Novel association between Helicobacter pylori infection and gastrointestinal stromal tumors (GIST) in a multi-ethnic population

Introduction

Gastrointestinal stromal tumors (GIST) are relatively rare tumors affecting approximately 5,000 newly diagnosed patients annually in the USA, but are the most common mesenchymal tumors of the gastrointestinal tract. GIST were called leiomyomas and leiomyosarcomas in the past, but recent studies have identified interstitial cells of Cajal as the cell of origin of GIST, distinguishing GIST from other mesenchymal GI tumors (1-3). c-KIT (CD117) is expressed in 80–100% and CD34 in 50–80% of GIST, respectively (1,4), and their expression is used to help diagnose GIST. DOG-1 and PKC_θ are commonly expressed by GIST as well and are particularly useful in the identification of c-KIT-negative stromal tumors (5-9). Previous reports have identified c-KIT and PDGFRA mutations in the vast majority of GIST (1,2).

Although there are multiple studies that examine the histology, pathogenesis, and treatment of GIST, existing data regarding the epidemiology of GIST are limited. Few publications to date have addressed potential ethnic differences in the incidence or prevalence of GIST. A population-based study of 292 GIST patients and 292 age- and sex-matched controls from the U.S. National Cancer Institute’s Surveillance, Epidemiology and End Results (SEER) registry database, found no statistically significant differences in geographic region, location of residence, or median income, but found significantly more white controls than cases compared with blacks and other non-whites (10). Another retrospective SEER study examined 1,458 malignant GIST cases from 1992 to 2000 in 12 SEER registry regions and found the highest incidence rate in Hawaii (1.06/100,000 person-years compared to the national age-adjusted incidence rate of 0.68/100,000 person-years) (11). This led us to examine the factors associated with Hawaii having the highest incidence of GIST among SEER regions in the USA.

We present the following article in accordance with the MDAR reporting checklist (available at http://dx.doi.org/10.21037/gist-20-2).

Methods

Epidemiology of GIST

The Hawaii SEER registry data were queried and all diagnoses of malignant gastrointestinal stromal sarcoma (ICD-O-3 code 8936) from 1998 to 2009 were identified. The Hawaii Tumor Registry (HTR) has been part of the U.S. SEER program since 1973.

Diagnosis of GIST was based on morphology (spindle, epithelioid, mixed) and immunohistochemical tumor markers (c-KIT, DOG1 or PDGFRA). Prior to 1998, the terms leiomyoma, leiomyosarcoma, neurofibromas, schwannomas were more commonly used to refer to GIST, however because of their location and lack of immunohistochemical staining, were largely indistinguishable from GIST. To avoid historical coding “bias”, sampling bias, or classification errors, tumors with these diagnoses (leiomyoma, leiomyosarcoma, neurofibroma, schwannoma) prior to 1998 were excluded from our data set. The HTR database includes patient demographics, tumor histology, primary site topography, tumor behavior, grade, and stage of disease at diagnosis, and immunohistochemistry in more recent cases.

The U.S. Census data (year 2000) was used to determine the ethnic composition of Hawaii’s population as a reference for the ethnicity of GIST cases (12). Pacific Islander is a term that includes Chamorro, Micronesian, Marshallese, Samoan, and Tongan ethnic groups. Ethnicity was self-reported in both the SEER and U.S. Census. The incidence rates for GIST were compared amongst ethnic groups reported to the HTR from 1998–2009. The incidence rates in non-white ethnic groups were compared to the incidence rate in whites.

Histological specimens

The Residual Tissue Repositories of two SEER registries in Hawaii and Iowa were queried for de-identified malignant GIST tissue cases (ICD-O-3 code 8936) cases from 1998–2017. Formalin-fixed and paraffin-embedded (FFPE) GIST tissue sections (cases; n=63) were collected from the HTR and Queen’s Medical Center. An additional eight malignant GIST cases from 2000–2015 were provided by the State Health Registry of Iowa due to the smaller number of whites with GIST in Hawaii. All GIST surgical resection specimens contained tumor bordered by non-malignant gastrointestinal tissue.

The controls were chosen from diagnostic esophagogastroduodenoscopy cases performed at the Queen’s Medical Center from 2015–2017 found to have gastritis without evidence of tumors. Multiple (4-8) pinch biopsy specimens from the gastric corpus and antrum were obtained from 65 control patients who were referred for evaluation of upper gastrointestinal symptoms or anemia, and were found to have endoscopic and histopathologic evidence of gastritis without evidence of gastric malignancies or peptic ulcer disease.

