Molecular subtypes of intestinal-type gastric cancer: Association with T-lymphocytes and formins

Despite the decline of gastric cancer (GC) incidence in Western countries over the last decade, it is still one of the most significant causes of cancer mortality worldwide. The traditional morphology-based grading systems, including the world health organization (WHO) and Lauren's grading systems, have limited applicability in managing treatment choices, as they poorly catch the molecular heterogeneity of GC. Thus, classifications based on molecular features are needed. Recent genome analyses have shown that GC consists of several molecular subtypes characterized by distinct alterations. In our study, we used tissue-based methods, i.e., immunohistochemistry and in situ hybridization, in the molecular classification of GC, emphasizing the intestinal subtype. Our results show that GC can be divided into four nonoverlapping subtypes based on Epstein-Barr virus (EBV) positivity, mismatch repair protein (MMR), and TP53 aberration status. In conclusion, GC molecular subtyping can be performed with a simple methodology applicable to clinical routine. Host immune response is an important predictive and prognostic factor in many cancer types, including GC. Detailed information on the accumulation of tumorinfiltrating T lymphocytes in the different molecular GC subtypes and their prognostic correlation is scarce. We analyzed the presence of CD3+, CD8+, and FOXP3+ (Forkhead box P3) T lymphocytes in the molecular subtypes of intestinal-type GC. We found that EBV+ cancers harbor increased lymphocyte infiltration and a high CD8+/FOXP3+ ratio. In addition, we found that high numbers of CD8+ and CD3+ T lymphocytes are associated with better survival, and their accumulation is an independent prognostic factor. Formin proteins regulate the actin cytoskeleton and cell migration and play an essential role in cancer call functions. However, the expression and clinical association of formins in GC remains largely undiscovered. Here we analyzed the expression of FHOD1 and FMNL1 formins in GC cell lines and clinical samples of intestinal- type GC. We found that FHOD1 expression in cancer cells correlated with high intratumoral CD8+ T lymphocyte infiltration. Reduced FHOD1 expression was seen in the tumors with aberrant TP53. FMNL1 expression in cancer cells was associated with the size of the tumors and the stage of the disease. The results demonstrate a link between FHOD1 and FMNL1 expression with biological features of GC. However, we did not find a correlation between formin expression and GC prognosis.Suolistotyyppisen mahasyövän molekyylityypit: Assosiaatio T-lymfosyyttien ja forminien kanssa Vaikka mahasyövän esiintyminen on viime vuosikymmeninä laskenut kehittyneissä maissa, se on edelleen yksi tärkeimmistä syöpäkuolemien aiheuttajista. Mahasyöpä on biologialtaan monimuotoinen. Tästä syystä perinteinen kudosmorfologiaan perustua luokittelu, kuten WHO:n tai Laurénin luokitus, hyödyttää hoitopäätöksien tekoa vain rajallisesti. Molekulaarisiin piirteisiin perustuvan luokituksen kehittäminen olisikin tärkeää. Genomianalyyseihin perustuva tutkimus on osoittanut, että mahasyöpä koostuu useista molekulaarisista alatyypeistä. Analyysimenetelmät ovat kuitenkin monimutkaisia ja vaativat erityisosaamista. Tutkimuksessamme käytimme kliiniseen käyttöön soveltuvia menetelmiä; immunohistokemiaa ja in situ-hybridisaatiota mahasyöpien luokitteluun. Tulosten perusteella yksinkertaisen algoritmin avulla mahasyövät voidaan luokitella eri alaryhmiin Ebstain-Barr virus-positiivisuuden, TP53 poikkeavuuden ja MMR-puutoksen perusteella. Kliinisesti eroaviin alaryhmiin tapahtuva jaottelu voidaan siis toteuttaa yksinkertaisella ja kliiniseen diagnostiikkaan soveltuvalla menetelmällä Immuunijärjestelmän toiminta on tärkeä syövän ennusteen kannalta. Yksityiskohtaista tietoa siitä, miten T solut hakeutuvat eri mahasyövän alatyyppeihin ja liittyvät taudin käyttäytymiseen ei toistaiseksi ole. Tässä tutkimuksessa analysoimme CD3, CD8 ja FOXP3 antigeenejä ilmentävien T-lymfosyyttien esiintymistä intestinaalisen mahasyövän alatyypeissä. Tulosten perusteella EBV+ syövissä on runsaimmin T-lymfosyyttejä ja korkein CD8+/FOXP3-suhde. Totesimme myös, että runsas T-lymfosyyttien määrä korreloi intestinaalisen mahasyövän parempaan ennusteeseen ja toimii itsenäisenä ennustetekijänä. Formiiniproteiinit säätelevät solujen aktiinitukirankaa ja solujen migraatiota, ja ovat tärkeitä syöpäsolujen toiminnassa. Kuitenkin formiinien esiintymistä ja tehtäviä mahasyövässä tunnetaan huonosti. Tässä tutkimuksessa analysoimme FHOD1 ja FMNL1 formiineja mahasyöpäsolulinjoissa ja intestinaalista alatyyppiä edustavien mahasyöpien kudosnäytteissä. Kasvainsolujen FHOD1:n ilmentyminen oli yhteydessä korkeaan intratumoraalisten T-lymfosyyttien määrään. FHOD1:n alentunutta ekspressiota nähtiin syövän alatyypissä, johon liittyy TP53 mutaatio. FMNL1:n ilmentyminen puolestaan korreloi kasvaimen kokoon ja taudin leviämisasteeseen. Tulosten perusteella FHOD1 ja FMNL1-formiinien ilmentyminen liittyy mahasyövän biologisiin piirteisiin, joskaan ilmentymisellä ei näytä olevan yhteyttä mahasyövän ennusteeseen

