**4. Mechanism of resistance to targeted therapies**

Large advances in treatment results achieved with targeted therapies in mRCC are remarkable, but still between a third and two-thirds of patients with mRCC have tumors refractory to anti-VEGF and mTOR inhibitors from the beginning of treatment and all patients develop drug resistance and relapse some time during the course of their disease. Research of the mecha‐ nisms of resistance is very important in planning the development of new targeted agents [3, 23,24]. Most of information about drug resistance in mRCC known today is from the preclinical studies or studies on patients with different types of cancer, where targeted therapies are being in clinical practice for longer time (e.g. breast cancer). This is partially due to the rapid approval of targeted agents in mRCC which surpassed understanding of the mechanisms of response and resistance [3].

Until now two types of resistance to targeted therapy have been determined, so called intrinsic and extrinsic resistance [3].


to anti-VEGF antibody were associated with increase in infiltrating CD11b + GR1 + myeloid

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The proposed mechanisms of resistance to inhibitors of mammalian target of rapamycin (mTOR) include the presence of redundant signaling pathways, presence of KRAS or BRAF mutations, loss of phosphatase and tension homologue deleted on chromosome ten (PTEN),

Intrinsic resistance to anti-angiogenic factors and mTOR inhibitors is widespread and leads to poor patient outcome. Alternative pathways should be considered in this patients such as targeting RAF and MEK or PI3K/AKT. Including patients with resistant tumors in clinical trials

All patients who initially have clinical benefit of targeted therapy eventually develop resistance to it and experience disease progression. This resistance, named extrinsic resist‐ ance (also known as secondary, evasive, acquired or adaptive resistance) has been ex‐ plained more in detail [23]. TKI and VEGF inhibitors both target components of VEGF signaling pathway. Thus the mechanisms involved will affect any of these targeted agents. Extrinsic resistance results from the acquisition of adaptive mechanisms to the action of angiogenesis inhibitors which ultimately results in evasion of the angiogenesis and reemer‐

Sprouting of new vessels has been detected in Xenograft RCC tumors resected shortly after the start of sunitinib. The development of resistance is constantly preceded by restoration of blood flow, which suggests that new vasculature is less dependent (but not necessary inde‐

Different pro-angiogenic factors involved in the mechanism of resistance to targeted agents have been recognized. In a mouse model of pancreatic neuroendocrine cancer, resistant tumors expressed high levels of FGF 1, 2, ephrin A1, angiopoetin and interleukin-8 [23,25]. Inhibition

Interleukin-8 (IL-8) is a potent pro-angiogenic factor. Up-regulation of IL-8 plays an important role in RCC resistance. In a xenograft model of RCC mimicking clinical resistance to sunitinib, increased IL-8 secretion from tumors was associated with reactivation of tumor angiogenesis and administration of IL-8 neutralizing antibody lead to re-sensitization to sunitinib. Elevated IL-8 expression was also found in patients with tumors who did not respond to sunitinib from the beginning [5,22]. IL-angiogenic signaling may functionally compensate for the inhibition

Angiopoetin 2 (Ang-2) is a plasma glycoprotein involved in angiogenesis and cancer neovas‐ cularization. It is thought to have a role in development of the resistance. Levels of Ang-2 decrease after the initiation of sunitinib treatment and increase after the resistance occurs [23].

of these proteins was shown to inhibit tumor growth of resistant RCC-s [25].

testing these new agents that target these pathways is strongly recommended [23].

cells, which expressed several pro-angiogenic factors [23].

**4.2. Extrinsic resistance**

pendent) of VEGF [25].

gence of tumor-related vasculature [3,25].

*4.2.1. Up-regulation of pro-angiogenic factors*

of VEGF/VEGFR-mediated angiogenesis [5].

low cellular levels of p27 or 4E-bp1 and overexpression of eIF4E [3].

**Table 2.** Phase III trials of targeted agents in mRCC

#### **4.1. Intrinsic resistance**

Intrinsic resistance (primary resistance) occurs when tumor does not respond to the targeted therapy from the beginning of the treatment. Lack of the clinical benefit, even a short-lasting one is observed in these patients. Roughly 25% of patients are resistant to therapy; no response is detected on first evaluation after 2-3 months [23]. This type of resistance has not been explained entirely jet.

