Abstract
We present 2 patients with metastatic colorectal cancer who had progressed despite treatment with first-line FOLFOX and second-line FOLFIRI combination chemotherapy regimens. After failing these fluoropyrimidine-based regimens, both patients received additional cytotoxic and targeted therapies with eventual disease progression. These therapies included capecitabine plus dabrafenib and trametinib, regorafenib monotherapy, and regorafenib with panitumumab. After exhausting available options, both patients were offered regorafenib with either 5-fluorouracil (5-FU) or capecitabine. These therapies are individually approved for the treatment of colorectal cancer but have not yet been studied in combination. This regimen produced stable disease in both patients with acceptable toxicity. One patient continued therapy for 17 months. Although these patients previously progressed during treatment with regorafenib, capecitabine or 5-FU, the combination had some activity in both cases of refractory metastatic colorectal cancer and may be considered in the palliative setting. In bedside-to-bench cell culture experiments performed after the clinical observations, we observed sensitivity of human colorectal cancer cell lines (N = 4) to single agent regorafenib or 5-FU and evidence of synergy with the combination therapy. Synergistic effects were noted in colorectal cancer cells with KRAS mutation, BRAF mutation, and p53 mutation, as well as mismatch repair deficient cells. Regorafenib suppressed Mcl-1 and Bcl-XL in treated cancer cells that may have contributed to the anticancer efficacy including in combination with 5-FU. The safety and efficacy of regorafenib with 5-FU or capecitabine in combination should be further investigated as a therapy for patients with refractory metastatic colorectal cancer, including individuals who had progressed on regorafenib monotherapy.
Introduction
Colorectal cancer (CRC) is the third most common cancer diagnosis in the United States and the most prevalent malignancy of the digestive tract.1 Worldwide, approximately 1.3 million patients are diagnosed with CRC, and 694,000 deaths are attributed to the disease each year.2 Screening tests are both widely available and effective at detecting this disease at an early stage. As a result of screening efforts, fewer than 25% of patients diagnosed with CRC in the United States have evidence of metastatic spread at initial presentation.1 Unfortunately, 50 to 60% of patient diagnosed with CRC eventually develop metastases, and the majority of these patients have unresectable disease.3
Whenever possible, the standard of care for patients with CRC is complete surgical resection for possible cure. This is typically followed by adjuvant chemotherapy in patients with stage III disease, stage IV disease with resectable metastases or stage II disease with high-risk features.4 If complete resection is not possible, however, patients are offered systemic chemotherapy. Sometimes in this setting neoadjuvant therapy results in downsizing of disease, making surgical resection possible with improved long-term outcomes. The mainstays of first and second line treatment for patients with stage IV unresectable metastatic CRC (mCRC) consists of fluoropyrimidine-based combination chemotherapy with FOLFOX (leucovorin, 5-FU, oxaliplatin), Cape-Ox (capecitabine, oxaliplatin), or FOLFIRI (leucovorin, 5-FU, irinotecan). Patients with tumors that retain wild-type KRAS/NRAS can benefit from the addition of monoclonal antibodies that target the epidermal growth factor receptor (EGFR), such as cetuximab or panitumumab. Bevacizumab, a monoclonal antibody against vascular endothelial growth factor (VEGF) receptor is generally offered to patients who develop progressive disease while undergoing treatment with an EGFR antagonist or as a component of initial therapy for KRAS/NRAS mutated tumors.
Although clinical guidelines clearly outline the first and second lines of therapy, there is little consensus regarding subsequent treatments. Many patients with CRC continue to enjoy good performance status and wish to continue treatment beyond the second line. Establishing a regimen for these patients that is both effective and tolerable is an important area of active research.
Early clinical trials of later-line treatment focused on the use of single agent 5-FU or capecitabine, either of which may be utilized as the backbone of standard first or second-line therapy. The results of these studies were generally underwhelming. In a phase III trial of 463 patients that progressed despite first-line therapy with IFL (irinotecan, 5-FU, leucovorin), 5-FU monotherapy was associated with a median time to progression (TTP) of only 2.7 months and failed to generate a single objective response.5 A second study evaluated the use of single agent capecitabine in 20 patients who had developed progressive disease while receiving a fluorouracil-based combination regimen.6 This produced similarly dismal results with zero patients responding to treatment and a median TTP of 2.8 months. More recently, treatment with TAS-102, an oral combination of trifluridine and tipiracil hydrochloride, has demonstrated a modest overall survival (OS) benefit over placebo in patients with advanced disease.7 Mayer et al. recently reported that median OS with TAS-102 was 7.1 months compared to 5.3 months with placebo (HR 0.68; 95% CI 0.58–0.81; P < 0.001).
