Decision-making and regret in patients with germline CDH1 variants undergoing prophylactic total gastrectomy.

INTRODUCTION: Prophylactic total gastrectomy (PTG) can eliminate gastric cancer risk and is recommended in carriers of a germline CDH1 pathogenic variant. PTG has established risks and potential life-long morbidity. Decision-making regarding PTG is complex and not well-understood. METHODS: Individuals with germline CDH1 pathogenic or likely pathogenic variants who underwent surveillance endoscopy and recommended for PTG were evaluated. Factors associated with decision to pursue PTG (PTGpos) or not (PTGneg) were queried. A decision-regret survey was administered to patients who elected PTG. RESULTS: Decision-making was assessed in 120 patients. PTGpos patients (63%, 76/120) were younger than PTGneg (median 45 vs 58 years) and more often had a strong family history of gastric cancer (80.3% vs 34.1%). PTGpos patients reported decision-making based on family history more often and decided soon after diagnosis (8 vs 27 months) compared with PTGneg. Negative endoscopic surveillance results were more common among PTGneg patients. Age >60 years, male sex and longer time to decision were associated with deferring PTG. Strong family history, a family member who died of gastric cancer and carcinoma on endoscopic biopsies were associated with decision to pursue PTG. In the PTGpos group, 30 patients (43%) reported regret which was associated with occurrence of a postoperative complication and no carcinoma detected on final pathology. CONCLUSION: The decision to undergo PTG is influenced by family cancer history and surveillance endoscopy results. Regret is associated with surgical complications and pathological absence of cancer. Individual cancer-risk assessment is necessary to improve pre-operative counselling and inform the decision-making process. TRIAL REGISTRATION NUMBER: NCT03030404.

Germline CDH1 Variants and Lifetime Cancer Risk.

Importance: Approximately 1% to 3% of gastric cancers and 5% of lobular breast cancers are hereditary. Loss of function CDH1 gene variants are the most common gene variants associated with hereditary diffuse gastric cancer and lobular breast cancer. Previously, the lifetime risk of gastric cancer was estimated to be approximately 25% to 83% and for breast cancer it was estimated to be approximately 39% to 55% in individuals with loss of function CDH1 gene variants. Objective: To describe gastric and breast cancer risk estimates for individuals with CDH1 variants. Design, Setting, and Participants: Multicenter, retrospective cohort and modeling study of 213 families from North America with a CDH1 pathogenic or likely pathogenic (P/LP) variant in 1 or more family members conducted between January 2021 and August 2022. Main Outcomes and Measures: Hazard ratios (HRs), defined as risk in variant carriers relative to noncarriers, were estimated for each cancer type and used to calculate cumulative risks and risks per decade of life up to age 80 years. Results: A total of 7323 individuals from 213 families were studied, including 883 with a CDH1 P/LP variant (median proband age, 53 years [IQR, 42-62]; 4% Asian; 4% Hispanic; 85% non-Hispanic White; 50% female). In individuals with a CDH1 P/LP variant, the prevalence of gastric cancer was 13.9% (123/883) and the prevalence of breast cancer among female carriers was 26.3% (144/547). The estimated HR for advanced gastric cancer was 33.5 (95% CI, 9.8-112) at age 30 years and 3.5 (95% CI, 0.4-30.3) at age 70 years. The lifetime cumulative risk of advanced gastric cancer in male and female carriers was 10.3% (95% CI, 6%-23.6%) and 6.5% (95% CI, 3.8%-15.1%), respectively. Gastric cancer risk estimates based on family history indicated that a carrier with 3 affected first-degree relatives had a penetrance of approximately 38% (95% CI, 25%-64%). The HR for breast cancer among female carriers was 5.7 (95% CI, 2.5-13.2) at age 30 years and 3.9 (95% CI, 1.1-13.7) at age 70 years. The lifetime cumulative risk of breast cancer among female carriers was 36.8% (95% CI, 25.7%-62.9%). Conclusions and Relevance: Among families from North America with germline CDH1 P/LP variants, the cumulative risk of gastric cancer was 7% to 10%, which was lower than previously described, and the cumulative risk of breast cancer among female carriers was 37%, which was similar to prior estimates. These findings inform current management of individuals with germline CDH1 variants.

Tumor-Intrinsic Enhancer of Zeste Homolog 2 Controls Immune Cell Infiltration, Tumor Growth, and Lung Metastasis in a Triple-Negative Breast Cancer Model.

