Current Therapeutic Progress of CDK4/6 Inhibitors in Breast Cancer.

The clinical use of selective cyclin-dependent kinase (CDK) 4/6 inhibitors has significantly improved the prognosis of patients with hormone receptor (HR)-positive human epidermal growth factor receptor 2 (HER2)-negative advanced or metastatic breast cancer (ABC/mBC), which almost achieved the double progression-free survival (PFS) in combination with endocrine therapy (ET) compared with ET alone. To date, there are 3 CDK4/6 inhibitors (palbociclib, ribocilcib and abemaciclib) approved by the US Food and Drug Administration (FDA) and European Medicines Agency (EMA) to treat patients with HR+/HER2-ABC/mBC in the first and later lines. The aim of this review is to summarize the current clinical use and ongoing clinical trials of CDK4/6 inhibitors, the published overall survival data, and the potential biomarkers and resistance to CDK4/6 inhibitors.

Introduction

Breast cancer (BC) is a worldwide health problem for women with almost 2.1 million new cases diagnosed and an estimated 0.6 million deaths every year.1 Although the overall 5-year survival has reached to 90%, the 5-year survival rate was only 25% in metastatic or advanced BC (mBC/ABC).2 Of all BC cases, ~ 70% of women were diagnosed as hormone receptor-positive (HR+), human epidermal growth factor receptor two-negative (HER2-) BC.3 Recent innovative therapeutic regimens have revealed that endocrine therapy (ET) plus targeted therapy such as everolimus, has improved the prognosis of ER+/HER2- mBC/ABC.4 Moreover, the combination of ET and cyclin-dependent kinase (CDK) 4/6 inhibitors has also shown significantly clinical benefit.5

In this review, we summarized the recent therapeutic progress of 3 CDK4/6 inhibitors, palbociclib, ribociclib and abemaciclib, which have been approved by US Food and Drug Administration (FDA) and European Medicines Agency (EMA) to treat patients with HR+/HER2- mBC/ABC, including their mechanism of action, approved indications, the published overall survival data, ongoing clinical trials and potential clinical use in future.

Mechanism of Action of CDK4/6 Inhibitors

Cell division is a common cell process under strict control in normal cells in case of unexpected proliferation, which is usually the cause of cancer. There are many pathways involved in regulating cell cycle and CDK family is one of the most important protein families in cell division regulation (Figure 1).6 In G1 phase of cell cycle, CDK4/6 interacts with cyclin D to form the cyclin D-CDK4/6 complex, which phosphorylates retinoblastoma (Rb).7,8 Inactivated Rb is tightly binding to E2F, the transcription factor, and phosphorylation of Rb releases E2F from Rb-E2F complex, followed by inducing upregulation of E2F target genes and initiating DNA synthesis, resulting in cell cycle entry into S phase.8–10 There are several instinct negative regulators of cyclin D-CDK4/6-Rb signaling pathway, which prevent cells from unchecked proliferation, such as the INK4 family of proteins (p16, p15, p18, and p19), cyclin inhibitory proteins (CIPs) and kinase inhibitory proteins (KIPs, p21 and p27).11–13

Figure 1

Mechanism of CDK4/6 inhibitors and possible combined therapy with CDK4/6 inhibitors. Activation of upstream signaling pathways, such as MAPK, PI3K and ER, promotes the formation of cyclin D-CDK4/6 complex, which phosphorylates Rb protein. With phosphorylation of Rb, E2F is dissociated from Rb-E2F complex.84 As a transcription factor, released E2F initiates DNA synthesis, resulting in cell cycle progressed into S phase from G1 phase. CDK4/6 inhibitors (palbociclib, ribociclib and abemaciclib) prevent the activation of CDK4/6 to cause cell cycle arrest at G1 phase. Combination therapeutic strategies mainly focus on blockade of upstream of cyclin D-CDK4/6 signaling, including blockade of ER by AIs, fulvestrant and tamoxifen, blockade MAPK by BRAF89 inhibitors (vemurafenib and dabrafenib) and MEK inhibitors (cobimetinib and trametinib),90 and blockade of PI3K pathway by alpelisib91 and everolimus.92

Abbreviations: CDK, cyclin-dependent kinase; MAPK, mitogen-activated protein kinase; PI3K, phosphoinositide 3-kinase; ER, estrogen receptor; AI, aromatase inhibitor.

