Purpose:

To assess the antitumor activity and safety of tipifarnib, a highly potent and selective farnesyltransferase inhibitor, we performed a phase II clinical trial in patients with advanced and refractory urothelial carcinoma harboring missense HRAS mutations.

Patients and Methods:

A total of 245 adult patients with previously treated, advanced urothelial carcinoma entered the molecular screening program including HRAS. Those with missense HRAS mutations or STK11:rs2075606 received oral tipifarnib 900 mg twice daily on days 1–7 and 15–21 of 28-day treatment cycles. The primary endpoint was progression-free survival at 6 months (PFS6).

Results:

We identified 16 (7%) missense HRAS mutations (G13R, 7; Q61R, 4; G12S, 3; G12C, 2) and 104 (46%) STK11:rs2075606 carriers. In 21 patients enrolled in the study, 14 and 7 patients had missense HRAS mutations and STK11:rs2075606, respectively. The most frequently observed adverse events included fatigue (86%) and hematologic toxicities. With a median follow-up of 28 months, 4 patients (19%) reached PFS6: 3 had missense HRAS mutations and one patient, enrolled as an STK11 carrier, had HRAS frameshift insertions at H27fs and H28fs rendering a nonsense HRAS mutation. The overall response rate by intent-to-treat analysis was 24% (4 missense and one nonsense frameshift HRAS mutation); no response was observed in patients with urothelial carcinoma with wild-type HRAS tumors. Five responses were observed in 12 evaluable patients of 15 with tumors carrying HRAS mutations.

Conclusions:

Oral tipifarnib resulted in a manageable safety profile and encouraging antitumor efficacy against treatment-refractory urothelial carcinoma containing HRAS mutations.

Translational Relevance

In a prospective phase II study, patients with metastatic, heavily pretreated urothelial carcinoma harboring missense HRAS mutations were treated with oral tipifarnib, an orally administered farnesyl transferase inhibitor. Tipifarnib showed a manageable safety profile and encouraging activity. While this study was primarily focused on single-agent tipifarnib, potential combinations with other agents, for example, immune checkpoint inhibitors, should be considered as a novel therapeutic strategy to further improve outcomes in the treatment of urothelial carcinoma.

Urothelial carcinoma, a malignant neoplasm involving the transitional epithelial lining of the urinary tract, is a highly prevalent disease, with an estimated 3,500 new cases and 1,200 deaths annually in Korea (1). In urothelial carcinoma, somatic mutations in the FGFR, RAS, and PI3K genes are suggested for prognostic markers as well as for therapeutic targets (2). These proteins play a pivotal role in the transduction of cell growth–stimulatory signals, and their mutation is known to lead to constitutive activation, resulting in uncontrolled cell proliferation. The high prevalence of mutated RAS genes, with HRAS in high-grade urothelial carcinoma being most prominent (3, 4), makes this pathway an attractive target for anticancer drug development. RAS targeting may be accomplished by multiple mechanisms. A promising way of interfering with RAS function is the inhibition of farnesyl transferase, the enzyme coupling an isoprenyl group to RAS proteins. By inhibiting RAS farnesylation, a blockade of the RAS-mediated signal transduction pathway is accomplished, with attenuation of cell growth (5).

Consequently, inhibition of RAS signaling using highly potent and selective farnesyl transferase inhibitors (FTI) was proposed as an effective therapeutic approach in multiple oncology indications (5). Tipifarnib (Kura Oncology) is an orally administered, highly potent, and selective nonpeptide FTI. Preclinical data indicate that tumor models carrying HRAS mutations are sensitive to FTI (6). In a phase II study of tipifarnib in subjects with metastatic urothelial carcinoma, although the presence of HRAS mutations was not evaluated, two responses were observed [overall response rate (ORR), 6%] in subjects with no prior chemotherapy treatment and a total of 13 study subjects achieved disease stabilization (7). Given the evidence of safety and antitumor activity of tipifarnib, we designed the present phase II study to test the hypothesis that treatment with tipifarnib may translate to clinical benefit in patients with relapsed and/or refractory metastatic urothelial carcinoma harboring HRAS mutations.

The protocol of this investigator-initiated, internally funded, single-center, prospective phase II trial was registered in advance to ClinicalTrials.gov (NCT02535650) and Clinical Research Information Service (http://cris.nih.go.kr/cris/en/, KCT0004115), and approved by the Samsung Medical Center (SMC) Institutional Review Board (IRB no. 2015-05-039). Study drug, tipifarnib, was kindly provided by Kura Oncology.

