Predictive Impact of Peripheral Blood Lymphocytes on clinicAL outcoMes During First-line CDK4/6 Inhibitors Plus Endocrine Therapy in Patients With Advanced Hormone REceptor-poSitive HER2-negative Breast Cancer: the Retrospective-prospective Multicenter Italian PALMARES-2 Study
Conditions: Breast Adenocarcinoma, Breast Cancer Stage IV, Breast Cancer, Metastatic, Breast Carcinoma, Breast Diseases, Breast Neoplasms, Breast Neoplasms, Male, Breast Cancer, Breast Cancer With Metastatic Bone Disease, Breast Cancers, Breast Neoplasm, Breast Tumors, HR+ HER2- Men, Pre/Postmenopausal Advanced Breast Cancer, HR+ Advanced or Metastatic Breast Cancer, HR+/HER2- Breast Cancer, HRpos Breast Neoplasms, HR-positive, HER2-negative Advanced Breast Cancer, HR-positive, HER2-negative and PIK3CA Mutation Advanced Breast Cancer, HR-positive Breast Cancer, Hormone Receptor-Positive Breast Cancer, Hormone Receptor Positive Breast Adenocarcinoma, Hormone Receptor Positive Breast Carcinoma, Hormone Receptor Positive Breast Neoplasms, Hormone Receptor Positive HER-2 Negative Breast Cancer, Hormone Receptor Positive Malignant Neoplasm of Breast, Hormone Receptor Positive Metastatic Breast Cancer, Hormone Receptor Positive, HER2 Negative Breast Cancer, Hormone Receptor Negative Breast Cancer, Hormone Receptor Positive, HER2-negative, Advanced Breast Cancer, Hormone Receptor Positive Breast Cancer, Hormone Receptor Positive (ER+/PR+, and Her2-) Metastatic Breast Cancer, Hormone Receptor Positive, HER2-negative Neoplasms, Hormone Receptor Positive, HER2-low Neoplasms, Hormone Receptor Positive (HR+), HER2-negative Breast Cancer, Hormone Receptor (HR)-Positive Breast Cancer, Hormone Receptor-Positive, HER2-Negative Metastatic Breast Cancer, Palbociclib, Ribociclib, Abemaciclib, CDK4/6 Inhibitor, CDK4/6 Inhibitors
Sponsor: Fondazione IRCCS Istituto Nazionale dei Tumori, Milano

 
Observational Retrospective-Prospective Study Protocol 
Study title: 
Predictive impact of Peripheral blood lymphocytes on clinicAL outcoMes during first-line CDK4/6 inhibitors 
plus endocrine therapy in patients with Advanced hormone REceptor-poSitive HER2-negative breast cancer: 
the retrospective-prospective multicenter Italian PALMARES-2 study   
Version:  
2.0, 31-10-2024 
Study Investigators 
Principal Investigator: Dr. Claudio Vernieri  
Co-investigators: Dr. Leonardo Provenzano, Dr.ssa Francesca Ligorio, Dr. Giacomo Mazzoli, Dr. Giuseppe 
Fotia, Dr.ssa Giovanna Armani, Dr.ssa Caterina Sposetti, Dr. Matteo De Monte, Dr. Luigi Mariani, Prof. 
Giancarlo Pruneri, Dr. Andrea Vingiani, Dr. Gianfranco Scaperrotta, Dr.ssa Catherine Depretto                         
Background and Rationale of the Study 
CDK4/6 inhibitors as the standard of care for the treatment of HR+/HER2- aBC 
Breast cancer (BC) is the most common malignancy in women worldwide, with a critical impact on public 
health due to its incidence, prevalence and global disease burden.1–6 Historically, BC is classified into three 
major histopathological categories based on the expression of Hormone Receptors (HR) and Human 
Epidermal growth factor Receptor 2 (HER2), with the HR+/HER2- subgroup accounting for the majority of 
cases (~65-70%), and it is responsible for the majority of BC-related deaths. Despite huge recent 
improvements in the treatment of this subtype of BC, it remains an almost invariably deadly disease.7,8 
The cyclin-dependent kinase (CDK) 4/6 inhibitors palbociclib, ribociclib and abemaciclib are the mainstay 
of treatment in combination with endocrine therapies (ET) for patients with advanced (unresectable locally 
advanced or metastatic) hormone receptor-positive human epidermal growth factor receptor 2 (HER2)-
negative breast cancer (HR+/HER2- aBC).9,10 CDK 4/6 inhibitors (CDK4/6i) induce cell cycle arrest in 
tumor cells by preventing the phosphorylation of retinoblastoma protein (RB). In its unphosphorylated form, 

   
       
        
                
 
 
 
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RB cannot dissociate from transcriptional factor E2F, which is consequently inhibited and unable to promote 
cell cycle progression.11 
In the last decade, the introduction of CDK4/6i, namely palbociclib, ribociclib and abemaciclib, in 
combination with endocrine therapies (ETs) remarkably improved patient progression-free survival (PFS) 
and, in some cases, also overall survival (OS).12–15  In particular, multicenter, international, phase III clinical 
trials, namely PALOMA-2, PALOMA-3, MONALEESA-2, MONALEESA-3, MONALEESA-7, 
MONARCH 2 and MONARCH 3, demonstrated that adding palbociclib, ribociclib or abemaciclib to 
standard ET is associated with a significant benefit, in terms of PFS, when compared to ET alone, in both 
endocrine sensitive and resistant patients.16–22 Based on these results, CDK4/6i plus ET are currently 
recommended by national and national and international guidelines as the treatment of first choice for 
patients with HR+/HER2- aBC who have not received previous lines of treatment for metastatic 
disease.9,10,23,24       
Determinants of CDK4/6i benefit as first-line treatment for HR+/HER2- aBC 
Subgroup analyses from pivotal trials failed to demonstrate a differential PFS benefit from the addition of 
CDK4/6i based on a specific clinical biomarker, since all the subgroups achieved a better PFS compared to 
standard arm. In particular, to investigate the efficacy of CDK4/6i in combination with ET in different 
clinical-pathologic subgroups of MBC, Gao et al performed a pooled analysis of seven pivotal registration 
trials submitted to the FDA in support of marketing applications for palbociclib, ribociclib, and abemaciclib. 
Again, they found that the addition of CDK4/6i to ET benefitted all the analysed subgroups. These results 
remarked CDK4/6i in combination with ET as first-line treatment for HR+/HER2- aBC in all subgroups of 
patients.25 
Therefore, the only predictive biomarker of response to CDK4/6i currently used is the presence of estrogen 
receptor on tumor cell membranes, as determined by immunohistochemistry (ER ≥1%).9 However, 
other biomarkers derived from tumor tissue emerged as potential predictors of response in this context.26–28 
One of the most investigated biomarkers is the retinoblastoma (Rb) protein, which is a direct target of 
CDK4/6 activity.29 Functional RB is essential for CDK4/6i to exert their effect, highlighting the loss of RB, 
as assessed by genomic loss or absence of the protein evaluated on immunohistochemistry, as potential 
biomarker of CDK4/6i resistance. However, this molecular alteration accounts for only a small percentage of 
cases (3-9%). Additionally, other cell-cycle alteration can affect the efficacy of this class of drugs. In 
particular, cyclin D1/E1 amplification, c-myc overexpression, as well as dysregulation of members of the 
CDKN2/INK family, which are endogenous inhibitors of CDK4/6 (p16, p15, p18, p19 protein, encoded by 
CDKN2A/ CDKN2A/CDKN2C/CDKN2D genes, respectively), are being explored as potential predictive 
biomarkers, with conflicting results.26,29 Similarly, other genomic alterations in other genes not primarily 

   
       
        
                
 
 