Triple-negative breast cancer (TNBC) is an aggressive and highly metastatic type of tumor. TNBC is often enriched in tumor-infiltrating neutrophils (TINs), which support cancer growth in part by counteracting tumor-infiltrating lymphocytes (TILs). Prior studies identified the enhancer of zeste homolog 2 (EZH2) as a pro-tumor methyltransferase in primary and metastatic TNBCs. We hypothesized that EZH2 inhibition in TNBC cells per se would exert antitumor activity by altering the tumor immune microenvironment. To test this hypothesis, we used CRISPR to generate EZH2 gene knockout (KO) and overexpressing (OE) lines from parent (wild-type-WT) 4T1 cells, an established murine TNBC model, resulting in EZH2 protein KO and OE, respectively. In vitro, EZH2 KO and OE cells showed early, transient changes in replicative capacity and invasiveness, and marked changes in surface marker profile and cytokine/chemokine secretion compared to WT cells. In vivo, EZH2 KO cells showed significantly reduced primary tumor growth and a 10-fold decrease in lung metastasis compared to WT cells, while EZH2 OE cells were unchanged. Compared to WT tumors, TIN:TIL ratios were greatly reduced in EZH2 KO tumors but unchanged in EZH2 OE tumors. Thus, EZH2 is key to 4T1 aggressiveness as its tumor-intrinsic knockout alters their in vitro secretome and in vivo primary tumor growth, TIN/TIL poise, and metastasis.

Cancer surveillance as an alternative to prophylactic total gastrectomy in hereditary diffuse gastric cancer: a prospective cohort study.

BACKGROUND: Loss of function variants in CDH1 are the most frequent cause of hereditary diffuse gastric cancer. Endoscopy is regarded as insufficient for early detection due to the infiltrative phenotype of diffuse-type cancers. Microscopic foci of invasive signet ring cells are pathognomonic of CDH1 and precede development of diffuse gastric cancer. We aimed to assess the safety and effectiveness of endoscopy for cancer interception in individuals with germline CDH1 variants, particularly in those who declined prophylactic total gastrectomy. METHODS: In this prospective cohort study, we included asymptomatic patients aged 2 years or older with pathogenic or likely pathogenic germline CDH1 variants who underwent endoscopic screening and surveillance at the National Institutes of Health (Bethesda, MD, USA) as part of a natural history study of hereditary gastric cancers (NCT03030404). Endoscopy was done with non-targeted biopsies and one or more targeted biopsy and assessment of focal lesions. Endoscopy findings, pathological data, personal and family cancer history, and demographics were recorded. Procedural morbidity, gastric cancer detection by endoscopy and gastrectomy, and cancer-specific events were assessed. Screening was defined as the initial endoscopy and all subsequent endoscopies were considered surveillance; follow-up endoscopy was at 6 to 12 months. The primary aim was to determine effectiveness of endoscopic surveillance for detection of gastric signet ring cell carcinoma. FINDINGS: Between Jan 25, 2017, and Dec 12, 2021, 270 patients (median age 46·6 years [IQR 36·5-59·8], 173 [64%] female participants, 97 [36%] male participants; 250 [93%] were non-Hispanic White, eight [3%] were multiracial, four [2%] were non-Hispanic Black, three [1%] were Hispanic, two [1%] were Asian, and one [<1%] was American Indian or Alaskan Native) with germline CDH1 variants were screened, in whom 467 endoscopies were done as of data cutoff (April 30, 2022). 213 (79%) of 270 patients had a family history of gastric cancer, and 176 (65%) reported a family history of breast cancer. Median follow-up was 31·1 months (IQR 17·1-42·1). 38 803 total gastric biopsy samples were obtained, of which 1163 (3%) were positive for invasive signet ring cell carcinoma. Signet ring cell carcinoma was detected in 76 (63%) of 120 patients who had two or more surveillance endoscopies, of whom 74 had occult cancer detected; the remaining two individuals developed focal ulcerations each corresponding to pT3N0 stage carcinoma. 98 (36%) of 270 patients proceeded to prophylactic total gastrectomy. Among patients who had a prophylactic total gastrectomy after an endoscopy with biopsy samples negative for cancer (42 [43%] of 98), multifocal stage IA gastric carcinoma was detected in 39 (93%). Two (1%) participants died during follow-up, one due to metastatic lobular breast cancer and the other due to underlying cerebrovascular disease, and no participants were diagnosed with advanced stage (III or IV) cancer during follow-up. INTERPRETATION: In our cohort, endoscopic cancer surveillance was an acceptable alternative to surgery in individuals with CDH1 variants who declined total gastrectomy. The low rate of incident tumours (>T1a) suggests that surveillance might be a rational alternative to surgery in individuals with CDH1 variants. FUNDING: Intramural Research Program, National Institutes of Health.

