The Stromal and Immune Landscape of Colorectal Cancer Progression during Anti-EGFR Therapy
Authors: Irene Catalano, Livio Trusolino
Journal: Cancer Cell (2019)

In this issue of Cancer Cell, Woolston et al. show that colorectal cancers that become refractory to initially effective anti-EGFR therapy contain an abundance of stromal and immune cells, irrespective of the contextual presence of resistance-conferring mutations. This reconfiguration puts forward therapeutic opportunities for patients who relapse on EGFR-targeting treatment. In this issue of Cancer Cell, Woolston et al. show that colorectal cancers that become refractory to initially effective anti-EGFR therapy contain an abundance of stromal and immune cells, irrespective of the contextual presence of resistance-conferring mutations. This reconfiguration puts forward therapeutic opportunities for patients who relapse on EGFR-targeting treatment. The EGFR antibodies cetuximab and panitumumab are used in patients with EGFR-expressing, KRAS, or NRAS wild-type metastatic colorectal cancer (mCRC) either in combination with standard chemotherapy, for first-line treatment, or as single agents when tumors become resistant to prior cytotoxic regimens. However, only 20% of individuals experience tumor regressions, and only an additional 30% have some extent of clinical benefit in terms of disease stabilization (Douillard et al., 2013Douillard J.Y. Oliner K.S. Siena S. Tabernero J. Burkes R. Barugel M. Humblet Y. Bodoky G. Cunningham D. Jassem J. et al.Panitumumab-FOLFOX4 treatment and RAS mutations in colorectal cancer.N. Engl. J. Med. 2013; 369: 1023-1034Crossref PubMed Scopus (1694) Google Scholar). This relatively low response rate is compounded by the dismal reality that subjects who initially respond typically become refractory to treatment in a period of months. In this issue of Cancer Cell, Woolston et al., 2019Woolston A. Khan K. Spain G. Barber L.J. Griffiths B. Gonzalez-Exposito R. Hornsteiner L. Punta M. Patil Y. Newey A. et al.Genomic and transcriptomic determinants of therapy resistance and immune landscape evolution during anti-EGFR treatment in colorectal cancer.Cancer Cell. 2019; 35 (this issue): 35-50Abstract Full Text Full Text PDF Scopus (129) Google Scholar offer a comprehensive picture of the identifying traits of primary and acquired resistance to cetuximab in a cohort of 35 mCRC patients (Figure 1). In a difference from previous studies, mostly conducted in a retrospective manner and focused on a small number of candidate biomarkers, here the authors embarked on a prospective trial whereby biopsies collected before initiation of single-agent cetuximab and at the time of disease progression were subjected to whole-exome and RNA-sequencing analyses and immunophenotyping. Lack of response to antibody treatment ab initio has been partly ascribed to the occurrence of mutations or amplifications in genes encoding other tyrosine kinase receptors or RAS downstream effectors, which, similar to mutationally activated RAS, trigger compensatory pathways sustaining EGFR-independent tumor growth (Bertotti et al., 2015Bertotti A. Papp E. Jones S. Adleff V. Anagnostou V. Lupo B. Sausen M. Phallen J. Hruban C.A. Tokheim C. et al.The genomic landscape of response to EGFR blockade in colorectal cancer.Nature. 2015; 526: 263-267Crossref PubMed Scopus (323) Google Scholar). Since these genetic abnormalities occur individually at very low frequency, their catalog has not yet saturated the space of mCRC with primary resistance to EGFR antibodies. Indeed, when Woolston et al., 2019Woolston A. Khan K. Spain G. Barber L.J. Griffiths B. Gonzalez-Exposito R. Hornsteiner L. Punta M. Patil Y. Newey A. et al.Genomic and transcriptomic determinants of therapy resistance and immune landscape evolution during anti-EGFR treatment in colorectal cancer.Cancer Cell. 2019; 35 (this issue): 35-50Abstract Full Text Full Text PDF Scopus (129) Google Scholar stratified global genomic data onto response annotation, they pinpointed previously unrecognized