Advanced malignancies treated with a combination of the VEGF inhibitor bevacizumab, anti-EGFR antibody cetuximab, and the mTOR inhibitor temsirolimus

BACKGROUND Bevacizumab and temsirolimus are active agents in advanced solid tumors. Temsirolimus inhibits mTOR in the PI3 kinase/AKT/mTOR pathway as well as CYP2A, which may be a resistance mechanism for cetuximab. In addition, temsirolimus attenuates upregulation of HIF-1α levels, which may be a resistance mechanism for bevacizumab. RESULTS The median age of patients was 60 years (range, 23-80 years). The median number of prior systemic therapies was 3 (range, 1-6). The maximum tolerated dose (MTD) was determined to be bevacizumab 10 mg/kg biweekly, temsirolimus 5 mg weekly and cetuximab 100/75 mg/m2 weekly. Grade 3 or 4 toxicities were seen in 52% of patients with the highest prevalence being hyperglycemia (14%) and hypophosphatemia (14%). Eighteen of the 21 patients were evaluable for response. Three patients were taken off the study before restaging for toxicities. Partial response (PR) was observed in 2/18 patients (11%) and stable disease (SD) lasting ≥ 6 months was observed in 4/18 patients (22%) (total = 6/18 (33%)). In 8 evaluable patients with squamous cell carcinoma of the head and neck (HNSCC) there were partial responses in 2/8 (25%) patients and SD ≥ 6 months in 1/8 (13%) patients (total = 3/8, (38%)). PATIENTS AND METHODS We analyzed safety and responses in 21 patients with advanced solid tumors treated with bevacizumab, cetuximab, and temsirolimus. CONCLUSION The combination of bevacizumab, cetuximab, and temsirolimus showed activity in HNSCC; however, there were numerous toxicities reported, which will require careful management for future clinical development.


Protocol 2012-0061
October 10, 2014 Page 2 Additionally it has been shown in preclinical models that inhibition of mTOR pathway by everolimus cooperates with EGFR inhibitors in human tumors sensitive and resistant to anti-EGFR drugs. 18 Multi-kinase targeting of the EGFR, VEGFR and mTOR may improve results with either agent alone. Thus, one attractive approach is to combine EGFR, VEGFR and mTOR inhibitors targeting downstream pathways.
Therefore combining a histone deacetylase inhibitor (valproic acid) to a VEGF inhibitor (bevacizumab) and an mTOR inhibitor (temsirolimus) targets multiple pathways in tumor progression and angiogenesis, representing a novel therapeutic approach in cancer treatment.
The next step is to determine how to combine these targeted agents safely, to establish appropriate dose and schedule for Phase II efficacy studies, and to provide preliminary data on target impact. Because these targeted agents have mostly non-overlapping toxicities, they may be amenable to escalation to full doses in combination. Angiogenesis has a fundamental role in tumor growth and metastasis. 7,19,20 Antiangiogenic agents, such as the monoclonal antibody bevacizumab, target the vascular endothelial growth factor (VEGF) pathway and have demonstrated clinical benefit for a variety of malignancies, including colorectal, lung, breast, ovarian, and renal cell cancer. 6,7,[21][22][23][24][25] Despite this progress, the biologic activity of these agents may be difficult to assess because they appear to be primarily cytostatic rather than cytotoxic, and when they are used as monotherapy, only a minority of patients have a major tumor response. 19,26 To advance the clinical testing of these agents, valid surrogate biomarkers of angiogenic activity are needed. 26 Such biomarkers would measure the agents' biological effects, determine the optimal dose, identify which patients are most likely to benefit from treatment, and monitor responses during treatment.
Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) is a noninvasive imaging technology that can be used to measure properties of tissue microvasculature. 27 DCE-MRI is sensitive to changes in blood volume and vascular permeability that can be associated with tumor angiogenesis, and consequently DCE-MRI is a promising biomarker for characterizing tumor response to antiangiogenic treatment. [27][28][29][30] Correlative studies performed in combination with therapeutic trials have demonstrated proof of concept for DCE-MRI as a biomarker. 27 One mechanism of tumor resistance to antiangiogenic therapy is upregulation of hypoxiainducible factor 1α (HIF-1 α), which mediates adaptive responses to hypoxic conditions. 3-11 HIF-1α inhibition in combination with antiangiogenic therapy is a promising strategy for targeting tumor resistance. 8,[12][13][14][15] Temsirolimus, a mammalian target of rapamycin (mTOR) inhibitor, has demonstrated the ability to inhibit VEGF production in vitro under both normoxic and hypoxic conditions through inhibition of hypoxia-stimulated hypoxia-inducible (HIF)-1α expression and transcriptional activation. 31 Combination treatment with bevacizumab and temsirolimus is therefore a potentially effective treatment deserving of clinical investigation.