 
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implicated in cell-cycle regulation could influence response to CDK4/6i, e.g. indirectly modulating CDK6 
expression such as in the case of FAT1 loss of function mutation.30 Furthermore, other cell-cycle 
independent, relatively common, genomic alterations, such as those regarding the PI3K pathway (AKT1/2/3 
mutations, PIK3CA mutation and PTEN loss) and the amplification of fibroblast growth factor receptor 
(FGFR1-3) genes, have demonstrated conflicting results in influencing the efficacy of CDK4/6 inhibitors. In 
particular, the co-inhibition of the cell-cycle through CDK4/6 inhibition and the blockade of PI3K pathway 
have already been demonstrated to synergize in preclinical models and in phase II studies. Conversely, 
FGFR1/2/3, as well as other receptor t     yrosine kinase (RTK) alterations like EGFR and MET, which are 
altered in less than 10% of cases, emerged as potential biomarkers of resistance to CDK4/6i, indicating that 
patients with these alterations might require alternative therapeutic strategies or combinations.26,31 
Furthermore, breast cancer intrinsic subtypes, as evaluated by PAM50 transcriptomic profiles, were observed 
to differently benefit from CDK4/6i in combination with ET. In particular, basal-like tumors are associated 
with poor prognosis and refractoriness to the addition of CDK4/6i to ET alone, as observed in a retrospective 
pooled analysis across the MONALEESA phase III studies (mPFS 3.7 with ribociclib vs 3.6 with 
placebo).32,33 
Prognostic biomarkers in HR+/HER2- aBC patients treated with first-line 
CDK4/6i plus ET combinations 
Despite the tremendous improvement in the prognosis of HR+/HER2- aBC patients, a minority (~10-15%) of 
patients treated with first-line ET+CDK4/6i still undergo early disease progression.34,35 This subgroup of 
patients could therefore benefit from treatment intensification, such as through the addition of a target 
therapy (e.g., PI3Ki, AKTi, mTORi, PARPi for selected populations), hormonal backbone enhancement 
(e.g., oral SERD), or even from the use of a completely different treatment strategy such as chemotherapy or 
novel antibody drug conjugates (ADCs). Conversely, another subgroup of patients demonstrated to derive 
long-term benefit from ET+CDK4/6i in pivotal trials, with 23.3% of patients enrolled in the MONARCH 3 
trial still on treatment after a median follow up of 72 months.36 These patients could potentially benefit from 
tailoring treatment in terms of de-escalation, sparing unnecessary toxicities without negatively impacting on 
survival outcomes. 
However, there are no studies conducted to date with the specific aim of identifying the subset of patients at 
high risk of early progression or, conversely, long-term benefit. Several clinical and biological factors 
demonstrated in clinical trials to have relevant impact on prognosis, and the addition of specific CDK4/6i 
differently improved prognosis across pivotal trials. Among these, endocrine resistance, presence of liver 
metastasis, tumor biology intended as luminal A- or B-like disease, menopausal status, de novo metastatic 
presentation, patient age and comorbidity.37–40 Mason et al conducted an individual-level analysis using 

   
       
        
                
 
 
 
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patient data from the 7 randomized controlled trials (RCTs) that lead CDK4/6i FDA approval, including a 
total of 2633 patients treated with CDK4/6i+ET in first- or second-line for metastatic disease. The prediction 
models for PFS and OS in the ET+CDK4/6i group had overall accuracies of 62% and 63%, respectively. 
Variables that were most likely to positively influence the model included disease burden, line of therapy, 
menopausal status, performance status and tumor grading.41 
Finding new biomarkers to be added to current models to improve prediction ability is crucial to allow their 
applicability in the clinical practice. These biomarkers should ideally be informative, not-redundant, 
reproducible, easy-to-access, low-cost and easily interpretable by clinicians. For these reasons, an optimal 
source of biomarkers could be represented by data extrapolated from diagnostic exams performed during 
patient clinical care, but often underreported in clinical trials. 
Based on this hypothesis, we already investigated the prognostic significance of peripheral blood immune 
cell counts in HR+/HER2- aBC patients treated with ET+CDK4/6i.42 In fact, although the main mechanism 
of CDK4/6i-mediated anticancer activity consists in cell autonomous anticancer effects, preclinical evidence 
indicates the CDK4/6i ability to stimulate antitumor immunity. To preliminary investigate if peripheral blood 
immune cell counts or composite immune parameters, as evaluated at baseline or during CDK4/6i therapy, 
are associated with efficacy of any-line ET+CDK4/6i, we performed an analysis in 638 HR+/HER2- aBC 
patients treated in six Italian centers. In this study (PALMARES study), we investigated if baseline or on-
treatment peripheral blood immune cells, such as lymphocytes, neutrophils, platelets or monocytes, or the 
composite scores neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR), monocyte-to-
lymphocyte ratio (MLR) or the Pan-Immune Inflammatory Value (PIV), are associated with patient PFS. 
Peripheral blood immune cell values were collected at baseline (T0), 2 weeks after treatment initiation (T1) 
and before fourth cycle initiation, approximately 12 weeks after treatment initiation (T2). We used Random 
Forest models to select clinically relevant covariates (line of therapy for advanced disease, ECOG PS, 
presence of liver metastases, number of metastatic sites, patient age, disease-free interval (DFI), percentage 
of Ki-67-positive cells in primary tumor specimens and presence of ER expression in primary tumor 
specimens) to adjust the role of peripheral blood immune cells in multivariable cox models. Median patient 
age in this cohort of patients was 61 years (IQR: 52-70 years). The majority of patients were postmenopausal 
(n=530; 83.1%), had an ECOG PS of 0 (n=464, 72.8%) and were treated with palbociclib (n=529; 82.9%). 
Approximately half of enrolled patients received CDK4/6i in the first-line setting (n=335; 52.5%) and had 
visceral disease (n=334; 52.4%). Among all the considered peripheral blood immune variables (neutrophils, 
platelets, lymphocytes and monocytes, the NLR, the PLR, the MLR and also the PIV), only baseline 
lymphocyte values and their precocious modulation (i.e. after 2 weeks of treatment), were independently 
associated with patient PFS.42 

   
       
        
                
 
 