Costs of Cancer Prevention: Physical and Psychosocial Sequelae of Risk-Reducing Total Gastrectomy.

PURPOSE: Risk-reducing surgery for cancer prevention in solid tumors is a pressing clinical topic because of the increasing availability of germline genetic testing. We examined the short- and long-term outcomes of risk-reducing total gastrectomy (RRTG) and its lesser-known impacts on health-related quality of life (QOL) in individuals with hereditary diffuse gastric cancer syndrome. METHODS: Individuals who underwent RRTG as part of a single-institution natural history study of hereditary gastric cancers were examined. Clinicopathologic details, acute and chronic operative morbidity, and health-related QOL were assessed. Validated questionnaires were used to determine QOL scores and psycho-social-spiritual measures of healing. RESULTS: One hundred twenty-six individuals underwent RRTG because of a pathogenic or likely pathogenic germline CDH1 variant between October 2017 and December 2021. Most patients (87.3%; 110/126) had pT1aN0 gastric carcinoma with signet ring cell features on final pathology. Acute (<30 days) postoperative major morbidity was low (5.6%; 7/126) and nearly all patients (98.4%) lost weight after total gastrectomy. At 2 years after gastrectomy, 94% (64/68) of patients exhibited at least one chronic complication (ie, bile reflux, dysphagia, and micronutrient deficiency). Occupation change (23.5%), divorce (3%), and alcohol dependence (1.5%) were life-altering consequences attributed to total gastrectomy by some patients. In patients with a median follow-up of 24 months, QOL scores decreased at 1 month after gastrectomy and returned to baseline by 6-12 months. CONCLUSION: RRTG is associated with life-changing adverse events that should be discussed when counseling patients with CDH1 variants about gastric cancer prevention. The risks of cancer-prevention surgery should not only be judged in the context of likelihood of death due to disease if left untreated, but also based on the real consequences of organ removal.

Protocol for isolation of signet ring cells from human gastric mucosa.

More than 90% of individuals with germline pathogenic CDH1 variants will harbor occult, microscopic foci of signet ring cell carcinomas capable of progressing to advanced diffuse-type gastric cancer. Here, we present a protocol for high viability suspension of signet ring cells from human gastric tissue. We describe the steps for gastric mucosa isolation and tissue dissociation. We then detail procedures for embedding cells into HistoGel for immunohistochemistry staining and additional applications such as flow cytometry and single-cell sequencing.

Association of CDH1 Germline Variants and Colon Polyp Phenotypes in Patients with Hereditary Diffuse Gastric Cancer.

Background and Aims: Germline CDH1 variants resulting in E-cadherin loss of function result in an increased risk of diffuse type gastric cancer and lobular type breast cancer. However, the risk of developing other epithelial neoplasms, specifically colorectal cancer, is unknown. Methods: Patients enrolled in a prospective natural history study of hereditary gastric cancer who underwent at least one colonoscopy were evaluated. Results: Out of 300 patients with CDH1 pathogenic or likely pathogenic variants, 85 underwent colonoscopy. More than half of patients (56%, 48/85) had at least one colorectal polyp. Most of those patients (83%, 40/48) had at least one precancerous polyp (adenoma or sessile serrated lesion). More than half (56%) of patients younger than age 45 had a colorectal polyp. Of those with polyps, the most frequent CDH1 variant type was canonical splice site (27%, 13/48) followed by nonsense (21%, 10/48). There was no association between CDH1 variant type and increased likelihood of colorectal polyps. Conclusions: In summary, a majority of CDH1 variant carriers who underwent colonoscopy had colorectal polyps detected, and most subjects were less than 45 years old. This study of colorectal cancer risk based on the prevalence of colorectal polyps in the CDH1 population requires further investigation to appropriately counsel patients on colorectal cancer screening. Clinical trial registry website: https://clinicaltrials.gov/. Clinical trial number: NCT03030404.

Micronutrient Supplementation and Bone Health After Prophylactic Total Gastrectomy in Patients With CDH1 Variants.