Regorafenib, a novel oral multikinase inhibitor, has shown antitumor activity in mCRC. It inhibits a broad array of oncogenic gene products and growth factor receptors, such as KIT, RET, RAF1, BRAF, BRAFV600E, VEGFR, PDGFR, and FGFR.8 Regorafenib impedes the growth of colorectal tumors that have progressed despite multiple lines of prior therapy,9-11 earning FDA approval for use as monotherapy in such circumstances in 2012.12 Below, we describe the cases of 2 patients who responded to treatment with a combination of regorafenib and either 5-FU or capecitabine. Based on these anecdotal case reports, we subsequently performed laboratory experiments with human colorectal cancer cell lines and were able to document synergistic growth inhibitory effects from the combination of regorafenib plus 5-FU toward multiple human colorectal cancer cell lines with KRAS mutation, BRAF mutation, p53 mutation, or mismatch repair deficiency.
Clinical Case Report
Patient 1
In 2011, a 58 year-old woman underwent a colonoscopy to localize the source of a lower GI bleed and was diagnosed with BRAF V600E mutated colon adenocarcinoma (Fig. 1). After discovering the primary tumor, a staging PET scan demonstrated several mildly FDG-avid pulmonary nodules in addition to lymphadenopathy throughout the chest and abdomen. Approximately 1 month later, she underwent an exploratory laparotomy with right hemicolectomy, salpingoophorectomy and lymph node dissection that confirmed the presence of metastatic disease in 7 of 20 lymph nodes.
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19 January 2016Figure 1. Colorectal cancer histopathology from Patient 1. (A). Invasive adenocarcinoma with mucinous and signet ring cell features from the colon resection in 2011. (B). Metastatic mucinous adenocarcinoma from the lung biopsy in 2011. (C). Metastatic adenocarcinoma with mucinous and signet ring cell features from aortocaval lymph node biopsy in 2012. All are Hematoxylin & Eosin stains, 400 × original magnification.
![]({"type":"image","src":"/na101/home/literatum/publisher/tandf/journals/content/kcbt20/2015/kcbt20.v016.i12/15384047.2015.1113355/20160119/images/medium/kcbt_a_1113355_f0001_oc.gif"})
Figure 1. Colorectal cancer histopathology from Patient 1. (A). Invasive adenocarcinoma with mucinous and signet ring cell features from the colon resection in 2011. (B). Metastatic mucinous adenocarcinoma from the lung biopsy in 2011. (C). Metastatic adenocarcinoma with mucinous and signet ring cell features from aortocaval lymph node biopsy in 2012. All are Hematoxylin & Eosin stains, 400 × original magnification.
The patient was initially treated with FOLFOX with bevacizumab and received 11 cycles before repeat PET/CT scan demonstrated progression of thoracic lymphadenopathy that was later biopsy-proven to contain mCRC. Her therapy was then transitioned to FOLFIRI plus cetuximab. Repeat imaging after completion of the third cycle revealed continued growth of these malignant thoracic lymph nodes, prompting another change in treatment. The patient subsequently received 2–8 months of sorafenib plus cetuximab, single agent regorafenib, regorafenib with panitumumab, dabrafenib with trametinib, and capecitabine plus both trametinib and dabrafenib. Each change of therapy was prompted by radiographic or serologic evidence of disease progression.
The patient's initial response to regorafenib monotherapy was difficult to assess due to a delay between the pretreatment CT scan in November, 2012 and initiation of therapy in January, 2013. Follow up PET/CT after approximately 5 weeks of therapy did demonstrate progressive disease, but it is possible that this growth occurred during the previous regimen. Tumor markers remained stable during the first 2 months of single agent regorafenib before rising convincingly in February of 2013. It should also be noted that during treatment with dabrafenib and trametinib, the patient was diagnosed with a pulmonary embolism (PE) with associated area of infarction and was started on anticoagulation.
After progressing on the aforementioned regimens, this patient was transitioned to regorafenib 160 mg PO daily for days 1–21 of a 28 day cycle with capecitabine 1000 mg PO twice daily for days 1–14 of a 21 day cycle starting in January 2014. At the time that this regimen was started, the patient was not eligible for any established clinical trials. Repeat imaging (Fig. 2) and tumor markers (Fig. 3) obtained approximately 3 weeks into treatment demonstrated stable disease. However, less than one month into therapy, she developed pneumonia that was further complicated by pulmonary hemorrhage into the area of previous infarction. Imaging obtained at that time revealed stable disease with a slight increase in size of thoracic lymphadenopathy, which may have been secondary to the underlying infection. Following a brief course in the ICU, the decision was made to withdraw care, and the patient expired.