 
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Interestingly, we found that patients with higher baseline lymphocyte counts and undergoing a lower on-
treatment reduction of lymphocytes had significantly longer PFS when compared to patients with lower 
baseline lymphocytes and undergoing a higher decrease of lymphocyte counts [median PFS 21.4 vs. 11 
months, respectively; adjusted hazard ratio (aHR): 0.82; 95% CI 0.73-0.93; p=0.0037]. For the first time, 
these results underlie the prognostic significance of blood lymphocytes in HR+/HER2- aBC patients treated 
with CDK4/6 inhibitors. 42 
Since the majority of HR+/HER2- aBC patients nowadays receive CDK4/6i in the first-line treatment setting, 
we performed a sub-analysis, in which we evaluated the association of baseline lymphocyte count (L_t0) and 
its precocious modulation (L_r1) with the PFS of patients treated with CDK4/6i in the first-line setting 
(n=335; 52.5%). Multivariable analysis revealed an independent and statistically significant association 
between higher L_r1 and reduced risk of disease progression; on the other hand, L_t0 was not associated 
with patient PFS. In this model, a higher DFI was associated with better PFS, while a higher number of 
metastatic sites, as well as higher ECOG PS, were associated with significantly worse PFS. We also adjusted 
the prognostic role of L_r1 for NLR_t0 in this patient subset. Of note, L_r1 was independently associated 
with patient PFS (HR: 0.83; 95% CI 0.71-0.99; p=0.0325), while the NLR_t0 was not (HR: 1.08; 95% CI 
0.86-1.35 (p=0.5085). Together, these data confirm an independent prognostic role of the precocious 
modulation of peripheral blood lymphocytes also in the first-line setting.42 
The differential expression of HER2 protein in HR+/HER2- tumors recently emerged as a predictive 
biomarker of response to novel anti-HER2 anticancer drugs, in particular the antibody-drug conjugates 
trastuzumab deruxtecan (T-DXd). Data from the phase III RCT DESTINY-Breast04 trial showed that, in 
patients with HR+/- HER2-low, as defined by an IHC score for HER2 of 1 or 2 in the absence of HER2 gene 
amplification by ISH analysis, aBC that is refractory to ETs (if HR+), and who received 1-2 prior lines of 
chemotherapy for metastatic disease, T-DXd is superior to several standard cytotoxic treatments in terms of 
PFS and OS.43 In addition, data from the phase III RCT DESTINY-Breast06 recently presented at the 
international congress ASCO 2024 demonstrated T-DXd to be superior in terms of PFS compared to 
treatment of physician choice (TPC) for patients with HR+/HER2- aBC and HER2-low status progressing on 
first-line ET+CDK4/6i therapy (mPFS 13.2 vs 8.1 months).44 
These new therapeutic opportunities defined two distinct subgroups among HR+/HER2- aBC tumors, 
namely HER2-low and HER2-0. However, it remains unclear if HER2-low status is also associated with 
different clinical outcomes (PFS, OS) as compared to HER2-0 status in patients receiving standard first-line 
ET plus CDK4/6i therapy. Several studies tried to investigate the prognostic role of HER2 status (low vs. 0) 
in patients with aBC. While studies suggested worse survival in HER2-low patients compared to HER2-0, 
the statistical significance was reached only in some of these studies.45–48 In addition, other studies did not 
show any prognostic difference between HER2-low and HER2-0 tumors. To address this clinical question, 

   
       
        
                
 
 
 
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we conducted a subanalysis on the PALMARES study cohort treated with first-line CDK4/6i plus ET. HER2 
status was available for 428 patients in the study cohort. We found that patients with HR+/HER2-low aBC 
had significantly worse PFS (mPFS 23.6 months vs. 32.3 months; p=0.014) and OS (mOS: 48.7 vs 58.3 
months; p=0.029) when compared to patients with HER2-0 aBC. Multivariable analysis confirmed an 
independent association between HER2-low status and worse PFS (aHR 1.42; 95% CI:1.07-1.89; p=0.016) 
and OS (aHR: 1.64; 95% CI: 1.08-2.48; p=0.02).48 However, another large study conducted by Mouabbi J. et 
al investigated the prognostic role of HER2 status in 1649 HR+/HER2- aBC patients treated with ET in 
combination with different targeted therapies (CDK4/6i, everolimus or alpelisib). In the sub-cohort of 919 
patients treated with CDK4/6i plus ET, there were no statistically significant differences between patients 
with HER2-low and HER2-0 aBC in terms of PFS (13.0 vs 11.6 months, p=0.273) and OS (32.4 vs 31.2 
months, p=0.222).49 The reported discrepancies among published studies, in addition to the increased value 
of the HER2-low status in the treatment of HR+/HER2- aBC, laid the foundations for further investigations 
on this potentially useful and easy-to-access biomarker. 
To summarize, our previous preliminary results, together with available literature, demonstrated that some 
biomarkers already available in clinical practice could be potentially used to distinguish different prognostic 
classes of HR+/HER2- aBC patients treated with first-line ET+CDK4/6i. However, larger studies are 
necessary to confirm available results and finally implement in clinical practice novel treatment biomarkers. 
Differences between the three CDK4/6i drugs in combination with ET and their 
impact on efficacy 
The three CDK4/6i molecules are characterized by minor differences in terms of chemical composition, but 
with significant consequences in clinical terms. In particular, ribociclib and abemaciclib, unlike palbociclib, 
have a higher affinity for CDK4 than CDK6. Palbociclib and ribociclib, on the other hand, have much 
bulkier substituents at the ATP-binding pocket than abemaciclib, resulting in the latter's lower selectivity for 
CDK4 and CDK6 and higher pleiotropic affinity towards other members of the cyclin-dependent kinase 
family. This justifies the higher off-target activity, as well as toxicity, of abemaciclib compared to the other 
two drugs.50 From a pharmacokinetic point of view, palbociclib and ribociclib have a higher affinity for 
glycoprotein P and the efflux transporter Breast Cancer Resistance Protein (BCRP), resulting in lower central 
nervous system (CNS) permeability than abemaciclib.51 Due to different pharmacokinetic properties, the 
three CDK4/6i are approved for administration in different fashion, with abemaciclib prescribed in a 
continuous schedule, that provides sustained inhibition of CDK4 and CDK6, while palbociclib and ribociclib 
given in intermittent dosing with a 3-weeks-on, 1-week-off schedule.50 
As a consequence of different pharmacokinetic and pharmacodynamics properties, palbociclib, ribociclib and 
abemaciclib are characterized by a different toxicity profile. In particular, while palbociclib and ribociclib 

   
       
        
                
 
 
 
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are characterized by a more frequent bone marrow toxicity, ribociclib treatment is associated with a higher 
rate of liver toxicity and prolongation of the corrected QT (QTc) tract on electrocardiographic tracing, while 
abemaciclib is associated with an increased rate of gastrointestinal disorders, particularly in terms of 
diarrhea.52,53 
Regarding differential clinical activity among the three CDK4/6i drugs, in pivotal studies not all molecules 
demonstrated a statistically significant benefit in overall survival (OS), which endpoint was only achieved 
for studies with ribociclib (MONALEESA-2, MONALEESA-3, MONALEESA-7 trials), while abemaciclib 
demonstrated this benefit only in the context of disease endocrine-resistant disease (MONARCH 2).13,14,19 
However, the efficacy of palbociclib, ribociclib and abemaciclib has never been directly compared in large 
clinical studies. In fact, the available published literature consists of small case series lacking sufficient 
power for efficacy comparisons, and available studies did not properly adjust for confounders to homogenize 
treatment cohorts.54–58 Only three large real-world comparisons of the three CDK4/6i were available in 
literature, two of these was recently presented at international congresses; however, none of these two studies 
were published in full to date. Pantano F. et al conducted a comparison among the three CDK4/6 on a cohort 
of 1184 patients using propensity score weighting for covariate adjustment, showing abemaciclib and 
ribociclib to be associated with better PFS when compared to palbociclib in the endocrine-sensitive setting, 
while abemaciclib was associated with better PFS when compared to both ribociclib and palbociclib in 
patients with endocrine-resistant disease. However, this analysis was limited by the relatively low number of 
enrolled patients, especially in the group of patients treated with abemaciclib (n=158).59 Another study 
presented at ASCO 2024 annual meeting included 1511 HR+/HER2- aBC patients treated with ET+CDK4/6i 
as first- or second-line of treatment, showing that fulvestrant plus ribociclib was associated with better PFS 
and OS when compared to both fulvestrant plus palbociclib and fulvestrant plus abemaciclib; no adjustment 
method was applied when comparing PFS among the three drug cohorts, despite significant imbalance in the 
three groups.60 Finally, Gehrchen et al conducted a real-world comparison between the three molecules on 
data extracted from a national Danish dataset. In the context of first-line subgroup of patients (N = 1554), 
they found better outcomes in terms of rwPFS of ribociclib and abemaciclib compared to palbociclib, also 
when adjusting with some clinical co-variates.61 However, the lack of availability of an appropriate amount 
of patients’ characteristics, such as biological ones, did not allow a proper adjustment between groups and 
potentially not correcting the confounding bias related to drug attribution by oncologists. This kind of 
problem underlines the need of deeply annotated, comprehensive datasets to conduct proper and trustworthy 
statistical analyses in the real-world setting. 
Another relevant issue is represented by the potential differential activity of the three molecules across 
clinical scenarios. In fact, results of published phase III RCTs reported a different magnitude of benefit 
among clinical settings with three drugs. As an example, in patients with endocrine-sensitive disease, all the 
three CDK4/6i in combination with ET improve PFS when compared to ET alone. However, only ribociclib 

