05). In conclusion, CLDN18.2-positive colorectal adenocarcinomas are frequently MMR deficient, BRAF V600E mutated, and demonstrate distinct histologic features. Future studies addressing the efficacy of zolbetuximab therapy in this subset of colorectal cancers are needed.
Keywords: Claudin 18.2, colorectal carcinoma, mismatch repair, microsatellite instability, HER2, mucinous adenocarcinoma, tumor infiltrating lymphocytes
INTRODUCTION
Colorectal carcinoma (CRC) is the third most common cancer and third leading cause of cancer-related deaths in the United States (1). While enhanced screening programs have contributed to an overall decline in incidence and improvement in prognosis of CRC, these broad trends mask evolving challenges faced by subsets of CRC patients (2,3). For example, the incidence of early onset CRC diagnosed in patients under 50 years of age has been significantly increasing since 2011 (4), associated with presentation at more advanced clinical stages, diminished responsiveness to standardized therapeutic approaches and a higher proportion of patients with genetic susceptibility to CRC development (5). Given the heterogeneity of CRC biology contributing to variable prognoses and therapeutic response rates, there is an ever-present need for new and emerging biomarkers with associated targeted therapies to help optimize clinical outcomes.
The claudin family of transmembrane proteins serve as important components of cellular tight junctions throughout the body, with different claudins expressed in different tissues (6,34). Claudin 18 (CLDN18) has two isoforms with CLDN18.1 specific to the lung and with CLDN18.2 normal expression restricted to within the lateral and basal aspects of gastric mucosal epithelial cells, helping maintain cell polarity, conduct cell signaling pathways, and contributing to their barrier function (34,35). Persistent, though downregulated, expression of CLDN18.2 can be seen on the surface of gastric and gastroesophageal junction adenocarcinoma cells. Recently, CLDN18.2 has emerged as a potential therapeutic target in solid tumors, particularly those of the upper gastrointestinal (GI) tract. CLDN18.2 expression is retained in a significant proportion of gastric and gastroesophageal adenocarcinomas, with varying degrees of aberrant expression also documented in carcinomas of the pancreas, lung, ovary, colorectum, liver, breast, and head and neck (6–10). In carcinogenesis, the CLDN18.2 protein contributes to tumor cell proliferation, differentiation, and migration (6,36). The highly restricted expression pattern of CLDN18.2 in normal tissues combined with its retained or aberrant expression in many carcinomas and its two available extracellular domains make CLDN18.2 an ideal tumor-associated antigen for targeted therapy (9,11). To date, multiple types of agents have been created to target CLDN18.2, including monoclonal antibodies, chimeric antigen receptor T (CAR-T) cells, and antibody-drug conjugates (6,12,13). At the time of publication, clinical trials interrogating the efficacy of multiple monoclonal antibodies and CAR-T cell therapies targeting CLDN18.2 are underway (NCT05472857, NCT06353152, NCT06005493, NCT05981235). The first anti-CLDN18.2 monoclonal antibody created was zolbetuximab, leading to multiple phase 2 and phase 3 clinical trials evaluating the efficacy of zolbetuximab paired with various chemotherapeutic regimens in the treatment of advanced gastric and gastroesophageal adenocarcinomas. The phase 2 FAST trial first revealed a statistically significant difference in progression free survival and overall survival compared to placebo groups, specifically for those patients with a higher expression of CLDN18.2, defined as >70% (14). These statistically significant improvements in progression free survival and overall survival were confirmed in subsequent phase 3 SPOTLIGHT and GLOW trials where a higher threshold for CLDN18.2 positivity (≥75%) was required for patient entry (15,16). Additional clinical trials remain in the enrollment phase, recruiting participants to evaluate the efficacy of two additional anti-CLDN18.2 monoclonal antibodies combined with other chemotherapy and immunotherapy regimens in the setting of metastatic or advanced solid tumors including gastric, esophageal, gastroesophageal junction, and pancreatic adenocarcinomas (NCT06005493 and NCT05981235).
As zolbetuximab is FDA approved for gastric and gastroesophageal junction adenocarcinoma, with additional CLDN18.2 targeted therapy approval likely in metastatic or advanced solid tumors, it is important to note that data correlating CLDN18.2 expression with any specific histopathologic, immunohistochemical, or molecular features for many tumors of the digestive tract, particularly colorectal adenocarcinomas, are limited. Review of the English-language literature to date reveals only five published studies in which CLDN18.2 immunohistochemistry (IHC) was performed on colorectal adenocarcinomas (7,9,17–19). However, each of these studies used different CLDN18.2 clones and positivity thresholds limiting comparability. Importantly, none of these studies utilized the phase 3 SPOTLIGHT and GLOW clinical trial scoring criteria for CLDN18.2 positivity, requiring ≥75% of tumor cells with moderate-to-strong membranous staining with the on-market Ventana clone (15,16).
The aim of this study was to correlate immunohistochemical expression of CLDN18.2 with clinicopathologic and molecular features in a large series of digestive tract adenocarcinomas, including a large cohort of colorectal adenocarcinomas.