Angiogenesis and Bevacizumab in the Treatment of Cancer
Angiogenesis, now a well-established aspect of cancer biology, is important for supplying a growing tumor with oxygen, nutrients, growth factors, hormones, proteolytic enzymes, and hemolytic factors, and is a critical step in the pathogenesis of metastasis. 7,20,32 Increased tumor vascularization and tumor expression of pro-angiogenic factors has been associated with advanced tumor stage and poor prognosis. 7 The vascular endothelial growth factor (VEGF) family of proteins and receptors play Protocol 2012-0061 October 10, 2014 Page 4 a pivotal role in tumor angiogenesis and in the pathogenesis of a wide range of human cancers. 33 Consequently, agents that inhibit the VEGF pathway, such as bevacizumab, have generated substantial interest in the treatment of malignancy.
Bevacizumab is a recombinant, humanized, anti-VEGF monoclonal antibody developed from a murine antibody to human VEGF by recombinant DNA technology and was selected for clinical development based on preclinical evidence showing high antiangiogenic and antitumor activity. 6,34 Phase I clinical trials demonstrated that bevacizumab was relatively non-toxic and that adding bevacizumab to standard chemotherapy regimens did not significantly exacerbate chemotherapyassociated toxicities. 35 When bevacizumab was approved by the FDA as a first-line treatment for metastatic colorectal cancer in February 2004, it became the first approval by the FDA of a therapy developed to target tumor angiogenesis. 36 Further clinical trials demonstrated clinical benefit in other malignancies, including lung, breast, ovarian, and renal cell cancer. 6,7,[21][22][23][24][25] A more detailed description of bevacizumab, including its mechanism of action, preclinical models, activity in colorectal cancer and other malignancies, and reported adverse events, is described in detail in section "Background Drug Information."

Biomarkers for Antiangiogenic Activity
The development of angiogenesis inhibitors has underscored the need to develop new biological markers. Traditional methods of assessing response with conventional imaging may be inadequate because antiangiogenic agents appear to be primarily cytostatic rather than cytotoxic, and when they are used as monotherapy, only a minority of patients have a major tumor response. 19,26 To advance the clinical testing of antiangiogenic agents, reliable markers which can predict which patients are more likely to respond to anti-VEGF therapy are needed, but such markers remain elusive. 26,37 Such biomarkers would measure the agents' biological effects, determine the optimal dose, identify which patients are most likely to benefit from treatment, and monitor responses during treatment.
Unfortunately, exploratory studies of the use of angiogenic factors in the serum, plasma, and urine as surrogate markers have been disappointing. 26 Measurements of VEGF, bFGF, VCAM-1, Eselectin, and matrix metalloproteinases do not consistently show correlation with clinical response. 26 The use of flow cytometry to quantify circulating endothelial cells and endothelial cell progenitors from the peripheral blood is another potential surrogate marker which is currently being studied. 19,26,37 3.3 Dynamic Contrast-Enhanced Magnetic Resonance Imaging (DCE-MRI) as an

Imaging Biomarker of Anti-angiogenic Activity
Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) performed at high Protocol 2012-0061 October 10, 2014 Page 5 temporal resolution following the administration of gadolinium (Gd)-chelated contrast medium is a noninvasive imaging technology that can be used to measure properties of tissue microvasculature.
DCE-MRI is sensitive to changes in blood volume and vascular permeability that can be associated with tumor angiogenesis, and consequently DCE-MRI is a promising biomarker for characterizing tumor response to antiangiogenic treatment. [27][28][29][30] The concept of an imaging biomarker is very appealing because it fulfills the need to assess tumor biology in vivo and to monitor the effects of treatment. An imaging biomarker can be measured non-invasively and repeatedly. By evaluating the entire tumor in vivo, an imaging biomarker can capture the heterogeneity of both the tumor and its response to treatment. [27][28][29][30] DCE-MRI is performed by obtaining sequential magnetic resonance images before, during, and following the injection of gadolinium (Gd)-chelated contrast medium. The properties of the tissue microvasculature measured by DCE-MRI include: • The volume transfer constant (K trans ), which is the rate of flux of contrast agent into the extracellular extravascular space within a given volume Temsirolimus is being developed as a cytostatic agent to delay the time to tumor recurrence or progression or to increase survival in patients with various malignancies. Key features of this agent include its good tolerability, unique mechanism of action, ability to arrest cells in the G 1 phase, and ability to induce apoptosis.