Association of tumor-infiltrating T lymphocytes with intestinal-type gastric cancer molecular subtypes and outcome

While host immune response is likely to be important for the prognosis of gastric cancer patients, detailed information on the T lymphocyte infiltration in different gastric cancer subtypes is lacking. Here, we studied the presence of CD3, CD8, and FOXP3 (Forkhead box p3) expressing T lymphocytes in a retrospective cohort of 190 intestinal gastric and gastroesophageal adenocarcinomas. The cancers represented four distinct molecular subtypes: Epstein-Barr virus–positive (EBV+), mismatch-repair-deficient (MMR-D), aberrant TP53, and the “other” subtype. The absolute numbers of CD3+, CD8+, and FOXP3+ T lymphocytes were analyzed in relation with these molecular subtypes and selected clinicopathological parameters. Overall, there was a large variation in the amount of infiltrating T lymphocyte in all molecular subtypes. Among the subtypes, EBV+ cancers differed from the other subtypes in increased lymphocyte infiltration and high CD8+/FOXP3+ ratio. While the TP53 aberrant subtype did not differ in the absolute amount of T lymphocyte, the ratio of CD8+/FOXP3+ and CD3+/FOXP3+ cells was highest in this subtype, possibly reflecting immunosuppression associated with genomic instability. Increased CD3+ and CD8+ T lymphocyte infiltrates were associated with better survival, and remained as independent prognostic factors in a multivariate analysis. This study is the first to investigate lymphocytic infiltration within four molecular subtypes of intestinal-type gastric cancer in a European cohort. The results provide an important addition to the current knowledge of T lymphocyte–dependent immune response in gastric cancer and its prognostic significance.Peer reviewe

HER2 overexpression is a putative diagnostic and prognostic biomarker for late-stage colorectal cancer in North African patients