In breast cancer and other malignancies, dysregulation of cyclin D1-CDK4/6-Rb signaling cascade was observed and promoted unchecked cell proliferation.14,15 Nearly 15% of breast cancers have been detected amplification of the cyclin D2 gene, CCND1, and expression of cyclin D1 at mRNA and protein has been determined to upregulated in up to 50% of primary ER+ breast cancers, and well-differentiated tumors.16,17 In breast cancer cell lines, induction of cyclin D initiate cell cycle process and increase the number of cell processing from G1 to S phase,18 and in vivo study showed that overexpression of cyclin D promoted abnormal mammary cell proliferation and promoting the development of mammary carcinomas in transgenic mice.19 Overexpression of Cyclin D1 is associated with poor prognosis in many cancers and is often associated with increased metastasis.16,20,21 Similarly, overexpression of CDK4 is positively correlated with high proliferative ability of tumor cells in sporadic breast carcinomas.22 Elevated activity of CDK6 was detected in five squamous cell carcinoma lines23 and inhibition of cyclin D3-CDK6 led to tumor cell apoptosis.24 All these evidences indicate that CDK4/6 and cyclin D could be the potential therapeutic targets in cancer.

Current Indications and Dosage of CDK4/6 Inhibitors

So far, three CDK4/6 inhibitors have been approved by both FDA and EMA: palbociclib (Ibrance, Pfizer, USA), ribociclib (Kisqali, Novartis, Switzerland) and abemaciclib (Verzenio, Lilly, USA).

The prescribing information between palbociclib and ribociclib is similar: in combination with an aromatase inhibitor (AI) for the treatment of patients with HR+/HER2-locally ABC/mBC as initial therapy in postmenopausal women or in combination with fulvestrant in women who have previously treated with ET.25,26 In women who have not yet reached menopause, luteinising hormone-releasing hormone agonists should also be given according to EMA approval.27,28 In addition, the indication of palbociclib has been expanded to male patients with HR+/HER2-mBC on the basis of real-world data from electronic health records and insurance claims on April 4, 2019, by FDA.29

The indication of abemaciclib from FDA is different from palbociclib and ribociclib: in combination with fulvestrant to treat women with HR+/HER2- mBC/ABC who have previously treated with ET; As monotherapy for the treatment of adult patients with HR+/HER2- mBC/ABC who have previously treated with ET and prior chemotherapy in the metastatic setting.30 Abemaciclib is the only CDK4/6 inhibitor that can be used as a monotherapeutic drug.

Palbociclib is started with 125 mg/day on a 3/1 schedule (21-day on, 7-day off); If patients were not resistant, the dose should be reduced to 100 mg/day and further reduced to the final dose of 75 mg.25 Ribociclib is also administrated on a 3/1 schedule (21-day on, 7-day off) at a dose of 600 mg/day; Dose reduction is allowed if patients were not resistant with the first dose reduction to 400 mg/day, and the final reduction to 200 mg/day.26 Abemaciclib is primarily prescribed with 200 mg twice daily continuously as a monotherapy. If combined with endocrine treatment, the start dose is 150 mg twice daily continuously. The first dose reduction is 100 mg twice daily, and the final dose is 50 mg twice daily.30 Palbociclib should be taken orally with food because the drug exposure is decreased with an empty stomach, which may reduce effectiveness.31 On the contrary, the absorption and exposure of ribociclib or abemaciclib are not affected by food intake.26,30

Treatment of HR+/HER2- ABC/mBC with CDK4/6 Inhibitors Plus ET

CDK4/6 Inhibitors and AIs in Treatment-Naïve HR+/HER2- ABC/mBC

There are 3 Phase III randomized clinical trials (RCTs) to prove the efficacy and safety of the CDK4/6 inhibitors combined with AIs as the first-line treatment of postmenopausal HR+/HER2- ABC/mBC (Table 1).32 The results are consistent among the 3 CDK4/6 inhibitors.