   
       
        
                
 
 
 
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in MONALEESA-2/3/7 trials also improved patient OS in the endocrine sensitive setting, while abemaciclib 
resulted in not statistically significant OS prolongation in the MONARCH 3 trial, neither OS improvement 
was observed with palbociclib in the PALOMA-2 trial. In premenopausal women, data from the 
MONALEESA-7 trial indicate that ribociclib specifically improves PFS and OS in combination with ET in 
this setting, whereas abemaciclib prolonged PFS and OS in premenopausal women enrolled in the 
MONARCH 2 trial.14,18,62 On the other hand, palbociclib resulted in PFS but not OS gain in premenopausal 
patients form the PALOMA-3 trial. Regarding tumor biology, exploratory analyses from RCTs showed that 
palbociclib and ribociclib improve PFS in patients with both luminal-A and luminal-B disease, while 
ribociclib also provided OS benefit in patients with luminal-B disease.32,33,63 In sub-analyses of the 
MONARCH 2/3 trials, abemaciclib improved PFS in patients with tumors lacking PgR expression, a feature 
that contributes to the definition of luminal B-like disease.20,64 These data suggest a differential activity of the 
three CDK4/6i drugs in settings that are often encountered during daily clinical practice. 
Efficacy of systemic therapies administered on progression to first-line 
ET+CDK4/6i 
While CDK4/6i in combination with endocrine therapy is established as the standard of care treatment for 
first-line treatment for virtually all HR+/HER2- aBC patients, both in endocrine-sensitive and endocrine-
resistant setting, the treatment algorithm for second and subsequent lines for HR+/HER2- aBC is an area of 
uncertainty.9,65 To add a further layer of complexity, the aBC scenario after CDK4/6i is extremely 
heterogeneous from a biological, genomic and clinical point of view, raising the necessity to find the best 
treatment pattern at the individual level, based on the combination of patient-specific characteristics. 
This urgent clinical unmet need is significantly relevant in the actual oncologic era, when a plethora of new 
treatment options demonstrated activity in HR+/HER2- aBC progressed after endocrine-based treatment. In 
detail, numerous trials demonstrated the efficacy of several agents to improve survival outcomes in second or 
subsequent lines after ET-alone first-line treatment. In particular, endocrine (e.g. fulvestrant), biological (e.g. 
everolimus in combination with exemestane, alpelisib in combination with fulvestrant) and chemotherapy 
agents (eribuline) demonstrated to prolong PFS upon ET-alone treatment progression in phase III RCTs.66–68 
However, with the advent of CDK4/6i as upfront treatment for HR+/HER2- aBC patients, further trials are 
needed to confirm the efficacy of these agents as second and/or subsequent lines of therapy. In fact, reduced 
efficacy of these agents has been observed whenCDK4/6i was added to the first-line treatment (e.g. 
fulvestrant monotherapy, everolimus in combination with exemestane).69,70 
Indeed, some therapeutic agents have already demonstrated their efficacy in CDK4/6i pre-treated population. 
In particular, oral SERDs showed ability to significantly prolong mPFS, as elacestrant already received US 
and European regulatory approval based on the EMERALD trial results showing PFS survival advantage 

   
       
        
                
 
 
 
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over standard second-line endocrine therapy.71 The inhibition of PTEN-AKT1- PI3K pathway also provided 
benefit when added to endocrine treatment alone as second-line therapy, leading to marketing authorization 
of the AKT-inhibitor capivasertib based on CAPITELLO-291 phase III trial results.72 While the phase II 
BYLieve trial substantially confirmed the activity of the combination of the PI3K-inhibitor alpelisib and 
fulvestrant in the post-CDK4/6i setting, results from the phase III randomized EPIK-B5 trial are awaited for 
alpelisib approval in this setting.73 
Antibody-Drug Conjugates (ADCs) recently emerged as a novel treatment strategy for solid tumors. 
Trastuzumab deruxtecan (T-DXd), an ADC composed by trastuzumab linked to the topoisomerase I inhibitor 
deruxtecan, is associated with a strong bystander effect, thus leading to clinically-meaningful antitumor 
activity also in malignancies bearing heterogeneous and/or weak HER2 expression such as in case of HER2-
low aBC. Indeed, DESTINY-Breast04 was a phase III RCT comparing T-DXd with chemotherapy of 
physician’s choice among 557 patients with HER2-low aBC who had already received one or two previous 
lines of chemotherapy. The majority of patients were HR+ (88.7%). The study met both the primary and 
secondary endpoints, proving that T-DXd prolongs both PFS and OS in pre-treated patients affected by 
HER2-low aBC.43 As already highlighted above, data from the phase III RCT DESTINY-Breast06 recently 
showed improved PFS for HR+/HER2-low aBC patients receiving T-DXd compared to TPC (capecitabine = 
59.8%), after progression on first-line ET+CDK4/6i therapy. Interestingly, a subsequent amendment also 
allowed the inclusion of patients with HR+/HER2ultralow disease, defined as weak and incomplete 
membrane expression of HER2 in <10% of neoplastic cells. PFS in this patient group was thus introduced as 
a secondary endpoint. The analysis in this subgroup, although smaller in size than that of the HR+/HER2-
low patients’ group (153 vs 753, respectively), confirmed the same results, with an mPFS of 13.2 vs 8.3 
versus TPC.44 
Patritumab deruxtecan (U3-1402; HER3-DXd) is a novel, first-in-class, HER3-directed ADC composed of a 
human monoclonal antibody to HER3 (patritumab) covalently bonded to an exatecan-derived topoisomerase 
I inhibitor payload via a cleavable linker. U31402-A-J101 trial (NCT02980341) was a global, open-label, 
phase I/II trial assessing the safety and the activity of HER3-DXd in HER3-expressing heavily pretreated 
aBC. In 113 patients with HER3-overexpressed or HER3-low HR+/HER2– aBC, the confirmed ORR was 
30.1% and DCR 80.5%, with a median PFS of 7.4 months. Of the 97 patients with HR+/HER2– disease, the 
confirmed ORR was 36.2% in patients with HR+/HER2-low and 28.2% in patients with HR+/HER2-0 
disease.74 
TROP-2 is a carcinoma-associated antigen overexpressed in different solid tumors, which plays a role in 
tumor progression by actively interacting with several key molecular signalling pathways. The randomized 
phase 3 trial TROPiCS-02 sacituzumab govitecan, a first in class ADC directed against TROP-2, 
demonstrated a survival advantage in patients with pre-treated HR+/HER2- aBC compared to chemotherapy 

   
       
        
                
 
 