INTRODUCTION: Patients with germline variants in CDH1 who undergo prophylactic total gastrectomy (TG) are at risk of altered nutrient and drug absorption due to modified gastrointestinal anatomy. Bone mineral density loss and micronutrient deficiencies have not been described previously in this patient population. METHODS: In this study we included 94 patients with germline CDH1 variants who underwent prophylactic TG between October 2017 and February 2022. We examined pre- and post-gastrectomy bone mineral density (BMD); serum biomarkers including calcium, phosphorus, alkaline phosphatase, and 25 (OH)-vitamin D; and postoperative adherence to calcium and multivitamin supplementation. RESULTS: Almost all patients (92/94, 98%) lost a substantial amount of weight post-TG, with an average weight loss of 26.5% at 12 months post-surgery. Serum biomarkers of mineral metabolism, namely calcium and phosphorus, did not change significantly after TG. However, average BMD was decreased in all patients at 12 months post-TG. Nonadherence to calcium supplementation was associated with a decrease in BMD. Nonadherence to multivitamin supplementation was associated with greater percent BMD loss in the femoral neck and total hip. CONCLUSIONS: Appropriate micronutrient supplementation and nutritional counseling pre- and postoperatively in patients undergoing prophylactic TG are important to mitigate the long-term effects of gastrectomy on bone health.

Defining features of hereditary lobular breast cancer due to CDH1 with magnetic resonance imaging and tumor characteristics.

Women with germline pathogenic variants in CDH1, which encodes E-cadherin protein, are at increased lifetime risk of invasive lobular carcinoma (ILC). The associated tumor characteristics of hereditary lobular breast carcinoma (HLBC) in this high-risk population are not well-known. A single-center prospective cohort study was conducted to determine the imaging and pathologic features of HLBC compared to population-based ILC using Surveillance, Epidemiology, and End Results (SEER) data. One hundred fifty-eight women with CDH1 variants were evaluated, of whom 48 (30%) also had an ILC diagnosis. The median age at CDH1 diagnosis was 45 years [interquartile range, IQR 34-57 years] whereas the median age at diagnosis of CDH1 with concomitant ILC (HLBC) was 53 [IQR 45-62] years. Among women with HLBC, 83% (40/48) were identified with CDH1 mutation after diagnosis of ILC. Among 76 women (48%, 76/158) undergoing surveillance for ILC with breast magnetic resonance imaging (MRI), 29% (22/76) had an abnormal MRI result with available biopsy data for comparison. MRI detected ILC in 7 out of 8 biopsy-confirmed cases, corresponding with high sensitivity (88%), specificity (75%), and negative predictive value (98%); however, false-positive and false-discovery rates were elevated also (25% and 68%, respectively). HLBC was most frequently diagnosed at age 40-49 years (44%, 21/48), significantly younger than the common age of diagnosis of ILC in SEER general population data (most frequent age range 60-69 years, 28%; p < 0.001). HLBC tumors were smaller than SEER-documented ILC tumors (median 1.40 vs. 2.00 cm; p = 0.002) and had a higher incidence of background lobular carcinoma in situ (88% vs. 1%; p < 0.001) as well as progesterone receptor positivity (95% vs. 81%, p = 0.032). These findings suggest that HLBC is often detected via conventional screening methods as an early-stage hormone receptor-positive tumor, thus the clinical benefit of intensive screening with MRI may be limited to a subset of women with germline CDH1 variants.

Early Immune Changes Support Signet Ring Cell Dormancy in CDH1-Driven Hereditary Diffuse Gastric Carcinogenesis.