alterations, including biallelic inactivation of NF1 (encoding a GTPase-activating protein that antagonizes RAS function) and KRAS and BRAF mutations endowed with attenuated enzymatic and transforming activity. Interestingly, hypomorphic BRAF and KRAS mutations co-existed in the same tumor or, when present singly, were associated with polysomy of the corresponding chromosome. This suggests that their individual contribution to cetuximab resistance is suboptimal and requires either a cooperative or a dosage effect for complete manifestation. Secondary resistance is often propelled by the clonal expansion of the same alterations responsible for primary resistance, with a preponderance of RAS pathway mutations. Such alterations may arise de novo on a stochastic basis, as a consequence of tumor genetic instability, or may pre-exist as minor subclones in the original tumor population because of genetic heterogeneity and become positively selected under drug pressure (Khan et al., 2018Khan K.H. Cunningham D. Werner B. Vlachogiannis G. Spiteri I. Heide T. Mateos J.F. Vatsiou A. Lampis A. Damavandi M.D. et al.Longitudinal liquid biopsy and mathematical modeling of clonal evolution forecast time to treatment failure in the PROSPECT-C phase II colorectal cancer clinical trial.Cancer Discov. 2018; 8: 1270-1285Crossref PubMed Scopus (141) Google Scholar). Genetic instability and heterogeneity explain why acquired resistance mutations are usually polyclonal and can be more accurately grasped by analysis of circulating tumor DNA (ctDNA)—which incorporates DNA fragments shed by the whole tumor—than by examination of solid biopsies—which, by definition, are subspatial snapshots of the entire lesion (Khan et al., 2018Khan K.H. Cunningham D. Werner B. Vlachogiannis G. Spiteri I. Heide T. Mateos J.F. Vatsiou A. Lampis A. Damavandi M.D. et al.Longitudinal liquid biopsy and mathematical modeling of clonal evolution forecast time to treatment failure in the PROSPECT-C phase II colorectal cancer clinical trial.Cancer Discov. 2018; 8: 1270-1285Crossref PubMed Scopus (141) Google Scholar). Woolston et al., 2019Woolston A. Khan K. Spain G. Barber L.J. Griffiths B. Gonzalez-Exposito R. Hornsteiner L. Punta M. Patil Y. Newey A. et al.Genomic and transcriptomic determinants of therapy resistance and immune landscape evolution during anti-EGFR treatment in colorectal cancer.Cancer Cell. 2019; 35 (this issue): 35-50Abstract Full Text Full Text PDF Scopus (129) Google Scholar report a number of genetic alterations of acquired resistance, including already-characterized mutations in components of the RAS pathway (Khan et al., 2018Khan K.H. Cunningham D. Werner B. Vlachogiannis G. Spiteri I. Heide T. Mateos J.F. Vatsiou A. Lampis A. Damavandi M.D. et al.Longitudinal liquid biopsy and mathematical modeling of clonal evolution forecast time to treatment failure in the PROSPECT-C phase II colorectal cancer clinical trial.Cancer Discov. 2018; 8: 1270-1285Crossref PubMed Scopus (141) Google Scholar) and a hitherto-unidentified amplification of FGF10 (encoding a ligand of the FGFR2 tyrosine kinase receptor). These genetic aberrations were detected in only a limited number of post-treatment biopsies but were mostly captured in ctDNA samples, further attesting to the pervasiveness of tissue-sampling bias. Of note, resistance mutations in ctDNA were calculated to occur in a minority of cells, in keeping with previous reports demonstrating the presence of recurrent but subclonal RAS pathway mutations in the blood of cetuximab-refractory mCRC patients (Bettegowda et al., 2014Bettegowda C. Sausen M. Leary R.J. Kinde I. Wang Y. Agrawal N. Bartlett B.R. Wang H. Luber B. Alani R.M. et al.Detection of circulating tumor DNA in early- and late-stage human malignancies.Sci. Transl. Med. 2014; 6: 224ra24Crossref PubMed Scopus (2991) Google Scholar, Khan et al., 2018Khan K.H. Cunningham D. Werner B. Vlachogiannis G. Spiteri I. Heide T. Mateos J.F. Vatsiou A. Lampis A. Damavandi M.D. et al.Longitudinal liquid biopsy and mathematical modeling of clonal evolution forecast time to treatment failure in the PROSPECT-C phase II colorectal cancer clinical trial.Cancer Discov. 