Concerning adenocarcinomas of the esophagogastric junction, neoadjuvant chemotherapy is regularly implemented, but patients’ response varies greatly, with some cases showing no therapeutic effect, being deemed as chemoresistant. Small, noncoding RNAs (miRNAs) have evolved as key players in biological processes, including malignant diseases, often promoting tumor growth and expansion. In addition, specific miRNAs have been implicated in the development of chemoresistance through evasion of apoptosis, cell cycle alterations, and drug target modification. We performed a retrospective study of 33 patients receiving neoadjuvant chemotherapy by measuring their miRNA expression profiles. Histologic tumor regression was evaluated using resection specimens, while miRNA profiles were prepared using preoperative biopsies without prior therapy. A preselected panel of 96 miRNAs, known to be of importance in various malignancies, was used to test for significant differences between responsive (chemosensitive) and nonresponsive (chemoresistant) cases. The cohort consisted of 12 nonresponsive and 21 responsive cases with the following 4 miRNAs differentially expressed between both the groups: hsa-let-7f-5p, hsa-miRNA-221-3p, hsa-miRNA-31-5p, and hsa-miRNA-191-5p. The former 3 showed upregulation in chemoresistant cases, while the latter showed upregulation in chemosensitive cases. In addition, significant correlation between high expression of hsa-miRNA-194-5p and prolonged survival could be demonstrated (
Since the discovery of microRNAs (miRNAs), which are small, noncoding RNAs with a length of 19–22 nucleotides [
Regarding human cancer in general, miRNAs have been found to act not only as oncogenes, promoting tumor growth and dissemination, but also as tumor suppressors, inhibiting tumor cell proliferation and migration and inducing apoptosis [
Concerning carcinomas of the upper gastrointestinal tract, especially gastric cancer and adenocarcinoma of the esophagogastric junction, a multitude of miRNAs have been identified as useful biomarkers: for example, upregulation of miRNA-17-5p, miRNA-20a, miRNA-106b, miRNA-150, and miRNA-93 has been reported to inhibit apoptosis and to promote cell cycle progression, whereas downregulation of miRNA-29 and miRNA-375 has been shown to increase cell growth and migration [
Complementing their diagnostic and prognostic significance, miRNAs have also been found to contribute to chemoresistance and/or chemosensitivity via regulation of apoptosis, DNA damage, and repair mechanisms, and epithelial-mesenchymal transition and modulation of drug targets, drug-metabolizing enzymes, and drug efflux transporters [
Following these observations and taking into account that data concerning chemoresistance in carcinomas of the esophagogastric junction are still scarce and many studies have focused on cell lines rather than tissue specimen, our study aimed to further contribute to the knowledge for this entity by comparing miRNA profiles in chemoresistant and chemosensitive cancer tissue.
Samples from formalin-fixed, paraffin-embedded (FFPE) tissue containing adenocarcinomas of the esophagogastric junction were included in the present study. All patients received neoadjuvant chemotherapy. Only tumor-containing samples of preoperative biopsies (without prior therapy) were used for miRNA analysis, while whole-resection specimens (postchemotherapeutic) were taken to determine the degree of histological regression and thereby treatment response. All cases were collected as part of routine clinical care at the University Hospital of Schleswig-Holstein, Campus Luebeck, during 1997–2013. All analyses performed were in accordance with the Declaration of Helsinki and had been approved by the local Ethics Committee beforehand (reference number 14-242A).
Samples were carefully examined by two researchers (CJ and JK) with a light microscope (Axioskop, Zeiss, Jena, Germany), and histologic tumor types were determined using the current WHO standard [
RNA for profiling of miRNA was isolated from FFPE tissue using the RecoverAll™ total nucleic acid isolation kit (Applied Biosystems, Carlsbad, California, USA). RNA concentrations were quantified using the NanoDrop Spectrophotometer (Nanodrop Technologies, Montchanin, New Castle, Delaware, USA). Afterwards, reverse transcription (RT) using amounts of 20 ng of total RNA by applying the miRCURY LNA™ Universal cDNA Synthesis Kit II (Exiqon, Vedbaek, Denmark), containing synthetic RNA Spike Ins, was performed. 5
In order to compensate for variations in quality of extracted RNA, extraction yield, and efficiency of reverse transcription, normalization of data was carried out using GenEx Software Version 6.1 (Trial Version; MultiD Analyses AB, Munich). The first normalization method used was the Normfinder algorithm which has been described in detail earlier [
To test for significant differences in miRNA expression profiles between responder and nonresponder groups, the Mann–Whitney-
To estimate differences in clinical features (age, gender, differentiation grade, nodal status, depth of infiltration, perineural invasion, lymphovascular invasion, and presence of distant metastases) between both groups and between clinical features and miRNA expression levels, the
To assess the prognostic value of miRNA expression, the median for each analyzed miRNA was calculated. Cases were then dichotomized, either showing an expression level above or below the median as described previously [
Data were adjusted for multiple testing using the Bonferroni procedure; after that, a
Overall, 24 cases were classified as tubular adenocarcinoma, 3 cases as poorly cohesive carcinoma, 2 cases as mucinous adenocarcinoma, and 4 cases as undifferentiated/unclassifiable according to the current WHO standard [
Pictures of histologic tumor regression: (a) regression grade 1a (no vital tumor); (b) regression grade 1b (<10% vital tumor cells); (c) regression grade 3 (>50% vital tumor cells). H&E staining, magnification 200x.
