The efficacy of angiogenesis inhibitors in cancer is limited by resistance mechanisms that are poorly understood. Notably, instead of inducing angiogenesis, some cancers vascularize by the non-angiogenic mechanism of vessel co-option. Here we show that vessel co-option is associated with a poor response to the anti-angiogenic agent bevacizumab in patients with colorectal cancer liver metastases. Moreover, we find that vessel co-option prevails in human breast cancer liver metastases, a setting where results with anti-angiogenic therapy have been disappointing. In our preclinical mechanistic studies, we show that cancer cell motility mediated by the Arp2/3 complex is required for vessel co-option in liver metastases in vivo and that combined inhibition of angiogenesis and vessel co-option is more effective than inhibiting angiogenesis alone in this setting. Vessel co-option is therefore a clinically relevant mechanism of resistance to anti-angiogenic therapy and combined inhibition of angiogenesis and vessel co-option may be a warranted therapeutic strategy.

Expression of endogenous markers of hypoxia for the HIF-1 and HIF-2 pathway is strongly associated with radiotherapy failure. Using immunohistochemical methods it is possible to identify subgroups of HNSCC patients who are highly curable with radiotherapy, or who are excellent candidates for clinical trials on hypoxia-targeting drugs in two distinct pathways.

Anti‐angiogenic therapies have shown limited efficacy in the clinical management of metastatic disease, including lung metastases. Moreover, the mechanisms via which tumours resist anti‐angiogenic therapies are poorly understood. Importantly, rather than utilizing angiogenesis, some metastases may instead incorporate pre‐existing vessels from surrounding tissue (vessel co‐option). As anti‐angiogenic therapies were designed to target only new blood vessel growth, vessel co‐option has been proposed as a mechanism that could drive resistance to anti‐angiogenic therapy. However, vessel co‐option has not been extensively studied in lung metastases, and its potential to mediate resistance to anti‐angiogenic therapy in lung metastases is not established. Here, we examined the mechanism of tumour vascularization in 164 human lung metastasis specimens (composed of breast, colorectal and renal cancer lung metastasis cases). We identified four distinct histopathological growth patterns (HGPs) of lung metastasis (alveolar, interstitial, perivascular cuffing, and pushing), each of which vascularized via a different mechanism. In the alveolar HGP, cancer cells invaded the alveolar air spaces, facilitating the co‐option of alveolar capillaries. In the interstitial HGP, cancer cells invaded the alveolar walls to co‐opt alveolar capillaries. In the perivascular cuffing HGP, cancer cells grew by co‐opting larger vessels of the lung. Only in the pushing HGP did the tumours vascularize by angiogenesis. Importantly, vessel co‐option occurred with high frequency, being present in >80% of the cases examined. Moreover, we provide evidence that vessel co‐option mediates resistance to the anti‐angiogenic drug sunitinib in preclinical lung metastasis models. Assuming that our interpretation of the data is correct, we conclude that vessel co‐option in lung metastases occurs through at least three distinct mechanisms, that vessel co‐option occurs frequently in lung metastases, and that vessel co‐option could mediate resistance to anti‐angiogenic therapy in lung metastases. Novel therapies designed to target both angiogenesis and vessel co‐option are therefore warranted. © 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Background: The anti-angiogenic Sorafenib is the only approved systemic therapy for advanced hepatocellular carcinoma (HCC). However, acquired resistance limits its efficacy. An emerging theory to explain intrinsic resistance to other anti-angiogenic drugs is ‘vessel co-option,’ ie, the ability of tumors to hijack the existing vasculature in organs such as the lungs or liver, thus limiting the need for sprouting angiogenesis. Vessel co-option has not been evaluated as a potential mechanism for acquired resistance to anti-angiogenic agents.Methods: To study sorafenib resistance mechanisms, we used an orthotopic human HCC model (n = 4-11 per group), where tumor cells are tagged with a secreted protein biomarker to monitor disease burden and response to therapy. Histopathology, vessel perfusion assessed by contrast-enhanced ultrasound, and miRNA sequencing and quantitative real-time polymerase chain reaction were used to monitor changes in tumor biology.Results: While sorafenib initially inhibited angiogenesis and stabilized tumor growth, no angiogenic ‘rebound’ effect was observed during development of resistance unless therapy was stopped. Instead, resistant tumors became more locally infiltrative, which facilitated extensive incorporation of liver parenchyma and the co-option of liver-associated vessels. Up to 75% (±10.9%) of total vessels were provided by vessel co-option in resistant tumors relative to 23.3% (±10.3%) in untreated controls. miRNA sequencing implicated pro-invasive signaling and epithelial-to-mesenchymal-like transition during resistance development while functional imaging further supported a shift from angiogenesis to vessel co-option.Conclusions: This is the first documentation of vessel co-option as a mechanism of acquired resistance to anti-angiogenic therapy and could have important implications including the potential therapeutic benefits of targeting vessel co-option in conjunction with vascular endothelial growth factor receptor signaling.