Table 1

Combination of CDK4/6 Inhibitors and ET in Patients with HR+/HER2- ABC/mBC

Study Name	Intervention	Phase	NCT	LOT	Meno Status	mPFS	ORR (%)	CBR (%)	mOS
PALOMA-134,35	Let ± Pal	II	NCT00721409	1st	Post	10.2 vs 20.2	33 vs 43	58 vs 82.6	34.5 vs 37.5
PALOMA-236,37	Let ± Pal	III	NCT01740427	1st	Post	14.5 vs 27.6	34.7 vs 42.1	70.3 vs 84.9	–
PALOMA-4	Let ± Pal	III	NCT02297438	1st (Asian)	Post	–	–	–	–
MONALEESA-239,93	Let ± Ribo	III	NCT01958021	1st	Post	16.0 vs 25.3	27.5 vs 40.7	72.8 vs 79.6	–
MONARCH340,41	NSAI ± Abema	III	NCT02246621	1st	Post	14.8 vs 28.2	34.5 vs 48.2	71.5 vs 78.0	–
PALOMA-342,43,94	Ful ± Pal	III	NCT01942135	1st + 2nd + later	Pre/peri/post	4.6 vs 9.5	9 vs 19	40 vs 67	28.0 vs 34.9
MONALEESA-344,45	Ful ± Ribo	III	NCT02422615	1st + 2nd	Post	12.8 vs 20.5	21.5 vs 32.4	62.8 vs 70.2	40.0 vs NR
MONARCH247,95	Ful ± Abema	III	NCT02107703	1st + 2nd	Pre/peri/post	9.3 vs 16.4	16.1 vs 35.2	56.1 vs 72.2	37.3 vs 46.7
MONALEESA-748,49	TAM/NSAI ± Ribo	III	NCT02278120	1st + 2nd	Pre	13.0 vs 23.8	30 vs 41	70 vs 79	40.9 vs NR
MONARCH plus96	Cohort A: NSAI ± AbemaCohort B: Ful ± Abema	III	NCT02763566	≥1st (Chinese)	Post	A: 14.7 vs NRB: 5.6 vs 11.5	A: 30.3 vs 56.0B: 7.5 vs 38.5	A: 62.5 vs 82.6B: 45.3 vs 77.9	–
Young PEARL97	Cape vs Pal + Exe + OFS	II	NCT02592746	1st + 2nd	Pre	14.4 vs 20.1	34 vs 37	66 vs 79	–
PEARL98	Cape vs Pal + ET	III	NCT02028507	Later	Post	8.0 vs 10.6	33.3 vs 26.7	–	–

Abbreviations: NCT, ClinicalTrials.gov identifier; LOT, line of therapy; mPFS, median progression-free survival (months); ORR, objective response rate; CBR, clinical benefit rate; mOS, median overall survival (months); Pal, palbociclib; Ribo, ribociclib; Abema, abemaciclib; Let, letrozole; NSAI, nonsteroidal aromatase inhibitor; Ful, fulvestrant; TAM, tamoxifen; Cape, capecitabine; Exe, exemestane; OFS, leuprolide; ET, endocrine therapy; Meno, menopausal; Pre, premenopausal; Peri, perimenopausal; Post, postmenopausal; NR, not reached; vs, versus.

Palbociclib was granted accelerated approval by FDA33 based just on the Phase II trial PALOMA-1/TRIO-18 results, which demonstrated that a remarkable prolongation in the median progression-free survival (PFS) from 10.2 months with letrozole alone to 20.2 months with palbociclib plus letrozole (hazard ratio [HR], 0.49, p = 0.0004),34 and the median overall survival (OS) was prolonged by 3 months (37.5 months versus [vs] 34.5 months HR = 0.897, p = 0.281).35 Since this accelerated approval is conditional, a phase III RCT termed PALOMA-2 (n = 666) was conducted to validate these promising results.36,37 The final PFS in addition of palbociclib to letrozole was 27.6 months compared to 14.5 months with letrozole alone (HR, 0.563; p < 0.001). The objective response rate (ORR) was 42.1% vs 34.7% (p = 0.06) and the clinical benefit rate (CBR) was significantly improved from 70.3% to 84.9% (p < 0.001).