3.6
The Role of Hypoxia-Inducible Factor-1 (HIF-1) as a Mechanism of Resistance to

Antiangiogenic Therapy
One mechanism of tumor resistance to antiangiogenic therapy is upregulation of hypoxiainducible factor 1α (HIF-1α), which mediates adaptive responses to hypoxic conditions. 3-11 The hypoxia-mediated increase in HIF-1α is critical to the establishment and progression of many common cancers via HIF-1-dependent activation of genes that allow cancer cells to survive and metastasize in the hostile hypoxic tumor environment. 12 Increased HIF-1α is associated with increased expression VEGF, aggressive tumor growth, and poor patient prognosis. 12 HIF-1α inhibition in combination with antiangiogenic therapy is a promising strategy for targeting tumor resistance. 8,[12][13][14][15]  Temsirolimus is an inhibitor of mTOR. Temsirolimus binds to an intracellular protein named FKBP-12 and the protein-drug complex inhibits the activity of mTOR which controls cell division.
Inhibition of mTOR activity results in G1 growth arrest in tumor cells that are treated with this agent. Histone modifications are one of the epigenetic mechanisms for regulation of gene expression.
Histones are found in the nucleosomes, which are the chromatin units in which DNA is wrapped around the core histone. Chromatin can become more accessible to transcription through nucleosome remodeling and histone alterations. Histone structure can be modified through acetylation/deacetylation mechanism to produce the "histone code." 43,44 It has been found that acetylation status of the histones correlates with transcriptional activity. This "code" can be reversed and the degree of this modification correlates with gene expression.
Histone acetyl transferases (HAT) and Histone deacetylases (HDACs) are the enzymes that mediate the acetylation and deacetylation of histones. Histone acetylation is associated with increased transcription whereas histone deacetylation is associated with decreased transcription. 45 Abnormal activity of HATs and HDACs resulting in aberrant gene transcription is commonly observed in cancer cells, especially on genes involved on cell cycle growth inhibition and differentiation which are generally repressed by HDACs.
Treatment with HDIs induces growth arrest, differentiation and apoptosis and also decreases angiogenesis presumably by inducing acetylation of histones and also of nonhistone proteins. 45,46 Valproic acid, an antiepileptic agent, has been shown to be an effective HDI, to reduce tumor weekly for 4 out of every 6 weeks. Patients who received bevacizumab had a higher median overall response rate (45 versus 35%, respectively) and prolonged median overall survival (20.3 versus 15.6 months, respectively), median progression-free survival (10.6 versus 6.2 months, respectively), and median duration of response (10.4 versus 7.1 months, respectively) than patients receiving placebo (every 2 weeks) in conjunction with the same combination regimen. Grade 3 or 4 hypertension occurred more frequently (12 versus 2%) in patients receiving bevacizumab rather than placebo in conjunction with the irinotecan/fluorouracil/leucovorin regimen.

Other Uses
Bevacizumab administered in combination with carboplatin and paclitaxel is approved for the paclitaxel versus carboplatin and paclitaxel alone and found a significant improvement in overall survival in those patients treated with bevacizumab.
Bevacizumab is being investigated for use in the treatment of breast cancer. In a phase III randomized trial, the addition of bevacizumab to capecitabine increased response rates but did not affect progression-free or overall survival in patients with previously treated metastatic breast cancer.
A phase III randomized trial comparing bevacizumab with paclitaxel versus paclitaxel alone for locally recurrent or metastatic breast cancer is under way.
Bevacizumab is being investigated for use in the treatment of renal cancer. In a randomized, double-blinded, phase II trial involving 116 patients with metastatic clear-cell renal cancer, those receiving high-dose bevacizumab had longer progression-free survival than those receiving placebo.
No difference in progression-free survival was observed in patients receiving low-dose bevacizumab compared with those receiving placebo, and no difference in overall survival was noted between the 3 groups. A phase III randomized trial comparing bevacizumab with interferon alfa-2b versus interferon alfa-2b alone for advanced renal cell cancer is under way.