 
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of physician choice in terms of PFS (5.5 vs 4.0 mo) and OS (14.4 vs 11.2 months). Interestingly, survival 
benefit was consistent across TROP-2 expression-level subgroups, suggesting that protein expression 
assessed as IHC cannot be a reliable biomarker to assess treatment efficacy.75 Datopotamab deruxtecan 
(Dato-DXd), another TROP-2 directed monoclonal antibody attached to a topoisomerase I inhibitor payload, 
was tested in a phase 3 randomized trial, the TROPION-Breast01 in 732 patients with HR+/HER2- aBC who 
have received one or two prior lines of chemotherapy in the advanced setting. Patients receiving Dato-DXd 
had significantly improved PFS versus chemotherapy (HR 0.63, 95% CI 0.52–0.76; p<0.0001), with a 
favourable toxicity profile compared to standard chemotherapy. While OS data were not mature, a trend for 
improved OS favouring Dato-DXd was observed.76,77 
BRCA1 and BRCA2 germline mutations occur in HR+/HER2- aBC, with a prevalence up to 5%. PARP 
inhibitors (PARPi) are drugs specifically designed to exploit the vulnerability of tumor cells linked to the 
presence of these mutations. Employing a synthetic lethality mechanism, PARPi demonstrated efficacy in 
several disease settings including ovarian, breast and pancreatic cancers. Specifically, two studies have 
investigated the use of PARPi in HR+/HER2- patients progressing to 2 or 3 lines of treatment (OlympiAD 
trial, evaluating olaparib, and EMBRACA, with talazoparib), demonstrating a similar benefit in terms of 
PFS, but not in the OS endpoint.78–80 
Finally, also the use of immunotherapy could represent a potential option for pre-treated HR+/HER2- aBC. 
The phase 2 PACE trial demonstrated potential activity of the immune-checkpoint inhibitor avelumab plus 
fulvestrant compared with fulvestrant alone or the combination of fulvestrant and palbociclib, in patients 
with HR+/HER2- aBC progressing after first-line ET+CDK4/6i, with a statistically significant advantage in 
terms of PFS (8.1 months for triplet vs 4.6 for doublet vs 4.8 for Fulvestrant alone), opening an opportunity 
for immunotherapy in selected patients in this setting.81 
Together, these data indicate that several therapeutic options are available, or may become available soon, 
for the treatment of HR+/HER2-aBC patients in different lines of therapy. However, few reliable predictive 
biomarkers actually exist for optimizing the treatment trajectory of individual patients and finally improving 
overall survival (OS). Since there is no accepted evidence on which is the best choice after CDK4/6i+ET for 
HR+/HER2-aBC patients, clinicians are left alone during the treatment decision, causing fragmentation in 
therapeutic practice between cancer centers and even among individual oncologists. 
The importance of real world data (RWD) in HR+/HER2- aBC patients treated 
with ET+CDK4/6i 
Recently, there has been a growing interest in observational real-world studies, due to their ability to observe 
the effects of a given treatment even outside the ideal clinical conditions that characterize RCTs. These types 
of studies also allow sub-analyses to be carried out in certain subgroups, usually underreported in RCTs, and 

   
       
        
                
 
 
 
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to study biomarkers potentially useful for establishing treatment efficacy. This is particularly relevant in the 
context of established clinical settings, such as HR+/HER2- aBC patients who are candidates for first-line 
therapy, where it is difficult, in terms of ethics and pharmaco-economics, to conduct comparison prospective 
randomized trials to address specific clinical questions. 
In this context, and in view of the long average duration of patients' exposure to this class of drugs, it is of 
primary importance to more accurately establish the real-world efficacy and toxicity of the three CDK4/6i.  
Different attempts have been made in this context. The largest real-world experience is represented by the 
real-world collection of data on patients treated with palbociclib in the P-REALITY-X trial. Data were 
extracted from the Flatiron US Flatiron Health Analytic Database and eventually adjusted with appropriate 
statistical methods, such as stabilized inverse probability of treatment weighting (sIPTW) to conduct 
different analyses such as the comparison of CDK4/6i+ET vs ET alone in the real-world setting, with special 
regard to OS. 82,83 Similar, even though smaller, experiences are reported in literature with the other two 
CDK4/6i.84,85 
These studies underlined the feasibility and utility of conducting real-world studies in the context of 
HR+/HER2- aBC. Specifically, this type of studies are essential to assess the effectiveness and safety of 
CDK4/6i in a real-world setting, including neglected subset of patients usually excluded from pivotal clinical 
trials.86 In addition, they represent a unique opportunity to conduct specific sub-analyses that respond to 
relevant questions directly emerged from clinical practice and academic research. Finally, RWD are a 
struggling source of data to discover and validate potential biomarkers, to finally include in predictive 
models potentially useful to distinguish class of patients associated with diverse phenotypes and/or 
prognosis. 
Unmet clinical needs in the treatment of HR+/HER2- aBC 
In absence of direct prospective evidence, it is of crucial importance to conduct large, population-based 
observational, real-world studies to provide evidences in the complex scenario of HR+/HER2- aBC. Of 
particular interest at the moment is the development and validation of new biomarkers for patients receiving 
ET+CDK4/6i in the first-line setting, in order to identify subgroups of patients with different prognoses, for 
which treatment escalation and de-escalation approaches can be considered. For this purpose, it would be of 
paramount importance to have a predictive model, able to define prognosis at the individual patient level, 
taking into account the specific characteristics and complex inter-relationships existing between the clinical 
and biological variables. Another critical issue is represented by the scenario of HR+/HER2- aBC 
progressing after first-line treatment with ET+CDK4/6i. Since several therapies are currently available, 
including novel promising anticancer drugs such ADCs, and in consideration of the large heterogeneity of 
these patients in terms of tumor biology and clinical behaviour, the development of new powerful, easy-to-

   
       
        
                
 
 
 
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access biomarkers and the integration of existing ones with novel signatures represents nowadays and urgent 
clinical need.  
Novel methodological approaches, including random forest, gradient boosting models and neural networks, 
thanks to their ability to extract significant and quantitative features potentially associated with different 
clinical outcomes from electronic health records (EHRs), medical images and biological data through the use 
of complex high-level predictive models, represents a promising approach to achieve these results. In this 
context, these approaches may help in optimizing the treatment sequence in HR+/HER2- aBC patients with 
the aim of optimizing patient OS. In particular, radiomics and pathomics are promising tools of applications 
in oncology, as they are capable of identifying relevant features not accessible by raw human eye from 
radiological images and from pathology slides, respectively. As an example, Khorrami M. et al built a CT 
scan-based radiomic model able to predict survival benefit in aBC with liver metastases treated with 
CDK4/6i in combination with endocrine therapy, reaching a notable accuracy (AUC = 0.77).87 
Master protocol study design 
PALMARES-2 is a retrospective/prospective, observational, multicenter, population-based study, aiming at 
providing real-world evidences on HR+/HER2- aBC patients treated with first-line CDK4/6i plus ET. 
The present study has the objective to collect data coming from different sources, i.e. RWD, medical images 
and biological samples, from patients treated with CDK4/6i as first-line of therapy for HR+/HER2- aBC. In 
consideration of the complexity of data collected and different objectives of the study, this master protocol 
foresees different sub-studies, which encompasses different methodologies for data collection, data 
extraction and analyses. The sub-studies will be described in the following sections. 

   
       
        
                
 
 
 
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Figure 1. PALMARES-2 graphical abstract. CBC: cell blood count; CT: computed tomography; 
ET+CDK4/6i: endocrine therapy plus cyclin-dependent kinase 4/6 inhibitor; FdG-PET: fluorodeoxyglucose 
positron emission tomography; FFPE: formalin-fixed paraffin-embedded; HE: hematoxylin-eosin; 
HR+/HER2- aBC: hormone receptor-positive, human epidermal growth factor receptor 2-negative advanced 
breast cancer; PD: progressive disease; RWD: real-world data. 
(1) Real world data (RWD) PALMARES-2 sub-study 
Objectives and endpoints of the sub-study: 
Primary objectives:        
- 
To test the difference in terms of OS between the group of patients receiving ribociclib and 
palbociclib; 
- 
To test the difference in terms of OS between the group of patients receiving abemaciclib and 
palbociclib. 
 