AIM: Colorectal cancer (CRC) is one of the leading cancers in the world. Even though its mortality and pathophysiology are well documented in the US and the European countries, it is seldom studied in North African population. Recent studies have shown link of HER2 overexpression in oesophageal and gastric cancers. The aim of this study is to assess the HER2 protein and mRNA expression and its correlation with tumor pathogenesis in Libyan CRC patients. METHODOLOGY: A total of 17 FFPE tissue blocks were collected from patients with primary CRC. The HER2 protein expression was assessed by immunohistochemistry and the mRNA expression was assessed using qRT-PCR. Survival analysis of the role of HER2 overexpression on rectal adenocarcinoma was carried out on additional 165 patients. RESULTS: From the CRC cohort, adenocarcinoma was found to be more frequent accounting for 88.2%, and 11.8% for mucinous adenocarcinomas. Almost 47% of the cases were positive for HER2 (score ≥ 2+) and about 50% adenocarcinoma cases with tumor grade II were positive for HER2. Moreover, 57.4% adenocarcinoma patients with grade-II tumor had undergone right hemicolectomy. Furthermore, significant correlation (p = 0.03) between the HER2 mRNA expression with the tumor grade was observed. In addition, poor overall all survival was observed with high HER2 expression in rectum adenocarcinoma. CONCLUSION: To our knowledge, this is the first study that HER2 overexpression correlates with more aggressive colorectal cancer in North African population. Our study shows that HER2 overexpression associates with right colon surgeries. Also, the correlation of mRNA and protein expression could warrant the implementation of a nationwide screening program for HER2 positivity in CRC patients. Taken together, stratifying patients according to HER2 expression can help in the diagnosis and prognosis of CRC patients from North African origin

Formin Proteins FHOD1 and INF2 in Triple-Negative Breast Cancer: Association With Basal Markers and Functional Activities

Basal-like breast cancer is an aggressive form of breast cancer with limited treatment options. The subgroup can be identified immunohistochemically, by lack of hormone receptor expression combined with expression of basal markers such as CK5/6 and/or epidermal growth factor receptor (EGFR). In vitro, several regulators of the actin cytoskeleton are essential for efficient invasion of basal-like breast cancer cell lines. Whether these proteins are expressed in vivo determines the applicability of these findings in clinical settings. The actin-regulating formin protein FHOD1 participates in invasion of the triple-negative breast cancer cell line MDA-MB-231. Here, we measure the expression of FHOD1 protein in clinical triple-negative breast cancers by using immunohistochemistry and further characterize the expression of another formin protein, INF2. We report that basal-like breast cancers frequently overexpress formin proteins FHOD1 and INF2. In cell studies using basal-like breast cancer cell lines, we show that knockdown of FHOD1 or INF2 interferes with very similar processes: maintenance of cell shape, migration, invasion, and proliferation. Inhibition of EGFR, PI3K, or mitogen-activated protein kinase activity does not alter the expression of FHOD1 and INF2 in these cell lines. We conclude that the experimental studies on these formins have implications in the clinical behavior of basal-like breast cancer.</p

Gastric cancer : immunohistochemical classification of molecular subtypes and their association with clinicopathological characteristics

Gastric cancer is traditionally divided into intestinal and diffuse histological subtypes, but recent molecular analyses have led to novel classification proposals based on genomic alterations. While the intestinal- and diffuse-type tumours are distinguishable from each other at the molecular level, intestinal-type tumours have more diverse molecular profile. The technology required for comprehensive molecular analysis is expensive and not applicable for routine clinical diagnostics. In this study, we have used immunohistochemistry and in situ hybridisation in molecular classification of gastric adenocarcinomas with an emphasis on the intestinal subtype. A tissue microarray consisting of 244 gastric adenocarcinomas was constructed, and the tumours were divided into four subgroups based on the presence of Epstein-Barr virus, TP53 aberrations and microsatellite instability. The intestinal- and diffuse-type tumours were separately examined. The distribution of EGFR and HER2 gene amplifications was studied in the intestinal-type tumours. Epstein-Barr virus positive intestinal-type tumours were more common in male patients (p = 0.035) and most often found in the gastric corpus (p = 0.011). The majority of the intestinal-type tumours with TP53 aberrations were proximally located (p = 0.010). All tumours with microsatellite instability showed intestinal-type histology (p = 0.017) and were associated with increased overall survival both in the univariate (p = 0.040) and multivariate analysis (p = 0.015). In conclusion, this study shows that gastric adenocarcinomas can be classified into biologically and clinically different subgroups by using a simple method also applicable for clinical diagnostics.Peer reviewe

