Epidermal growth factor receptor and other tissue biomarkers in gastrointestinal cancers

Personalised medicine has plays an increasing role in the treatment of cancer. New therapeutic molecules are being developed, but their compatibility for each patient has to be tested before starting the treatment by examining the appropriate tissue biomarkers expressed in the tumour. These biomarkers can be utilised not only in treatment selection but also in predicting treatment efficacy and patient survival. They can also be used to classify tumours into specific molecular subtypes that have distinct characteristics related to tumour behaviour, response to cancer treatments and prognosis of the patients. In order to implement these classifications in clinical practice, instead of time-consuming sequencing-based methods, the methods have to be simple enough and easy to interpret. Gastrointestinal cancers are among the most prevalent malignancies and often lead to death. Monoclonal antibodies against the epidermal growth factor receptor (EGFR) can be used in the treatment of RAS wild-type metastatic colorectal cancer. It has been shown that in addition to RAS mutation testing, determining the EGFR gene copy number (GCN) of the tumours can aid in selecting the patients likely to benefit from the antibody treatment. In oesophagogastric cancer, EGFR GCN has not yet been shown to have a predictive role, although the overexpression of HER2, which belongs to the same receptor family as EGFR, is used as a biomarker to predict response to anti-HER2 antibody treatment. In this thesis, the prevalence of EGFR amplification was observed to be at a similar level with the prevalence of HER2 amplification specifically among the intestinal-type oesophagogastric adenocarcinomas from 220 patients. This implies that it might be useful to examine whether EGFR GCN analysis could function as a biomarker predicting anti-EGFR treatment response in the intestinal-type tumours. In addition, in this thesis, tissue microarray was used to detect the different molecular subtypes of oesophagogastric cancers from 244 patients by staining methods applicable to clinical practice. Comparative studies detecting EGFR GCN in primary colorectal tumours and their metastases are scarce. In this thesis, corresponding primary and metastatic tumours from 80 patients were examined. EGFR GCN was observed to decrease between the primary and metastatic tumours during anti-EGFR treatment but to remain stable or even increase among patients not receiving treated with anti-EGFR antibodies. This EGFR GCN change may be relevant regarding the clinical response to anti-EGFR treatment. Preoperative chemoradiotherapy can be used in the treatment of rectal cancer patients to enable a complete resection of the tumour or reduce the risk of local recurrence. However, treatment response among patients is variable. Thus, a suitable biomarker could be helpful in predicting response or stratifying patients into separate treatment groups according to their prognosis. In this thesis, CIP2A expression was examined in rectal cancer tissue samples from 210 patients. Low CIP2A expression level was observed to associate with a better response among patients treated with long-course chemoradiotherapy. Affirming results were obtained in cell culture studies, where the suppression of CIP2A expression was observed to render the cells more sensitive to irradiation than the