Patients

The study included adult (≥20 years of age) patients with a metastatic urothelial carcinoma for which no available systemic therapy existed. They were required to have a history of disease progression during or after at least one course of previous systemic chemotherapy for metastatic disease. Molecular criteria for eligibility included missense, nonsynonymous HRAS mutations and/or the STK11:rs2075606 (T>C) single nucleotide variant (SNP) within the archival tumor. Initially, we enrolled only patients with missense HRAS mutations; however, because the main target of an FTI in urothelial carcinoma is yet unknown, we considered also the investigation of the antitumor activity of tipifarnib in patients whose tumors carry the rs2075606 variant at position 1220321 (NC_000019.9:g.1220321T>C) in the STK11 gene. Other criteria for eligibility included age of 20 years or older, an Eastern Cooperative Oncology Group performance status of 0 or 1, no antitumor therapy within 4 weeks, normal organ functions, and at least one measurable tumor mass by RECIST v1.1. All patients gave written informed consent consistent with the Declaration of Helsinki and the principles of Good Clinical Practice.

Study procedures

Patients with HRAS and/or STK11 variants were selected through the SMC Oncology Biomarker study (ClinicalTrials.gov, NCT01831609), and the details of the methodology for mutational and transcriptional profiling were described previously (3). In brief, genomic DNA was extracted from tumor samples and analyzed using the Ion Ampliseq Cancer Hotspot Panel v2 (Thermo Fisher Scientific Korea). The panel examines 2,855 mutations and polymorphisms in 50 commonly mutated oncogenes and tumor suppressor genes, including HRAS and STK11.

Tipifarnib was administered orally at a starting dose of 900 mg twice daily on days 1–7 and 15–21 of 28-day treatment cycles. Treatment continued until disease progression, unacceptable adverse events, or consent withdrawal. Safety was evaluated clinically every week in the first cycle then every 4 weeks, if no significant adverse events had occurred, and graded using the Common Terminology Criteria for Adverse Events (CTCAE v 4.03). Patients who experienced neutropenic fever, grade 3 or 4 hematologic toxicities, or grade 3 nonhematologic toxicities had treatment delayed until toxicity resolved to grade 2 or less, and subsequently their dose was reduced to 600 mg twice daily. If recovery required more than 4 weeks or the toxicity was recurrent following two dose reductions, study treatment was discontinued permanently. Tumor assessment according to the RECIST was done at screening and every 8 weeks, or sooner if deemed necessary by the investigators. If a measurable radiological response was observed at any time, the same radiological assessment was repeated after 4 weeks to confirm the response. After disease progression or study discontinuation for any reason, patients were monitored for progression, survival, and for information relating to subsequent therapies and their general safety and efficacy.

Statistical analysis

The primary endpoint of the study was to investigate the efficacy of tipifarnib in patients with metastatic urothelial carcinoma harboring HRAS mutations and/or STK11:rs2075606 as assessed by progression-free survival rate at 6 months (PFS6). Secondary endpoints included ORR, the median PFS, overall survival (OS), and safety profile of tipifarnib. PFS was defined as the time from study entry until death, disease progression, or date of last contact, whichever occurred first. If a patient survived progression-free for at least 6 months, the patient was deemed to have reached PFS6. PFS, OS, and ORR were calculated with corresponding 95% confidence interval (CI). Patient demographics and other baseline characteristics were summarized using conventional descriptive statistics.

To determine sample size, we assumed that ineffective therapies in pretreated urothelial carcinoma have a PFS6 less than 10%. To differentiate between a 10% and 30% PFS6 rate, a total of 18 patients was required using a two-stage phase II design (8). If 2 or more patients were PFS6 in the first stage of accrual of 11 patients, 7 additional patients were enrolled. The design provided 80% power to detect a difference between 10% and 30% PFS6 at one-sided significance level of 0.087. Statistical analyses were performed using SPSS 13 for Windows (SPSS).

Between November 2015 and July 2019, a total of 245 patients with urothelial carcinoma gave an informed consent for the Oncology Biomarker study; 21 were excluded due to inadequate or missing tumor samples for analysis (Fig. 1). Among 224 patients screened, 16 (7%) had missense HRAS mutations (G13R, n = 7; Q61R, n = 4; G12S, n = 3; G12C, n = 2) and 104 (46%) patients had STK11:rs2075606 (Fig. 1; Supplementary Fig. S1). On the basis of the interim evaluation after the accrual of the first-stage patients and, upon observation of the lack of efficacy in patients with STK11:rs2075606, investigators decided that only patients with HRAS mutations were eligible for further inclusion. Finally, a total of 21 eligible patients entered the study and received tipifarnib (Table 1). The median age at study entry was 64 years (range, 51–77). The median number of prior systemic chemotherapy was 2 (range, 1–4). Two patients received prior therapy with immune checkpoint inhibitors (SMC-15 and SMC-20).

Figure 1.

Study diagram.

Table 1.

Baseline characteristics of all enrolled patients (n = 21).