Adverse Events Effects of Bevacizumab
Because the bevacizumab therapeutic regimen includes the use of fluorouracil and other anti-neoplastic agents, the usual cautions, precautions, and contraindications of these drugs also should be considered. Hypertension. Hypertension and severe hypertension have been reported in patients receiving bevacizumab. Acute increases in blood pressure have been associated with initial or subsequent infusions of bevacizumab; some cases were associated with clinical sequelae.
Hypertension was more common in patients with previous history of hypertension and may respond to antihypertensive therapy. Hypertension also occurred more frequently in patients who received higher dosages (e.g., 10 mg/kg). Permanently discontinue the drug in patients who develop hypertensive crisis. Temporary suspension is recommended in patients with severe hypertension that is not controlled with medical management.
Proteinuria. Increased incidence and severity of proteinuria have been reported in patients receiving bevacizumab. In clinical trials, proteinuria ranged in severity from clinically silent to nephrotic syndrome. Discontinue the drug in patients with nephrotic syndrome. Patients receiving bevacizumab should be monitored for the development or worsening of proteinuria. The safety of continued treatment in patients with moderate to severe proteinuria has not been evaluated; such patients should be monitored regularly until improvement and/or resolution is observed. In most clinical studies, bevacizumab was interrupted for proteinuria exceeding 2 g per 24 hours and resumed when proteinuria declined below this level.
Congestive Heart Failure. Congestive heart failure has been reported in patients receiving bevacizumab. Some patients also were receiving or had previously received anthracyclines and/or left chest wall irradiation. The safety of continuation or resumption of bevacizumab in patients who develop cardiac dysfunction has not been studied.

Infusion Reactions.
In clinical studies, infusion reactions with the first bevacizumab dose were uncommon (less than 3%). Severe infusion reactions occurred in 0.2% of patients receiving

Temsirolimus
The observed antitumor and immunosuppressive properties of rapamycin analogs are due to their ability to disrupt the mTOR-dependent signaling pathway. 48 mTOR, a member of the phosphatidylinositide 3'-kinase (PI3K)-related family, is located predominantly in the nuclear fraction of both neoplastic and normal cells. 49 mTOR activation triggers resting cells to increase the translation of a subset of mRNAs whose proteins are required for cell cycle progression from G 1 to S phase.
mTOR regulates essential signal transduction pathways and is involved in the coupling of growth stimuli with cell cycle progression. Experimental data indicate that mTOR acts downstream of the PI3K/Akt pathway and is phosphorylated in response to mitogenic signals. 48 Early studies reported that mTOR was dedicated to initiating mRNA translation in response to favorable nutrient environments. 50 In fact, cells treated with rapamycin undergo changes that are strikingly similar to those observed during conditions of starvation. These include mTOR inactivation, down regulation of translation, G 1 arrest, accumulation of glycogen stores and altered transcription patterns. 50 More recent studies have demonstrated that mTOR is involved in regulating many aspects of cell growth, including organization of the actin cytoskeleton, membrane traffic, protein degradation, protein kinase C (PKC) signaling, ribosome biogenesis, and transcription. 51 Temsirolimus reacts with the ubiquitous intracellular FK506-binding protein 12 (FKBP12), forming a Temsirolimus/FKBP12 complex that is a potent inhibitor of the highly conserved kinase mTOR. 52 Inhibition of mTOR leads to suppression of several downstream signaling effectors, including the ribosomal subunit p70 S6k and the eukaryotic initiation factor 4 binding protein 1 (4E-BP1). 53 These two proteins play key roles in ribosomal biogenesis and cap-dependent translation, respectively. 