Glucose transporter-1 protein (GLUT1) and carbonic anhydrase IX (CAIX) are regulated by hypoxia inducible factor-1 (HIF-1) and have been studied as putative intrinsic cellular markers for hypoxia. This study directly compares CAIX and GLUT1 with pimonidazole binding in a prospective series of bladder cancer patients and also studies the prognostic significance of the markers, in combination with vascularity and proliferation, in a retrospective series of bladder cancer patients treated in a phase II trial of radical radiotherapy with carbogen and nicotinamide (ARCON). A total of 21 patients with a diagnosis of transitional cell carcinoma of the bladder received 0.5 g m À2 pimonidazole. Serial tumour sections were stained for pimonidazole, GLUT1 and CAIX and compared. Tissue sections obtained from a series of 64 patients previously treated for invasive bladder cancer using ARCON were stained for GLUT1 and CAIX together with Ki-67 and CD31/34. There was a good geographical colocalisation of both intrinsic markers with pimonidazole and a highly significant agreement in individual patients; correlation coefficients were 0.82 (P ¼ 0.0001) for GLUT1 and 0.74 (Po0.0001) for CAIX. In both series of patients, the intrinsic hypoxia markers were highly correlated with each other and a correlation with proliferation was also evident in the retrospective study. In univariate and multivariate analyses, GLUT1 and CAIX were independent predictors for overall and cause specific survival. The hypoxia markers did not predict for local control or metastases-free survival although higher Ki-67 indices showed a trend towards local failure. The data suggest that both hypoxia modification and accelerated treatment may be valid treatment options in bladder cancer.

This study indicates a key role for the EGFR receptor in determining the proliferative cellular response to fractionated radiotherapy in HNSCC. It also shows that we can select the dose-fractionation regime that has the greatest chance of benefiting the patient. These results also encourage further development of EGFR targeting combined with fractionated radiotherapy in HNSCC.

Triple negative breast cancer (TNBC) encompasses molecularly different subgroups, with a subgroup harboring evidence of defective homologous recombination (HR) DNA repair. Here, within a phase 2 window clinical trial, RIO trial (EudraCT 2014-003319-12), we investigate the activity of PARP inhibitors in 43 patients with untreated TNBC. The primary end point, decreased Ki67, occured in 12% of TNBC. In secondary end point analyses, HR deficiency was identified in 69% of TNBC with the mutational-signature-based HRDetect assay. Cancers with HRDetect mutational signatures of HR deficiency had a functional defect in HR, assessed by impaired RAD51 foci formation on end of treatment biopsy. Following rucaparib treatment there was no association of Ki67 change with HR deficiency. In contrast, early circulating tumor DNA dynamics identified activity of rucaparib, with end of treatment ctDNA levels suppressed by rucaparib in mutation-signature HR-deficient cancers. In ad hoc analysis, rucaparib induced expression of interferon response genes in HR-deficient cancers. The majority of TNBCs have a defect in DNA repair, identifiable by mutational signature analysis, that may be targetable with PARP inhibitors.

Primary systemic therapy (PST) for operable breast cancer enables the identification of in vivo biological markers that predict response to treatment. A total of 118 patients with T2 -4 N0 -1 M0 primary breast cancer received six cycles of anthracycline-based PST. Clinical and radiological response was assessed before and after treatment using UICC criteria. A grading system to score pathological response was devised. Diagnostic biopsies and postchemotherapy surgical specimens were stained for oestrogen (ER) and progesterone (PgR) receptor, HER-2 and cell proliferation (Ki-67). Clinical, radiological and pathological response rates were 78, 72 and 38%, respectively. There was a strong correlation between ER and PgR staining (Po0.0001). Higher Ki-67 proliferation indices were associated with PgRÀ tumours (median 28.3%, PgR þ 22.9%; P ¼ 0.042). There was no relationship between HER-2 and other biological markers. No single pretreatment or postchemotherapy biological parameter predicted response by any modality of assessment. In all, 10 tumours changed hormone receptor classification after chemotherapy (three ER, seven PgR); HER-2 staining changed in nine cases. Median Ki-67 index was 24.9% before and 18.1% after treatment (P ¼ 0.02); the median reduction in Ki-67 index after treatment was 21.2%. Tumours displaying 475% reduction in Ki-67 after chemotherapy were more likely to achieve a pathological response (77.8 vs 26.7%, P ¼ 0.004).