Gastric cancer: immunohistochemical classification of molecular subtypes and their association with clinicopathological characteristics

Gastric cancer is traditionally divided into intestinal and diffuse histological subtypes, but recent molecular analyses have led to novel classification proposals based on genomic alterations. While the intestinal- and diffuse-type tumours are distinguishable from each other at the molecular level, intestinal-type tumours have more diverse molecular profile. The technology required for comprehensive molecular analysis is expensive and not applicable for routine clinical diagnostics. In this study, we have used immunohistochemistry and in situ hybridisation in molecular classification of gastric adenocarcinomas with an emphasis on the intestinal subtype. A tissue microarray consisting of 244 gastric adenocarcinomas was constructed, and the tumours were divided into four subgroups based on the presence of Epstein-Barr virus, TP53 aberrations and microsatellite instability. The intestinal- and diffuse-type tumours were separately examined. The distribution of EGFR and HER2 gene amplifications was studied in the intestinal-type tumours. Epstein-Barr virus positive intestinal-type tumours were more common in male patients (p = 0.035) and most often found in the gastric corpus (p = 0.011). The majority of the intestinal-type tumours with TP53 aberrations were proximally located (p = 0.010). All tumours with microsatellite instability showed intestinal-type histology (p = 0.017) and were associated with increased overall survival both in the univariate (p = 0.040) and multivariate analysis (p = 0.015). In conclusion, this study shows that gastric adenocarcinomas can be classified into biologically and clinically different subgroups by using a simple method also applicable for clinical diagnostics.</p

Association of tumor-infiltrating T lymphocytes with intestinal-type gastric cancer molecular subtypes and outcome

While host immune response is likely to be important for the prognosis of gastric cancer patients, detailed information on the T lymphocyte infiltration in different gastric cancer subtypes is lacking. Here, we studied the presence of CD3, CD8, and FOXP3 (Forkhead box p3) expressing T lymphocytes in a retrospective cohort of 190 intestinal gastric and gastroesophageal adenocarcinomas. The cancers represented four distinct molecular subtypes: Epstein-Barr virus-positive (EBV+), mismatch-repair-deficient (MMR-D), aberrant TP53, and the "other" subtype. The absolute numbers of CD3+, CD8+, and FOXP3+ T lymphocytes were analyzed in relation with these molecular subtypes and selected clinicopathological parameters. Overall, there was a large variation in the amount of infiltrating T lymphocyte in all molecular subtypes. Among the subtypes, EBV+ cancers differed from the other subtypes in increased lymphocyte infiltration and high CD8+/FOXP3+ ratio. While the TP53 aberrant subtype did not differ in the absolute amount of T lymphocyte, the ratio of CD8+/FOXP3+ and CD3+/FOXP3+ cells was highest in this subtype, possibly reflecting immunosuppression associated with genomic instability. Increased CD3+ and CD8+ T lymphocyte infiltrates were associated with better survival, and remained as independent prognostic factors in a multivariate analysis. This study is the first to investigate lymphocytic infiltration within four molecular subtypes of intestinal-type gastric cancer in a European cohort. The results provide an important addition to the current knowledge of T lymphocyte-dependent immune response in gastric cancer and its prognostic significance

FHOD1 and FMNL1 formin proteins in intestinal gastric cancer: correlation with tumor-infiltrating T lymphocytes and molecular subtypes