Secondary objectives:     

   
       
        
                
 
 
 
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- 
To test the difference in terms of OS between the group of patients receiving abemaciclib and 
ribociclib. 
- 
To determine the real-world progression-free survival (rwPFS), time to next treatment or death 
(TTNT-D), time to chemotherapy or death (TTC-D) and OS in the real-world setting; 
- 
To test the difference in terms of rwPFS between the group of patients receiving ribociclib and 
palbociclib; 
- 
To test the difference in terms of rwPFS between the group of patients receiving abemaciclib and 
palbociclib; 
- 
To test the difference in terms of rwPFS between the group of patients receiving abemaciclib and 
ribociclib; 
- 
To test the difference in terms of  TTNT-D between the group of patients receiving ribociclib and 
palbociclib; 
- 
To test the difference in terms of TTNT-D between the group of patients receiving abemaciclib and 
palbociclib; 
- 
To test the difference in terms of TTNT-D between the group of patients receiving abemaciclib and 
ribociclib; 
- 
To test the difference in terms of TTC-D between the group of patients receiving ribociclib and 
palbociclib; 
- 
To test the difference in terms of TTC-D between the group of patients receiving abemaciclib and 
palbociclib; 
- 
To test the difference in terms of TTC-D between the group of patients receiving abemaciclib and 
ribociclib; 
- 
To test the difference in terms of rwPFS, TTNT-D and TTC-D among the three CDK4/6i in 
clinically-relevant subgroups, such as endocrine sensitive and resistant patients, premenopausal 
women, patients with poorer ECOG PS, patients harboring luminal-B like disease, patients with liver 
metastases; 
- 
To test the difference in terms of rwPFS, TTNT-D, TTC-D and OS among the three CDK4/6i in 
older women (defined as >75 years old); 
- 
To test the effect of histology (non special type vs lobular vs other) on rwPFS, TTNT-D, TTC-D; 
- 
To test the difference in terms of rwPFS, TTNT-D, TTC-D and OS among the three CDK4/6i in 
patients with lobular breast cancer histology; 
- 
To test the difference in terms of rwPFS, TTNT-D, TTC-D and OS among the three CDK4/6i 
according to disease-free interval (DFI); 
- 
To test the difference in terms of rwPFS, TTNT-D, TTC-D and OS among the three CDK4/6i 
according to endocrine resistance status, including primary versus secondary resistance; 

   
       
        
                
 
 
 
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- 
To test the difference in terms of rwPFS, TTNT-D, TTC-D and OS among the three CDK4/6i 
according to different ER expression levels; 
- 
To determine the rwPFS, TTNT-D, TTC-D and OS in the subgroup of patients with brain 
metastases; 
- 
To determine the rwPFS, TTNT-D, TTC-D and OS in the subgroup of patients with oligometastatic 
disease and in the subgroup of patients with high metastatic burden; 
- 
To test the effect on rwPFS, TTNT-D, TTC-D and OS of available metabolic biomarkers (e.g. body 
mass index, glycemia); 
- 
To test the difference in terms of rwPFS, TTNT-D, TTC-D and OS among the three CDK4/6i in 
patients harboring germline mutations, including BRCA1, BRCA2, PALB2, and in patients with 
homologous recombination deficiency; 
- 
To test the difference in terms of rwPFS, TTNT-D, TTC-D and OS among the three CDK4/6i in 
patients harboring genomic somatic alterations, like PIK3CA and ESR1 status; 
- 
To test the association between blood lymphocyte counts at baseline, or their precocious on-
treatment modifications (T1: 2 weeks after treatment initiation, and T2: before fourth cycle initiation, 
approximately 12 weeks after treatment initiation) and rwPFS, TTNT-D, TTC-D and OS of 
HR+/HER2- patients treated with first-line ET plus CDK4/6i; 
- 
To evaluate the association between peripheral blood crude cell counts, namely whole blood cells, 
neutrophils, monocytes and platelets, as evaluated at baseline and as precocious on-treatment 
modifications, and patient rwPFS, TTNT-D, TTC-D and OS; 
- 
To evaluate the association between baseline composite parameters, such as the Neutrophil-to-
Lymphocyte ratio (NLR), the platelet-to-lymphocyte ratio (PLR), the monocyte-to-lymphocyte ratio 
(MLR), or the Pan-Immune Inflammation Value (PIV), and patient PFS; 
- 
To evaluate the association between precocious on-treatment modifications (i.e., 2 weeks and ~12 
weeks after treatment initiation) of NLR, PLR, MLR and PIV, and patient PFS; 
- 
To evaluate the association between HER2 status (HER2-low vs. HER2-0; HER2 2+ with negative 
ISH vs. HER2 1+ vs. HER2-0) in the primary tumor and rwPFS, TTNT-D, TTC-D and OS; 
- 
To evaluate the association between HER2 status (HER2-low vs. HER2-0; HER2 2+ with negative 
ISH vs. HER2 1+ vs. HER2-0) in one metastatic site (subjected to biopsy before CDK 4/6 inhibitor 
initiation), and rwPFS, TTNT-D, TTC-D and OS; 
- 
To explore pattern of progression of HR+/HER2- aBC after ET plus CDK4/6i treatment; 
- 
To test the effect on rwPFS, TTNT-D, TTC-D and OS of mastectomy in patients with de novo 
HR+/HER2- aBC. 

   
       
        
                
 
 
 
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Study Population 
Inclusion criteria:  
- 
Pre- and Post-menopausal patient (woman OR man)                  
- 
Age≥18 years 
- 
Diagnosis of aBC. HR+/HER2- aBC, as defined as at least 1% estrogen receptor (ER) and/or 
progesterone receptor (PgR) positivity at IHC. HER2 negativity is defined on the basis of an IHC 
score of 0, 1+, or 2+ with absence of gene amplification at in situ hybridization (ISH) analyses. 
- 
Have received or are candidate to receive treatment with palbociclib, ribociclib or abemaciclib in 
combination with endocrine therapy as first-line treatment for HR+/HER2- aBC. 
Exclusion criteria: 
- 
Less than 3 months of follow up from the CDK4/6i start to the date of data cut-off; 
- 
Have received CDK4/6i as monotherapy; 
- 
Have received CDK4/6i as adjuvant treatment for localized disease. 
Data collection 
The study involves the collection of RWD, i.e. obtained directly from the medical records, electronic or 
paper, of the patients enrolled in the study. Therefore, no additional patient assessment will be required 
specifically for study purposes. Data will be collected locally within the center participating in the study, 
subject to patient consent, and then reported on a platform in .xlsx format. 
The types of data collected within this sub-study include: 
- 
Demographic data: date of birth, gender, menopausal status; 
- 
Neoplasm history data: de novo vs metachronous presentation; date of diagnosis of localised disease; 
prior treatment for localised disease, including type and duration of prior hormonal, chemotherapy 
and CDK4/6i treatment; number and type of metastatic sites; 
- 
CDK4/6i treatment data: drug used; backbone of hormone therapy and date of initiation of hormone 
therapy versus initiation of CDK4/6i; use of LHRH analogue; 
- 
Tumor biology data: hormone receptor status, grading, proliferation index at biopsy site of 
metastasis and, if not available, relative to primary tumor; OncotypeDX; 
- 
Data on haematochemical parameters: complete bloodcounts and some biochemical parameters such 
as LDH, blood glucose and CA15.3, collected at baseline, 15 days after starting CDK4/6i, after 30 
days and at treatment progression; 
- 
Genomic data: genomic alterations available to the patient, performed according to clinical practice; 
this section includes both somatic mutations and germline DNA alterations.  