Characteristics of adenocarcinoma of the esophagogastric junction according to the responder or nonresponder status.
Characteristics | Responders | Nonresponders |
|
---|---|---|---|
Total |
21 | 12 | |
Gender | |||
Male | 18 | 8 | 0.198 |
Female | 3 | 4 | |
WHO classification | |||
Tubular | 17 | 7 |
|
Poorly cohesive | 2 | 1 | |
Mucinous | 2 | 0 | |
Undifferentiated | 0 | 4 | |
Others | |||
Differentiation grade | |||
Well (G1) | 0 | 0 |
|
Moderately (G2) | 19 | 7 | |
Poor (G3) | 2 | 5 | |
pT (low) | |||
pT0 | 2 | 0 | 0.136 |
pT1a | 2 | 0 | |
pT1b | 5 | 0 | |
pT2 | 3 | 1 | |
pT (high) | |||
pT3 | 8 | 10 | |
pT4a | 1 | 1 | |
pT4b | 0 | 0 | |
pN | |||
pN0 | 12 | 6 | 0.203 |
pN1 | 3 | 4 | |
pN2 | 6 | 1 | |
pN3 | 0 | 1 | |
|
|||
Present | 6 | 5 | 0.443 |
Absent | 15 | 7 | |
Perineural invasion | |||
Present | 1 | 3 | 0.087 |
Absent | 20 | 9 | |
Distant metastases | |||
Present | 4 | 1 | 0.409 |
Absent | 17 | 11 |
LVSI: lymphovascular space invasion; bold lettering in
Correlation between both groups showed that undifferentiated carcinomas and poorly differentiated carcinomas (differentiation grade 3) were more common in the nonresponder group, while responders showed a higher proportion of tubular adenocarcinomas (
Neoadjuvant chemotherapy was administered in all cases, and 3 patients received radiation therapy in addition (both belonging to the responder group with irradiation doses of 45 Gy, 59 Gy, and 66 Gy, respectively).
Concerning the nonresponder group, neoadjuvant chemotherapy consisted in most cases of 5-fluorouracil combined with leucovorin, oxaliplatin, and docetaxel (so-called FLOT regimen; 8/12 patients). The remaining patients received either 5-fluorouracil in combination with cisplatin (2 cases) or epirubicin combined with oxaliplatin (2 cases).
In the responder group, a combination of cisplatin and 5-fluorouracil was administered in most cases (12/21 patients). Only 4 patients received treatment using the FLOT regimen, and 2 were treated with 5-fluorouracil in combination with leucovorin and etoposide. The remaining three patients received chemotherapy without 5-fluorouracil containing oxaliplatin, etoposide, and irinotecan.
Overall, the following 4 miRNAs were differentially expressed between responder and nonresponder groups: hsa-let-7f-5p, hsa-miRNA-191-5p, hsa-miRNA-221-3p, and hsa-miRNA-31-5p.
Concerning the different normalisation methods, only minor variations were detected: applying the Normfinder algorithm, hsa-let-7f-5p, hsa-miRNA-191-5p, and hsa-miRNA-31-5p were differentially expressed (
Throughout all normalization methods, there was higher expression of hsa-let-7f-5p, hsa-miRNA-221-3p, and hsa-miRNA-31-5p in the nonresponder group, while hsa-miRNA-191-5p showed higher expression in the responder group. The respective miRNA expression patterns (Ct values) are depicted in Figure
Boxplots and Ct values according to differentially expressed miRNAs in responder and nonresponder groups: (a) Ct values for hsa-let-7f-5p; (b) Ct values for hsa-miRNA-191-5p; (c) Ct values for hsa-miRNA-221-3p; (d) Ct values for hsa-miRNA-31-5p. Lower Ct values indicate higher miR expression, while higher Ct values indicate lower miR levels.
