Converging and evolving immuno-genomic routes toward immune escape in breast cancer.

The interactions between tumor and immune cells along the course of breast cancer progression remain largely unknown. Here, we extensively characterize multiple sequential and parallel multiregion tumor and blood specimens of an index patient and a cohort of metastatic triple-negative breast cancers. We demonstrate that a continuous increase in tumor genomic heterogeneity and distinct molecular clocks correlated with resistance to treatment, eventually allowing tumors to escape from immune control. TCR repertoire loses diversity over time, leading to convergent evolution as breast cancer progresses. Although mixed populations of effector memory and cytotoxic single T cells coexist in the peripheral blood, defects in the antigen presentation machinery coupled with subdued T cell recruitment into metastases are observed, indicating a potent immune avoidance microenvironment not compatible with an effective antitumor response in lethal metastatic disease. Our results demonstrate that the immune responses against cancer are not static, but rather follow dynamic processes that match cancer genomic progression, illustrating the complex nature of tumor and immune cell interactions.

Extramedullary multiple myeloma patient-derived orthotopic xenograft with a highly altered genome: combined molecular and therapeutic studies.

Extramedullary multiple myeloma (EMM) has an overall survival of 6 months and occurs in 20% of multiple myeloma (MM) patients. Genetic and epigenetic mechanisms involved in EMM and the therapeutic role of new agents for MM are not well established. Besides, well-characterized preclinical models for EMM are not available. Herein, a patient-derived orthotopic xenograft (PDOX) was generated from a patient with an aggressive EMM to study in-depth genetic and epigenetic events, and drug responses related to extramedullary disease. A fresh punch of an extramedullary cutaneous lesion was orthotopically implanted in NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ(NSG) mouse. The PDOX mimicked histologic and phenotypic features of the tumor of the patient. Cytogenetic studies revealed a hyperploid genome with multiple genetic poor-prognosis alterations. Copy number alterations (CNAs) were detected in all chromosomes. The IGH translocation t(14;16)(q32;q23)IGH/MAF was already observed at the medullary stage and a new one, t(10;14)(p?11-12;q32), was observed only with extramedullary disease and could be eventually related to EMM progression in this case. Exome sequencing showed 24 high impact single nucleotide variants and 180 indels. From the genes involved, only TP53 was previously described as a driver in MM. A rather balanced proportion of hyper/hypomethylated sites different to previously reported widespread hypomethylation in MM was also observed. Treatment with lenalidomide, dexamethasone and carfilzomib showed a tumor weight reduction of 90% versus non-treated tumors, whereas treatment with the anti-CD38 antibody daratumumab showed a reduction of 46%. The generation of PDOX from a small EMM biopsy allowed us to investigate in depth the molecular events associated with extramedullary disease in combination with drug testing.

The temporal mutational and immune tumour microenvironment remodelling of HER2-negative primary breast cancers.

The biology of breast cancer response to neoadjuvant therapy is underrepresented in the literature and provides a window-of-opportunity to explore the genomic and microenvironment modulation of tumours exposed to therapy. Here, we characterised the mutational, gene expression, pathway enrichment and tumour-infiltrating lymphocytes (TILs) dynamics across different timepoints of 35 HER2-negative primary breast cancer patients receiving neoadjuvant eribulin therapy (SOLTI-1007 NEOERIBULIN-NCT01669252). Whole-exome data (N = 88 samples) generated mutational profiles and candidate neoantigens and were analysed along with RNA-Nanostring 545-gene expression (N = 96 samples) and stromal TILs (N = 105 samples). Tumour mutation burden varied across patients at baseline but not across the sampling timepoints for each patient. Mutational signatures were not always conserved across tumours. There was a trend towards higher odds of response and less hazard to relapse when the percentage of subclonal mutations was low, suggesting that more homogenous tumours might have better responses to neoadjuvant therapy. Few driver mutations (5.1%) generated putative neoantigens. Mutation and neoantigen load were positively correlated (R2 = 0.94, p = <0.001); neoantigen load was weakly correlated with stromal TILs (R2 = 0.16, p = 0.02). An enrichment in pathways linked to immune infiltration and reduced programmed cell death expression were seen after 12 weeks of eribulin in good responders. VEGF was downregulated over time in the good responder group and FABP5, an inductor of epithelial mesenchymal transition (EMT), was upregulated in cases that recurred (p < 0.05). Mutational heterogeneity, subclonal architecture and the improvement of immune microenvironment along with remodelling of hypoxia and EMT may influence the response to neoadjuvant treatment.

Noncanonical TGFß Pathway Relieves the Blockade of IL1ß/TGFß-Mediated Crosstalk between Tumor and Stroma: TGFBR1 and TAK1 Inhibition in Colorectal Cancer.