No. (SMC-)Age (years)GenderPrimary tumor originPrior therapyMeasurable lesionsHRAS (VAF, %)STK11
01 75 Bladder Lymph node G13R (56.9) rs2075606 
02 73 Renal pelvis Lymph node WT rs2075606 
03 73 Bladder Lung G13R (56.7) rs2075606 
04 56 Renal pelvis Lung G13R (55)  
05 60 Bladder Liver WTa rs2075606 
06 58 Bladder Lung G13R (55.7)  
07 64 Bladder Liver G12S (4.5) rs2075606 
08 65 Ureter Lymph node WT rs2075606 
09 68 Bladder Lymph node G13R (17.1) rs2075606 
10 66 Ureter Liver, lung WT rs2075606 
11 51 Bladder Lung Q61R (26.8) rs2075606 
12 74 Renal pelvis Lung WT rs2075606 
13 57 Renal pelvis Lung G13R (10) rs2075606 
14 64 Ureter Lung WT rs2075606 
15 77 Renal pelvis Lung Q61R (23.1) rs2075606 
16 73 Renal pelvis Liver WT rs2075606 
17 60 Bladder Lung, liver G12C (55.3) rs2075606 
18 63 Renal pelvis Lung G12S (51.1)  
19 59 Bladder Lung Q61R (42.6) rs2075606 
20 64 Renal pelvis Lung G13R (17.6)  
21 67 Bladder Lymph node Q61R (3.9) rs2075606 
No. (SMC-)Age (years)GenderPrimary tumor originPrior therapyMeasurable lesionsHRAS (VAF, %)STK11
01 75 Bladder Lymph node G13R (56.9) rs2075606 
02 73 Renal pelvis Lymph node WT rs2075606 
03 73 Bladder Lung G13R (56.7) rs2075606 
04 56 Renal pelvis Lung G13R (55)  
05 60 Bladder Liver WTa rs2075606 
06 58 Bladder Lung G13R (55.7)  
07 64 Bladder Liver G12S (4.5) rs2075606 
08 65 Ureter Lymph node WT rs2075606 
09 68 Bladder Lymph node G13R (17.1) rs2075606 
10 66 Ureter Liver, lung WT rs2075606 
11 51 Bladder Lung Q61R (26.8) rs2075606 
12 74 Renal pelvis Lung WT rs2075606 
13 57 Renal pelvis Lung G13R (10) rs2075606 
14 64 Ureter Lung WT rs2075606 
15 77 Renal pelvis Lung Q61R (23.1) rs2075606 
16 73 Renal pelvis Liver WT rs2075606 
17 60 Bladder Lung, liver G12C (55.3) rs2075606 
18 63 Renal pelvis Lung G12S (51.1)  
19 59 Bladder Lung Q61R (42.6) rs2075606 
20 64 Renal pelvis Lung G13R (17.6)  
21 67 Bladder Lymph node Q61R (3.9) rs2075606 

Abbreviations: F, female; M, male; VAF, variant allele frequency; WT, wild-type.

aThe patient had frameshift insertions in HRAS: NM_005343:exon2:c.84_85insT:p.F28fs, NM_176795:exon2:c.84_85insT:p.F28fs, M_001130442:exon2:c.84_85insT:p.F28fs (VAF 54.9%; 309/573), NM_005343:exon2:c.80_81insC:p.H27fs, NM_176795:exon2:c.80_81insC:p.H27fs, NM_001130442:exon2:c.80_81insC:p.H27fs (VAF 44.4%; 258/573).

Safety

At final analysis (September 25, 2019), 2 of the 21 patients remained on treatment. The most frequent reason for treatment discontinuation was disease progression (n = 14; Fig. 1). A total of 97 tipifarnib cycles (median, 3; range, 1–18) were delivered. Safety was evaluable in all 21 patients in whom at least one dose of tipifarnib was given. Twenty-one (100%) patients had a treatment-related adverse event (Table 2); 14 patients (67%) experienced grade 3 to 5 events. While difficult to differentiate from the symptoms of the underlying disease, one patient (SMC-17) died of pneumonitis and massive hemoptysis during the second cycle of study participation. The most frequently observed adverse events included fatigue (86%) and hematologic toxicities. Ten patients received blood transfusion during the study treatment. Of note, although all patients were pretreated with cytotoxic chemotherapy and the incidence of hematologic toxicities was substantial, only three episodes of febrile neutropenia occurred. Dose reduction was required in 12 patients. Twelve patients had a treatment delay of one week or more at some time during treatment. The most common adverse events leading to a dose reduction and/or treatment delay were neutropenia and fatigue. The median dose intensity of tipifarnib was 792 mg/week, which corresponded to 88% of the planned dose.

Table 2.

Maximum grade adverse events (n = 21).