Similarly, the efficacy of ribociclib and abemaciclib plus AI was confirmed by MONALEESA-2 and MONARCH 3, respectively. MONALEESA-2 is a phase III RCT enrolled 668 postmenopausal women with HR+/HER2-ABC as first-line treatment38,39 with a median follow-up of 26.4 months. The final PFS was 25.3 months with combination of ribociclib with letrozole and 16.0 months with letrozole alone (HR, 0.568; p = 9.63 × 10–8). The ORR (ribociclib plus letrozole vs letrozole alone) was 40.7% vs 27.5 % (p = 9.18 × 10–5) and the CBR is 79.6% vs 72.8 %. The aim of MONARCH 3 is to validate the clinical effectiveness and safety of abemaciclib plus a nonsteroidal AI in 493 postmenopausal women with previously untreated HR+/HER2-ABC.40,41 Both ORR (48.2% vs 34.5%. p = 0.02) and PFS (28.2 vs 14.8 months; HR 0.54; p = 0.000002) were significantly improved in the combination arm compared with placebo control.

In summary, as the initial treatment of postmenopausal BC patients, the efficacy was similar among the 3 CDK4/6 inhibitors, and the prolonged PFS was more than one year in palbociclib/abemaciclib + AIs while there was only 9-month prolongation of PFS with ribociclib + letrozole. The OS data were highly expected to achieve the significant benefit in combination treatment group.

CDK4/6 Inhibitors Plus Fulvestrant in Previously ET Treated mBC/ABC

PALOMA-3 was a phase III RCT study to investigate palbociclib plus fulvestrant in pre- or postmenopausal patients with disease progression during previous endocrine therapy. The combination of palbociclib and fulvestrant significantly prolonged PFS to 9.5 months compared with 4.6 months in fulvestrant alone (HR: 0.46, 0.36–0.59; p < 0.001). ORR was increased from 9% to 19% in the intent-to-treat (ITT) patients.42 The OS did not show any statistical difference although 6.9-month absolute improvement was achieved in combination group compared with fulvestrant group (34.9 vs 28.0 months, HR, 0.81; p = 0.09).43 Further subgroup analysis demonstrated that, the OS was extended from 29.7 to 39.7 (HR, 0.72; 95% CI, 0.55 to 0.94; absolute improvement, 10.0 months) in patients who were sensitive to previous ET.43

MONALEESA-3 is a phase III RCT study to test the clinical efficacy of ribociclib + fulvestrant as first- or second-line treatment in postmenopausal HR+/HER2- mBC patients. Unlike the PALOMA-3, MONALEESA-3 included ET treatment-naïve patients (about 50% of population) or relapsed >12 months from completion of adjuvant ET. PFS was improved by the addition of ribocilib from 12.8 to 20.5 months (HR: 0.593, 0.415–0.802; p < 0.001). The ORR was also improved from 21.5 % to 32.4% (p < 0.001).44 The OS data was not reached in the ribociclib plus fulvestrant arm while the OS was 40 months in the fulvestrant alone arm (HR: 0.724, p = 0.00455) and the relative risk of death was reduced by 28%.45

MONARCH 2 aimed to study abemaciclib plus fulvestrant in HR+/HER2-mBC patients who had progressed with prior endocrine therapy. The results manifested that PFS was significantly prolonged to 16.4 months in the abemaciclib + fulvestrant arm compared with 9.3 months in the fulvestrant arm (HR: 0.553, p < 0.001). The ORR was again increased from 16.1% to 35.2% (p < 0.001) in ITT population. Further subgroup analysis demonstrated that the benefit was consistent across all subgroups.46 The significant OS data was achieved with 46.7 months in combination of abemaciclib and fulvestrant arm and 37.3 months in fulvestrant arm (HR: 0.757; 95% CI: 0.606–0.945; p = 0.01). The absolute prolongation of OS is 9.4 months and the improvement in OS was consistent across all stratification factors. Moreover, the median time to second disease progression (23.1 vs 20.6 months), chemotherapy (50.2 vs 22.1months), and chemotherapy-free survival (25.5 vs 18.2 months) was also significantly extended in the abemaciclib + fulvestrant arm.47