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19 January 2016Figure 2. Radiographic response to regorafenib and capecitabine in patient 1. Computerized tomography of a CRC implant was obtained on October 11, 2013 (left). This was approximately 2 months prior to initiating capecitabine plus regorafenib. Follow up imaging on January 24, 2014 after 1 month on therapy (right) demonstrated stable disease.
![]({"type":"image","src":"/na101/home/literatum/publisher/tandf/journals/content/kcbt20/2015/kcbt20.v016.i12/15384047.2015.1113355/20160119/images/medium/kcbt_a_1113355_f0002_b.gif"})
Figure 2. Radiographic response to regorafenib and capecitabine in patient 1. Computerized tomography of a CRC implant was obtained on October 11, 2013 (left). This was approximately 2 months prior to initiating capecitabine plus regorafenib. Follow up imaging on January 24, 2014 after 1 month on therapy (right) demonstrated stable disease.
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19 January 2016Figure 3. Serologic and radiographic response to prior systemic therapies and regorafenib with capecitabine in patient 1. CEA (carcinoembryonic antigen), CA19-9 (carbohydrate antigen 19-9), are displayed as a function of time. The different treatment regimens are as indicated.
![]({"type":"image","src":"/na101/home/literatum/publisher/tandf/journals/content/kcbt20/2015/kcbt20.v016.i12/15384047.2015.1113355/20160119/images/medium/kcbt_a_1113355_f0003_oc.gif"})
Figure 3. Serologic and radiographic response to prior systemic therapies and regorafenib with capecitabine in patient 1. CEA (carcinoembryonic antigen), CA19-9 (carbohydrate antigen 19-9), are displayed as a function of time. The different treatment regimens are as indicated.
Overall survival from the time of diagnosis was approximately 33 months, or 1 month after starting regorafenib with capecitabine. This patient did not experience toxicities commonly associated with regorafenib or capecitabine, namely hypertension, proteinuria, mucositis, gastrointestinal distress, cytopenias or hand-foot skin reaction. As such, she did not require any dose reductions or delays during this short course of therapy.
Patient 2
A 48 year-old man was diagnosed with KRAS G12A mutated adenocarcinoma of the rectum in 2010 during surgery to repair a perforated sigmoid colon (Fig. 4). At that time, the primary tumor was resected and a colostomy was placed. The patient subsequently received 9 cycles of adjuvant FOLFOX followed by 25 fractions of radiation therapy. In August 2011, he underwent elective colostomy reversal, but the postoperative course was complicated by intra-abdominal abscesses, abdominal wall infection, and small bowel obstruction. These complications required multiple surgeries, including extensive debridement of the abdominal wall, drainage of abscesses, transverse colostomy, diverting loop ileostomy, and diverting loop jejunostomy.
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19 January 2016Figure 4. Invasive adenocarcinoma from colon resection for patient 2 in 2010. Hematoxylin & Eosin stain, 400 × original magnification.
![]({"type":"image","src":"/na101/home/literatum/publisher/tandf/journals/content/kcbt20/2015/kcbt20.v016.i12/15384047.2015.1113355/20160119/images/medium/kcbt_a_1113355_f0004_oc.gif"})
Figure 4. Invasive adenocarcinoma from colon resection for patient 2 in 2010. Hematoxylin & Eosin stain, 400 × original magnification.
Shortly after these surgeries, the patient was noted to have a rising CEA level and PET/CT suggested the presence of a poorly defined mass at the prior surgical site. As this clinical picture was most concerning for recurrence, the patient received 5 cycles of FOLFIRI and bevacizumab with good serologic response. Following the 5th cycle, his treatment was interrupted due to hospitalization for candidemia followed by sepsis from a central line-associated bacteremia. The patient was again evaluated in clinic on November 2012, at which time his CEA level had again increased. He subsequently received another 6 cycles of FOLFIRI with bevacizumab. Unfortunately, follow-up PET/CT revealed multiple new FDG-avid pulmonary nodules and a recurrent mass at the anastomotic site. The patient then received 3 cycles of single agent regorafenib. However, his tumor markers continued to rise while on therapy, and repeat imaging demonstrated progressive disease.
