In this study, we report the discovery of two genomic subtypes of GC using profiles initially derived from GC cell lines. Since cancer cell lines are devoid of stroma, vasculature and immune cells, we reasoned that comparing signatures between cell lines would be more likely to reflect intrinsic differences between tumor cells, minimizing potentially confounding effects from neighboring non-cancer tissues. The validity of the cell line based approach is supported by similar studies in other solid tumors28, 29
, where major patterns of tumor heterogeneity have also been shown to be recapitulated by cell lines. We acknowledge that this approach does have caveats - for example, it may miss rare subtypes represented by only one or two lines, or subtypes for which it is difficult to derive immortalized cell lines. The use of cell lines is also unlikely to fully recapitulate the biology of tumor/microenvironment interactions, which can also influence disease prognosis and treatment response. Nevertheless, we believe the intrinsic subtypes discovered in this study represents an important first step in establishing a basic foundational taxonomy of GC, one to which additional layers of genomic complexity can be subsequently added.
One notable aspect of our study was that unlike previous comparative molecular studies for GC30, 31
, we chose to use unsupervised approaches for subclass discovery. We did so because a) the major distinctions in the molecular heterogeneity of GC might be unrelated to presently known classification systems or phenotypes, and b) using current classification systems, reproducibility among pathologists is only about 70%32, 33
and we were concerned that this lack of inter-observer concordance might compromise supervised analysis. Testing several different prediction algorithms, we confirmed that the intrinsic subtypes exhibited stable and reproducible classification performance in cell lines and primary tumors, thus demonstrating that the subtypes are statistically robust. Using a strict filtering criteria (FDR<0.002), we then identified a genomic classifier of 171 genes exhibiting differential regulation between the subtypes. Biological curation of the classifier confirmed that the intrinsic subtypes are associated with very different gene expression features, cellular processes and biological pathways. Taken collectively, these finding are consistent with the intrinsic subtypes being very distinct and possibly representing distinct lineages.
The clinical relevance of the intrinsic subclasses is supported by the finding that it can act as an independent predictor of clinical survival in multiple patient cohorts, even after controlling for tumor stage. In this regard, it is interesting to contrast the intrinsic subclasses against Lauren’s histological subtypes (intestinal and diffuse), to which the intrinsic classes are partially associated. Intestinal cancers are classically characterized by glandular differentiation on a background of gastric atrophy or intestinal metaplasia, while diffuse cancers typically appear as rows of single mononuclear “signet ring” cells with little cell adhesion. These apparently distinct features, however, are not always discernable in clinical samples where inter-observer variation and unclassifiable or “mixed” subtypes are not uncommonly reported. In our study, patients stratified by Lauren’s histopathology did not exhibit significantly different survival outcomes, while patients discordant between the intrinsic subclasses and Lauren’s exhibited survival patterns that support the intrinsic genomic taxonomy. Our data thus suggests that the intrinsic subclasses can provide information about the predominant lineage in GC samples that may not be precisely distinguished by morphology, and that this information is clinically relevant.
Besides gene expression, we also employed two genes in the classifier (LGALS4 and L1-Cadherin (CDH17)) as immunohistochemical markers for the G-INT intrinsic subtype. LGALS4 and CDH17 have been previously reported to be differentially regulated across subsets of gastric tumors14
and cell line34
, and expressed in intestinal metaplasia35, 36
. CDH17 was recently reported as a prognostic factor in early-stage GC37
, a marker of poor prognosis in another study36
, and a potential therapeutic target in experimental models38
. In our study, we specifically chose to compare the 2-marker positive group to the 2-marker negative group to confidently distinguish between the G-INT and G-DIF cancers. Interestingly, we have also observed that the one-third of 1-marker positive patients also appeared to exhibit an improved survival trend compared to the 2-marker negative group (CDH17, p=0.08 adjusted for stage; LGALS4, p=0.07 adjusted for stage). This result suggests that some of the 1-marker positive cancers may also be G-INT cancers as well (Supplementary Figure S4
). At this stage, it is likely premature to definitely conclude that LGALS4 and CDH17 necessarily represent the best markers for the G-INT subtype. Identifying the optimal combination of immunohistochemical markers to distinguish between the intrinsic subtypes should thus be a focus for future research.
G-INT lines were more sensitive to 5-FU and oxaliplatin than G-DIF cell lines, but were also more resistant to cisplatin. Although the absolute magnitude of these in vitro
differential sensitivities appears modest (3–5 fold), these differences could still prove clinically meaningful given the relatively small therapeutic windows associated with cytotoxic chemotherapy. Indeed, supporting the clinical relevance of these differences, we observed a significant interaction between the intrinsic subtypes and differential benefit from adjuvant 5-FU therapy in retrospective patient cohorts ( and Supplementary Table S4
). These findings suggest that in addition to patient prognosis, the intrinsic subtypes could potentially be used to guide treatment selection. However, we also emphasize that our results at this stage should be interpreted in the context of an early discovery study, requiring further validation efforts. For example, while we observed a trend of stage-adjusted survival benefit in G-INT patients due to 5-FU adjuvant therapy, this benefit was not statistically significant relative to surgery alone ( (combined, p=0.06) and Supp Table S4
(gene expression, p=0.18; TMA, p=0.11). There may be multiple reasons for this – compared to large Phase III trials such as INT-011639
, which evaluated adjuvant 5-FU based chemoradiation in GC, our patient cohorts are smaller in size, non-randomized, and being retrospective in nature, quality of surgery and treatment regimens were also not strictly enforced. As such, the interaction of intrinsic subclass with treatment response should definitely be further validated in additional studies (see below). Nevertheless, given the association the intrinsic subtypes with Lauren’s, it is worth noting that in INT-011640
, a ten-year update subgroup analyses revealed that all GC subsets benefited from 5-FU therapy except
for cases with diffuse histology. Moreover, in JCOG 991241
which established S-1 monotherapy as a first-line palliative chemotherapy option in Japan, benefit of irinotecan/cisplatin over 5-FU based monotherapy was observed in diffuse but not intestinal GCs. Our findings in this study are thus consistent with exploratory subgroup analysis of these two large GC clinical trials.
Our hypothesis-generating study requires further validation studies. Most immediately, the interaction between the intrinsic subtypes and treatment outcomes will be retrospectively validated in larger patient cohorts including tissue from a randomized phase 3 Japanese trial in the first line palliative treatment of advanced gastric cancer (PI: Akira Tsuburaya). Future prospective research efforts will focus on validating the predictive value of the GC intrinsic subclasses in the metastatic setting. In most countries, combination therapy with a fluropyrimidine/platinum combination therapy with or without epirubicin remains standard first line palliative chemotherapy(3)
. We have thus initiated a phase 2 study, Genomic Guided Gastric cancer (3G) trial with mandatory pre-treated tumor biopsies from which patients will be allocated to S1/oxaliplatin or S1/cisplatin based upon their genomic profiles (NCT01100801, http://clinicaltrialsfeeds.org
In conclusion, the quest for a clinically meaningful GC taxonomy has motivated several attempts to classify GC on the basis of clinical, histologic, and molecular features. With the exception of tumor stage however, few previously-proposed classification strategies have been shown to provide additional prognostic or predictive value. It is possible that the intrinsic subtypes reported here in this study may represent a promising step in establishment of a clinically relevant genomic taxonomy of GC.