Taken together, all these 3 RCTs have demonstrated that CDK4/6 inhibitors plus fulvestrant prolonged the PFS in HR+/HER2- ABC/mBC patients who had progressed on endocrine therapy (Table 1). The meaningful prolongation of OS was observed in MONALEESA-3 and MONARCH 2 but not in PALOMA-3. Ribociclib and abemaciclib in MONALEESA-3 and MONARCH 2 were used only as first- and second-line treatment of ABC/mBC.44,46 Palbociclib in PALOMA-3 was used to treatment ABC/mBC in any line, including first line (25%), second line (39%) and later lines.42 In addition, the phase III trial MONALEESA-7, which investigated ribociclib + ET + goserelin in pre-menopausal women, has proved that ribociclib + ET + goserelin significantly prolonged both PFS and OS in patients with HR+/HER2-ABC.48,49 The inclusive patients were allowed to receive up to only one previous line of chemotherapy and no previous ET for ABC in MONALEESA-7. With a comparison of the inclusion criteria and OS data, the results indicated that earlier intervention with CDK4/6 inhibitor might be positively associated with more benefits achieved, especially in OS benefits.

CDK4/6 Inhibitors in Neoadjuvant Therapy of HR+/HER2-BC

The major objective of neoadjuvant therapy is to historically shrink inoperable lesions to make them operable and facilitate breast conservation without significant increase in local recurrence. The clinical efficacy of CDK4/6 inhibitors plus ET was also explored in neoadjuvant therapy of HR+/HER2-BC patients (Table 2).

Table 2

The (Neo)adjuvant Therapy of CDK4/6 Inhibitor in HR+/HER2-BC

Study Name	Intervention	Phase	NCT	Primary Endpoint
Neoadjuvant
NeoPalAna50	Pal + Ana + Goserelin	II	NCT01723774	Complete cell cycle arrest rate
NeoPAL99	Pal + Let vs CT	II	NCT02400567	Evaluation of the number of patients with a Residual Cancer Burden (RCB) 0-I index as a measure of efficacy
PALLET51	Let vs Let then Let + Pal vs Pal then Let + Pal vs Let + Pal	II	NCT02296801	Measurement of the proliferation marker Ki67
CORALLEEN52	Pal + Let vs CT	II	NCT03248427	Rate of ROR-low according to the Prosigna test
FELINE	NSAI ± Ribo	II	NCT02712723	Rate of PEPI score 0 at surgery
NEOLBC	Ribo + Let vs Let vs CT	II	NCT03283384	Difference in complete cell cycle arrest
neoMONARCH53		II	NCT02441946	Percent Change from Baseline to 2 Weeks in Ki67 Expression
CheckMate 7A8	Pal/Abema + Ana vs Pal/Abema + Ana + Niv vs Pal/Abema + Ana then Pal/Abema + Ana + Niv	II	NCT04075604	Dose Limiting ToxicityResidual Cancer Burden RCB (0–1) rate by central assessment
Adjuvant
PALLAS	SET ± Pal	III	NCT02513394	iDFS
POLAR	SET ± Pal	III	NCT03820830	iDFS
PENELOPE-B	Pal vs Placebo	III	NCT01864746	iDFS
EarLEE-2	SET ± Ribo	III	NCT03081234	iDFS
NATALEE	ET ± Ribo	III	NCT03701334	iDFS
ADAPTcycle	Ribo +ET vs CT	III	NCT04055493	iDFS
monarchE	SET ± Abema	III	NCT03155997	iDFS

Abbreviations: iDFS, invasive disease-free survival; PEPI, pre-operative Endocrine Prognostic Index; CT, chemotherapy; ROR, risk of relapse; Ana, anastrozole; Niv, nivolumab; SET, standard endocrine therapy; CT, chemotherapy.