Correlation between miRNA expression profiles and tumor differentiation grade found that poorer differentiation (G3) was significantly associated with decreased levels of hsa-miRNA-200a-3p and elevated levels of hsa-miRNA-21-5p, hsa-miRNA-222-3p, hsa-miRNA-25-3p, and hsa-let-7d-5p (
Complete survival data were available for 29 patients with a mean follow-up period of 44.52 months (range 1–100 months). During follow-up, 12 patients (41.38%) died. After adjusting for multiple testing, significant differences in survival according to high or low expression of miRNAs were detected only for hsa-miRNA-194-5p with a
Appropriate survival curves and mean survival times including 95% confidence interval are shown in Figure
Kaplan–Meier curve showing survival differences according to high or low expression of hsa-miRNA-194-5p with a
Average survival times according to high or low expression of hsa-miRNA-194-5p.
Average survival (months) | SD | 95% CI |
| |
---|---|---|---|---|
hsa-miRNA-194-5p | ||||
High | 97.67 | 1.91 | 93.93–101.4 |
|
Low | 32.69 | 7.89 | 17.22–48.16 |
The mechanisms underlying chemotherapy and multidrug resistance in human cancer are polymorphic [
The miRNA signature we discovered seems to differ from previously published data; other studies found that especially in esophageal carcinoma—in addition to miRNA-221—miRNA-141, miRNA-200c, miRNA-148a, miRNA-296, miRNA-23, miRNA-223, and miRNA-27a are substantially contributing to chemoresistance [
Hsa-let-7f-5p is located on the long arm of chromosome 9 (9q22.3) and shows involvement in immune cell differentiation, angiogenesis, and cellular growth arrest [
Hsa-miRNA-221-3p is well characterized, and its function and involvement in human cancer has been extensively described. It is commonly known as an onco-miRNA, promoting tumor proliferation, invasion, dissemination, and metastasis [
These findings are difficult to compare with our study population as both responders and nonresponders had been treated using a combination chemotherapy containing 5-fluorouracil in most cases (28/33 cases).
Downregulation of hsa-miRNA-31-5p has been described in a variety of human cancers, for instance, triple-negative breast carcinoma [
Data concerning its association with therapy response are more controversial: while some studies report that overexpression promotes chemoresistance in gastric and ovarian cancer [
Throughout the literature, hsa-miRNA-191-5p is described as having oncogenic properties, leading to increased tumor cell proliferation, invasion, and inhibition of apoptosis [
It remains to be seen whether these results can be reproduced in larger studies or if a similar effect can be shown in other cancer entities. It may be conceivable that our findings reflect—as it is possibly the case with both hsa-let-7f-5p and hsa-miRNA-31-5p—an effect which is only discernible in a defined subset of tumors or specific tumor entities.
Due to the small case number in our study and the often limited availability of both pretherapeutic biopsies and resection specimen in a single institution, we additionally consulted a web database (GEO database) to supplement our data. Here, we could find only one additional study analyzing chemotherapy response in a very small cohort (8 cases) of upper gastrointestinal carcinomas, namely, stomach cancer, proposing a miRNA signature used for predicting chemotherapy outcome [
In addition, we correlated the miRNA expression with patients’ overall survival and could demonstrate that overall survival was significantly correlated with expression levels of hsa-miRNA-194-5p (
Our results imply that miRNAs might play an important role in the evolution of chemoresistance and/or chemosensitivity in adenocarcinomas of the esophagogastric junction. Nevertheless, as—due to the restricted availability of both pre- and posttherapeutic tissue samples in a single institution—the number of patients in our study was limited, statistical results should be interpreted with caution. In addition, only tumor tissue was compared without establishing baseline levels of miRNA expression in nonneoplastic mucosa.
Regardless of the abovementioned limitations, our results contribute to other studies postulating that miRNAs might be a pretherapeutic means to predict therapy response in the future and better stratify patients who benefit from neoadjuvant therapy or who might not benefit at all and therefore could be spared of adverse effects of noneffective treatment strategies.
It remains to be seen if the miRNA signature we established for adenocarcinomas of the esophagogastric junction can be reproduced in future studies or for different tumor entities.
Cycle threshold
MicroRNA.
The data used to support the findings of this study are available from the corresponding author upon request.
Part of this study has been presented as a scientific poster at the ASCP Annual Meeting 2017 (Chicago, Illinois).
The authors declare that they have no conflicts of interest.
Christina Just and Juliana Knief contributed equally to this work.