2018; 8: 1270-1285Crossref PubMed Scopus (141) Google Scholar). Altogether, these findings suggest that tumor relapse is engendered by polyclonal mutuality, with an ecosystem of different subclones contributing to therapeutic resistance. However, we cannot exclude that when subclonal alterations are present at a very low allele frequency the impact on resistance may remain sub-threshold, and other (non-genetic) determinants could subsidize or substitute for DNA mutations to reduce responsiveness to EGFR inhibition. Woolston et al., 2019Woolston A. Khan K. Spain G. Barber L.J. Griffiths B. Gonzalez-Exposito R. Hornsteiner L. Punta M. Patil Y. Newey A. et al.Genomic and transcriptomic determinants of therapy resistance and immune landscape evolution during anti-EGFR treatment in colorectal cancer.Cancer Cell. 2019; 35 (this issue): 35-50Abstract Full Text Full Text PDF Scopus (129) Google Scholar strongly embrace the assumption that progression on cetuximab can be also fostered by non-mutational mechanisms and extend their investigation by delineating the transcriptomic profiles of matched sensitive and post-therapy resistant tumors. First, they confirm that a subgroup of tumors with gene expression traits reminiscent of those portrayed by the transient-amplifying precursors of the normal intestine (assigned to the so-called CMS2 consensus transcriptional subtype) were enriched for cetuximab-responsive cases. Then, the authors show that the majority of tumors with acquired resistance to cetuximab (including some harboring subclonal mutations) underwent a gene expression transition toward a stroma-rich (CMS4) phenotype featuring high content of carcinoma-associated fibroblasts (CAFs) and increased expression of CAF-derived growth factors such as TGF-β, HGF, and FGF family ligands. Consistent with the observed association between stromal abundance and drug resistance, the CAF secretome was found to exert a protective activity against cetuximab. These results highlight a key role for transcriptionally regulated growth factors in conveying survival cues that safeguard CRC tumors from the effects of EGFR blockade, in agreement with previous findings (Zanella et al., 2015Zanella E.R. Galimi F. Sassi F. Migliardi G. Cottino F. Leto S.M. Lupo B. Erriquez J. Isella C. Comoglio P.M. et al.IGF2 is an actionable target that identifies a distinct subpopulation of colorectal cancer patients with marginal response to anti-EGFR therapies.Sci. Transl. Med. 2015; 7: 272ra12Crossref PubMed Scopus (86) Google Scholar). Intriguingly, Woolston et al., 2019Woolston A. Khan K. Spain G. Barber L.J. Griffiths B. Gonzalez-Exposito R. Hornsteiner L. Punta M. Patil Y. Newey A. et al.Genomic and transcriptomic determinants of therapy resistance and immune landscape evolution during anti-EGFR treatment in colorectal cancer.Cancer Cell. 2019; 35 (this issue): 35-50Abstract Full Text Full Text PDF Scopus (129) Google Scholar describe a more copious representation of cytotoxic T lymphocytes and dendritic cells, increased expression of a T cell-associated inflammatory signature, and upregulation of immune checkpoints in CMS4-like, TGF-β-high resistant tumors. This result corroborates a retrospective study documenting heightened infiltration of cytotoxic, effector memory, and regulatory T cells in CRC tumors treated with cetuximab and chemotherapy (Van den Eynde et al., 2018Van den Eynde M. Mlecnik B. Bindea G. Fredriksen T. Church S.E. Lafontaine L. Haicheur N. Marliot F. Angelova M. Vasaturo A. et al.The link between the multiverse of immune microenvironments in metastases and the survival of colorectal cancer patients.Cancer Cell. 2018; 34: 1012-1026.e3Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). However, the coexistence of elevated TGF-β activity and immune inflammation is unexpected, as TGF-β is known to impair T cell function and promote T cell physical exclusion (Tauriello et al., 2018Tauriello D.V.F. Palomo-Ponce S. Stork D. Berenguer-Llergo A. Badia-Ramentol J. Iglesias M. Sevillano M. Ibiza S. Cañellas A. Hernando-Momblona X. et al.TGFβ drives immune evasion in genetically reconstituted colon cancer metastasis.Nature. 