In the case of the resistance to VEGF inhibitors and TKI-s pre-existing pro-angiogenic factors, such as fibroblast growth factor-2 promote tumor angiogenesis. Pre-existence of pro-angio‐ genic factors compensate for the inhibition of VEGF signaling and thus allow angiogenesis to continue [3,23]. Pre-existing inflammatory cells may also contribute to the angiogenesis by expressing pro-angiogenic factors. In pre-clinical trials mRCC tumors that were not responsive to anti-VEGF antibody were associated with increase in infiltrating CD11b + GR1 + myeloid cells, which expressed several pro-angiogenic factors [23].

The proposed mechanisms of resistance to inhibitors of mammalian target of rapamycin (mTOR) include the presence of redundant signaling pathways, presence of KRAS or BRAF mutations, loss of phosphatase and tension homologue deleted on chromosome ten (PTEN), low cellular levels of p27 or 4E-bp1 and overexpression of eIF4E [3].

Intrinsic resistance to anti-angiogenic factors and mTOR inhibitors is widespread and leads to poor patient outcome. Alternative pathways should be considered in this patients such as targeting RAF and MEK or PI3K/AKT. Including patients with resistant tumors in clinical trials testing these new agents that target these pathways is strongly recommended [23].

### **4.2. Extrinsic resistance**

All patients who initially have clinical benefit of targeted therapy eventually develop resistance to it and experience disease progression. This resistance, named extrinsic resist‐ ance (also known as secondary, evasive, acquired or adaptive resistance) has been ex‐ plained more in detail [23]. TKI and VEGF inhibitors both target components of VEGF signaling pathway. Thus the mechanisms involved will affect any of these targeted agents. Extrinsic resistance results from the acquisition of adaptive mechanisms to the action of angiogenesis inhibitors which ultimately results in evasion of the angiogenesis and reemer‐ gence of tumor-related vasculature [3,25].

Sprouting of new vessels has been detected in Xenograft RCC tumors resected shortly after the start of sunitinib. The development of resistance is constantly preceded by restoration of blood flow, which suggests that new vasculature is less dependent (but not necessary inde‐ pendent) of VEGF [25].

### *4.2.1. Up-regulation of pro-angiogenic factors*

**4.1. Intrinsic resistance**

**Objective Response Rate**

31% Sunitinib 6% IFN

3% Placebo

31% Bev+IFN 13% Placebo+IFN

10% Sorafenib 2% Placebo

19% Axitinib 9% Sorafenib

1% Everolimus 0% Placebo

8.6% Tem 4.8% IFN 8.1% Tem+IFN

**Table 2.** Phase III trials of targeted agents in mRCC

**Pazopanib** 30% Pazopanib

**Sunitninib**

194 Renal Tumor

**Bevacisumab**

**Sorafenib**

**Axitinib**

**Everolimus**

**Temsirolimus**

**Progression Free Survival**

11 months Sunitinib 5 months IFN P<0.0001

9.2 months Pazopanib 4.2 months Placebo

10.2 months Bev+IFN 5.4 months Placebo+IFN

5.5 months Sorafenib 2.8 months Placebo

6.7 months Axitinib 4.7 months Sorafenib

4.9 months Everolimus 1.9 months Placebo

P<0.0001

P<0,0001

P<0,01

P<0.001

P<0,0001

and IFN)

3.7 months Tem 1.9 months IFN 3.7 months Tem+IFN P=0.0001 (between Tem **Overall Survival**

26.4 months Sunitinib 21.8 months IFN P=0.051

21.1 months Pazopanib 18.7 months Placebo

23.3 months Bev+IFN 21.3 months Placebo

17.8 months Sorafenib 15.2 months Placebo

14.8 months Everolimus 14.4 months Placebo

+IFN P=0.13

P=0.15

P=0.177

and IFN)

10.9 months Tem 7.3 months IFN 8.4 months Tem+IFN P=0.08 (between Tem

Not reported

**Most Common Adverse Events of Experimental**

Diarrhea, fatique, nausea, stomatitis, vomiting

Diarrhea, hypertension, hair color change, nausea,

Fatique, pyrexia, anorexia, bleeding, asthenia

Diarrhea, rash, fatigue, hand-foot syndrome

Diarrhea, hypertension, fatigue, decreased apetite,

Stomatitis, rash, fatigue, asthenia, diarrhea

Asthenia, rash, anemia, nausea, anorexia

**Drug**

anorexia

nausea

explained entirely jet.