DNA extraction and purification

For cases and controls, sequential sections (5 µm thick) were cut from the tissue samples and mounted on glass microscope slides. For cases, one of the slices was stained with hematoxylin and eosin to verify the presence of tumor surrounded by adjacent normal gastrointestinal tissue. For controls, one of the slices was stained with hematoxylin and eosin to verify the presence of gastric tissue. The tissue specimens were scraped from 2–3 sequential unstained slides for each case and control. Both tumor and normal surrounding gastrointestinal tissue was collected for the GIST cases. The DNA was extracted from the paraffin-embedded tissue sections as previously described (13). Briefly, paraffin-embedded sections were placed in 180 µL ATL lysis buffer from the DNAeasy kit (Qiagen, Germany), followed by incubation at 100 °C for 30 minutes. The tubes were briefly centrifuged to remove condensate. Twenty µL of proteinase K was added to the content of the microfuge tubes, and the tubes were incubated at 65 °C for 16 hours. The lysed emulsion was further purified with the DNAeasy spin-column (Qiagen). DNA was finally recovered in a single elution step with 100 µL AE solution from the DNAeasy kit (Qiagen).

Detection of Helicobacter pylori (H. pylori)

The following polymerase chain reaction (PCR) primer pairs were used to perform PCR amplification of H. pylori-specific genes from tumor DNA:

16S rRNA: 109 bp (14)

F: 5'-CTGGAGAGACTAAGCCCTCC-3'
R: 5'-ATTACTGACGCTGATTGTGC-3'

HP1177: 187 bp (14)

F: 5'-ACGAACGCGCAAAAACTTTA-3'
R: 5'-TTGCCATTCTCATCGGTGTA-3'

vacA: 136/163 bp (14)

F: 5'-ATGGAAATACAACAAACACAC-3'
R: 5'-CAACAATGGCTGGAATGAT-3'

ureB: 132 bp (15)

F: 5'-CCCATTTGACTCAATGCGATG-3'
R: 5'-TGGGATTAGCGAGTATGTCGG-3'

iceA1: 218 bp (16)

F: 5'-ATCATAAAGACGGCCGCAAAGAT-3'
R: 5'-AT(A/G)GGGTCATATTGATAACA(A/G)CC-3'

iceA2: 247 bp (16)

F: 5'-CGCTGTTTTTCTAGCGGTGTTTTA-3'
R: 5'-CATTGATCT(A/G)TGTTTGTATGCTTC-3'

(iceA1 and iceA2 are allelic variants of iceA. Detection of either allele was considered positive for iceA).

Human beta-globulin was used as a PCR control for the extraction of human DNA from the tissues:

bgl [beta-globulin (control)]: 110 bp (17)

F: 5'-ACACAACTGTGTTCACTAGC-3'

R: 5'-CAACTTCATCCACGTTCACC-3'

PCR was performed on purified tumor DNA in 20 µL goTaq green PCR buffer (PROMEGA, USA) containing forward and reverse primers. PCR was performed using a GeneAmp PCR System 9700 (Applied Biosystems, USA) with the following conditions: 6 minutes of preincubation at 94 °C followed by 40 cycles of 30 seconds each at 94 °C, 30 seconds at 55 °C and 30 seconds at 72 °C. Final extension was performed for 7 minutes at 72 °C. The PCR products were resolved on 2% agarose gels, and, the respective bands were cut out of the agarose gels followed by DNA isolation and purification. Sequencing of the PCR products extracted from the gels was performed to verify the PCR products. The purified PCR products were sequenced using the same primers above and the resulting sequences matched using Nucleotide BLAST with matches yielding significance at P<0.05. Both negative and positive (patients with positive H. pylori infection by Giemsa stain of gastric biopsies) controls were utilized for all PCR assays. The uniformity of DNA quality was determined by detection of the housekeeping gene bgl by PCR. The PCR detection of the H. pylori-specific genes was performed in duplicate for each sample.

Approval for this research was granted by the Western Institutional Review Board (protocol no. 1168906) and University of Hawaii Human Subjects Committee (2014-302). This study conformed to the provisions of the Declaration of Helsinki (as revised in 2013). Due to the retrospective nature of the data and biospecimen collection, and de-identification of all personal health information, individual consent for this retrospective analysis was waived.

Statistical analysis

In addition to descriptive statistical analyses, bivariate analyses were performed to compare data; chi-square tests were used to determine the statistical significance between the frequency of GIST in various minority ethnic groups. Crude and unconditional logistic regression analyses were performed using SPSS v22 (IBM Corp., Armonk, NY, USA) to calculate risk [odds ratio (OR); 95% confidence interval (CI)] of detection of H. pylori genes in all 71 GIST cases (from Hawaii and Iowa) versus the 66 controls (from Hawaii). All 95% CI were two-sided. P<0.05 was considered statistically significant.