PURPOSE: The aim of the study is blocking the recruitment of a protective stroma by altering the crosstalk between normal stromal cells and tumor cells for stripping tumors of the protection conferred by the microenvironment. EXPERIMENTAL DESIGN: A transcriptomic analysis of cocultured normal colonic fibroblasts and colorectal tumor cells was performed. We focused on the study of molecules that mediate the communication between both compartments and that entail fibroblasts' activation and the alteration of the sensitivity to chemotherapy. We identified targets for the blocking of the tumor-stroma interaction. Finally, we tested, in vivo, the blockade of the tumor-stroma interaction in orthotopic models derived from patients and in models of acquired resistance to oxaliplatin. RESULTS: IL1ß/TGFß1 are the triggers for fibroblasts' recruitment and conversion into carcinoma-associated fibroblasts (CAF) in colorectal cancer. CAFs then secrete proinflammatory factors that alter sensitivity in tumor cells, activating JAK/STAT and PI3KCA/AKT pathways. Blocking such crosstalk with a neutralizing IL1ß antibody and a TGFBR1 inhibitor is relieved by the TAK1-mediated activation of the noncanonical TGFß pathway, which induces a change in the cytokine/chemokine repertoire that maintains a sustained activation of AKT in tumor cells. TAK1 plus TGFBR1 inhibition blocks IL1ß/TGFß1-mediated fibroblast activation, decreasing the secretion of proinflammatory cytokines. In turn, tumor cells became more sensitive to chemotherapy. In vivo, the combination of a TAK1 inhibitor plus TGFBR1 inhibitor reduced the metastatic capacity of tumor cells and the recruitment of fibroblasts. CONCLUSIONS: Our findings provide a translational rationale for the inhibition of TAK1 and TGFBR1 to remove the chemoprotection conferred by CAFs.

SHP2 is required for growth of KRAS-mutant non-small-cell lung cancer in vivo.

RAS mutations are frequent in human cancer, especially in pancreatic, colorectal and non-small-cell lung cancers (NSCLCs)1-3. Inhibition of the RAS oncoproteins has proven difficult4, and attempts to target downstream effectors5-7 have been hampered by the activation of compensatory resistance mechanisms8. It is also well established that KRAS-mutant tumors are insensitive to inhibition of upstream growth factor receptor signaling. Thus, epidermal growth factor receptor antibody therapy is only effective in KRAS wild-type colon cancers9,10. Consistently, inhibition of SHP2 (also known as PTPN11), which links receptor tyrosine kinase signaling to the RAS-RAF-MEK-ERK pathway11,12, was shown to be ineffective in KRAS-mutant or BRAF-mutant cancer cell lines13. Our data also indicate that SHP2 inhibition in KRAS-mutant NSCLC cells under normal cell culture conditions has little effect. By contrast, SHP2 inhibition under growth factor-limiting conditions in vitro results in a senescence response. In vivo, inhibition of SHP2 in KRAS-mutant NSCLC also provokes a senescence response, which is exacerbated by MEK inhibition. Our data identify SHP2 inhibition as an unexpected vulnerability of KRAS-mutant NSCLC cells that remains undetected in cell culture and can be exploited therapeutically.

Carcinoma-associated fibroblasts affect sensitivity to oxaliplatin and 5FU in colorectal cancer cells.

The importance of tumor microenvironment (TME) as a relevant contributor to cancer progression and its role in the development of de novo resistance to targeted therapies has become increasingly apparent. However, the mechanisms of microenvironment-mediated drug resistance for nonspecific conventional chemotherapeutic agents, such as platinum compounds or antimetabolites, are still unclear.Here we describe a mechanism induced by soluble factors released by carcinoma-associated fibroblasts (CAFs) that induce the translocation of AKT, Survivin and P38 to the nucleus of tumor cells. These changes are guided to ensure DNA repair and the correct entrance and exit from mitosis in the presence of chemotherapy. We used conditioned media (CM) from normal-colonic fibroblasts and paired CAFs to assess dose response curves of oxaliplatin and 5-fluorouracil, separately or combined, compared with standard culture medium. We also evaluated a colony-forming assay and cell death to demonstrate the protective role of CAF-CM. Immunofluorescence confirmed the translocation of AKT, P38 and Survivin to the nucleus induced by CAF-soluble factors. We also have shown that STAT3 or P38 inhibition provides a promising strategy for overcoming microenvironment-mediated resistance. Conversely, pharmacologic AKT inhibition induces an antagonistic effect that relieves a cMET and STAT3-mediated compensatory feedback that might explain the failure of AKT inhibitors in the clinic so far.

A monotonic and prognostic genomic signature from fibroblasts for colorectal cancer initiation, progression, and metastasis.

UNLABELLED: The differential gene expression patterns between normal colonic fibroblasts (NCF), carcinoma-associated fibroblasts from primary tumors (CAF-PT), and CAFs from hepatic metastasis (CAF-LM) are hypothesized to be useful for predicting relapse in primary tumors. A transcriptomic profile of NCF (n = 9), CAF-PT (n = 14), and CAF-LM (n = 11) was derived. Prediction Analysis of Microarrays (PAM) was used to obtain molecular details for each fibroblast class, and differentially expressed transcripts were used to classify patients according to recurrence status. A number of transcripts (n = 277) were common to all three types of fibroblasts and whose expression level was sequentially deregulated according to the transition: NCF→CAF-PT→CAF-LM. Importantly, the gene signature was able to accurately classify patients with primary tumors according to their prognosis. This capacity was exploited to obtain a refined 19-gene classifier that predicted recurrence with high accuracy in two independent datasets of patients with colorectal cancer and correlates with fibroblast migratory potential. The prognostic power of this genomic signature is strong evidence of the link between the tumor-stroma microenvironment and cancer progression. Furthermore, the 19-gene classifier was able to identify low-risk patients very accurately, which is of particular importance for stage II patients, who would benefit from the omission of chemotherapy, especially T4N0 patients, who are clinically classified as being at high risk. IMPLICATIONS: A defined stromal gene expression signature predicts relapse in patients with colorectal cancer.