METHODS
Study Group
The test cohort consisted of tissue microarrays (TMAs) of 805 adenocarcinomas of the digestive system that included 576 colorectal adenocarcinomas, 155 gastroesophageal (stomach, esophagogastric junction, and esophagus) adenocarcinomas, and 74 pancreatic ductal adenocarcinomas, as previously described (20). The colorectal adenocarcinomas in the test cohort were enriched in mismatch repair (MMR) deficient tumors and included a consecutive series of MMR proficient tumors between 2011 through 2012 and a consecutive series of MMR deficient tumors resected between 2010 through 2015. The validation cohort consisted of TMAs of a consecutive series of 599 colorectal adenocarcinomas irrespective of MMR status from the Ontario Familial Colon Cancer Registry (OFCCR) (21). Review of the clinical records and pathology slides confirmed the primary site of tumor origin for each case included in the TMAs. The TMAs consisted of 1.0 mm tissue cores with representative areas from each adenocarcinoma selected from paraffin blocks based on hematoxylin and eosin (H&E)-stained sections. Each tumor was represented by 2 or 3 cores on the TMA.
The study was approved by the University of Pittsburgh Institutional Review Board/Ethical Board (IRB#20040335) and the Mayo Clinic Arizona Institutional Review Board (IRB18-011309).
Immunohistochemistry and Molecular Analysis
CLDN18.2 immunohistochemistry (IHC) was performed on all TMAs using the Ventana CLDN18 (43-14A clone) RxDx assay (Roche Diagnostic Solutions, Tucson, AZ). The 43-14A antibody clone was used to assess CLDN18.2 expression for selecting patients for targeted therapy with zolbetuximab in both the SPOTLIGHT and GLOW clinical trials (15,16). CLDN18.2 positive expression was defined using the SPOTLIGHT and GLOW clinical trials scoring criteria requiring ≥75% of tumor cells with moderate-to-strong membranous staining using the on-market Ventana clone 43-14A. The overall percentage of positive tumor cells and intensity of staining was evaluated by visual estimation by two pathologists (KM and ReKP). In the test cohort, MMR IHC including MLH1, PMS2, MSH2, and MSH6 was performed on all adenocarcinomas, as previously described (22). HER2 testing with both IHC using the Dako Hercep Test (Carpinteria, CA) and HER2 FISH using the PathVysion HER2 (ERBB2) DNA probe (Abbott Molecular, Des Plaines, IL) was performed on all gastroesophageal adenocarcinomas, as previously described (23). HER2 IHC for all other adenocarcinomas was performed using the anti-HER2 4B5 rabbit monoclonal primary antibody (Ventana, Tucson, AZ). HER2 scoring for all gastroesophageal and pancreatic adenocarcinomas was performed using the College of American Pathologists/American Society of Clinical Pathology/American Society of Clinical Oncology scoring criteria for gastric cancer (24). HER2 IHC scoring for colorectal adenocarcinomas was performed using the HERACLES criteria outlined by Sartore-Bianchi et al. (25). SATB2 (clone EP281, Cell Marque, Rocklin, CA) and CDX2 (clone CDX2-88, Biogenex, Fremont, CA) immunohistochemical stains were performed on all available colorectal adenocarcinomas from the test cohort, as previously described (22).
BRAF V600E and KRAS exon 2 and 3 mutation analysis was performed on a subset of consecutively resected colorectal carcinomas in the test cohort regardless of stage, as previously described (26). BRAF V600E mutation analysis was also performed on all colorectal carcinomas that demonstrated loss of MLH1 and PMS2 expression by IHC.
Histopathologic Evaluation of Colorectal Carcinoma
Histologic examination of all pathology slides from 465 surgically resected colorectal carcinomas from the test cohort were reviewed by one pathologist (ReKP). The following histologic features were recorded for each case: histologic grade, angiolymphatic invasion, perineural invasion, mucinous histology, signet ring cell histology, medullary histology, tumor budding, Crohn’s-like reaction by visual assessment of H&E stains, and tumor infiltrating lymphocytes by visual assessment of H&E stains. Tumor grade was assessed for all tumors, including those with mucinous differentiation, using a modification of College of American Pathologists (CAP) grading scheme into low-grade (well/moderately, >50% gland formation) and high-grade (<50% gland formation), regardless of MMR status (27). Tumor budding was assessed using the International Tumor Budding Consensus Conference (ITBCC) (28,29). Tumor buds were defined as isolated cancer cells or a cluster of <5 neoplastic cells, and high tumor budding was defined as 10 or more buds per 0.785 mm2. The presence of tumor infiltrating lymphocytes within tumor epithelium was visually assessed on H&E stained sections using the criteria outlined by Williams et al. (30). Crohn’s-like peritumoral lymphocytic reaction was scored using criteria outlined by Ueno et al. and defined as large lymphoid aggregates present at the tumor periphery with at least 1 lymphoid aggregate measuring >1 mm in diameter (31).