Intrinsic resistance (primary resistance) occurs when tumor does not respond to the targeted therapy from the beginning of the treatment. Lack of the clinical benefit, even a short-lasting one is observed in these patients. Roughly 25% of patients are resistant to therapy; no response is detected on first evaluation after 2-3 months [23]. This type of resistance has not been

In the case of the resistance to VEGF inhibitors and TKI-s pre-existing pro-angiogenic factors, such as fibroblast growth factor-2 promote tumor angiogenesis. Pre-existence of pro-angio‐ genic factors compensate for the inhibition of VEGF signaling and thus allow angiogenesis to continue [3,23]. Pre-existing inflammatory cells may also contribute to the angiogenesis by expressing pro-angiogenic factors. In pre-clinical trials mRCC tumors that were not responsive

Different pro-angiogenic factors involved in the mechanism of resistance to targeted agents have been recognized. In a mouse model of pancreatic neuroendocrine cancer, resistant tumors expressed high levels of FGF 1, 2, ephrin A1, angiopoetin and interleukin-8 [23,25]. Inhibition of these proteins was shown to inhibit tumor growth of resistant RCC-s [25].

Interleukin-8 (IL-8) is a potent pro-angiogenic factor. Up-regulation of IL-8 plays an important role in RCC resistance. In a xenograft model of RCC mimicking clinical resistance to sunitinib, increased IL-8 secretion from tumors was associated with reactivation of tumor angiogenesis and administration of IL-8 neutralizing antibody lead to re-sensitization to sunitinib. Elevated IL-8 expression was also found in patients with tumors who did not respond to sunitinib from the beginning [5,22]. IL-angiogenic signaling may functionally compensate for the inhibition of VEGF/VEGFR-mediated angiogenesis [5].

Angiopoetin 2 (Ang-2) is a plasma glycoprotein involved in angiogenesis and cancer neovas‐ cularization. It is thought to have a role in development of the resistance. Levels of Ang-2 decrease after the initiation of sunitinib treatment and increase after the resistance occurs [23]. Sphingosine kinase (S1P) is also supposed to play a role in the resistance. S1P is an enzyme that catalyzes the formation of sphingosine-1-phosphate which is associated with cell prolif‐ eration, survival and angiogenesis. Plasma levels of S1P decrease after the start of sunitinib treatment and increase again upon the development of resistance. In pre-clinical models administering neutralizing antibodies against S1P to mice, delayed the growth of sunitinibresistant tumors [23].

concluded that reversible changes in gene expression within the tumor cells and/or their microenvironment could be the possible mechanism of reversible resistance. He implanted sorafenib-resistant RCC into mice and after implantation tumors regained the sensitivity to

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In the case of sunitinib, the proposed mechanism of resistance is the activation or up-regulation of alternative angiogenic signals (e.g. FGF-s, ephrins, andiopoetins) while in the case of sorafenib this mechanism seem to be recruitment of pro-angiogenic bone marrow-derived cells and monocytes. Recruitment of perycites that help to maintain vessels permeable and func‐ tional and prevent endothelial cells from being affected by antiangiogenic therapies, is the proposed mechanisms of resistance to pasopanib. In the case of bevacisumab resistance the increased potential of tumor cells to invade without the need of neovascularization is supposed

Overcoming the resistance to first line therapy is one of the aims of administering the second line and beyond. Several factors play important role in selection of second line strategy: clinical