   
       
        
                
 
 
 
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 (2) Toxicity – Comorbidity sub-study 
The “toxicity – comorbidity” sub-study aims to add a key piece to the real-world evidence of the three 
CDK4/6i, allowing for the first time a systematic comparison of the real-world toxicities of palbociclib, 
ribociclib and abemaciclib. 
Objectives and endpoints of the sub-study 
Primary objective: 
- 
To compare the real-world incidence of severe toxicity, defined as a grade 3 or higher adverse event 
reported by the clinician, occurring during treatment, among the three CDK4/6i.  
Secondary objectives: 
- 
To compare the real-world incidence of overall toxicity, defined as any-grade adverse event 
reported by the clinician, occurring during treatment, among the three CDK4/6i; 
- 
To assess the incidence of dose reduction conditioning toxicity; 
- 
To assess the incidence of toxicity requiring discontinuation of treatment or switch to another 
molecule of the CDK4/6i class; 
- 
To test the impact of dose reduction, drug switch and/or CDK4/6i discontinuation on survival 
outcomes (rwPFS, TTNT-D, TTC-D and OS); 
- 
To assess the incidence of HIV infection among HR+/HER2- aBC patients treated with first-line 
CDK4/6i+ET; 
- 
To assess the impact of HIV infection on survival outcomes (rwPFS, TTNT-D, TTC-D and OS) 
among HR+/HER2- aBC patients treated with first-line CDK4/6i+ET; 
- 
To assess the incidence of alterations in liver or kidney function parameters such as aspartate 
transaminase (AST), alanine transaminase (ALT), total bilirubin, lactic dehydrogenase (LDH), 
leukocyte alkaline phosphatase (ALP), creatininemia; 
- 
To assess the incidence of alterations in electrocardiographic tracing definedas an increase in the 
corrected QT (according to the Fridericia criterion) greater than 480 msec; 
- 
To assess the incidence of adverse events in groups of patients with comorbidities; 
- 
To assess the incidence of adverse events in groups of patients taking concomitant therapies at the 
start of CDK4/6i treatment. 
Study Population 
The same inclusion and exclusion criteria adopted for the RWD sub-study will be adopted for the present 
sub-study, for the only exception of the following inclusion criteria: 
- 
Availability of safety data during >50% of the time of study treatment; 

   
       
        
                
 
 
 
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- 
Availability of baseline patient information about comorbidities and concomitant medications. 
Data collection 
The data collection of this sub-study will take place in a similar manner to that of the RWD sub-study. In 
particular, the planned information will be collected directly from the patients' EHRs and collected in a 
separate .xlsx file, adopting the same pseudonymized code used for the RWD cohort. In detail, this sub-study 
will involve the collection of: 
- 
All adverse events occurring during treatment, indicating the maximum degree reached; 
- 
Dose reduction, indicating the level of reduction and the cycle at which this dose reduction occurred; 
- 
Any interruption of treatment due to toxicity or switch to another CDK4/6i; 
- 
Specific haematochemical parameters useful for assessing the progress of hepato-renal function, in 
particular AST, ALT, total bilirubin, LDH, ALP, creatininemia; these parameters will be collected at 
treatment baseline, after 15 days and 3 months after starting CDK4/6i (i.e. before starting the fourth 
cycle of therapy); 
- 
Electrocardiographic parameters, in particular specific conduction alterations or arrhythmias and 
QTc value according to Fridericia, collected at baseline, after 15 and after 30 days from start; 
- 
Patient comorbidity, understood as concomitant disease at the start of treatment with CDK4/6i 
ascertained by the physician and classified in macro-areas (e.g. cardiological, respiratory, metabolic, 
infectious, chronic diseases such as diabetes mellitus type 2); 
- 
Concomitant therapies, initiated and ongoing before the start of treatment with CDK4/6i, reported as 
drug classes (e.g. steroid therapy, antibiotic therapy, proton pump inhibitors); 
- 
Blood glucose and body mass index at baseline of CDK4/6i treatment. 
 (3) Medical imaging sub-study 
Objectives and endpoints of the sub-study 
Primary objective: 
- 
To build a model able to predict prognosis of HR+/HER2- aBC patients treated with first-line 
ET+CDK4/6i using radiomics features extracted from baseline radiological images; 
- 
To build a model able to predict prognosis of HR+/HER2- aBC patients treated with first-line 
ET+CDK4/6i using radiomics features extracted from baseline radiological images; 
- 
To combine radiomics and pathomics features with clinical data to build a model able to predict 
prognosis of HR+/HER2- aBC patients treated with first-line ET+CDK4/6i; 
Secondary objectives: 

   
       
        
                
 
 
 
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- 
To combine radiomics features with clinical data, pathomics, genomics and transcriptomics features 
to build a model able to predict prognosis of HR+/HER2- aBC patients treated with first-line 
ET+CDK4/6i; 
- 
To build a model able to classificate HR+/HER2- aBC patients treated with first-line ET+CDK4/6i 
in early and late progressors using radiomics features extracted from baseline radiological images; 
- 
To build a model able to predict prognosis of HR+/HER2- aBC patients treated with first-line 
ET+CDK4/6i using baseline radiomics features and early radiomics modifications detected from first 
on-treatment radiological evaluation; 
- 
To build a model able to predict molecular characteristics, such as PIK3CA and ESR1 mutational 
status, of HR+/HER2- aBC patients treated with first-line ET+CDK4/6i using radiomics features 
extracted from baseline radiological images; 
- 
To build a model able to predict molecular characteristics, such as PIK3CA and ESR1 mutational 
status, of HR+/HER2- aBC patients treated with first-line ET+CDK4/6i using radiomics features 
extracted from digitalized HE slides; 
- 
To build a model able to predict intrinsic BC subtypes, defined by PAM50 panel on transcriptomic 
analyses, of HR+/HER2- aBC patients treated with first-line ET+CDK4/6i using radiomics features 
extracted from baseline radiological images; 
- 
To build a model able to predict intrinsic BC subtypes, defined by PAM50 panel on transcriptomic 
analyses, of HR+/HER2- aBC patients treated with first-line ET+CDK4/6i using radiomics features 
extracted from digitalized HE slides. 
Study Population 
The same inclusion and exclusion criteria adopted for the RWD sub-study will be adopted for the present 
sub-study, for the only exception of the following inclusion criterion: 
- 
Availability of at least one of the following two data: 
o A baseline Computerized Tomography (CT) scan or 18F-Fluorodeoxyglucose Positron 
Emission Tomography (FdG-PET) 
o A digitalized pathology slides derived from biopsy of metastatic sites and/or from primary 
tumor biopsy and/or from surgery specimen. 
Data collection 
This sub-study encompasses the collection of medical images of patients enrolled in the PALMARES-2 
RWD sub-study. Two type of images will be collected: 
- 
Radiological images: CT scan, or FdG-PET scan, performed at the baseline of CDK4/6i, i.e. up to 
three months before the treatment start until one week after the start; the CT scan should include at 

   
       
        
                
 
 
 
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least the thorax and abdomen scanning; both pre- and post-contrast acquisition will be collected. CT 
scan, or FdG-PET, performed as the first radiological evaluation will be collected as well to assess 
early radiomics modifications and their effect on survival outcomes. For the purposes of the 
“Subsequent lines” sub-study (as detailed below, in the (5) section), CT scan, or FdG-PET scan, 
performed at the baseline of each of the subsequent line of therapy will be collected as well. 
- 
Digitalized pathology slide: hematoxylin and eosin (HE) slides coming from diagnostic biopsy or 
surgical specimens will be digitalized and collected in a pre-defined folder. Both slides from 
primary tumor and from metastatic sites that underwent biopsy assessment will be collected, if 
available. If more than one metastatic biopsy has been performed during the course of patient’s 
disease, all the HE slides should be collected, if available. If a patient did not undergo neoadjuvant 
treatment, diagnostic biopsy of the breast or surgical tumor slide could be collected equivalently; 
otherwise, the diagnostic biopsy performed before treatment initiation should be taken. 
 (4) Translational sub-study 
Objectives and endpoints of the sub-study 
Primary objective: 
- 
To find the mutational status of a gene, or a subset of genes, associated with differential survival 
outcomes (rwPFS, TTNT-D, TTC-D and OS) of HR+/HER2- aBC patients treated with first-line 
ET+CDK4/6i; 
- 
To find transcriptomic differential gene expression, or differential expressed pathways, associated 
with differential survival outcomes (rwPFS, TTNT-D, TTC-D and OS) of HR+/HER2- aBC treated 
with first-line ET+CDK4/6i. 
Secondary objectives: 
- 
To test the effect of PAM50 intrinsic subtype on survival outcomes (rwPFS, TTNT-D, TTC-D and 
OS) of HR+/HER2- aBC patients treated with first-line ET+CDK4/6i; 
- 
To combine genomics and transcriptomics features with clinical data and radiomics and pathomics 
features, to build a model able to predict prognosis of HR+/HER2- aBC patients treated with first-
line ET+CDK4/6i; 
- 
To test the association between agnostic features included in radiomics model and transcriptomic 
pathways and mutational status. 