Quantitative digital pathology segmentation using QuantCRC
One whole section H&E slide of each colorectal adenocarcinoma was digitized using Leica Aperio GT450 at 40X magnification at their respective institutions. Digitized H&E images were uploaded to the Aiforia Create deep learning cloud-based platform (Aiforia Technologies, Helsinski, Finland). Each image was manually annotated by one author (RiKP) to outline the areas of invasive adenocarcinoma. Non-neoplastic mucosa and precursor adenoma were specifically excluded from the area of analysis. QuantCRC was applied to the annotated tumor area, as previously described (32,33). In brief, as part of a prior study, the QuantCRC was trained on 24,157 annotations made on 559 colorectal carcinomas not used in this study. The segmentation algorithm employs convolutional neural networks to segment the image in a stepwise manner. First, the tumor area is segmented into carcinoma, tumor budding/poorly differentiated clusters (TB/PDC), stroma, mucin, necrosis, fat, and smooth muscle. The second layer segments stroma into immature (loose, often myxoid stroma with haphazardly arranged plump fibroblasts and collagen fibers), mature (densely collagenous areas with scattered fibroblasts, often with parallel collagen fibers), and inflammatory (dense clusters of chronic inflammatory cells obscuring stromal cells) subtypes. The third layer segments carcinoma into low-grade, high-grade, and signet ring cell carcinoma (SRCC). The fourth layer identifies tumor infiltrating lymphocytes (TILs) within tumor epithelium. Fat and smooth muscle were subtracted from the tissue area to generate the tumor area. Fifteen QuantCRC parameters were recorded for each tumor including: %tumor, %stroma, tumor:stroma ratio, %TB/PDC within the tumor, %mucin within the tumor, %necrosis within the tumor bed, %high-grade, %SRCC, TILs per mm2 of tumor, %immature stroma (tumor area), %inflammatory stroma (tumor area), %mature stroma (tumor area), %immature stroma (stromal region), %inflammatory stroma (stromal region), and %mature stroma (stromal region). The QuantCRC analysis was performed blinded to the immunohistochemical and histopathologic variables.
Statistical Analysis
Comparisons of patient clinical/pathologic characteristics were done using Wilcoxon rank-sum test for continuous variables and Pearson Chi-square tests for categorical variables. All statistics were assessed using two-sided tests with P-values <0.05 considered statistically significant. Statistical analyses were performed using SPSS (for Windows 27, IBM, Armonk, NY).
RESULTS
CLDN18.2 expression in adenocarcinomas of the digestive system and correlation with HER2 and MMR status within the test cohort
There was a statistically significant difference in the rates of CLDN18.2 expression between colorectal adenocarcinoma and gastroesophageal and pancreatic ductal adenocarcinomas. CLDN18.2 was positive in 39% (61/155) of gastroesophageal adenocarcinomas (Figure 1), 38% (28/74) of pancreatic ductal adenocarcinomas, and 5% (26/576) of colorectal adenocarcinomas (p<0.001).
Figure 1.
Claudin 18.2 (CLDN18.2) positive pancreatic ductal and gastric adenocarcinomas.
A and B. Pancreatic ductal adenocarcinoma (A, H&E 4x) with associated CLDN18.2 immunohistochemistry (IHC) showing strong membranous staining in 95% of tumor cells (B, CLDN18.2 4x).
C and D. Gastric adenocarcinoma, gland forming type (C, H&E 4x) with associated CLDN18.2 IHC showing strong membranous staining in 100% of tumor cells (D, CLDN18.2 4x).
E and F. Gastric adenocarcinoma, diffuse type (E, H&E 4x) with associated CLDN18.2 IHC showing strong membranous staining in 100% of tumor cells (F, CLDN18.2 4x).
HER2 was positive in 11% (10/89) of CLDN18.2-negative and 8% (5/59) of CLDN18.2-positive gastroesophageal adenocarcinomas (Table 1). Only, 1% (6/550) of CLDN18.2-negative colorectal adenocarcinoma were HER2 positive, and no CLDN18.2-positive colorectal adenocarcinomas were HER2 positive. There was no statistically significant difference in HER2 expression between CLDN18.2-positive and CLDN18.2-negative gastroesophageal adenocarcinomas or colorectal adenocarcinomas. None of the pancreatic adenocarcinomas were HER2 positive.
Table 1.
Correlation of Claudin 18.2 (clone 43.14A) and HER2 and MMR status within the test cohort
| Tumor Type | Claudin 18.2 Result | HER2 Result | P-value | MMR Result | P-value | ||
|---|---|---|---|---|---|---|---|
| Negative | Positive | Proficient | Deficient | ||||
| Gastroesophageal adenocarcinoma | Negative (%) | 79 (89) | 10 (11) | 0.6 | 89 (96) | 4 (4) | 0.9 |
| Positive (%) | 54 (92) | 5 (8) | 58 (95) | 3 (5) | |||
| Pancreatic ductal adenocarcinoma | Negative (%) | 46 (100) | 0 | 1.0 | 46 (100) | 0 | 1.0 |
| Positive (%) | 28 (100) | 0 | 28 (100) | 0 | |||
| Colorectal adenocarcinoma | Negative (%) | 544 (99) | 6 (1) | 0.6 | 415 (75) | 135 (25) | <0.001 |
| Positive (%) | 26 (100) | 0 | 7 (27) | 19 (73) | |||
*HER2 negative includes HER2 0-1+ by immunohistochemistry or 2+ with negative HER2/CEP17 FISH. HER2 positive includes 3+ by immunohistochemistry or 2+ with positive HER2/CEP17 FISH.