In October 2013, combination therapy with regorafenib 160 mg PO daily for days 1–21 of 28 day cycle plus infusional 5-FU every 2 weeks at 2400 mg/m2 over 46 hours, bolus 5-FU 400 mg/m2 on day 1 and leucovorin 250 mg/m2 on day 1 was initiated. At the time that this therapy was offered to the patient, he did not qualify for any clinical trials. After starting treatment, the patient's tumor markers stabilized, and CT scan obtained on December 2013 revealed slight enlargement of abdominal lymph nodes but stability of known abdominal disease (Fig. 5). Approximately 3 months into treatment, however, his CEA level began to slowly increase (Fig. 6A). Repeat PET/CT scan showed a new focus of metastatic disease within the abdomen, FDG-avid hepatic lesions, and pulmonary nodules. The recurrent tumor at the surgical anastomosis had also extended into the bladder, causing unilateral hydronephrosis. The patient opted to continue this regimen for a number of reasons, including a lack of other feasible or attractive treatment options for standard or experimental therapy. He was tolerating dual therapy with 5-FU and regorafenib well and felt that it may have been restraining the rate of tumor growth despite evidence of metastatic spread. The patient ultimately completed a total of 11 cycles of regorafenib plus 5-FU. Unfortunately, he developed septic shock secondary to an infection involving a necrotizing metastatic lesion and expired. The patient's overall survival from the date of diagnosis was 57 months, and 17 months after starting regorafenib with 5-FU.
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19 January 2016Figure 5. Radiographic response to regorafenib and capecitabine in patient 2. CT scan obtained in October 2013, which is less than 1 month before starting regorafenib with 5-FU is displayed on the left. The image on the right depicts repeat CT approximately after 2 months of therapy, in December 2013.
![]({"type":"image","src":"/na101/home/literatum/publisher/tandf/journals/content/kcbt20/2015/kcbt20.v016.i12/15384047.2015.1113355/20160119/images/medium/kcbt_a_1113355_f0005_b.gif"})
Figure 5. Radiographic response to regorafenib and capecitabine in patient 2. CT scan obtained in October 2013, which is less than 1 month before starting regorafenib with 5-FU is displayed on the left. The image on the right depicts repeat CT approximately after 2 months of therapy, in December 2013.
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19 January 2016Figure 6. Serologic and radiographic response to prior systemic therapies and regorafenib plus capecitabine in patient 2. CEA (carcinoembryonic antigen), CA19-9 (carbohydrate antigen 19-9) are displayed as a function of time in the patient's treatment course. The various treatment regimens given are also shown.
![]({"type":"image","src":"/na101/home/literatum/publisher/tandf/journals/content/kcbt20/2015/kcbt20.v016.i12/15384047.2015.1113355/20160119/images/medium/kcbt_a_1113355_f0006_oc.gif"})
Figure 6. Serologic and radiographic response to prior systemic therapies and regorafenib plus capecitabine in patient 2. CEA (carcinoembryonic antigen), CA19-9 (carbohydrate antigen 19-9) are displayed as a function of time in the patient's treatment course. The various treatment regimens given are also shown.
While receiving treatment with regorafenib and 5-FU, this patient did experience modest treatment-associated toxicity. During the first 2 cycles, he sustained grade 1 mucositis and hyperbilirubinemia. This hepatic dysfunction progressed to grade 2 following the ninth cycle of treatment but stabilized after the dose of regorafenib was reduced from 160 mg to 80 mg PO daily and infusional 5-FU was reduced to 80%. The patient experienced intermittent grade 2 diarrhea and thrombocytopenia. He also had one episode of grade 2 neutropenia with nadir absolute neutrophil count (ANC) of 1110/ μL. This patient required several treatments delays due to hospitalization for both neutropenic and non-neutropenic sepsis in December 2013, February 2014, April 2014, May 2014, September 2014 and March 2015. Interestingly, at the time the patient was initially treated with regorafenib and 5-FU, his CEA rose when therapy was interrupted due to his infections, and ceased to rise when the therapy was resumed (Fig. 6B). The patient did not experience any clinically significant hypertension, proteinuria, or other adverse effects of the regorafenib and 5-FU treatment. The patient had multiple indwelling catheters and intravenous central lines during his course, and so we do not believe his sepsis episodes were causally related to his dual oral chemotherapy regimen.
Materials and Methods for Preclinical Experiments
Cell culture
HT29 and SW480 cells were obtained from the American Type Culture Collection. HCT116 and HCT116 p53−/− cells were generously provided by Dr. Bert Vogelstein (Johns Hopkins University, Baltimore, MD). All cell lines were grown in ATCC recommended media supplemented with 10% fetal bovine serum, 100 units/mL of penicillin and 100 ug/mL of streptomycin in a humidified incubator at 5% CO2 and 37°C.