Results

One hundred and forty-seven patients with a confirmed histologic diagnosis of malignant GIST were reported in the Hawaii SEER database from 1998–2009 (Table 1). Fifty-six percent of GIST were in the stomach, 34.0% in the small intestine, and 3.5% in the colon (Table 1). There was no significant difference in the prevalence of GIST between men or women of any ethnic group (Table 1). Fifty-four point four percent of GIST were staged as localized, 17.7% regional, 22.4% with distant metastases, and 5.4% unstaged at the time of diagnosis (Table 1). Ethnicity was reported for all individuals except for 0.7% (no ethnicity reported) of GIST subjects. Compared to whites, malignant GIST incident rates were significantly greater in Japanese (P=4.3×10^–4), Chinese (P=4.7×10^–5), Korean (P=1.5×10^–7), Vietnamese (P=2.6×10^–9), Pacific Islander (P=3.3×10^–7), and Filipino (P=0.03) ethnic groups in Hawaii (Table 2). The gastric and Asian predominance of GIST in the multi-ethnic Hawaii population was reminiscent of a previous study that strongly linked H. pylori infection to gastric adenocarcinoma in Hawaii Asians (18). Multiple studies have found a high coincidence of GIST and non-GIST cancers (mostly adenocarcinomas) in the stomachs and gastroesophageal junctions of the same patients, suggesting common risk factors for the two types of tumors (19-23). These data led us to look for an association between H. pylori infection and GIST.

Paraffin-embedded GIST tumor specimens were available for 71 GIST and 65 gastritis controls. Of the GIST cases, 11 (15.5%) ranged in age 21 to 49 years, 13 (18.3%) were 50 to 59 years, 14 (19.7%) were 60 to 69 years, 21 (29.6%) were 70 to 79 years, and 12 (16.9%) were >80 years (Table 3). Of the controls, 16 (24.6%) ranged in age 21 to 49 years, 11 (16.9%) were 50 to 59 years, 19 (29.2%) were 60 to 69 years, 12 (18.5%) were 70 to 79 years, and 7 (10.8%) were >80 years (Table 3). Females comprised 49.3% and 60% of GIST patients and controls, respectively (Table 3). Asians made up the majority of both GIST and controls at 42 (59.2%) and 43 (66.2%) (P=0.40), respectively (Table S1). The remainder of GIST and controls were comprised of Hawaiians 9 (12.7%) and 5 (7.7%) (P=0.34), whites 14 (19.7%) and 13 (20.0%) (P=0.97), Pacific Islanders 5 (7.0%) and 4 (6.2%) (P=0.85), and “other” (ethnicity not reported) 1 (1.4%) and 0 (0.0%) (P=0.34), respectively (Table S1). Of the primary GIST, 49 (69.0%) were gastric in origin, 16 (22.5%) occurred in the small intestines, 2 (2.8%) in the colon, 2 (2.8%) in the rectum and 2 (2.8%) were detected outside the stomach and intestines (liver, mesentery) (Table 3). Among small intestine GIST (n=16), 5 were duodenal (31.3%), 3 ileal (18.8%), 1 overlapped different segments of small intestine (6.3%), and 7 were not otherwise specified (43.8%).

H. pylori infection was detected by positive PCR amplification of five H. pylori genes (HP1177, 16S rRNA, iceA, ureB, vacA1). For individual H. pylori genes, HP1177 was detected in 21 GIST and 1 control (P<0.0001), 16S rRNA in 18 GIST and 2 controls (P=0.0003), iceA in 25 GIST and 9 controls (P=0.0041), ureB in 15 GIST and 5 controls (P=0.028), and vacA in 14 GIST and 4 controls (P=0.021) (Table S2). Of the total 71 patients with GIST, 48 (67.6%) were positive for at least one H. pylori gene, compared with only 13 (20.0%) of 65 controls [unadjusted OR: 8.3 (95% CI: 3.8–18.3); P<0.0001] (Table 4). Furthermore, 23 (32.4%) cases were positive for at least two H. pylori genes, compared with only 6 (9.2%) controls [unadjusted OR: 4.7 (95% CI: 1.8–12.5); P=0.002] (Table 4). For the eight GIST cases from Iowa, 5 (62.5%) were positive for at least one H. pylori gene (Table 3). Excluding the eight patients from Iowa did not alter the significance between GIST and H. pylori genes compared with controls.

Of the GIST cases that were positive for one or more H. pylori genes (n=48), 32 (66.7%) were gastric, 11 (22.9%) were found in the small intestines, 1 (2.1%) in the colon, 2 (4.2%) occurred in the rectum, and 2 (4.2%) were extra-intestinal (liver, mesentery) (Table S1). On further analysis of the 11 small intestine GIST cases positive for one or more H. pylori genes, 4 (36.4%) were duodenal, 3 ileal (27.3%), 3 (27.3%) overlapping small intestinal segments and 1 (9.1%) not otherwise specified.