Sequential use of targeted agents is currently the standard of care for mRCC patients. This approach enables patients to get most benefit from these agents avoiding the excessive toxicity associated with combination therapy [26-28]. Targeted agent in the second line can have the same or different mechanism of action as first-line one. Limited data suggest that the use of a TKI after the failure of another TKI is reasonable and that there is not complete cross-resistance of these agents. The hypothesis behind this is that although TKIs share the same mechanism of action, their molecular targets are different. Despite this, the evidence of this approach is not strong; prospective, phase III trials are missing. Changing mechanism of action can have several advantages: greater chance of overcoming resistance while decreasing the probability of cumulative toxicity [4,25]. Toxicities of TKI-s and mTOR inhibitors for example, differ considerably. Frequent grade 3 toxicities encountered in patients on TKI-s are hand-foot syndrome, diarrhoea, fatigue, hypertension, neutropenia and leukopenia, while grade 3 toxicities in patients treated with mTOR inhibitors are rash, stomatitis, pneumonitis, anemia

The almost historical treatment strategy where changing of mechanism of action proved to be effective was TKI-s following cytokines. Currently this approach is not of clinical use anymore, because most of the patients get molecular targeted agent in the first line; however it is likely that some patients will have been treated with a cytokine previously. Phase III trials demon‐

sorafenib [4,25].

to be the mechanism [4,9,25].

and infection [25-30].

**5.1. TKI-s following cytokine therapy**

**5. Overcoming the resistance**

*4.2.7. Mechanism of resistance to different targeted agents*

evidence, toxicity issues and individual patient profile [4,25,26].

### *4.2.2. Down-regulation of angiostatic factors*

Down-regulation of angiostatic factors is another mechanism of resistance to TKI-s. Treatment with sunitinib and sorafenib results in the increased expression of several IFN-inducible genes including the angiostatic chemokines CXCL 10 and CXCL 11 and tumor suppressor genes. Following the development of resistance, the expression of IFN-γ and several of IFN-inducible genes is reduced. Down regulation of these factors is associated with the development of resistance to sunitinib and sorafenib [23].

### *4.2.3. Recruitment of bone marrow-derived cells*

Recruitment of bone marrow-derived cells which can result in the development of new blood vessels is another possible mechanism of resistance. In pre-clinical studies recruitment of CD11b + GR1 + myeloid cells cells resulted in resistance development. There is also evidence that tumor vasculature can be protected from anti-angiogenic therapy by increased pericyte coverage [23].

### *4.2.4. Development of invasion without angiogenesis*

Invasion of tumor in normal tissue and recruitment of normal tissue vasculature protect the tumors from anti-angiogenic therapy. It has been reported that the tumor of a patient experi‐ encing disease progression during antiangiogenic therapy had invaded the surrounding tissue and there had been increase of the vascularization from the normal tissue to the center of the tumor [23].

### *4.2.5. Resistance to m-TOR inhibitors*

Resistance to mTOR inhibitors is far less explained. It is supposed to be the result of activation of feedback loops that promote the activation of molecular signaling pathways of survival, increased activity of mTOR-complex 2, up-regulation of insulin-like growth factor and increase in the ERK/MAPK pathway signaling [4,24,25].

### *4.2.6. Reversible resistance*

Preclinical studies revealed that resistance to VEGF targeted therapies can be reversible. Hammers and colleagues grafted skin metastases of mRCC patient who had become resistant to sunitinib into mice and these xenogafts regained sensitivity and responded to sunitinib. Histology of original skin metastasis and xenograft revealed that a reversible epithelial-tomesenchymal transition could be responsible for acquired resistance to sunitinib. Zhang concluded that reversible changes in gene expression within the tumor cells and/or their microenvironment could be the possible mechanism of reversible resistance. He implanted sorafenib-resistant RCC into mice and after implantation tumors regained the sensitivity to sorafenib [4,25].

### *4.2.7. Mechanism of resistance to different targeted agents*

In the case of sunitinib, the proposed mechanism of resistance is the activation or up-regulation of alternative angiogenic signals (e.g. FGF-s, ephrins, andiopoetins) while in the case of sorafenib this mechanism seem to be recruitment of pro-angiogenic bone marrow-derived cells and monocytes. Recruitment of perycites that help to maintain vessels permeable and func‐ tional and prevent endothelial cells from being affected by antiangiogenic therapies, is the proposed mechanisms of resistance to pasopanib. In the case of bevacisumab resistance the increased potential of tumor cells to invade without the need of neovascularization is supposed to be the mechanism [4,9,25].