cells with normal CIP2A expression.Epidermaalinen kasvutekijäreseptori ja muita kudosbiomarkkereita ruoansulatuskanavan syövissä Yksilöllistetty lääketiede on yhä merkittävämmässä osassa myös syöpätautien hoidossa. Syövän hoitoon kehitetään jatkuvasti uusia lääkemolekyylejä, joiden soveltuvuus kullekin potilaalle on ennen hoidon aloitusta selvitettävä kasvaimen ilmentämien molekulaaristen biomarkkereiden avulla. Biomarkkereita voidaan käyttää paitsi hoitojen valitsemisen apuna niin myös syövän käyttäytymisen ja potilaiden ennusteen arvioimiseen. Biomarkkereiden avulla tietty syöpätyyppi voidaan myös luokitella molekulaarisiin alatyyppeihin, joilla on toisistaan poikkeavia kasvaimen käyttäytymiseen, ennusteeseen ja hoitovasteisiin liittyviä ominaisuuksia. Molekulaaristen luokittelun käytännön soveltamiseen tarvitaan kuitenkin riittävän yksinkertaisia ja helposti tulkittavissa olevia menetelmiä aikaa vievien sekvennointitutkimusten sijaan. Ruoansulatuskanavan syövät ovat yleisimpiä pahanlaatuisia kasvaimia ja johtavat usein kuolemaan. Levinneen suolistosyövän hoidossa voidaan käyttää epidermaaliseen kasvutekijäreseptoriin (EGFR) kohdistuvaa vasta-ainehoitoa, mikäli kasvaimessa ei ole osoitettavissa Ras-geenimutaatiota. Aikaisemmin on todettu, että Ras-geenitestin ohella EGFR:n geenikopiomäärän selvittämisen avulla voidaan valikoida pelkkää geenitestiä paremmin hoidosta todennäköisesti hyötyvät potilaat. Maha- ja ruokatorvisyövässä suurentuneen EGFR:n geenikopiomäärän ennusteellista merkitystä ei kuitenkaan ole vielä pystytty osoittamaan, vaikka samaan reseptoriperheeseen kuuluvan HER2:n geenimonistuman tiedetään ennustavan siihen kohdistuvan vasta-ainehoidon tehoa. Väitöstutkimuksessa EGFR:n geenimonistuman yleisyyden todettiin 220 potilaan aineistossa olevan nimenomaan intestinaalisen alatyypin maha- ja ruokatorvisyövissä HER2:n monistuman tasoa, joten sen selvittämisestä voisi olla hyötyä tutkittaessa EGFR-vasta-ainehoidon tehoa maha-ja ruokatorvisyövän hoidossa. Lisäksi maha- ja ruokatorvisyöpien molekulaaristen alatyyppien tunnistamista selvitettiin 244 potilaan näytteistä koostetun kudosmikrosirun avulla, ja tunnistamisen todettiin onnistuvan myös kliiniseen diagnostiikkaan soveltuvien menetelmien avulla. EGFR:n geenikopiomäärää alkuperäisen paksusuolikasvaimen ja sen lähettämän etäpesäkkeen välillä vertailevia tutkimuksia on tehty niukasti. Väitöstutkimuksessa verrattiin 80 potilaan primaari- ja metastaattisia kasvaimia. EGFR:n kopiolukumäärän todettiin pienenevän vasta-ainehoitoa saaneilla potilailla mutta pysyvän samana tai jopa suurentuvan muuta hoitoa saaneiden potilaiden etäpesäkkeissä. Kopiolukumäärän muutoksella saattaisi olla merkitystä vasta-ainehoidon tehon kannalta. Peräsuolipotilaiden hoidossa voidaan käyttää ennen leikkausta annettavaa kemosädehoitoa, jonka avulla pyritään mahdollistamaan kasvaimen täydellinen poisto. Osa potilaista jää kuitenkin ilman merkittävää hoitovastetta tai saa haitallisia sivuvaikutuksia, joten soveltuvasta biomarkkerista voisi olla hyötyä hoidon kohdentamisen parantamisessa. CIP2A:n ilmentymistä tutkittiin 210 potilaan peräsuolisyöpänäytteissä, ja matalan ilmenemistason todettiin olevan yhteydessä parempaan vasteeseen pitkän kemosädehoidon saaneilla potilailla. Samansuuntainen tulos saatiin syöpäsoluviljelmien sädetyskokeissa, joissa sädetyksen todettiin estävän enemmän CIP2A:ta ilmentämättömien kuin sitä normaalisti ilmentävien solujen kasvua