2018; 554: 538-543Crossref PubMed Scopus (998) Google Scholar). How can cetuximab-resistant tumors concomitantly display high levels of immune suppressive TGF-β and an active immune microenvironment? One possibility is that increased immune infiltration precedes the CMS2-to-CMS4 transition. This would be coherent with the notion that cetuximab triggers IgG1 antibody-mediated immunogenic cell death (Pozzi et al., 2016Pozzi C. Cuomo A. Spadoni I. Magni E. Silvola A. Conte A. Sigismund S. Ravenda P.S. Bonaldi T. Zampino M.G. et al.The EGFR-specific antibody cetuximab combined with chemotherapy triggers immunogenic cell death.Nat. Med. 2016; 22: 624-631Crossref PubMed Scopus (170) Google Scholar) and with the observation that EGFR pathway activity in lung cancer prompts immune escape, which is counteracted by EGFR inhibition (Akbay et al., 2013Akbay E.A. Koyama S. Carretero J. Altabef A. Tchaicha J.H. Christensen C.L. Mikse O.R. Cherniack A.D. Beauchamp E.M. Pugh T.J. et al.Activation of the PD-1 pathway contributes to immune escape in EGFR-driven lung tumors.Cancer Discov. 2013; 3: 1355-1363Crossref PubMed Scopus (955) Google Scholar). If this is the case, immune stimulation would be a direct consequence of productive cetuximab treatment rather than a hallmark of cetuximab resistance, and it would be interesting to see whether cetuximab-sensitive tumors at maximal response to EGFR blockade have already undergone the inflammatory shift shown by resistant tumors. One could even push this reasoning to the extreme: strengthened TGF-β activity might be a delayed adaptive mechanism to contrast cetuximab-induced immune cell deployment. In this scenario, immunotherapy is expected to synergize with cetuximab in the early phases of response rather than after the emergence of resistance. The application of genomic technologies has enabled the identification of clinically actionable DNA alterations in RAS wild-type mCRC tumors that fail or cease to respond to EGFR antibodies, including those illustrated in this study. By providing fresh evidence that acquired resistance to cetuximab also entails a stromagenic and immune-inflamed phenotype, results from Woolston et al., 2019Woolston A. Khan K. Spain G. Barber L.J. Griffiths B. Gonzalez-Exposito R. Hornsteiner L. Punta M. Patil Y. Newey A. et al.Genomic and transcriptomic determinants of therapy resistance and immune landscape evolution during anti-EGFR treatment in colorectal cancer.Cancer Cell. 2019; 35 (this issue): 35-50Abstract Full Text Full Text PDF Scopus (129) Google Scholar have important ramifications for the biological understanding of CRC evolution under EGFR blockade and introduce potential opportunities for targeting a novel repertoire of non-mutational vulnerabilities. L.T. is supported by Fondazione AIRC, Associazione Italiana per la Ricerca sul Cancro, Investigator Grant 18532; AIRC 5x1000 2018 program 21091; AIRC/CRUK/FC AECC Accelerator Award 22795; Transcan, TACTIC; European Union H2020 COLOSSUS; and Fondazione Piemontese per la Ricerca sul Cancro-ONLUS, 5x1000 Ministero della Salute 2011, 2014, 2015, and 2016. L.T. receives research grants from Symphogen, Servier, Pfizer, and Merus, and he is in the speakers' bureau of Eli Lilly, AstraZeneca, and Merck KGaA. Genomic and Transcriptomic Determinants of Therapy Resistance and Immune Landscape Evolution during Anti-EGFR Treatment in Colorectal CancerWoolston et al.Cancer CellJuly 08, 2019In BriefWoolston et al. show that in metastatic colorectal cancer cetuximab resistance can be conferred by genetic mechanisms, such as NF1 loss or RAS/RAF alterations, or by transcriptomic changes that induce a stroma-rich phenotype. They also provide a rationale for combining cetuximab with immunotherapy. 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