MMR, mismatch repair
There was a statistically significant difference in MMR status between CLDN18.2-negative and CLDN18.2-positive colorectal adenocarcinomas (Table 1). MMR deficiency was identified in 73% (19/26) of CLDN18.2-positive colorectal adenocarcinomas and 25% (135/415) of CLDN18.2-negative colorectal adenocarcinomas (p<0.001). There was no statistically significant difference in MMR status between CLDN18.2-negative and CLDN18.2-positive gastroesophageal adenocarcinomas (p=0.9). None of the pancreatic adenocarcinomas were MMR deficient.
Clinicopathologic features of CLDN18.2 positive colorectal carcinoma within the test cohort
The demographic features of our colorectal carcinoma test cohort were relatively similar between the CLDN18.2-positive and CLDN18.2-negative tumors in the test cohort with no difference in patient age or gender (Table 2). There was a statistically significant difference in tumor site between the CLDN18.2-positive and CLDN18.2-negative cohorts. CLDN18.2-positive tumors were more frequently located in the cecum, ascending colon, hepatic flexure, and transverse colon compared to CLDN18.2-negative tumors (90% vs. 51%, p<0.001). There was no significant difference between the CLDN18.2-positive and CLDN18.2-negative cohorts regarding clinical stage at presentation (p=0.16) and clinical outcome characterized as dead of disease (p=0.16).
Table 2.
Clinicopathologic features of colorectal carcinomas stratified by Claudin 18.2 (clone 43.14A) expression within the test cohort.
| Clinicopathologic Features | Claudin 18.2 Negative N (%) |
Claudin 18.2 Positive N (%) |
P-value |
|---|---|---|---|
| No. of Patients (%) | 550 (95) | 26 (5) | NA |
| Gender, Male/Female | 258 (47) / 292 (53) | 9 (35) / 17 (65) | 0.22 |
| Median Age in Years (IQR) | 68 (20) | 73 (19) | 0.15 |
|
Tumor Site
Cecum Right/Transverse Colon Left/Sigmoid Colon or Rectum |
99 (18) 181 (33) 267 (49) |
8 (31) 15 (58) 3 (12) |
<0.001 |
|
Tumor Site
Cecum Ascending Hepatic Flexure Transverse Splenic Flexure Descending Sigmoid Rectosigmoid Rectum |
99 (18) 134 (24) 10 (2) 37 (7) 16 (3) 35 (6) 104 (19) 32 (6) 80 (15) |
8 (31) 9 (35) 3 (12) 3 (12) 0 3 (12) 0 0 0 |
<0.001 |
|
Stage
I II III IV |
91 (17) 186 (34) 181 (33) 92 (17) |
3 (12) 14 (54) 7 (27) 2 (8) |
0.19 |
|
Disease status at last follow-up
Number with follow-up Dead of disease |
468 118 (25) |
21 3 (14) |
0.16 |
|
MMR Pattern Intact (MMR proficient) MLH1/PMS2 loss MSH2/MSH6 loss MSH6 loss PMS2 loss |
415 (75) 114 (21) 9 (2) 6 (1) 6 (1) |
7 (27) 14 (54) 2 (8) 0 3 (12) |
<0.001 |
| BRAF V600E mutation | 76 / 433 (18) | 11 / 22 (50) | <0.001 |
| KRAS mutation | 137 / 390 (35) | 2 / 14 (14) | 0.11 |
| CDX2 Positive | 494 / 542 (91) | 16 / 26 (62) | <0.001 |
| SATB2 Positive | 471 / 539 (87) | 20 / 25 (80) | 0.3 |
|
No. of cases with histopathologic
re-review |
441 / 550 (80) | 24 / 26 (92) | NA |
| High tumor grade | 52 (12) | 9 (38) | 0.004 |
| Mucinous differentiation | 136 (31) | 18 (75) | <0.001 |
| Signet ring cell differentiation | 13 (3) | 3 (12.5) | <0.001 |
| Medullary differentiation | 24 (5) | 5 (21) | 0.016 |
| High TILs by H&E | 195 (44) | 16 (67) | <0.001 |
| Crohn’s like reaction by H&E | 133 (30) | 11 (46) | 0.11 |
| Angiolymphatic invasion | 244 (55) | 11 (46) | 0.39 |
| Perineural invasion | 111 (25) | 3 (12.5) | 0.36 |
| High tumor budding | 123 (28) | 3 (12.5) | 0.09 |
IQR, interquartile range; MMR, mismatch repair; TILs, tumor infiltrating lymphocytes; H&E, hematoxylin and eosin
There was a statistically significant difference in MMR patterns between the CLDN18.2-positive and CLDN18.2-negative colorectal adenocarcinomas (Table 2). MMR deficiency was identified in 73% (19/26) of CLDN18.2-positive tumors and 25% (135/550) of CLDN18.2-negative tumors, respectively (p<0.001). Fourteen of 19 (74%) CLDN18.2-positive tumors showed MLH1 and PMS2 loss by MMR IHC. The remaining five MMR-deficient, CLDN18.2-positive tumors showed either MSH2/MSH6 loss (2/19, 10%) or isolated PMS2 loss (3/19, 16%). BRAF V600E mutations were identified in 50% (11/22) of tested CLDN18.2-positive colorectal adenocarcinomas and 18% (76/433) of tested CLDN18.2-negative colorectal adenocarcinomas (p<0.001). There was no statistically significant difference in rates of KRAS mutation stratified by CLDN18.2 expression.