Western blots
Cells were treated with 5-FU (Hospira) and/or Regorafenib (MedKoo) at the indicated dosages and times, rinsed with cold PBS twice and lysed with RIPA buffer (Sigma-Aldrich) containing fresh protease inhibitor cocktail (Life Technologies). The amount of protein in cell lysates was quantified with Pierce™ BCA Protein Assay Kit (Life Technologies) and diluted with NuPAGE® LDS Sample Buffer (Life Technologies) and denatured at 95°C for 10 minutes. Approximately 25–30 μg protein of each sample was resolved by SDS-PAGE and polypeptides were electrophoretically transferred to Immobilon-FL PVDF membranes (EMD Millipore). Membranes were blocked with 10% non-fat milk for 1 hr at room temperature. Then membranes were incubated overnight at 4°C with primary antibodies: rabbit anti-human ERK, p-ERK (T202/Y204), Mcl-1, Bcl-xL, PUMA from Cell Signaling Technology, and mouse anti-human Ran from BD Biosciences. Membranes were washed thoroughly and incubated with species specific secondary antibodies conjugated with Alexa Fluor® 680 or IRDye® 800CW (Li-Cor Biosciences) for 1.5 hr at room temperature. Membranes were washed thoroughly and imaged with Odyssey Imaging System (Li-Cor Biosciences).
Cell viability assay
Changes in cell viability were evaluated by measuring ATP level using CellTiter-Glo® luminescence assay (Promega, Fitchburg, WI). 6000 cells were seeded into a 96-well flat-bottomed plate and incubated for 24 hr. Then cells were treated as desired and CellTiter-Glo reagent was added and incubated at room temperature for 10 min protected from light. The plate was imaged with IVIS imaging system (Xenogen). The luminescence of each well was quantified using LivingImage3.2 software. Synergistic activity was determined by calculating Combination Index (CI) using the CompuSyn software (combosyn.com). CI <1 , =1 and >1 indicate synergism, additive effect and antagonism respectively.
Results from preclinical experiments
In order to further investigate the potential for a synergistic effect of regorafenib with 5-FU toward colorectal cancer, we performed growth inhibition assays using the individual agents alone and in combination (Figs. 7–9).
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19 January 2016Figure 7. Effect of regorafenib on colorectal cancer cell line viability. (A) 48h dose curves with a panel of 4 human colorectal cancer cell lines. (B) Table of experimentally derived regorafenib IC50 values.
![]({"type":"image","src":"/na101/home/literatum/publisher/tandf/journals/content/kcbt20/2015/kcbt20.v016.i12/15384047.2015.1113355/20160119/images/medium/kcbt_a_1113355_f0007_b.gif"})
Figure 7. Effect of regorafenib on colorectal cancer cell line viability. (A) 48h dose curves with a panel of 4 human colorectal cancer cell lines. (B) Table of experimentally derived regorafenib IC50 values.
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19 January 2016Figure 8. Effect of 5-FU on colorectal cancer cell line viability. (A) 48 h dose curves with a panel of 4 human colorectal cancer cell lines. (B) Table of experimentally derived 5-FU IC50 values.
![]({"type":"image","src":"/na101/home/literatum/publisher/tandf/journals/content/kcbt20/2015/kcbt20.v016.i12/15384047.2015.1113355/20160119/images/medium/kcbt_a_1113355_f0008_b.gif"})
Figure 8. Effect of 5-FU on colorectal cancer cell line viability. (A) 48 h dose curves with a panel of 4 human colorectal cancer cell lines. (B) Table of experimentally derived 5-FU IC50 values.
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19 January 2016Figure 9. Regorafenib synergizes with chemotherapy. (A) Colorectal cancer cells were treated with the indicated concentrations of regorafenib in combination with 5-FU for 48 h. Viability is visualized on a rainbow scale using the CellTiter Glo reagent as described in materials and methods. (B) Combination index (CI) values of regorafenib in combination with 5-fluorouracil for 48 h in various non-constant ratios against colon cancer cell lines. Combination index <1, =1 and >1 indicates synergism, additivity or antagonism, respectively. Red numbers indicate synergy (CI<1).