The phase II NeoPalAna trial50 is a single-arm study to test the antiproliferative activity of anastrozole plus palbociclib in clinical stage II/III ER+ BC. Primary endpoint is the rate of complete cell cycle arrest (CCCA, defined as Ki-67 < 2.7% after 15 days of combination therapy). The results of this trial demonstrated that the rate of CCCA was significantly higher than that on Day 1 (87% vs 26%; p < 0.001), suggesting that palbociclib is an effective anti-proliferative medication for early-stage BC. However, the pathological complete response (pCR) was not evaluated in this study. Another phase II randomized trial PALLET51 demonstrated that adding palbociclib to letrozole significantly suppressed malignant HR+ BC cell proliferation (Ki-67) but did not increase the clinical response rate and pCR rate.

CORALLEEN is an open-label, multicenter, randomized, Phase 2 trial to evaluate the proportion of patients with PAM50 low-risk-of-relapse (ROR) disease at surgery after neoadjuvant treatment with ribociclib plus letrozole versus chemotherapy.52 This study has enrolled 106 women with the luminal B subtype of BC at the stage I-IIIA. The low-ROR was 46.9% (95% CI 32.5–61.7) in ribociclib plus letrozole group and 46.1% (95% CI 32.9–61.5) in chemotherapy group, and the pCR rate (2.0% vs 5.8%) and ORR (57.2% vs 78.8%) did not show any significant difference. The neoMONARCH53 is a phase II study to evaluate the clinical efficacy of abemaciclib plus anastrozole in the neoadjuvant setting. This study enrolled 223 postmenopausal women with HR+/HER2− primary breast tumor (≥1 cm) and the primary endpoint is the change in Ki67 from baseline to 2 weeks after treatment (CCCA). More patients in the combination arm versus anastrozole alone achieved CCCA (68% vs 14%, p < 0.001), and pCR was 4% in ITT patients. Taken together, all the 3 CDK4/6 inhibitors have manifested their advantage in CCCA, but these clinical trials have not observed any advantages on pCR compared with ET alone or chemotherapy. Phase III head-to-head clinical trials are still needed to confirm the biological and clinical activity of CDK4/6 inhibitors in the neoadjuvant setting. In the adjuvant setting, there are several ongoing phase III trials (): PALLAS (NCT02513394), PENELOPE-B (NCT01864746), EarLEE-2 (NCT03081234), NATALEE (NCT03701334) and monarchE (NCT03155997), etc. (Table 2). The promising results are expected to benefit more patients with early BC.

CDK4/6 Inhibitors in HER2-Positive BC

HER2-positive (HER2+) tumors account for 15–20% of all BC. HER2+ BC is associated with biological aggressiveness and poor outcomes.54 Cell cycle is also alternated in HER2+ BC.55 Studies have demonstrated that both cyclin D1 and CDK4 are essential for HER2+ murine mammary tumor development.56–58 Combination of palbociclib and trastuzumab demonstrates a synergistic effects on 3 HER2-amplified BC cell lines.59 On a basis of these preclinical data, clinical trials are conducted to explore the clinical activity of CDK4/6 inhibitors in HER2+ BC.

A multicohort, open-label, single-arm, phase II study NA-PHER2 aimed to investigate the combination of palbociclib, fulvestrant and trastuzumab in patients with HR+/HER2+ BC in the neoadjuvant setting.60 Ki67 expression was reduced from 31.9% to 4.3% at Week 2 (n = 25, p < 0.001) and 12.1% in the triple combination group at time of surgery (n = 22, p = 0.013). The clinical objective response was 29 of 30 patients (97%, 95% CI 83–100) immediately before surgery and 8 (27%; 95% CI 12–46) patients had a pathological complete response in the arm of palbociclib + fulvestrant + trastuzumab.