All gradesGrades 3 or 4
Neutropenia 14 (67%) 4 (19%) 
Febrile neutropenia  3 (14%) 
Anemia 16 (76%) 8 (38%) 
Thrombocytopenia 10 (48%) 6 (30%) 
Anorexia 9 (43%) 1 (5%) 
Nausea 7 (33%) 2 (10%) 
Vomiting 5 (24%) 
Stomatitis 3 (14%) 
Constipation 3 (14%) 
Diarrhea 3 (14%) 
Fatigue 18 (86%) 3 (14%) 
Pruritus 2 (10%) 
Rash 3 (14%) 
Pain 6 (30%) 
Transaminase increase 1 (5%) 
Creatinine increase 3 (14%) 1 (5%) 
All gradesGrades 3 or 4
Neutropenia 14 (67%) 4 (19%) 
Febrile neutropenia  3 (14%) 
Anemia 16 (76%) 8 (38%) 
Thrombocytopenia 10 (48%) 6 (30%) 
Anorexia 9 (43%) 1 (5%) 
Nausea 7 (33%) 2 (10%) 
Vomiting 5 (24%) 
Stomatitis 3 (14%) 
Constipation 3 (14%) 
Diarrhea 3 (14%) 
Fatigue 18 (86%) 3 (14%) 
Pruritus 2 (10%) 
Rash 3 (14%) 
Pain 6 (30%) 
Transaminase increase 1 (5%) 
Creatinine increase 3 (14%) 1 (5%) 

Efficacy

With a median follow-up duration of 28 months (range, 4–47 months), the median PFS and OS in this study population were 4.7 months (95% CI, 2.5–5.6 months) and 6.1 months (95% CI, 5.0–7.2 months), respectively (Fig. 2). We observed that 4 patients (19%; 95% CI, 2%–36%) reached PFS6: 3 patients (SMC-11, -15, and -18) had missense HRAS mutations and one patient (SMC-05), enrolled as an STK11 SNP carrier, had HRAS frameshift rendering a nonsense HRAS mutation. Among the 21 patients enrolled in the study, 16 were evaluable for objective clinical response; 5 patients were excluded for consent withdrawal (n = 4) and early death (n = 1). In an intent-to-treat principle, these patients were considered as nonresponders. As a result, 5 patients achieved an objective response (ORR, 24%; 95% CI, 6%–42%). An additional 4 patients (19%) had stable disease, leading to the disease control rate of 43%. Among 14 patients with missense HRAS mutations, 4 (29%; 95% CI, 5%–52%) achieved an objective response. One additional response was observed in the patient carrying a nonsense HRAS mutation resulting in an ORR of 33% (95% CI, 7%–59%) for the overall population of patients with urothelial carcinoma carrying HRAS mutations. On the contrary, only one objective response (14%; 95% CI, 0%–40%) was observed in 7 patients who were STK11 SNP carriers. SMC-05 was the one with frameshift insertions at HRAS H27fs and H28fs resulting in nonsense HRAS; thus, no responses were observed in patients with urothelial carcinoma with wild-type HRAS tumors (Fig. 3). The duration of clinical response in responders was 8.8 months (95% CI, 3.8–13.8 months). In total, 8 of the 15 patients with tumor HRAS mutations experienced some degree of tumor size reduction. All but one of these patients presented with lung target lesions. When we compared the PFS between patients with HRAS mutations (i.e., missense or frameshift) and those with wild-type HRAS, the difference was significant (median, 5.1 vs. 0.8 months; HR, 0.262; 95% CI, 0.087–0.793). On the contrary, no significant difference in the PFS was observed between patients with STK11 SNP carriers or not (HR, 0.387; 95% CI, 0.088–1.701).

Figure. 2.

Progression-free (green line) and overall (orange line) survivals of the enrolled patients (n = 21).

Figure. 2.

Progression-free (green line) and overall (orange line) survivals of the enrolled patients (n = 21).

Close modal
Figure. 3.

Change in target lesion burden per independent radiology review committee by best overall response in all response-evaluable patients. A, Treatment duration for the enrolled patients (n = 21). Objective responses are indicated. B, Waterfall plot of maximal change in tumor size in the evaluable patients (n = 16). The patient numbers are indicated (#).

Figure. 3.

Change in target lesion burden per independent radiology review committee by best overall response in all response-evaluable patients. A, Treatment duration for the enrolled patients (n = 21). Objective responses are indicated. B, Waterfall plot of maximal change in tumor size in the evaluable patients (n = 16). The patient numbers are indicated (#).

Close modal

Among 19 patients who discontinued study treatment, the median time from tipifarnib failure to death was 2.1 months (95% CI, 0.6–3.7). Nine patients received a further systemic treatment after tipifarnib discontinuation, mostly in the context of clinical trials: taxanes (n = 6), immune checkpoint inhibitors (n = 3), and pemetrexed (n = 1). To explore predictive factors for clinical response to tipifarnib, we performed a logistic regression analysis using known clinical and laboratory parameters. ORR was not influenced by age, gender, performance status, the number of prior chemotherapy regimens, metastatic sites, or baseline laboratory parameters. We also tested whether the development of clinical responses was modified by interaction between the effects of parameters; the first-level interaction term between these variables was entered into a separate multivariate model but we found no interaction between them. For exploratory purposes, we compared the somatic mutational landscape with The Cancer Genome Atlas (TCGA) data (9). After excluding potential germline variations, 141 samples had at least one actionable genetic aberration, and we found similar frequencies of major oncogenic driver mutations between ours and the TCGA (n = 131) cohort (Supplementary Fig. S2). Furthermore, when we evaluated the patients' genomic profiles, we did not identify any genomic correlates that were directly associated with clinical response to tipifarnib (Supplementary Fig. S3).