![]({"type":"image","src":"/na101/home/literatum/publisher/tandf/journals/content/kcbt20/2015/kcbt20.v016.i12/15384047.2015.1113355/20160119/images/medium/kcbt_a_1113355_f0009_oc.gif"})
Figure 9. Regorafenib synergizes with chemotherapy. (A) Colorectal cancer cells were treated with the indicated concentrations of regorafenib in combination with 5-FU for 48 h. Viability is visualized on a rainbow scale using the CellTiter Glo reagent as described in materials and methods. (B) Combination index (CI) values of regorafenib in combination with 5-fluorouracil for 48 h in various non-constant ratios against colon cancer cell lines. Combination index <1, =1 and >1 indicates synergism, additivity or antagonism, respectively. Red numbers indicate synergy (CI<1).
The results suggest that colorectal cancer cell lines are sensitive to single agent regorafenib (Fig. 7) or 5-FU (Fig. 8), and each of the cell lines tested demonstrated evidence for synergy under some experimental conditions with combination index values of <1.0 (Fig. 9).
Additional experiments were performed to assess alterations in specific biomarkers that may correlate with observed sensitivity patterns (Fig. 10).
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19 January 2016Figure 10. Combinational effect of 5-FU and regorafenib on colorectal cancer cells. Analysis of Mcl-1, Bcl-XL, pERK, and PUMA with Ran loading control as analyzed by western blotting in HT29 (panel A), p53-null (panel B), SW480 (panel C), and wild-type HCT116 cells (panel D) treated with 5-FU, regorafenib, or the combination at the indicated doses for 24 hr.
![]({"type":"image","src":"/na101/home/literatum/publisher/tandf/journals/content/kcbt20/2015/kcbt20.v016.i12/15384047.2015.1113355/20160119/images/medium/kcbt_a_1113355_f0010_b.gif"})
Figure 10. Combinational effect of 5-FU and regorafenib on colorectal cancer cells. Analysis of Mcl-1, Bcl-XL, pERK, and PUMA with Ran loading control as analyzed by western blotting in HT29 (panel A), p53-null (panel B), SW480 (panel C), and wild-type HCT116 cells (panel D) treated with 5-FU, regorafenib, or the combination at the indicated doses for 24 hr.
Our analysis demonstrates that Mcl-1 and Bcl-xL were inhibited in HT29 (panel A), p53-null (panel B), SW480 (panel C), and wild-type HCT116 cells (panel D) by 5-FU and regorafenib. Regorafenib appears to be a potent inhibitor of Mcl-1 expression and the reduced expression was still present when 5-FU was added in combination across the panel of cell lines tested. pERK was induced in SW480 (panel C) and HCT116 (panel D) by combination treatment of 5-FU and regorafenib at 24 hr. PUMA was induced in HCT116 p53 null cells (panel B) but not in HT29 (panel A) by 5-FU and regorafenib at 24 hr.
Discussion
Both patients were diagnosed with colorectal cancer and underwent surgical resection followed by first-line FOLFOX and second-line FOLFIRI. After developing progressive disease on these regimens, each patient received an assortment of targeted agents and cytotoxic chemotherapeutics without sustained efficacy. Having exhausted all approved standard therapies without available clinical trial options, these patients ultimately received regorafenib in combination with capecitabine or 5-FU.
The first patient had stable disease for at least one month after starting therapy with regorafenib and capecitabine. Disease control was reflected in both radiographic and serologic measures of CRC activity. The duration of this positive response to treatment was cut short, however, by severe pneumonia and pulmonary hemorrhage resulting in the patient's demise. It remains uncertain whether this fatal bleed occurred as a treatment-related adverse event because the patient was concurrently receiving therapeutic-dose enoxaparin for her pulmonary embolus. Although regorafenib is associated with thrombocytopenia and an increased tendency toward hemorrhage,13 this patient had an adequate platelet count as well as a history of PE for which she was receiving anticoagulation. In addition, this bleed arose within the site of her prior pulmonary infarction, which was likely more friable due to a superimposed infection.
A more prolonged period of disease control following therapy was observed in the second patient, who received regorafenib with 5-FU for approximately 17 months. This patient had evidence of stable disease for at least 2 months before starting to accumulate new metastatic foci. Despite developing progressive disease, the patient opted to continue on this treatment, and for the next 11 months, his tumor markers remained largely suppressed. The patient ultimately passed away 17 months following his first dose of regorafenib with 5-FU due to a disease-related complication. By comparison, the natural history of untreated CRC predicts a median survival of only 4–6 months following progression on second line treatment.14-16 It therefore appears that this regimen continued to substantially deter the rate of tumor growth, even after development of progressive disease on therapy.