CDX2 expression was statistically significantly different between the CLDN18.2-positive and CLDN18.2-negative colorectal adenocarcinomas (Table 2). CDX2 positivity was present in 91% (494/542) of CLDN18.2-negative tumors, whereas it was only present in 62% (16/26) of CLDN18.2-positive tumors (p<0.001). There was no statistically significant difference in SATB2 expression stratified by CLDN18.2 expression in colorectal adenocarcinoma. CLDN18.2-positive tumors were statistically significantly more likely to show high tumor grade (38%; p=0.004), mucinous differentiation (75%; p<0.001), signet ring cell differentiation (12.5%; p<0.001), medullary differentiation (21%; p=0.016) and high tumor infiltrating lymphocytes (67%; p<0.001) by visual H&E assessment (Figure 2).
Figure 2.
Claudin 18.2 (CLDN18.2) positive colorectal adenocarcinomas.
A-C. CLDN18.2 with strong membranous staining in 100% of tumor cells (A, CLDN18.2 4x) in a mucinous colorectal adenocarcinoma (B, H&E 20x) from a patient with confirmed Lynch syndrome demonstrated isolated PMS2 loss by immunohistochemistry (C, PMS2 10x).
D-F. CLDN18.2 with strong membranous staining in 100% of tumor cells (D, CLDN18.2 4x) in a poorly differentiated colorectal adenocarcinoma with mucinous and signet ring cell features (E, H&E 20x) from a patient with sporadic mismatch repair deficiency with PMS2 and MLH1 loss by immunohistochemistry (F, MLH1 10X).
G-I. CLDN18.2 with strong membranous staining in approximately 80% of tumor cells (G, CLDN18.2 4x) in a colorectal adenocarcinoma with medullary features (H, H&E 20x) from a patient with sporadic mismatch repair deficiency with PMS2 and MLH1 loss by immunohistochemistry (I, MLH1 10x).
Quantitative digital pathology (QuantCRC) features of CLDN18.2 positive colorectal carcinoma within the test cohort
Similar to visual H&E assessment, quantitative digital pathology analysis using QuantCRC also showed marked differences in histologic features between CLDN18.2-positive and CLDN18.2-negative tumors (Table 3). CLDN18.2-positive tumors were statistically significantly more likely to have increased %tumor (p=0.003), tumor:stroma ratio (p=0.004), %mucin (p=0.01), %SRCC (p=0.003), TILs per mm2 (p=0.01), and %inflammatory stroma in stromal area (p=0.002) in comparison to CLDN18.2-negative tumors (Figure 3). CLDN18.2-positive tumors were statistically significantly more likely to have decreased %stroma (p=0.008), %TB/PDC (p=0.03), %immature stroma in tumor bed (p<0.001), and %immature stroma in stromal area (p=0.004) in comparison to CLDN18.2-negative tumors.
Table 3.
QuantCRC features stratified by Claudin 18.2 (clone 43.14A) expression within the test cohort
| QuantCRC features | Claudin 18.2 Negative Median (IQR) N=308 |
Claudin 18.2 Positive Median (IQR) N=21 |
P-value |
|---|---|---|---|
| %Tumor | 48.8 (20.3) | 61.9 (19.3) | 0.003 |
| %Stroma | 40.5 (21.2) | 31.8 (18.2) | 0.008 |
| Tumor:Stroma Ratio | 1.2 (1.2) | 1.8 (1.6) | 0.004 |
| %Mucin | 0.9 (13.9) | 18.8 (50.1) | 0.01 |
| %Necrosis | 4.5 (7.0) | 3.8 (5.5) | 0.2 |
| %High grade | 13.2 (24.2) | 15.6 (44.3) | 0.8 |
| %SRCC | 0.1 (0.4) | 0.5 (1.4) | 0.003 |
| %TB/PDC | 0.9 (2.4) | 0.6 (0.9) | 0.03 |
| TILs per mm2 | 28.4 (45.1) | 54.3 (180.2) | 0.01 |
| %immature stroma (tumor bed) | 23.8 (14.5) | 14.5 (11.1) | <0.001 |
| %inflammatory stroma (tumor bed) | 3.2 (5.5) | 5.6 (11.7) | 0.06 |
| %mature stroma (tumor bed) | 8.5 (9.8) | 6.7 (4.5) | 0.09 |
| %immature stroma (stromal area) | 62.5 (21.6) | 56.4 (15.9) | 0.004 |
| %inflammatory stroma (stromal area) | 8.4 (13.0) | 16.0 (27.3) | 0.002 |
| %mature stroma (stromal area) | 22.1 (17.4) | 22.5 (16.4) | 0.9 |
IQR, interquartile range; SRCC, signet ring cell carcinoma, TB/PDC, tumor budding/poorly differentiated clusters; TILs, tumor infiltrating lymphocytes
Figure 3.