PATRICIA II study (NCT02448420) is a prospective, multicenter, open-label phase II trial. The objective of this study is to test the role of palbociclib and trastuzumab plus or minus letrozole in HER2+ mBC. The primary endpoint of this study is PFS at 6 months and secondary endpoint is the safety profile, including the cardiac safety, the overall tumor ORR and the OS. PATINA (NCT02947685) is an open-label, randomized phase III trial that aimed to assess the prolongation of PFS with the addition of palbociclib to HER2-directed therapy and ET compared with HER2-directed therapy and ET in HR+/HER2+ ABC/mBC. The inclusive patients are those who have completed standard first-line treatment with dual HER2-directed therapy and a taxane. Results of this study are expected in 2021.

monarcHER is a multicenter, randomized, three-arm, open-label, phase II trial evaluating the clinical efficacy of abemaciclib plus trastuzumab ± fulvestrant compared with standard chemotherapy plus trastuzumab in women with HR+/HER2+ ABC/mBC (NCT02675231). The primary endpoint is PFS and secondary endpoints include OS, ORR, CBR, quality of life, pain control and pharmacokinetics. The initial results are expected in 2021.

An ongoing phase II trial aims to evaluate the effects of palbociclib and trastuzumab on HER2+ patients with brain metastasis (NCT02774681). The primary endpoint of this single-arm study is to measure the radiographic response rate in the central nervous system (CNS). The secondary endpoints are PFS, OS, ORR, time to CNS progression and toxicity.

There is another ongoing phase I/II trial investigating ribociclib plus trastuzumab or T-DM1 for HER2+ ABC/mBC (NCT02657343). This study includes three cohorts: ribociclib + trastuzumab + fulvestrant for HR+/HER2+ BC, ribociclib plus T-DM1 and ribociclib plus trastuzumab. The primary outcomes are the maximum tolerated dose or recommended phase II dose. The secondary outcomes are the plasma concentration of ribociclib, ORR, PFS, OS, frequency of biomarkers and frequency of adverse events.

The role of CDK4/6 inhibitors in patients with HER2+ BC is still under exploration. To date, most ongoing trials are phase II studies and NA-PHER2 is the only one that has been reported its results. Therefore, more phase III RCTs are warranted to figure out the effectiveness of CDK4/6 inhibitors in patients with HER2+ BC in future.

Biomarkers and Resistance of CDK4/6 Inhibitors

As mentioned above, the discovery of CDK4/6 inhibitors has improved the prognosis of HR+ BC and may also benefit HER2+ BC and other solid tumors. However, not all patients respond to the CDK4/6 inhibitors and even patient sensitive to CDK4/6 inhibitors might develop the acquired resistance.61 Mechanism of resistance to CDK4/6 inhibitors is still unclear and predicting biomarkers have not been identified. This review summarized the potential mechanisms and biomarkers in both preclinical and clinical data.

Loss of Rb

Rb, as the target of CDK4/6, is considered as one of the most important biomarkers of sensitivity to CDK4/6 target therapy.62 Preclinical studies have proved that loss of Rb function was detected in palbociclib resistance cell lines.63 In mBC patients who had received the treatment with palbociclib or ribociclib, somatic Rb1 mutations were detected when disease was progressed,64 suggesting that Rb mutation might be associated with acquired resistance to CDK4/6 inhibitors. However, only three patients reported in this publication. A large study is warranted to confirm this conclusion.

Overexpression of Cyclin E1

In addition to cyclin D-CDK4/6 complex, cyclin E-CDK2 complex could also release E2E via phosphorylating Rb.65 Expression of Cyclin E1 was upregulated in CDK4/6 inhibitor resistance cell lines66 and overexpression of Cyclin E1attenuated the inhibitory effects of CDK4/6 on cell cycle progress.67 Biomarker analysis of PALOMA-3 manifested that high expression of CCNE1 mRNA demonstrated a shorter PFS in patients received palbociclib plus fulvestrant treatment68 while this was not found in PALOMA-2 study,69 indicating that mRNA level of cyclinE1 was an effective biomarker in previously treated HR+/HER2-mBC.

p16 Amplification

p16INK4A is an intrinsic tumor-suppressor which can bind to CDK4/6 to disrupt the formation of cyclin D-CDK4/6 complex.70–72 Overexpression of p16 is observed during oncogenic stress. When p16 overexpression is concurrent with loss of Rb, resistance to CDK4/6 inhibitor was gained as a result of Rb dysfunction.73 In the presence of Rb, overexpression of p16 showed resistance to CDK4/6 inhibitor due to diminished CDK4.61,74 However, the results of PALOMA-1 did not show any significant difference in PFS in the loss of p16/CCND1 amplification cohort compared with the unselected cohort.75 Similar results were also obtained from the biomarker analysis of PALOMA-2 and PALOMA-3.68,69 Therefore, it is controversial to use p16 amplification as a biomarker.