This prospective study found that tipifarnib in patients with urothelial carcinoma with HRAS mutations resulted in a manageable safety profile and encouraging efficacy, even in those refractory to immune checkpoint inhibitors. We included patients with tumors with wild-type HRAS but carrying the polymorphism rs2075606 (T>C) in the STK11 intron into this study, and we now believe that tipifarnib is likely not effective in this group of patients. Our study showed the incidence of missense HRAS mutations in urothelial carcinoma to be 7% and, on the basis of historical controls with heavily pretreated metastatic urothelial carcinoma, tipifarnib is worthy of further investigation based on the PFS6 of 21% and ORR of 33% in patients with HRAS mutations. At the same time, the rate of early discontinuation due to consent withdrawal was thought to be related to adverse events of tipifarnib that could be substantial in some patients. The dose regimen employed in this study was based on a phase I trial (10). A recent report indicates that 600 mg twice daily administered in alternating weeks may be better tolerated than 900 mg and translates to a higher level of activity of tipifarnib in patients with squamous head and neck cancer with HRAS mutations (11). Thus, it may be possible that better tolerability of the 600 mg twice daily alternating week regimen could translate to increased antitumor activity of tipifarnib in the urothelial carcinoma population as well.

Urothelial carcinoma is a genomically heterogeneous disease, with high frequencies of significantly mutated genes in receptor kinase signaling such as MAPK, PI3K/AKT, FGFR/RAS, and TP53/RB1/MDM2 pathways closely related to tumor progression and evolution (9, 12, 13). The high prevalence of mutated RAS genes, found in 30% of all human cancers, makes this pathway an attractive target for antitumor drug development (14). However, HRAS is the least frequently found RAS mutation in human cancers (15), with most at one of three mutational hotspots: G12, G13, and Q61. Although targeted inhibition of RAS-based signaling is not yet available clinically (16), FTI is known to decrease RAS translocation to membranes and reduce its ability to mediate activation of downstream effectors in addition to targeting alternative farnesylated proteins (17, 18). With increasing experimental evidence supporting RAS isoform and mutation differences, as well as cell-specific and genetic context–specific differences, there is growing speculation that there will not be one simple anti-RAS therapeutic approach for all RAS-mutant cancers. Instead, cancer type–specific therapeutic strategies must be determined for different subsets of RAS mutations (15). As the membrane association of HRAS is utterly dependent on protein farnesylation (14), tumors containing HRAS mutations appear to be interesting targets for FTIs. In addition to HRAS mutations, we had patients with wild-type HRAS and STK11:rs2075606. STK11 gene encodes for LKB1, a farnesylated regulatory protein that is an important human tumor suppressor gene known to be involved in G1 cell-cycle arrest associated with TP53 and the PTEN pathway (19, 20). Loss-of-function mutations in TP53 and STK11 dramatically enhance the progression to highly invasive and metastatic cancers (21). In a retrospective study in patients with malignant pleural mesothelioma, the SNP variation rs2075606 in STK11 gene clustered in PI3K/AKT pathways was correlated with poor prognosis (22). Unfortunately, we found tipifarnib is not effective in tumors with STK11:rs2075606. It is interesting that we observed one clinical response in a patient with a tumor carrying nonsense HRAS frameshift insertions at H27fs and H28fs. This response was intriguing because these frameshifts do not appear to translate to a missense HRAS protein. We currently do not know whether the response could be due to other mutations or the regulation of wild-type farnesylated protein that are targets of tipifarnib. Other substrates for farnesyl transferase, including RhoB, Rheb, mTOR/Raptor, Rac1, CENP-E/CENP-F, and lamins A/B are also related to tumor initiation and progression, and a wealth of studies in recent years have demonstrated that these proteins are all enrolled in crucial signaling pathways that regulate the malignant transformation, proliferation, apoptosis, invasion of tumor cells, and tumor angiogenesis (23–25). FTIs target different downstream effectors according to host–tumor interactions, histologic tumor type, and stage of the tumor, and their antitumor effects are quite heterogeneous from a prominent antiangiogenic to an antiproliferative and an apoptotic effect in different tumors (26). Furthermore, it has been recently shown that the chemokine CXCL12 is downregulated by tipifarnib and that RHOE and PRICKLE2 could potentially mediate this effect (27, 28). With the increasing use of personalized medicine that will include profiling of the mutation status of tumors that will be coupled to pathway profiling to identify driving oncogenic mechanisms, it is possible that FTIs will eventually produce benefits in patients with disease that is driven through oncogenic HRAS function (29).