BackgroundGastric cancer (GC) is the third most common cause of cancer death. Intestinal type GC is a molecularly diverse disease. Formins control cytoskeletal processes and have been implicated in the progression of many cancers. Their clinical significance in GC remains unclear. Here, we characterize the expression of formin proteins FHOD1 and FMNL1 in intestinal GC tissue samples and investigate their association with clinical parameters, GC molecular subtypes and intratumoral T lymphocytes.MethodsThe prognostic significance of FHOD1 and FMNL1 mRNA expression was studied with Kaplan-Meier analyses in an online database. The expression of FHOD1 and FMNL1 proteins was characterized in GC cells, and in non-neoplastic and malignant tissues utilizing tumor microarrays of intestinal GC representing different molecular subtypes. FHOD1 and FMNL1 expression was correlated with clinical parameters, molecular features and T lymphocyte infiltration. Immunohistochemical expression of neither formin correlated with survival.ResultsKaplan-Meier analysis associated high FHOD1 and FMNL1 mRNA expression with reduced overall survival (OS). Characterization of FHOD1 and FMNL1 in GC cells showed cytoplasmic expression along the actin filaments. Similar pattern was recapitulated in GC tissue samples. Elevated FMNL1 was associated with larger tumor size and higher disease stage. Downregulation of FHOD1 associated with TP53-mutated GC tumors. Tumor cell FHOD1 expression strongly correlated with high numbers of tumor-infiltrating CD8 + lymphocytes.ConclusionsFHOD1 and FMNL1 proteins are expressed in the tumor cells of intestinal GC and significantly associate with clinical parameters without direct prognostic significance. FHOD1 correlates with high intratumoral CD8 + T lymphocyte infiltration in this cohort.</p

ALDH1A1-related stemness in high-grade serous ovarian cancer is a negative prognostic indicator but potentially targetable by EGFR/mTOR-PI3K/aurora kinase inhibitors

Poor chemotherapy response remains a major treatment challenge for high-grade serous ovarian cancer (HGSC). Cancer stem cells are the major contributors to relapse and treatment failure as they can survive conventional therapy. Our objectives were to characterise stemness features in primary patient-derived cell lines, correlate stemness markers with clinical outcome and test the response of our cells to both conventional and exploratory drugs. Tissue and ascites samples, treatment-naive and/or after neoadjuvant chemotherapy, were prospectively collected. Primary cancer cells, cultured under conditions favouring either adherent or spheroid growth, were tested for stemness markers; the same markers were analysed in tissue and correlated with chemotherapy response and survival. Drug sensitivity and resistance testing was performed with 306 oncology compounds. Spheroid growth condition HGSC cells showed increased stemness marker expression (including aldehyde dehydrogenase isoform I; ALDH1A1) as compared with adherent growth condition cells, and increased resistance to platinum and taxane. A set of eight stemness markers separated treatment-naive tumours into two clusters and identified a distinct subgroup of HGSC with enriched stemness features. Expression of ALDH1A1, but not most other stemness markers, was increased after neoadjuvant chemotherapy and its expression in treatment-naive tumours correlated with chemoresistance and reduced survival. In drug sensitivity and resistance testing, five compounds, including two PI3K-mTOR inhibitors, demonstrated significant activity in both cell culture conditions. Thirteen compounds, including EGFR, PI3K-mTOR and aurora kinase inhibitors, were more toxic to spheroid cells than adherent cells. Our results identify stemness markers in HGSC that are associated with a decreased response to conventional chemotherapy and reduced survival if expressed by treatment-naive tumours. EGFR, mTOR-PI3K and aurora kinase inhibitors are candidates for targeting this cell population. (c) 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.Peer reviewe

PRISM : recovering cell-type-specific expression profiles from individual composite RNA-seq samples

Motivation: A major challenge in analyzing cancer patient transcriptomes is that the tumors are inherently heterogeneous and evolving. We analyzed 214 bulk RNA samples of a longitudinal, prospective ovarian cancer cohort and found that the sample composition changes systematically due to chemotherapy and between the anatomical sites, preventing direct comparison of treatment-naive and treated samples. Results: To overcome this, we developed PRISM, a latent statistical framework to simultaneously extract the sample composition and cell-type-specific whole-transcriptome profiles adapted to each individual sample. Our results indicate that the PRISM-derived composition-free transcriptomic profiles and signatures derived from them predict the patient response better than the composite raw bulk data. We validated our findings in independent ovarian cancer and melanoma cohorts, and verified that PRISM accurately estimates the composition and cell-type-specific expression through whole-genome sequencing and RNA in situ hybridization experiments.Peer reviewe