TK1

Thymidine kinase-1 (TK1) is a key regulator of cell cycle and highly expresses in S/G2 phase to catalyze DNA precursor synthesis.76 Serum TK1 level and activity were increased in solid tumors, including lung, colorectal and breast cancer.77 In primary BC patients, high TK1 levels and activity are associated with large tumor size and poor prognosis.78,79 In patients with HR+/HER2-mBC, lower baseline TK1 activity was correlated with a longer PFS and decrease of TK1 activity after one month of treatment was also linked to a significantly better PFS,80 indicating that TK1 is a meaningful biomarker and a potential therapeutic target in HR+/HER2-mBC. ECLIPS is a prospective, pharmacogenetic study to identify the predictive biomarkers which are responsive/resistant to palbociclib plus ET (letrozole or fulvestrant).81 The results showed that the number of copies/mL of TK1 was significantly increased before treatment compared to that after 3 months of treatment (1200 vs 3350 copies/mL, p = 0.01) in patients with disease progression, suggesting that TK1 mRNA copies/mL is correlated to acquired resistance to CDK4/6 inhibitors.

Loss of FAT1

FAT1 is a tumor-suppressor belonging to the cadherin superfamily and interacts with the β-catenin and Hippo signaling pathways.75 Loss of FAT1 has been reported to promote cancer progression.82 With gene sequencing of 1501 HR+/HER2-BC patients, FAT1 mutation accounted for ~ 2% in primary and ~ 6% in metastatic tumors.83 Preclinical data demonstrated that FAT1 loss-induced upregulation of CDK6 expression through Hippo pathway, resulting in resistance to CDK4/6 inhibitors.66 Gene analysis results from 348 ER+/HER2-BC patients, who have previously treated with CDK4/6 inhibitors, showed that loss of FAT1 was linked to poor prognosis of CDK4/6 inhibitor therapy and shorter PFS (2.4 months) compared with FAT1 wild type arm (PFS: 10.1 months; p = 2.2 × 10−11.66 Therefore, loss of FAT1 might be an effective predictor of CDK4/6 inhibitor resistance.

Besides the potential biomarkers mentioned above, results from ECLIPS demonstrated that the number of copies/mL of CDK9 was significantly increased before treatment compared to that after 3 months of treatment (3800 vs 7500 copies/mL, p = 0.03) in HR+/HER2-mBC patients with disease progression.81 Interestingly, biomarker analysis of PALOMA-2 found that high level of PD-1 showed less benefit from the combination of palbociclib and letrozole compared to low PD-1expression.69 Results from PALOMA studies have demonstrated that CCND1, CDK4 and CDK6 did not indicate any predictive effects on resistance to CDK4/6 inhibitors.1,34,68 Therefore, future studies should focus on identification of the effective biomarkers of sensitivity/resistance to CDK4/6 inhibitors.

Conclusion and Future Prospection

With the introduction of CDK4/6 inhibitor, a longer PFS and better CBR and ORR were achieved in patients with HR+/HER2- ABC/mBC, and the benefit of OS was also observed in the patient previously treated with ET. Ongoing clinical trials focus on treatment with CDK4/6 inhibitors in the early stage of HR+/HER2- and HER2+ BC. Since cell cycle regulation of CDK4/6 was not only observed in breast cancer, but also in the other cancers,84 several phase I/II trials have been reported the preliminary clinical efficacy of CDK4/6 inhibitors in non-BC such as head and neck squamous cell carcinoma,85 mantle cell lymphoma,86 glioblastoma,87 germ cell tumor.88 Therefore, CDK4/6 inhibitor is hopeful to expand to treat patients with other tumors in addition to breast cancer and more and more cancer patient will gain benefit from CDK4/6 inhibitor therapy in future.