Stage IA gastric adenocarcinoma, characterized by foci of intramucosal signet ring cells (SRC), is found in nearly all asymptomatic patients with germline pathogenic CDH1 variants and hereditary diffuse gastric cancer syndrome (HDGC). The molecular steps involved in initiating malignant transformation and promoting SRC dormancy in HDGC are unknown. Here, whole-exome bulk RNA sequencing (RNA-seq) of SRCs and adjacent non-SRC epithelium (NEP) was performed on laser-capture microdissected (LCM) regions of interest found in risk-reducing total gastrectomy specimens from patients with HDGC (Clinicaltrials.gov ID: NCT03030404). In total, 20 patients (6 male, 14 female) with confirmed HDGC were identified. Analysis of differentially expressed genes (DEG) demonstrated upregulation of certain individual EMT and proliferation genes. However, no oncogenic pathways were found to be upregulated in SRCs. Rather, SRC regions had significant enrichment in pathways involved in T-cell signaling. CIBERSORTx predicted significant increases in the presence of regulatory T cells (Treg) specific to SRC regions. IHC confirmed an increase in FOXP3+ cells in SRC foci, as well as elevations in CD4+ T cells and HLA-DR staining. In summary, the tumor immune microenvironment is microscopically inseparable from stage IA gastric SRCs using a granular isolation technique. An elevation in CD4+ T cells within SRC regions correlates with clinically observed SRC dormancy, while Treg upregulation represents a potential immune escape mechanism. IMPLICATIONS: Characterization of the tumor-immune microenvironment in HDGC underscores the potential for the immune system to shape the transcriptional profile of the earliest tumors, which suggests immune-directed therapy as a potential cancer interception strategy in diffuse-type gastric cancer.

Frequent cleft lip and palate in families with pathogenic germline CDH1 variants.

Pathogenic and likely pathogenic (P/LP) germline variants in the tumor suppressor gene CDH1 (E-cadherin) result in increased lifetime risk of diffuse-type gastric cancer and lobular breast cancer. CDH1 variants are also associated with hereditary cleft lip and palate (CLP), the mechanism of which is not well understood. We sought to determine the prevalence of CLP in families who carry P/LP CDH1 variants. Patients with P/LP CDH1 variants who were enrolled in a prospective clinical trial were reviewed (NCT03030404). The cohort included 299 individuals from 153 families that had 80 unique P/LP variants in CDH1. The rate of CLP was 19% (29/153) in families reporting CLP in at least one family member, and 2.7% (8/299) among individuals with confirmed germline CDH1 P/LP variants. There were 22 unique variants in CDH1 among the 29 families that reported CLP, or a CLP rate of 27.5% per variant (22/80). 10 of the variants were not previously reported to be associated with CLP. We observed that 24% (7/29) of CLP-associated gene variants involved large-scale (≥1 exon) deletions. Among families with CLP, 69% (20/29) had a member diagnosed with gastric cancer, and 79% (23/29) had a member with breast cancer, which were similar to rates observed in non-CLP families (p >0.3 for both). Our analysis suggests that the prevalence of CLP in families with germline CDH1 P/LP variants was high in this large cohort, and there was no genotype-phenotype pattern. Genetic testing for CDH1 variants should be considered in families with CLP and history of either diffuse-type gastric or lobular breast cancer.

Metformin reduces PD-L1 on tumor cells and enhances the anti-tumor immune response generated by vaccine immunotherapy.

BACKGROUND: PD-L1 is one of the major immune checkpoints which limits the effectiveness of antitumor immunity. Blockade of PD-L1/PD-1 has been a major improvement in the treatment of certain cancers, however, the response rate to checkpoint blockade remains low suggesting a need for new therapies. Metformin has emerged as a potential new drug for the treatment of cancer due to its effects on PD-L1 expression, T cell responses, and the immunosuppressive environment within tumors. While the benefits of metformin in combination with checkpoint blockade have been reported in animal models, little remains known about its effect on other types of immunotherapy. METHODS: Vaccine immunotherapy and metformin were administered to mice inoculated with tumors to investigate the effect of metformin and TMV vaccine on tumor growth, metastasis, PD-L1 expression, immune cell infiltration, and CD8 T cell phenotype. The effect of metformin on IFN-γ induced PD-L1 expression in tumor cells was assessed by flow cytometry, western blot, and RT-qPCR. RESULTS: We observed that tumors that respond to metformin and vaccine immunotherapy combination show a reduction in surface PD-L1 expression compared with tumor models that do not respond to metformin. In vitro assays showed that the effect of metformin on tumor cell PD-L1 expression was mediated in part by AMP-activated protein kinase signaling. Vaccination results in increased T cell infiltration in all tumor models, and this was not further enhanced by metformin. However, we observed an increased number of CD8 T cells expressing PD-1, Ki-67, Tim-3, and CD62L as well as increased effector cytokine production after treatment with metformin and tumor membrane vesicle vaccine. CONCLUSIONS: Our data suggest that metformin can synergize with vaccine immunotherapy to augment the antitumor response through tumor-intrinsic mechanisms and also alter the phenotype and function of CD8 T cells within the tumor, which could provide insights for its use in the clinic.