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

Background Gastric 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. Methods The 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. Results Kaplan-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. Conclusions FHOD1 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.Peer reviewe

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

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

Protein phosphatase 2A (PP2A) inhibitor CIP2A indicates resistance to radiotherapy in rectal cancer

Preoperative (chemo)radiotherapy, (C)RT, is an essential part of the treatment of rectal cancer patients, but tumor response to this therapy among patients is variable. Thus far, there are no clinical biomarkers that could be used to predict response to (C)RT or to stratify patients into different preoperative treatment groups according to their prognosis. Overexpression of cancerous inhibitor of protein phosphatase 2A (CIP2A) has been demonstrated in several cancers and is frequently associated with reduced survival. Recently, high CIP2A expression has also been indicated to contribute to radioresistance in head and neck squamous cell carcinoma, but few studies have examined the connection between CIP2A and radiation response regarding other malignancies. We have evaluated CIP2A protein expression levels in relation to tumor regression after preoperative (C)RT and survival of rectal adenocarcinoma patients. The effects of CIP2A knockdown by siRNA on cell survival were further investigated in colorectal cancer cells exposed to radiation. Patients with low-CIP2A-expressing tumors had more frequently moderate or excellent response to long-course (C)RT than patients with high-CIP2A-expressing tumors. They also had higher 36-month disease-specific survival (DSS) rate in categorical analysis. In the multivariate analysis, low CIP2A expression level remained as an independent predictive factor for increased DSS. Suppression of CIP2A transcription by siRNA was found to sensitize colorectal cancer cells to irradiation and decrease their survival in vitro. In conclusion, these results suggest that by contributing to radiosensitivity of cancer cells, low CIP2A protein expression level associates with a favorable response to long-course (C)RT in rectal cancer patients.</p

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

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

EGFR gene copy number decreases during anti-EGFR antibody therapy in colorectal cancer

Epidermal growth factor receptor (EGFR) gene copy number (GCN) increase is associated with a favorable anti-EGFR antibody treatment response in RAS wild-type metastatic colorectal cancer. However, there are limited and comparative data regarding the EGFR GCN in primary colorectal cancer tumors and corresponding metastases or the effect of anti-EGFR antibody treatment on EGFR GCN in recurrent disease. In addition, little is known about the potential EGFR GCN changes during anti-EGFR therapy in comparison with other treatment regimens. EGFR GCN was analyzed by EGFR immunohistochemistry-guided silver in situ hybridization in primary and corresponding recurrent local or metastatic tumors from 80 colorectal cancer patients. GCN levels were compared between KRAS wild-type patients having received anti-EGFR therapy and patients having received other forms of treatment after primary surgery. The EGFR GCN decrease between primary and recurrent tumors was more pronounced among the anti–EGFR-treated patients than among patients not treated with anti-EGFR therapy (P = .047). None of the patients experiencing an EGFR GCN increase of at least 1.0 between the primary and recurrent tumors were treated with anti-EGFR antibodies. When including only patients with distant metastases, an EGFR GCN decrease of at least 1.0 was more common among the anti–EGFR-treated patients than among patients not treated with anti-EGFR therapy (P = .028). Our results suggest that anti-EGFR antibody treatment is associated with EGFR GCN decrease between the primary and recurrent colorectal adenocarcinomas, whereas no GCN change is observed among patients receiving other forms of treatment after primary surgery.</p

Programmed death-ligand 1 and tumor-infiltrating lymphocytes (TILs) – low TIL density may predict poorer long-term prognosis in T1 laryngeal cancer

We evaluated the prognostic role of programmed death-ligand 1 (PD-L1) and tumor-infiltrating lymphocytes (TILs) in T1 glottic laryngeal squamous cell carcinoma (LSCC). T1 glottic LSCC patients (n = 174) treated at five Finnish university hospitals between 2003 and 2013 were included. Tissue microarray (TMA) blocks were used for PD-L1 immunohistochemistry. TILs were scored from intratumoral and stromal regions in whole tissue sections. Of 174 patients, 92 (53%) had negative, 66 (38%) intermediate, and 16 (9%) high PD-L1 levels. Of 80 patients whose TILs were analyzed, 50 (63%) had low and 30 (38%) high stromal TIL density. Patients with a local recurrence or a new primary tumor of the larynx had lower TIL density than had other patients (p = 0.047). High PD-L1 expression with low stromal TIL density was associated with inferior 5-year disease-specific survival (85% vs. 100%, p = 0.02). In conclusion, in patients treated for T1 glottic LSCC, low stromal TIL density was associated with local recurrences and new primary tumors of the larynx. High PD-L1 expression with low stromal TIL density may be associated with worse survival in T1 glottic LSCC.Peer reviewe