Guanylate cyclase 2C (
GUCY2C
;
GUC
)expression and the tumor immune microenvironment (TIME) in gastrointestinal (GI) cancers.
Authors: Sackstein Paul , Krause Harris Benjamin , Elliott Andrew , Lenz Heinz-Josef , Battaglin Francesca , Prakash Ajay , Lou Emil , Oberley Matthew James , Seeber Andreas , Goldberg Richard M. , Grothey Axel , Shields Anthony F. , Marshall John , Goel Sanjay , Weinberg Benjamin Adam 
Published: October 2024

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Background:
An immunosuppressive TIME can result in reduced efficacy of immune checkpoint inhibitors (ICIs) in GI tumors.
GUC
is preferentially expressed in colorectal (CRC), gastroesophageal junction (GEJ) and gastric adenocarcinoma (GA). There is an ongoing trial of a GUC-directed bispecific T cell-engager in advanced GI cancers. We sought to better characterize the TIME in
GUC
-high GI cancers to understand the immunogenicity of this novel target.
Methods:
CRC (N = 15,285), GEJ (N = 3,276) and GA (N = 2,420) tumors were tested at Caris Life Sciences (Phoenix, AZ) with NextGen Sequencing on DNA (592 genes or whole exome) and RNA (whole transcriptome). PD-L1 expression was assessed by IHC (22C3: TPS 1% [CRC] or 28-8: 2+, 80% [GEJ, GA]). A combination of IHC and NGS was used to assess mismatch repair and microsatellite instability ([MSI] high/low) status.
GUC
-high (H) and -low (L) expression (transcripts per million, TPM) was defined for each molecularly defined subtype as top and bottom quartile, respectively. Cell infiltration was estimated by QuantiSEQ. Mann-Whitney U and /Fisher’s exact tests were applied as appropriate, and significance was determined as p adjusted for multiple comparisons (Q<0.05). Real-world overall survival (OS) was obtained from insurance claims and Kaplan-Meier estimates were calculated for molecularly-defined patients.
Results:
GUC
expression was highest in CRC (22.0 TPM) followed by GEJ (4.9) and GA (4.4).
GUC
expression was higher in metastatic v primary tumors for CRC (24.0 v 21.7 TPM); the opposite was observed for GEJ (2.5 v 5.9) and GA (2.7 v 5.1, Q<.001 all).
GUC
expression was increased in left- v right-sided CRC (24.5 vs 19.0, Q<.001). Consensus molecular subtype 1 (CMS1) CRC had lower
GUC
expression compared to other CMS subtypes (CMS1: 9.5, CMS2: 28.8, CMS3: 20.2, CMS4: 21.9).
GUC
-H had a lower prevalence of PD-L1+ as compared to GUC-L for CRC MSI-H (5.2 v 46.4%), CRC MSS (.8 v 7.1%) and GEJ (26.6 v 45.7%, Q<.001 all) but not for GA (36.7 v 21.9%, Q=0.15). Across all tumor types, there were decreased M1 macrophages (mac) and increased M2 mac, neutrophils (NEU) and natural killer cell (NK) infiltrate in
GUC
-H v -L (Table).
GUC
-H tumors were associated with increased OS v
GUC
-L in CRC MSS (26.9 v 23.8 months, Q=.003) and GEJ tumors (13.9 v 10.8 months, Q=.021).
Conclusions:
GUC
is expressed across GI cancers and
GUC
-H tumors are characterized by an “immune cold” TIME but longer OS. These results should be taken into consideration while evaluating the clinical utility of GUC as a novel immunotherapy target in advanced GI cancers that historically have not responded to ICI.

GA
GEJ
CRC MSI
CRC MSS

Immune Infiltrate

Mac. M1

Mac. M2

NEU

NK cells

Mac. M1

Mac. M2

NEU

NK cells

Mac. M1

Mac. M2

NEU

NK cells

Mac. M1

Mac. M2

NEU

NK cells

GUCY2C
-L(% infiltrate)

5.10
3.2
4
4.9
5.1
2.8
4.5
4.3
8.4
1.1
3.5
2.5
5
2.5
4.9
2.8

GUCY2C
-H(% infiltrate)

3.4
4.4
6.7
6.1
3.6
3.5
7.2
5.4
6.3
2.8
6
3.8
3.8
3.1
7.1
3.9