54 The extent of phosphorylation of these two downstream proteins (p70 S6 kinase and 4E-BP1) may therefore serve as indicators of temsirolimus biologic activity in vivo. Inhibition of the synthesis of ribosomal proteins and elongation factors, required to accelerate the process of cell division, are thought to contribute to the anti-proliferative effects of rapamycin analogs. 55 While temsirolimus inhibits the translation of only a subset of mRNAs, inhibition of mTOR can lead to a substantial decrease (~15%) in overall protein synthesis. 56 Pharmacokinetics. Cytochrome P450 3A4 is the major isozyme responsible for the formation of five Temsirolimus metabolites. Sirolimus, an active metabolite of temsirolimus, is the principal metabolite in humans following intravenous treatment. The remainder of the metabolites Protocol 2012-0061 October 10, 2014 Page 12 account for less than 10% of radioactivity in the plasma. In human liver microsomes temsirolimus was an inhibitor of CYP2D6 and 3A4. There was no effect observed in vivo when temsirolimus was administered with desipramine (a CYP2D6 substrate), and no effect is anticipated with substrates of CYP3A4 metabolism. Temsirolimus is cleared predominately by the liver and elimination of the drug is primarily via the feces.
Uses. Temsirolimus is used as a single-agent for the treatment of advanced renal cell carcinoma in patients. The current indication for temsirolimus is based principally on the results of a multi-center, randomized, open label clinical trial involving 626 patients with poor prognosis, previously untreated, metastatic renal-cell carcinoma. 57 Patients in this study needed at least three of six predictors of short term survival. These predictors included: a serum lactate dehydrogenase level of more than 1.5 times the upper limit of normal (ULN) range, a hemoglobin level below the lower limit of the normal range, a corrected serum calcium level of more than 10 mg/dL, a time from initial diagnosis of renal cell carcinoma to randomization of less than one year, a Karnofsky performance score of 60-70, or metastases in multiple organs. Prior to therapy, patients were stratified for prior nephrectomy status within three geographic regions and were randomly assigned (1:1:1) to receive IFN-α alone (n=207), temsirolimus alone (25mg weekly; n=209), or the combination arm (n=210 Predicted modeling of IC 50 (humans receiving doses as low as 10 mg) suggests that whole blood concentrations would be above the range of 1 ng/mL throughout the entire 1-week dose interval and above 5 ng/mL for the majority of this time period. It is expected that mTOR inhibition will be attained with a 25 mg dose.
Clinical pharmacokinetic data are available in patients with cancer receiving temsirolimus both IV daily x 5 days every 2 weeks, once weekly schedules, and orally daily × 5 days every 2 weeks.
These data indicate that there is no appreciable drug accumulation between cycles and that distribution is extensive. With increasing dose, exposure (AUC) increases in a less than proportional fashion. The mean volume of distribution at steady state (V dss ) is large (57 L after 2 mg IV dose; 900 L following a 250 mg IV dose) and increases with dose. Exposure to the hydrolytic product sirolimus is substantial with mean values of approximately 1.5-2.3-fold greater than those seen with temsirolimus following IV administration. Clearance (CL) of temsirolimus from whole blood increases with increasing dose from approximately 5.2 L/h after a 2 mg dose to 100 L/h after a 250 mg dose.
Intersubject variability in CL at a given dose was modest and ranged from 16-27%. The terminal halflife (t 1/2 ) following temsirolimus doses of 25 to 250 mg is approximately 15 hours.
Pharmacokinetic results from the initial phase I study showed that the AUC increased proportionally with doses up to 150 mg, but doses higher than 300 mg yielded high AUCs and low CL in some patients. 58  suggesting that mTOR inhibition may be of particular relevance in poor prognosis RCC. 59 A phase II study has been reported in patients with metastatic melanoma. 61 33 patients were treated with temsirolimus, 250 mg IV weekly. Only one patient had a partial response, and median time to progression was 10 weeks.