Discussion

In our study, we found GIST cases in Hawaii were overrepresented in Asians and Pacific Islanders compared with whites. Recent studies found a higher prevalence of H. pylori infection in non-whites than whites in Hawaii (24), which may help explain the higher incidence rates for GIST in Asians and Pacific Islanders than whites in our study.

Previous studies demonstrated strong associations between H. pylori infection and two other types of gastric cancers: adenocarcinoma (18) and MALT lymphoma (25). The association between H. pylori infection and gastric adenocarcinoma led the International Agency for Research on Cancers to declare H. pylori a carcinogen (26). One study, published only in abstract form, did report an association between GIST and H. pylori infection (27). In this study, of 71 malignant GIST cases and 65 non-GIST controls, gastrointestinal specimens were positive for H. pylori infection by Giemsa stain in 62.9% and 30%, respectively (P=0.013). Our study utilized PCR detection of H. pylori infection in gastrointestinal tissue specimens since this has been shown to be more sensitive than histochemical methods for H. pylori infection (28,29) and allows unequivocal distinction between various Helicobacter species, such as H. helmanii, that can also reside in the stomach (30). In the regression analyses performed on our subjects, GIST cases showed large ORs for H. pylori infection detected by PCR compared with controls. Due to the much lower number of GIST cases in whites than Asians in Hawaii, we obtained 8 GIST specimens from eight white patients from the University of Iowa to determine if this could be due to sampling error. The majority of the GIST cases from Iowa were positive for H. pylori infection as well. Even when the subset of 63 GIST cases and 66 matched (age, gender, ethnicity) controls from Hawaii were analyzed separately, the ORs for the association between H. pylori and GIST remained large and significant. Thus, our results strongly suggest an association between gastrointestinal H. pylori infection and GIST.

With regards to the controls, we selected patients referred for evaluation of upper gastrointestinal symptoms or anemia, who were found to have endoscopic and histopathologic evidence of gastritis without evidence of gastric malignancies or peptic ulcer disease after careful consideration. We did consider patients with known peptic ulcer disease as controls but previous studies have shown an inverse correlation between duodenal ulcers and gastric cancer, and a direct correlation between gastric ulcers and gastric cancer (31-34). Thus, including duodenal ulcer patients as controls would have potentially skewed the ORs for H. pylori infection in favor of GIST cases. Furthermore, using patients with gastric ulcers as controls would possibly have diminished the likelihood ratios for H. pylori infection and GIST, but may have included patients at risk for both gastric ulcers and GIST.

In our study, H. pylori genes were detected not just in gastric GIST but in small intestinal, colonic, and extra-intestinal GIST as well. This is not unexpected since H. pylori has been detected in the small and large intestines of patients with gastrointestinal diseases and even in the liver of patients with liver cancers (35-37). Nevertheless, the majority of primary GIST tumors in Hawaii occur in the stomach, where H. pylori infection is most prevalent. Gastric adenocarcinoma is much more frequent than gastric MALT lymphoma and GIST, likely due to the fact that the latter malignancies originate in the submucosal layers of the stomach which underlie the epithelium and therefore are more distant from H. pylori and carcinogens.

In the USA, there is no recommendation for gastric cancer screening in the general population due to the relatively low overall rate of gastric cancer. However, clinicians should be aware of the increased frequency of GIST, in addition to gastric adenocarcinoma and MALT lymphoma, in patients at increased risk for or with H. pylori infection. As GIST arise within the gastrointestinal submucosa, they first appear as raised lesions covered by normal appearing mucosa and frequently require endoscopic ultrasound with fine needle aspiration for further characterization (38). Future studies should determine if eradication of H. pylori infection decreases the rate of GIST.

Acknowledgments

Funding: Centers of Biomedical Research Excellence Program (award P20 RR018727 from the National Center for Research Resources, National Institutes of Health); NIH, NCI, Contract Nos. HHSN261201000037C and HHSN261201000032C, Surveillance, Epidemiology and End Results (SEER) Program; The Queen’s Medical Center Translational Seed Grant Program; Masami Horio Family Grant.

Footnote

Reporting Checklist: The authors have completed the MDAR reporting checklist. Available at http://dx.doi.org/10.21037/gist-20-2

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at http://dx.doi.org/10.21037/gist-20-2). Dr. CFL reports grants from National Cancer Institute, during the conduct of the study. The other authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. Approval for this research was granted by the Western Institutional Review Board (protocol No. 1168906) and University of Hawaii Human Subjects Committee (2014-302). This study conformed to the provisions of the Declaration of Helsinki (as revised in 2013). Due to the retrospective nature of the data and biospecimen collection, and de-identification of all personal health information, individual consent for this retrospective analysis was waived.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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