Contributing to the relatively limited clinical research in HRAS-mutant urothelial carcinoma, there has also been an underestimation of the complexities of HRAS. Evidence that different amino acid substitutions at any one hotspot can have differential HRAS structure, biochemistry and oncogenic potencies as well as distinct functional consequences adds an additional layer of complexity, suggesting that mutation-selective therapeutic strategies might be needed (15). Furthermore, there are striking cancer type–specific and isoform-distinct differences in the observed frequencies of specific HRAS missense mutations at the three hotspots. As crosstalk and feedback activations are also commonly observed in HRAS-mediated signaling pathways, a simultaneous inhibition of multiple RAS downstream pathways could provide benefits to a subgroup of HRAS-mutant patients with cancer and prevent cancer cells from switching to alternative survival pathways and escaping (5), and that effective strategies to stratify patients for precision therapy is likely required for effective anti-RAS therapy (21). In addition to a small sample size, this study is limited due to the difficulty in relating the study findings with the patients' current tumor status because all tissue specimens were archival and collected before the start of tipifarnib therapy. However, it has been reported that there was a consistency in the type of RAS mutations among different tumor samples obtained from the same patients (2), which is in agreement with published data suggesting that the majority of recurrences in urothelial carcinoma are considered to be clonally related (30). Furthermore, although there had been reports suggesting significant differences in the prevalence of several recurrently mutated genes (3, 31), we found no difference in the frequency of HRAS mutations between urothelial carcinoma arising from bladder and upper tract.

An improved understanding of molecular carcinogenesis has led to the development of novel agents designed to target critical signaling pathways. Recent reports suggested that, in patients with urothelial carcinoma with either FGFR gene alterations (32) or mRNA overexpression (33), FGFR inhibitors yielded 24% to 40% ORR. While the FGFR-targeting strategy is promising, reports state that FGFR and HRAS mutations are mutually exclusive or occur very rarely together (2, 34, 35). Urothelial carcinoma with HRAS mutations may represent a different subgroup requiring a novel therapeutic approach. Although HRAS mutations are found in only a few cases (7%) with urothelial carcinoma, screening for genetic aberrations by using multiplexed sequencing is now broadly conducted across many cancer types, and it may increase the likelihood for a patient to benefit from inhibitors targeting HRAS. In addition to the molecularly targeted agents, several novel therapeutics including immune checkpoint inhibitors and enfortumab vedotin (36, 37), were approved by the FDA for patients with urothelial carcinoma. With the emergence of multiple active agents, development of combination regimens has become a more attractive strategy to improve clinical outcome for urothelial carcinoma. A possible caveat of the combinations is the potentially higher incidence of adverse events.

This study shows that tipifarnib, an FTI targeting HRAS mutations, is effective for pretreated, metastatic urothelial carcinoma. Because tipifarnib prevents the processing of newly synthesized proteins, but does not cause the removal of prenyl groups from proteins already processed, it would be cytostatic, but not cytotoxic (38). As a single agent, tipifarnib appears to have modest clinical effects that are not sufficient to induce a long-term tumor inhibition (26). As multiple pathways are important for the proliferation, invasion, and metastases of malignant cells, and because combination therapies are often far more effective than single-agent regimens, the FTIs may complement other antitumor agents that may or may not affect RAS pathways (26). While this study was primarily focused on single-agent tipifarnib, potential combinations with other agents, for example, immune checkpoint inhibitors, should be considered as a novel therapeutic strategy to further improve outcomes in this devastating disease.

A. Gualberto reports other from Kura Oncology (employment) during the conduct of the study; other from Kura Oncology (ownership) outside the submitted work; in addition, author is an inventor on a patent for methods of treating cancer with farnesyltransferase inhibitors, assigned to Kura Oncology. C. Scholz reports personal fees from Kura Oncology (employee and stock owner of Kura Oncology) during the conduct of the study. No potential conflicts of interest were disclosed by the other authors.

H.W. Lee: Formal analysis, writing-original draft, writing-review and editing. J.K. Sa: Formal analysis, writing-original draft, writing-review and editing. A. Gualberto: Conceptualization, funding acquisition. C. Scholz: Conceptualization. H.H. Sung: Investigation, writing-review and editing. B.C. Jeong: Investigation. H.Y. Choi: Investigation. G.Y. Kwon: Investigation, writing-review and editing. S.H. Park: Conceptualization, supervision, funding acquisition, investigation, methodology, writing-original draft, project administration, writing-review and editing.