Adverse Effects of Temsirolimus
In a randomized, open label phase III of interferon alfa alone, temsirolimus alone, and temsirolimus and interferon alfa, a total of 616 patients were treated. Of these patients, 208 received Temsirolimus 25mg weekly. The most common adverse reactions that occurred with a frequency greater than 30 percent were rash, asthenia, mucositis, nausea, edema, and anorexia. The most common laboratory abnormalities that occurred with a frequency of greater than 30 percent were anemia, hyperglycemia, Pregnancy. Temsirolimus is Pregnancy Category D. Temsirolimus administered daily as an oral formulation caused embryo-fetal and intrauterine toxicities in rats and rabbits at human subtherapeutic exposures. Embryo-fetal adverse effects in rats consisted of reduced fetal weight and reduced ossifications, and in rabbits included reduced fetal weight, omphalocele, bifurcated sternabrae, notched ribs, and incomplete ossifications.
In rats, the intrauterine and embryo-fetal adverse effects were observed at the oral dose of 2.7mg/m 2 /day (approximately 0.04-fold the AUC in cancer patients at the human recommended dose).
In rabbits, the intrauterine and embryo-fetal adverse effects were observed a the oral dose of >=
The drug is an immunoglobulin containing human framework (i.e., IgG1 heavy and kappa light constant regions) and murine Fv regions. Cetuximab binds specifically to the extracellular domain of the human epidermal growth factor receptor (EGFR, HER1, c-ERbB-1) on both normal and tumor cells and competitively blocks the cellular action of EGF and other ligands (e.g., transforming growth factor [TGF]-a). EGFR is a transmembrane glycoprotein that belongs to the subfamily of type I receptor tyrosine kinases, which include EGFR (HER1), HER2, HER3, and HER4. While EGFR is expressed in many normal epithelial tissues (e.g., skin, hair follicle), overexpression of the glycoprotein is detected in human carcinomas (e.g., colon, rectum). Binding of cetuximab to EGFR blocks phosphorylation and activation of receptor-associated kinases resulting in inhibition of cell growth, induction of apoptosis (programmed cell death), and decreased matrix metalloproteinase and vascular endothelial growth factor production.
Evidence from in vitro tests and in vivo animal studies has suggested that cetuximab may inhibit growth and survival of tumor cells that overexpress EGFR, while such antitumor effects were not observed in human cancer xenografts that lacked the EGFR expression. In animal studies, addition of cetuximab to irinotecan or to a combination chemotherapy regimen of irinotecan and fluorouracil resulted in an increased antitumor effect when compared with chemotherapy alone. Following administration of the recommended regimen of cetuximab (initial loading dose, followed by weekly maintenance doses), steady state cetuximab concentrations are achieved by the third weekly infusion; the mean half-life of cetuximab following multiple dosing is 114 hours. The major route of clearance from the circulation is believed to be through internalization of the cetuximab EGFR complex on hepatocytes and skin.
Cetuximab has been used in the following cancers: Colorectal Cancer.Cetuximab is used in combination with irinotecan for the treatment of metastatic colorectal cancer that is refractory to irinotecan-based chemotherapy in patients with tumors that express the epidermal growth factor receptor (EGFR). Cetuximab also is used as a single  and these manifestations should be promptly investigated. If interstitial lung disease is confirmed, cetuximab should be discontinued, and appropriate therapy instituted.
Electrolyte Effects. Electrolyte abnormalities, sometimes severe, including hypomagnesemia, hypocalcemia, and hypokalemia, have occurred in patients receiving cetuximab. Interim analysis of data for 244 patients in controlled clinical trials shows that the incidence of overall or severe (grade 3 or 4) hypomagnesemia was increased in patients receiving cetuximab (alone or in combination with chemotherapy) compared with those receiving best supportive care or chemotherapy alone. About half of patients receiving cetuximab experienced hypomagnesemia, and 10-15% experienced severe hypomagnesemia. The onset of electrolyte abnormalities may occur from days to months following initiation of cetuximab therapy. Electrolyte repletion therapy should be administered as necessary and, in severe cases, intravenous replacement therapy is required. Because the time to resolution of electrolyte abnormalities is not established, continued monitoring following completion of cetuximab therapy is recommended.
Dermatologic Effects. Acneiform rash (e.g., multiple follicular-or pustular-appearing lesions) has been reported in 89% of patients receiving cetuximab in clinical studies; severe (grade 3 or 4) acneiform rash has been reported in 11% of patients. Acneiform rash occurred most frequently on the face, upper chest, and back but could extend to the extremities. Acneiform rash generally appears within the first 2 weeks of cetuximab therapy and may resolve following discontinuance of cetuximab; however, manifestations have persisted beyond 28 days in nearly 50% of cases. Other adverse dermatologic effects, including skin drying/fissuring and inflammation (e.g., blepharitis, cheilitis, cellulitis, cyst), also have been reported. Adverse dermatologic effects may result in infectious complications;Staphylococcus aureus sepsis and abscesses requiring incision and drainage have been reported in patients receiving cetuximab in clinical studies.
Patients who develop severe acneiform rash should receive reduced cetuximab dosages.
Patients who experience adverse dermatologic effects while receiving cetuximab should be monitored for development of inflammatory or infectious complications, and appropriate therapy instituted.
Treatment with topical and/or oral antibiotics should be considered; topical corticosteroids are not recommended.
Protocol 2012-0061 October 10, 2014 Page 20 Nail Disorder. Paronychial inflammation (particularly of the great toes and thumbs) has been reported in 14% of patients receiving cetuximab.