Regorafenib, an oral multikinase inhibitor, is believed to limit the growth of CRC cells through inhibition of multiple tyrosine kinases that facilitate angiogenesis, oncogenesis, tumor growth/proliferation signaling, and cancer cell survival. In 2012, a phase 1b study reported its antitumor activity and safety in patients with mCRC who had disease progression despite a median of 4 previous lines of treatment.9 Single agent regorafenib produced at least stable disease in 74% of these heavily pretreated patients.9 These findings were validated by the international, multicenter phase III CORRECT trial, which randomized 760 patients with mCRC to best supportive care (BSC) with regorafenib or placebo.11 Grothey et al. reported a significant increase in median OS with regorafenib compared to placebo (6.4 months vs 5.0 months; HR 0.77; 95% CI 0.64 – 0.94, P ( 0.0052) and a significant but modest improvement in progression free survival (PFS) (1.9 months vs. 1.7 months, P ( 0.000001). This survival benefit was likely due to a cytostatic rather than a cytotoxic effect of regorafenib, as tumor shrinkage was seldom observed.
Since regorafenib has been shown to possess antitumor activity against mCRC, its role in combination with fluoropyrimidine-based therapy is currently in area of investigation. A case report by Lu et al. showed that the combination of FOLFIRI and regorafenib may be a promising salvage therapy for patients with refractory mCRC. The patient received dose escalations of irinotecan based on UGT1A1 genotyping analysis and had a PFS of over 6 months.17 Lu et al. reported that the only adverse events on FOLFIRI with regorafenib were grade 2 hand-foot syndrome and grade 1 fatigue.17 A phase 1b study evaluating the effect of regorafenib in combination with FOLFOX or FOLFIRI as first- and second-line therapy for mCRC found that regorafenib can be safely administered with 5-FU, oxaliplatin, and irinotecan without significant toxicities.18 Schultheis et al. found no significant effect on 5-FU pharmacokinetics with the addition of regorafenib to FOLFOX or FOLFIRI. Moreover, 33 of 38 (87%) evaluable patients achieved disease control (partial response or stable disease) for a median of 126 d.18
The phase II CORDIAL trial investigating the efficacy of regorafenib plus FOLFOX as first-line treatment for mCRC did not show improvement in PFS or overall response rate compared to historical controls.19 However, Argiles et al. noted that the addition of regorafenib to FOLFOX was well-tolerated and 6 patients received at least one component of the study regimen for at least 1 year, including 2 patients on regorafenib with 5-FU (bolus and infusion) and 2 patients on regorafenib with infusional 5-FU. We observed a similar phenomenon with the second case in which the patient with refractory mCRC remained on regorafenib and 5-FU for 17 months. This relatively long duration of treatment contrasts with data from studies of other first-line therapy for mCRC (5.5 months for FOLFOX and 6.0 months for FOLFIRI).20 The combination of regorafenib and FOLFOX may help patients continue on treatment and maintain tumor control for longer than might be achieved with chemotherapy or targeted therapy alone. The authors speculated that regorafenib may spare some patients from certain toxicities associated with cytotoxic chemotherapy, thereby allowing them to continue treatment.
For treatment-refractory mCRC, the goal of therapy is to balance disease control and prolong survival with maintaining an acceptable quality of life for the patient. Any treatment-associated toxicity that negatively affects quality of life may easily outweigh the potential benefits. The most common adverse reactions to regorafenib include palmar-plantar erythrodysesthesia, fatigue, hypertension, mucositis, and diarrhea. A slight increased risk of thrombocytopenia11 and bleeding13 has also been reported. Both patients experienced some degree of fatigue and diarrhea. The second patient experienced grade 2 hyperbilirubinemia after 9 cycles of therapy, which resolved after both a dose reduction in regorafenib and 5-FU. He also had grade 2 neutropenia that spontaneously resolved after 5 d. Although both patients developed severe infections, the second patient expired from septic shock that was thought to be disease-related and not due to treatment. The second patient tolerated therapy for approximately 17 months. These two cases suggest that the combination of regorafenib with 5-FU or capecitabine is well-tolerated and does not substantially worsen toxicity when compared to known side effects of each individual component.
As a multikinase inhibitor targeting various signaling pathways implicated in CRC, regorafenib is being studied in combination with various chemotherapeutic and targeted agents. There is a phase II multicenter trial comparing the efficacy of FOLFIRI with regorafenib or placebo in second-line therapy for mCRC (NCT01298570). Another phase II placebo-controlled trial is evaluating the safety and efficacy of ruxolitinib, a JAK inhibitor, in combination with regorafenib in relapsed or refractory mCRC (NCT02119676). The REGAL-1 trial for mCRC is a phase 1b study investigating the safety and clinical activity of combining regorafenib with PF-03446962, a fully humanized monoclonal antibody to activin receptor like kinase 1 (ALK1) with antiangiogenic activity (NCT02116-894).