QuantCRC performed on Claudin 18.2 (CLDN18.2)-positive colorectal adenocarcinomas.
Digitized H&E whole slides and segmentation was performed in four steps (column headers). First, the whole section tumor image was segmented into carcinoma (green), stroma (light blue), mucin (dark blue), TB/PDC (red), necrosis (brown), smooth muscle (purple), and fat (yellow). Second, the stroma was further segmented into immature (teal), mature (green), and inflammatory (gray). The carcinoma was further segmented by differentiation into low-grade (purple), high-grade (orange), and signet ring cell (light green). Lastly, tumor infiltrating lymphocytes were detected within tumor epithelium (TILs) (blue dots). Whole sections from the same three colorectal adenocarcinoma depicted in Figure 2 are shown. Row 1: colorectal adenocarcinoma with 59.7% mucin content and with increased lymphocytes per mm2 of tumor epithelium (TILs) (213.7 TILs per mm2) on QuantCRC. Row 2: colorectal adenocarcinoma with 56.9% mucin content and 43.7% signet ring cell differentiation. Row 3: colorectal adenocarcinoma with medullary features with 59.9% high-grade component and increased %inflammatory stroma within the stromal region (16.2%).
Abbreviations: TB/PDC, tumor budding/poorly differentiated clusters; B, entire tumor bed; ST, stromal region; TILs, tumor infiltrating lymphocytes.
Colorectal carcinoma validation (OFCCR) cohort confirmed the association of CLDN18.2 expression with histologic and molecular features
Similar to the test cohort, there was a statistically significant difference in tumor site between the CLDN18.2-positive and CLDN18.2-negative tumors in the validation cohort (Supplementary Table 1). Overall, 71.4% of CLDN18.2-positive tumors were located in the cecum, ascending, and transverse colon and only 34.7% of CLDN18.2-negative tumors were found within these three sites (p=0.013). Within the validation cohort, there was a statistically significant difference in MMR status between the CLDN18.2-positive and CLDN18.2-negative colorectal adenocarcinomas (Supplementary Table 2). MMR deficiency was identified in 90% (9/10) of CLDN18.2-positive tumors and 18% (89/501) of CLDN18.2-negative tumors, respectively (p<0.001). BRAF V600E mutations were identified in 44% (4/9) of tested CLDN18.2-positive colorectal adenocarcinomas and 15% (66/443) of tested CLDN18.2-negative colorectal adenocarcinomas (p=0.03). Quantitative pathologic analysis using QuantCRC also showed differences in histologic features between CLDN18.2-positive and CLDN18.2-negative tumors within the validation cohort (Supplementary Table 3). CLDN18.2-positive tumors were statistically significantly more likely to have increased tumor:stroma ratio (p=0.02) and %mucin (p=0.02) in comparison to the CLDN18.2-negative tumors. CLDN18.2-positive tumors were also statistically significantly more likely to have decreased %stroma (p=0.02) and %immature stroma in tumor bed (p=0.003) in comparison to the CLDN18.2-negative tumors.
DISCUSSION
Our study is the largest series of CLDN18.2-immunostained colorectal adenocarcinomas in the English-language literature to date and the only study that employs CLDN18.2 interpretation methodologies established in the phase 3 SPOTLIGHT and GLOW clinical trials. Whereas earlier studies analyzed CLDN18.2 expression within narrow subsets of colorectal adenocarcinoma patients, such as those with signet ring cell carcinomas (19) or colitis-associated carcinomas (17), our study is the first to evaluate CLDN18.2 expression in relation to extensive histologic, immunohistochemical, and molecular analysis of comprehensive cohorts of colorectal adenocarcinomas, including consecutive cases from a tissue registry. Through this analysis, we are the first to show that CLDN18.2-positive colorectal adenocarcinomas are frequently MMR deficient, BRAF V600E mutated, proximally located tumors with histologic features typical of MMR deficient adenocarcinomas, specifically high tumor grade, mucinous differentiation, signet ring cell differentiation, medullary differentiation, high tumor infiltrating lymphocytes, increased inflammatory stroma, and CDX2 loss. While some of these features mirror those identified in earlier studies of CLDN18.2-positive colorectal adenocarcinomas (17,19), it is important to note that previous publications lacked MMR status evaluation of their patient cohorts.