EGFR Testing. Assessment for epidermal growth factor receptor (EGFR) expression should
be performed by laboratories with demonstrated proficiency in the specific technology being utilized.
Improper assay performance, including use of suboptimally fixed tissue, failure to utilize specified reagents, deviation from specific assay instructions, and failure to include appropriate controls for assay validation, may lead to unreliable results.
Therapy Monitoring. Patients should be monitored periodically for hypomagnesemia, and accompanying hypocalcemia and hypokalemia, during and following the completion of cetuximab therapy. Monitoring should be continued for at least 8 weeks following completion of therapy, which corresponds to the observed half-life and persistence of cetuximab.

Immunologic Effects. Non-neutralizing anticetuximab antibodies were detected in about 5%
of patients (28/530) receiving cetuximab; the median time to onset was 44 days. Although the incidence of antibody development has not been established, there appears to be no relationship between the appearance of antibodies to cetuximab and the safety and efficacy of the drug.

Valproic Acid
Valproic acid is administered orally. It is FDA approved for treatment of seizure, mood stabilization in bipolar disorder, and migraine prophylaxis. Valproic acid is FDA approved for seizure, bipolar disorder and migraine prophylaxis. It is commercially available and will be prescribed for patients.
Reported adverse events and potential risks: COMMON >10%: inducers and/or inhibitors prior to enrollment on the protocol, it is strongly recommended that the patient stops the drug and waits at least 5 half-lives of said drug before initiating therapy on protocol.
5.2.9 Colorectal cancer patients with known KRAS mutation (for the arm combining bevacizumab, temsirolimus and cetuximab) 5.2.10 Patients who have had major surgery within 6 weeks of enrollment in the study.
6.0 Treatment Plan 6.1 As two or more drugs being given in combination are being tested in this protocol, there is a need to explore a variety of dose levels.
6.2 A MTD is defined as the dose level below the dose at which 2 of 6 patients experience drug-related dose limiting toxicity (DLT) in the first cycle.
6.3 Dose escalation will proceed as described below in Table 1 through Table 3 depending on the arm on which the patient is enrolled.
6.3.1 Whenever a dose level is determined to be above the MTD, dose escalation will halt. Three additional patients will be entered at the next lowest dose level if only 3 patients were treated previously at that dose.

6.4
This protocol will utilize a standard 3+3 Phase I escalation design with three patients per cohort in an effort to obtain three evaluable patients. Three patients will be entered Protocol 2012-0061 October 10, 2014 Page 23 at each dose level in order to obtain adequate correlative data in addition to the safety data. Three patients will be treated per dose level and evaluated for toxicity. Dose escalation will then proceed as follows:

Number of Patients with DLT at a Given Dose
Level Escalation Decision Rule 0 out of 3 Enter 3 patients at the next dose level.
>2 out of 3 Dose escalation will be stopped. Three (3) additional patients will be entered at the next lowest dose level if only 3 patients were treated previously at that dose.
1 out of 3 Enter 3 more patients at this dose level.
• If 0 of these 3 patients experience DLT, proceed to the next dose level.
• If 1 or more of this group suffer DLT, then dose escalation is stopped, and next lowest dose is declared as a maximum tolerated dose (MTD).
• If 2 or more of this group suffer DLT, then dose escalation is stopped. Three (3) additional patients will be entered at the next lowest dose level if only 3 patients were treated previously at that dose.

6.5
The MTDs identified will be expanded by up to 10 additional patients to further evaluate toxicity and correlative data.

6.6
There will be no intra-patient dose escalation, and no patients will be enrolled in the next dose level until three patients enrolled at the previous dose level have completed at least four weeks of therapy. If a DLT is observed in one of the three patients after one cycle, then dose escalation will not proceed until six patients in the cohort have Protocol 2012-0061 October 10, 2014 Page 24 been assessed for toxicity after one cycle.