In support of the concept of evaluating potential activity of combination regorafenib and 5-FU therapy for patients with refractory mCRC, we have observed synergistic effects of the 2-drug combination against multiple human colorectal cancer cell lines. Synergistic effects were observed with the addition of regorafenib to 5-FU in colorectal cancer cell lines with KRAS mutation (SW480 and HCT116 cell lines), BRAF mutation (HT29 cell line), p53 mutation (SW480 and HT29 cell lines), p53 deletion (p53-null HCT116) or wild-type p53 status (wild-type p53 HCT116 cell line). These observations suggest that such a combination should be tested across a range of colorectal cancer subtypes harboring different driver gene mutations. Of note, while it is known that mismatch repair deficient colorectal cancers are generally more resistant to 5-FU than microsatellite stable tumors, the HCT116 mismatch repair deficient cells used here were sensitive to the regorafenib plus 5-FU combination regardless of whether p53 was wild-type or deleted. Thus a clinical study would not need to exclude patients with mismatch repair deficiency and advanced disease without other options for therapy. One of the limitations of the present study is that we did not evaluate the potential for synergy between regorafenib and 5-FU in colorectal cancer cells that were already resistant to the individual agents.
Potent inhibition of Mcl-1 expression was observed in regorafenib-treated colorectal cancer cells and the reduced expression was still present when 5-FU was added in combination across the panel of cell lines tested. Reduced Mcl-1 and Bcl-XL (although reduction of Bcl-XL was less robust and noted primarily in the combination therapy conditions) could be a contributing factor to the observed sensitivity patterns. pERK was induced in SW480 (panel C) and HCT116 (panel D) by combination treatment of 5-FU and regorafenib at 24 hr. pERK induction is perhaps not surprising given recent results.21 We did not assess very early time points where regorafenib has been shown to inhibit pERK.21 PUMA was induced in HCT116 p53 null cells (panel B) but not in HT29 (panel A) by 5-FU and regorafenib at 24 hr. The differences between our results and others published recently are not entirely clear for the HCT116 wild-type cells although different antibodies were used.22 Analysis of PARP cleavage suggested that the observed combinatorial synergistic effects were not explained by classical apoptotic effects (data not shown). We suspect that a combination of growth arrest and cell death, including apoptosis was involved in the observed effects on cell viability with the 2-drug regimen. We have previously observed both cytostatic effects as well as cytotoxic effects of sorafenib that is related to regorafenib.23 It may be of interest in the future to determine whether regorafenib can act as a radiosensitizer either alone or in combination with 5-FU. It may be of interest in the future to explore (and compare) whether the recently approved TAS102 drug displays similar combinatorial synergy with regorafenib as we observed with 5-FU.
Patients who are treated with regorafenib have various side effects including fatigue, mucositis, abnormal liver enzymes, hand-foot syndrome. While the patients described in the case reports tolerated full dose therapy with regorafenib fairly well, we should note that the observation of combinatorial efficacy and synergy with 5-FU may extend to lower doses of the drugs. One of the potential benefits of drug synergy is the possibility of using lower doses of both drugs to lessen the chance for toxicity as may be observed with higher doses when the drugs are given as single agents. This can clearly be tested further in preclinical (including in vivo experiments) as well as clinical studies.
The clinical observation of stable disease with the combination of regorafenib and 5-FU in the case reports led to laboratory experiments demonstrating, under certain experimental conditions, synergistic activities of the combination regimen in various human colorectal cancer cell lines. The preclinical observations that derived from the clinical experience may support a scientific rationale for future clinical research. For a future prospective clinical trial based on the described clinical and subsequent preclinical observations, this would be an example of bedside-to-bench and back translational research.
Despite the multitude of clinical trials, there has not been active clinical investigation in the safety and efficacy of combining regorafenib with capecitabine or 5-FU in treatment-refractory mCRC. This regimen holds considerable theoretical appeal, as fluoropyrimidines are the backbone of effective first and second-line therapy. Regorafenib, on the other hand, has been proven to prolong OS in patients who failed initial therapy and may blunt the side effects of cytotoxic chemotherapy. Further investigation into this possible later-line regimen is certainly warranted given the observation of stable disease from the combination of regorafenib and 5-FU or capecitabine despite progression on prior therapy with either agent alone.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
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