This work was supported by Samsung Medical Center (Seoul, Korea) Research Fund (OTX0002441) and in part by Kura Oncology, Cambridge, MA, USA. Study drug (tipifarnib) was kindly provided by Kura Oncology.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1.
Jung
KW
,
Won
YJ
,
Kong
HJ
,
Lee
ES
. 
Cancer statistics in Korea: incidence, mortality, survival, and prevalence in 2016
.
Cancer Res Treat
2019
;
51
:
417
30
.
2.
Kompier
LC
,
Lurkin
I
,
van der Aa
MN
,
van Rhijn
BW
,
van der Kwast
TH
,
Zwarthoff
EC
. 
FGFR3, HRAS, KRAS, NRAS and PIK3CA mutations in bladder cancer and their potential as biomarkers for surveillance and therapy
.
PLoS One
2010
;
5
:
e13821
.
3.
Lee
JY
,
Kim
K
,
Sung
HH
,
Jeon
HG
,
Jeong
BC
,
Seo
SI
, et al
Molecular characterization of urothelial carcinoma of the bladder and upper urinary tract
.
Transl Oncol
2018
;
11
:
37
42
.
4.
Iyer
G
,
Al-Ahmadie
H
,
Schultz
N
,
Hanrahan
AJ
,
Ostrovnaya
I
,
Balar
AV
, et al
Prevalence and co-occurrence of actionable genomic alterations in high-grade bladder cancer
.
J Clin Oncol
2013
;
31
:
3133
40
.
5.
Takashima
A
,
Faller
DV
. 
Targeting the RAS oncogene
.
Expert Opin Ther Targets
2013
;
17
:
507
31
.
6.
Brunner
TB
,
Hahn
SM
,
Gupta
AK
,
Muschel
RJ
,
McKenna
WG
,
Bernhard
EJ
. 
Farnesyltransferase inhibitors: an overview of the results of preclinical and clinical investigations
.
Cancer Res
2003
;
63
:
5656
68
.
7.
Rosenberg
JE
,
von der Maase
H
,
Seigne
JD
,
Mardiak
J
,
Vaughn
DJ
,
Moore
M
, et al
A phase II trial of R115777, an oral farnesyl transferase inhibitor, in patients with advanced urothelial tract transitional cell carcinoma
.
Cancer
2005
;
103
:
2035
41
.
8.
Jung
SH
,
Lee
T
,
Kim
K
,
George
SL
. 
Admissible two-stage designs for phase II cancer clinical trials
.
Stat Med
2004
;
23
:
561
9
.
9.
Cancer Genome Atlas Research Network
. 
Comprehensive molecular characterization of urothelial bladder carcinoma
.
Nature
2014
;
507
:
315
22
.
10.
Lara
PN
 Jr
,
Law
LY
,
Wright
JJ
,
Frankel
P
,
Twardowski
P
,
Lenz
HJ
, et al
Intermittent dosing of the farnesyl transferase inhibitor tipifarnib (R115777) in advanced malignant solid tumors: a phase I California Cancer Consortium trial
.
Anticancer Drugs
2005
;
16
:
317
21
.
11.
Ho
A
. 
Preliminary results from a phase 2 trial of tipifarnib in squamous cell carcinomas (SCCs) with HRAS mutations [abstract]
. In:
Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics
; 
2019
Oct 26–30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 
2019
;
18
(12 Suppl):Abstract nr PR08.
12.
Knowles
MA
,
Hurst
CD
. 
Molecular biology of bladder cancer: new insights into pathogenesis and clinical diversity
.
Nat Rev Cancer
2015
;
15
:
25
41
.
13.
Kim
PH
,
Cha
EK
,
Sfakianos
JP
,
Iyer
G
,
Zabor
EC
,
Scott
SN
, et al
Genomic predictors of survival in patients with high-grade urothelial carcinoma of the bladder
.
Eur Urol
2015
;
67
:
198
201
.
14.
Appels
NM
,
Beijnen
JH
,
Schellens
JH
. 
Development of farnesyl transferase inhibitors: a review
.
Oncologist
2005
;
10
:
565
78
.
15.
Hobbs
GA
,
Der
CJ
,
Rossman
KL
. 
RAS isoforms and mutations in cancer at a glance
.
J Cell Sci
2016
;
129
:
1287
92
.
16.
Cox
AD
,
Fesik
SW
,
Kimmelman
AC
,
Luo
J
,
Der
CJ
. 
Drugging the undruggable RAS: mission possible?
Nat Rev Drug Discov
2014
;
13
:
828
51
.
17.
Lancet
JE
,
Karp
JE
. 
Farnesyltransferase inhibitors in hematologic malignancies: new horizons in therapy
.
Blood
2003
;
102
:
3880
9
.
18.
Berndt
N
,
Hamilton
AD
,
Sebti
SM
. 
Targeting protein prenylation for cancer therapy
.
Nat Rev Cancer
2011
;
11
:
775
91
.
19.
Tiainen
M
,
Ylikorkala
A
,
Makela
TP
. 
Growth suppression by Lkb1 is mediated by a G(1) cell cycle arrest
.
Proc Natl Acad Sci U S A
1999
;
96
:
9248
51
.
20.
Karuman
P
,
Gozani
O
,
Odze
RD
,
Zhou