6.7
Patients will continue treatment until their disease worsens, their side effects become too severe, or the patient's physician feels it is not in the patient's best interest to continue. A patient may also be discontinued for an intercurrent illness that prevents further administration of treatment. A patient may also choose to discontinue enrollment in the protocol at any time.
6.8 Pre-medication, precautions, route, and schedule for each medication are described in Table "Dosing Regimen." 6.9 If a response has been observed in a particular tumor type with the study drug or drug combination, then the study may be expanded to include a total of 14 participants with that tumor type. All patients will be treated at the highest current dose level. All enrolled participants will be considered in the DLT analysis. If at any time more than or equal to one third of the participants at a dose level experience DLT, that dose is considered to be above the MTD.
For the purpose of adding up to 14 additional participants, a tumor response is defined as one or more of the following: (1) stable disease for more than or equal to 4 months, (2) decrease in measurable tumor (sentinel lesions) by more than or equal to 20% by RECIST criteria, (3) decrease in tumor markers by more than or equal to 25% (for example, a >/= 25% decrease in CA125 for patients with ovarian cancer), or (4) a partial response according to the Choi criteria, i.e. decrease in size by 10% or more, or a decrease in the tumor density, as measured in Hounsfield units (HU), by more than or equal to 15% (28).
6.10 Up to 3 additional patients may be added to a cohort for evaluation of correlative studies. These patients will be considered in the DLT analysis.
Patients will be assigned to the 3 different treatment combinations mentioned below at the discretion of the treating physician.
Protocol 2012-0061 October 10, 2014 Page 25    contrast-enhanced magnetic resonance imaging (DCE-MRI), tumor biopsy, and peripheral blood markers. During expansion, at any time > 33% of patients have a DLT, the expansion cohort will be terminated. If the dose expansion is terminated due to >33% of patients having a DLT, then an additional 10 patients may be entered at the next lowest dose level, for the purpose of exploratory analysis with additional optional correlative studies, including but not limited to dynamic contrastenhanced magnetic resonance imaging (DCE-MRI), tumor biopsy, and serum/plasma cytokines. For these additional 10 patients, we will monitor toxicity using the same criteria used in the initial expansion cohort.
13.6 Among the patients who undergo DCE-MRI, the tissue microvascular parameters (including K trans , v e , k ep ) will be measured on a continuous scale at the three specified time points. For each of these parameters, an exploratory analysis of change from baseline will be conducted which will include mean, median, standard deviation, and 95% confidence limits.
13. 7 We estimate that the number of patients required to find the MTD of this drug combination and to obtain adequate correlative studies is approximately 50-60 patients per arm. The number of patients that may be enrolled on the trial, given 11 total dose levels forBevacizumab/Temsirolimus and Cetuximab arm and 10 total dose levels each for Bevacizumab/Temsirolimus and Valproic Acid arm and Bevacizumab/Temsirolimus arm, is 186 (if six patients are enrolled at each dose level). Also, predicting that we will discover 3 MTDs (one for each arm) and allowing for expansion of 10 additional patients at those MTDs, we expect an absolute maximum of 216 patients for the trial. In order to enroll 216 evaluable patients, we may need to screen up to 300 patients since we expect a percentage of these patients to be screen failures or withdrawals. The estimated accrual rate is 1-5 patients per arm per month.

Dose Delays and Modifications
14.1 If a patient experiences a toxicity which is known to be related to one drug in the regimen, then that drug may be de-escalated to the prior dose level after the patient recovers to </= Grade 1 toxicity.
14.2 If a patient experiences a toxicity for which it is unclear which drug is the cause of Protocol 2012-0061 October 10, 2014 Page 35 the toxicity, then the drug which was dose escalated at the current dose level may be de-escalated to the prior dose level after the patient recovers to </= Grade 1 toxicity.
14.3 If a patient experiences a toxicity at the first dose level, and if the toxicity is known to be related to one drug in the regimen, then a dose reduction of 50% of that drug is permitted after the patient recovers to </= Grade 1 toxicity.
14.4 If a patient experiences a toxicity at the first dose level, and if it is unclear which drug is the cause of the toxicity, then a dose reduction of 50% of all drugs in the regimen is permitted after the patient recovers to </= Grade 1 toxicity.
14.5 If grade III toxicity occurs (DLT), dose reduction by 50% is allowed after patient recovers. The drug that will be reduced is the one that the physician feels has most likely caused the toxicity. If the drug that caused the toxicity is not known, the patient will be dose reduced to the previous dose level.

Correlative Studies
15.1 Correlative studies will be mandatory for those patients choosing to enroll in the expansion cohorts of up to 10 patients at the MTDs pending funding.

Dynamic Contrast-Enhanced Magnetic Resonance Imaging (DCE-MRI)
15.2.1 Dynamic contrast-enhanced magnetic resonance Imaging (DCE-MRI) will be performed at the following time points: 1. Baseline (within one week before day 1 treatment), 2. Acute phase (48 hours +/-6 hours after first infusion of bevacizumab and temsirolimus); 3. Chronic phase (at end of cycle 1 on day 20). DCE-MRI will be performed at high temporal resolution and will obtain sequential magnetic resonance images before, during, and following the injection of gadolinium (Gd)-chelated contrast medium. Following the acquisition of localizer images, T1 mapping data will be obtained using a multiple flip angle fast spoiled gradient echo sequence in a plane that includes the target lesion(s) as well as a reference vessel. Following the T1 mapping acquisition, the DCE-MRI scans will be obtained using the same pulse sequence and from the same acquisition volume before, during, and following bolus administration of 0.1 mmol/kg gadopentetate dimeglumine at 3 ml/s followed by a 20 ml saline flush also given at 3 ml/s. The total DCE-MRI acquisition will last about 8 minutes. All DCE-MRI data will be analyzed