Little is known about CLDN18.2 expression in colorectal carcinoma, and review of the English-language literature to date reveals a total of 5 studies published between 2008 and 2021 in which CLDN18 IHC was performed on colorectal adenocarcinomas (7,9,17–19). Importantly, Iwaya et al. demonstrated that CLDN18 immunohistochemical expression in colorectal carcinoma is only associated with increased CLDN18.2 expression by reverse transcriptase PCR with no cases showing CLDN18.1 expression by reverse transcriptase PCR (17). This indicates that CLDN18 immunohistochemical expression correlates specifically with overexpression of the CLDN18.2 isoform in colorectal carcinoma. Prior to our current study, a total of 928 cases of colorectal adenocarcinoma were assessed for CLDN18.2 expression, with 53 cases (5.7%) reported as positive (range, 0.9% to 38%). Six hundred and twenty-six (67%) of these 928 cases were TMA material (7,9,18). It is challenging to draw significant conclusions from these studies, as there is marked variability in study methodology, most notably in CLDN18 IHC clones and in CLDN18 positivity thresholds. CLDN18 clones utilized included two rabbit polyclonal CLDN18.2 antibodies (clone 38-8100 from Invitrogen and an undisclosed clone from ThermoFisher) (7,9), one rabbit monoclonal CLDN18 antibody (clone EPR19203 from Abcam) (17), and a polyclonal CLDN18 antibody (undisclosed clone) from Invitrogen/Zymed (18,19). Importantly, none of these studies utilized the positivity threshold (≥75% moderate-to-strong membranous staining) or Ventana claudin 18 clone (43-14A clone) used to define CLDN18.2 positivity in the recently published phase 3 clinical trials (15,16). CLDN18.2 positivity thresholds included any staining (9,19), any staining in ≥10% of tumor cells (18), at least weak (1+) staining in >5% of tumor cells (7) and at least moderate (2+) staining in ≥10% of tumor cells (17). Complete CLDN18.2 clone details were disclosed in only two of five studies (9,17). Disregarding variabilities in the definition of CLDN18.2 positivity, 3 of these 5 studies did evaluate CLDN18.2 expression in relation to limited clinicopathologic parameters (17–19). Interestingly, two authors found statistically significant relationships between CLDN18.2 positivity and MUC5AC expression in colorectal adenocarcinomas (17,18), with significantly decreased expression of CDX2 (18) and SATB2 (17) in CLND18.2-positive tumors. Other clinicopathologic features associated with CLDN18.2 positivity in these studies included colitis-associated carcinomas (17), signet ring cell differentiation (17,19), mucinous differentiation (17), and poorer prognosis (18).
The increased frequency of CLDN18.2 positivity in MMR deficient colorectal adenocarcinomas is of great interest, as it brings to the forefront the question of zolbetuximab efficacy when combined with immune checkpoint inhibitors, a first-line treatment choice in MMR deficient colorectal adenocarcinomas requiring neoadjuvant or adjuvant therapy. To date, only one trial (ILUSTRO) has evaluated the efficacy of zolbetuximab combined with pembrolizumab in the treatment of advanced gastric and gastroesophageal adenocarcinomas; however, this study restricted evaluation of this therapeutic combination to a patient cohort receiving third-line or later therapy whereas the comparison cohort, patients receiving combined zolbetuximab and mFOLFOX6, were on their first line of treatment (11,38). In this study, zolbetuximab with pembrolizumab showed limited efficacy in objective response rate and progression free survival for patients, whereas the combination of zolbetuximab with mFOLFOX6 showed significant improvement in both measures. Interestingly, the TWINPEAK trial (NCT05482893) is currently enrolling advanced gastric, gastroesophageal, and pancreatic ductal adenocarcinomas, with a subset of their planned treatment arms including a combination of an anti-CLDN18.2 antibody paired with pembrolizumab alone or paired with pembrolizumab and standard of care chemotherapy. The results of this trial may be of interest as we contemplate the potential efficacy of zolbetuximab in patients with MMR deficient colorectal adenocarcinoma, who are often treated with immune checkpoint inhibitors.
Beyond efficaciousness as a predictive biomarker, investigations of CLDN18.2 expression as a prognostic biomarker have been conducted in cohorts of gastric and gastroesophageal adenocarcinoma patients, with more limited interrogation of pancreatic ductal adenocarcinoma. Initial evaluations of the clinical implications of CLDN18.2 expression in gastric and gastroesophageal adenocarcinomas found CLDN18.2 positivity was associated with shorter overall survival (39,40) and significantly more frequent in diffuse type adenocarcinomas (35,41,42), mucinous tumors (43), high grade tumors (41), HER2-positive tumors (35) and EBV-associated tumors (42,43). However, the majority of these associations have been refuted in subsequent meta-analysis (44) and in more recent literature mirroring CLDN18.2 interpretation methodologies set forth in the phase 3 SPOTLIGHT and GLOW clinical trials (15,16). When utilizing the Ventana claudin 18 clone (43-14A clone) and a positivity threshold of moderate-to-strong membranous staining in ≥75% of tumor cells, multiple authors have found no relationship between CLDN18.2 positivity and diffuse subtype, high tumor grade, or prevalence of other established biomarkers including HER2, MMR, and PD-L1 (43,45). Furthermore, numerous authors utilizing myriad positivity thresholds have failed to demonstrate any relationship between CLDN18.2 positivity and overall survival (11,35,43,45). Notably, one study utilizing the Ventana clone and phase 3 clinical trial positivity thresholds did find statistically significant relationships between CLDN18.2-positivity and EBV-associated tumors, nodal involvement, high stage disease, age <70 years, peritoneal involvement, and lower incidence of liver metastasis in gastric and gastroesophageal adenocarcinomas (45).