XC
,
Zhu
H
,
Shaw
R
, et al
The peutz-jegher gene product LKB1 is a mediator of p53-dependent cell death
.
Mol Cell
2001
;
7
:
1307
19
.
21.
Fang
B
. 
RAS signaling and anti-RAS therapy: lessons learned from genetically engineered mouse models, human cancer cells, and patient-related studies
.
Acta Biochim Biophys Sin
2016
;
48
:
27
38
.
22.
Lo Iacono
M
,
Monica
V
,
Righi
L
,
Grosso
F
,
Libener
R
,
Vatrano
S
, et al
Targeted next-generation sequencing of cancer genes in advanced stage malignant pleural mesothelioma: a retrospective study
.
J Thorac Oncol
2015
;
10
:
492
9
.
23.
Wang
J
,
Yao
X
,
Huang
J
. 
New tricks for human farnesyltransferase inhibitor: cancer and beyond
.
Medchemcomm
2017
;
8
:
841
54
.
24.
Raponi
M
,
Belly
RT
,
Karp
JE
,
Lancet
JE
,
Atkins
D
,
Wang
Y
. 
Microarray analysis reveals genetic pathways modulated by tipifarnib in acute myeloid leukemia
.
BMC Cancer
2004
;
4
:
56
.
25.
Lebowitz
PF
,
Prendergast
GC
. 
Non-Ras targets of farnesyltransferase inhibitors: focus on Rho
.
Oncogene
1998
;
17
:
1439
45
.
26.
Agarwal
AG
,
Somani
RR
. 
Farnesyltransferase inhibitor in cancer treatment
. In:
Ozdemir
O
, editor.
Current cancer treatment: novel beyond conventional approaches
.
London, UK
:
InTechOpen
; 
2011
. p.
810
.
27.
Gualberto
A
,
Scholz
C
,
Mishra
V
,
Janes
MR
,
Kessler
L
,
Cutsem
EV
, et al
Mechanism of action of the farnesyltransferase inhibitor, tipifarnib, and its clinical applications [abstract]
. In:
Proceedings of the American Association for Cancer Research Annual Meeting 2019
; 2019 Mar 29–Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 
2019
;79(13 Suppl):Abstract nr CT191.
28.
Gualberto
A
,
Scholz
C
,
Mishra
V
,
Janes
MR
,
Kessler
L
. 
RHOE, CXCL12 and CXCR3 may identify complete responses in acute myeloid leukemia patients treated with tipifarnib
.
EHA Library
2019
;
266619
:
PS1002
.
29.
Mattingly
RR
. 
Activated ras as a therapeutic target: constraints on directly targeting ras isoforms and wild-type versus mutated proteins
.
ISRN Oncol
2013
;
2013
:
536529
.
30.
Sidransky
D
,
Frost
P
,
Von Eschenbach
A
,
Oyasu
R
,
Preisinger
AC
,
Vogelstein
B
. 
Clonal origin of bladder cancer
.
N Engl J Med
1992
;
326
:
737
40
.
31.
Sfakianos
JP
,
Cha
EK
,
Iyer
G
,
Scott
SN
,
Zabor
EC
,
Shah
RH
, et al
Genomic characterization of upper tract urothelial carcinoma
.
Eur Urol
2015
;
68
:
970
7
.
32.
Loriot
Y
,
Necchi
A
,
Park
SH
,
Garcia-Donas
J
,
Huddart
R
,
Burgess
E
, et al
Erdafitinib in locally advanced or metastatic urothelial carcinoma
.
N Engl J Med
2019
;
381
:
338
48
.
33.
Schuler
M
,
Cho
BC
,
Sayehli
CM
,
Navarro
A
,
Soo
RA
,
Richly
H
, et al
Rogaratinib in patients with advanced cancers selected by FGFR mRNA expression: a phase 1 dose-escalation and dose-expansion study
.
Lancet Oncol
2019
;
20
:
1454
66
.
34.
Ouerhani
S
,
Elgaaied
AB
. 
The mutational spectrum of HRAS, KRAS, NRAS and FGFR3 genes in bladder cancer
.
Cancer Biomark
2011–12
;
10
:
259
66
.
35.
Jebar
AH
,
Hurst
CD
,
Tomlinson
DC
,
Johnston
C
,
Taylor
CF
,
Knowles
MA
. 
FGFR3 and ras gene mutations are mutually exclusive genetic events in urothelial cell carcinoma
.
Oncogene
2005
;
24
:
5218
25
.
36.
Lotan
Y
,
Meng
X
. 
Critical treatment choices for patients with platinum-refractory urothelial carcinoma
.
Lancet Oncol
2020
;
21
:
11
3
.
37.
Rosenberg
JE
,
O'Donnell
PH
,
Balar
AV
,
McGregor
BA
,
Heath
EI
,
Yu
EY
, et al
Pivotal trial of enfortumab vedotin in urothelial carcinoma after platinum and anti-programmed death 1/programmed death ligand 1 therapy
.
J Clin Oncol
2019
;
37
:
2592
600
.
38.
Suzuki
N
,
Urano
J
,
Tamanoi
F
. 
Farnesyltransferase inhibitors induce cytochrome c release and caspase 3 activation preferentially in transformed cells
.
Proc Natl Acad Sci U S A
1998
;
95
:
15356
61
.