Similar limitations in positivity thresholds and clone utilization plague the limited number of publications evaluating CLDN18.2 expression in pancreatic ductal adenocarcinomas (46–50). Regardless, multiple authors have shown that CLDN18.2 positivity in pancreatic ductal adenocarcinomas is significantly associated with well-differentiated tumors (47,50) and improved overall survival (47,49,51). Lyu et al. confirmed CLDN18.2 positivity serves as an independent predictor of a more favorable prognosis in pancreatic ductal adenocarcinoma in the single study to date that utilized the interpretation methodologies set forth in the phase 3 SPOTLIGHT and GLOW clinical trials (51). Though only evaluated in one study to date, no relationship between CLDN18.2 expression and HER2 status has been observed in pancreatic ductal adenocarcinomas (49).
Of course, this study is not without its limitations. The main limitation of this study is the use of TMA material in evaluation of CLDN18.2 expression. While TMA material allowed for judicious tissue use in evaluating a powerfully large number of digestive tract adenocarcinomas with CLDN18.2 immunohistochemistry, the small tumor cores may not be representative of the entire tumors. This is of particular significance when investigating both tumors and immunohistochemical stains known to be heterogeneous, as is the case for MMR deficient CRC (52) and CLDN18.2 IHC (42,45,46,48). To combat this limitation, duplicates or triplicates of tumor cores were included within the TMA preparations to better account for tumor heterogeneity. However, we cannot entirely exclude the possibility that TMA use introduced error in immunohistochemical interpretation and/or histopathologic evaluation stemming from the innate heterogeneity of the tumor types and immunohistochemical stains being evaluated. Future studies evaluating CLDN18.2 expression in whole tissue sections of colorectal adenocarcinoma may be of utility in lending additional evidence in support of these findings initially established in TMA material. Given the relatively high prevalence of CLDN18.2 positivity in MMR deficient colorectal adenocarcinomas in our study, future studies evaluating the efficacy of zolbetuximab therapy in this subset of colorectal cancers are warranted.
In summary, our test and validation cohorts have demonstrated that CLDN18.2 is significantly more frequently positive in gastroesophageal and pancreatic ductal adenocarcinomas than in colorectal adenocarcinomas. Similar to prior studies, we confirmed no relationship between CLDN18.2 expression and HER2 or MMR status in gastroesophageal adenocarcinomas or pancreatic ductal adenocarcinomas (43,45,49). We are the first to show that CLDN18.2-positive colorectal adenocarcinomas are frequently MMR deficient, BRAF V600E mutated, proximally located tumors with morphologic and immunohistochemical features typical of MMR deficient tumors.
Supplementary Material
Funding Statement
Rish K. Pai is supported in part by funding from the National Cancer Institute (NCI), National Institutes of Health (NIH) (award U01 CA167551). The Colon Cancer Family Registry (CCFR, www.coloncfr.org) is supported in part by funding from the National Cancer Institute (NCI), National Institutes of Health (NIH) (award U01 CA167551). Support for case ascertainment was provided by the Ontario Cancer Registry (Canada). The content of this manuscript does not necessarily reflect the views or policies of the NCI, NIH or any of the collaborating centers in the Colon Cancer Family Registry (CCFR), nor does mention of trade names, commercial products, or organizations imply endorsement by the US Government, any cancer registry, or the CCFR.
Footnotes
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Disclosures: ReKP is a consultant for Alimentiv, and this relationship is unrelated to the content of this manuscript. RiKP is a consultant for Alimentiv, Allergan, and Verily, and these relationships are unrelated to the content of this manuscript. RCG received a graduate scholarship from Pfizer and provided consulting or advisory roles for Astrazeneca, Tempus, Eisai, Incyte, Knight Therapeutics, Guardant Health, and Ipsen, unrelated to the content of this manuscript. All other authors have no conflict of interest relevant to the content of this manuscript.
Ethics Approval and Consent to Participate
The present study was approved by the institutional review boards of both the University of Pittsburgh and Mayo Clinic Arizona. This study was conducted using retrospective, de-identified clinical data, and patient consent was not required.
Data Availability Statement
The CCFR data can be requested by collaboration with the CCFR at www.coloncfr.org. The UPMC data are available from the authors on reasonable request.
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Associated Data
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Supplementary Materials
Data Availability Statement
The CCFR data can be requested by collaboration with the CCFR at www.coloncfr.org. The UPMC data are available from the authors on reasonable request.