   
       
        
                
 
 
 
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Study Population 
The same inclusion and exclusion criteria adopted for the RWD sub-study will be adopted for the present 
sub-study, for the only exception of the following inclusion criterion: 
- 
Availability of adequate tumor sample (see Data Collection below) from biopsy of metastatic sites 
and/or from primary tumor biopsy and/or from surgery specimen. 
Data collection 
This sub-study encompasses the collection of tumor samples of patients enrolled in the PALMARES-2 RWD 
sub-study. At least one formalin-fixed paraffin-embedded tumor block will be collected from diagnostic 
biopsy or surgical specimens, performed for diagnostic purposes as per clinical practice. Both tumor samples 
from primary tumor and from metastatic sites underwent biopsy will be collected, if available. If more than 
one metastatic biopsy has been performed during the course of patient’s disease, all the non-stained slides 
and/or tumor blocks should be collected, if available. If a patient did not undergo neoadjuvant treatment, 
diagnostic biopsy of the breast or surgical tumor slide could be collected equivalently; otherwise, the 
diagnostic biopsy performed before treatment initiation should be taken. 
(5) Second and further lines sub-study 
Objectives and endpoints of the sub-study 
Primary objective: 
- 
To build a model based on clinical, radiomics and pathomics data to select the best treatment option 
in terms of OS in HR+/HER2- aBC patients progressing after first-line ET+CDK4/6i. 
Secondary objectives: 
- 
To build a model based on clinical data to select the best treatment option in terms of OS in 
HR+/HER2- aBC patients progressing after first-line ET+CDK4/6i. 
- 
To build a model based on clinical, radiomics, pathomics, genomics and transcriptomics data to 
select the best treatment option in terms of OS in HR+/HER2- aBC patients progressing after first-
line ET+CDK4/6i. 
- 
To assess the efficacy in terms of rwPFS and OS of second and subsequent line of therapy in a real-
world setting of  HR+/HER2- aBC patients progressing after first-line ET+CDK4/6i. 
Study Population 
The same inclusion and exclusion criteria adopted for the RWD sub-study will be applied for the present 
sub-study, for the only exception of the following inclusion criteria: 

   
       
        
                
 
 
 
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- 
Clinical, biochemical or radiological progression on CDK4/6i+ET first-line treatment 
- 
Second-line therapy start after CDK4/6i+ET progression 
- 
Data availability about at least one of the subsequent lines, including at least the treatment line of 
therapy, treatment start date, treatment progression date or last follow up, type of treatment and drug 
used. 
Data collection 
The study involves the collection of RWD of patients enrolled in the PALMARES-2 RWD study, who 
started a second line therapy after CDK4/6i progression. Data will be collected at the study centers in the 
same manner of RWD sub-study. Data collected within this sub-study will include the type of treatment 
performed (i.e. ET-based therapy, chemotherapy, other) and line of therapy for metastatic disease for which 
the treatment has been performed; treatment start date and progression date. Data derived from medical 
images and biological samples collected in the context of sub-study (3) and (4) will be used to pursue the 
objectives of this sub-study. 
Study procedures 
Informed consent process and patients’ enrollment 
Patients who are alive at the time of study conduction will be asked to sign the informed consent for the 
study partecipation and for treatment of personal data for research purposes, during the first clinical visit. 
Patients with available material to conduct analyses for the translational sub-study will be asked to the use of 
data generated from available material for the scientific purposes planned for the present study. It will also 
be asked to consent to receive unexpected genetic-hereditary information, should it emerge from the analysis 
of tumor samples.  
For patients who are not alive at the moment of study initiation and data collection, the study will adhere to 
the UE-Reg 2016/679 - GDPR rules for the protection of personal data. In accordance with the Declaration 
of Helsinki and other applicable regulations, a subject, or his/her legal representative, has the right to 
withdraw from the study at any time and for any reason without prejudice to his/her future medical care.88 
Data protection procedures 
Patients are assigned by the local investigator a unique progressive identifier code, that will be used for all 
the tasks implied in the study. The pseudonymization procedure will ensure the unique identification of 
patients still keeping adequate data security. This pseudonymized code will used to save medical images 

   
       
        
                
 
 
 
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once shared with the Sponsor center. The same code used to mark biological samples at the moment of the 
shipment to the centralization for analyses conduction. 
Clinical data collection and management 
Clinical real world data will be collected by centers in ad hoc document in .xlsx format. At pre-specified data 
cut-off (see sections below), data collected will be shared with the Sponsor for data analyses. Investigators at 
study centers could be required to modify data in case of data inconsistencies encountered during data 
analyses. 
All consecutive HR+ HER2- aBC patients treated with first-line ET plus CDK 4/6i as per clinical practice in 
the time interval between January 2017 and December 2030 will be enrolled retrospectively and 
prospectively. Data will be collected in a continuous manner and will be shared with the Sponsor center on 
an annual basis, at pre-specified data cut-offs. Data cut-offs will occur annually, at certain cutoffs falling on 
1st January of each year. Taking into account the time interval and the centers involved, approximately 2000 
patients will be included at the first data cut-off of 1st January 2024. Those patients who have already 
concluded the treatment at the time of study inclusion will be evaluated for clinical outcomes (i.e. PFS, OS) 
and biological parameters of interest will be retrospectively collected. Patients who are still receiving ET 
plus CDK4/6i at the time of inclusion in the study will be evaluated for the same variables until the end of 
the study. At the time of data collection and analysis, these patients will be censored for the events of interest 
(e.g. progression, death) if these events have not occurred yet.  
   
 
Figure 2. PALMARES-2 study expected timeline and enrollment. EC: ethical committee; pts: patients. 
 

   
       
        
                
 
 
 
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Medical images collection and sharing 
The medical images, collected within the timeframe specified in the appropriate section (see section 
“Medical images sub-study”), will be collected locally in the individual centers, pseudonymised and 
uploaded to an ad hoc secure sharepoint. A folder will be set up for each individual center participating in 
this sub-study, so that only the center uploading the images and the Sponsor center itself will be able to 
access them. 
Tumor samples collection, storage and shipment 
For the “Translational sub-study” purposes, tumor samples related to patients enrolled in the RWD sub-study 
will be considered. If multiple tumor samples are available, the ones to be used for the study has to be chosen 
as specified in the section “Translational sub-study”. Tumor materials have to be shipped to the Sponsor 
center at the moment of the conduction of translational analyses, attributing the same pseudonymized code 
used for the other sub-study. The remaining unused tumor tissue will be sent back to the pathology 
laboratory at the end of the study. 
Statistical considerations 
Statistical design 
Based on pivotal trials showing a statistically significant OS advantage for ribociclib and abemaciclib, but 
not for palbociclib in the endocrine resistant setting, and clinically meaningful OS advantage for ribociclib 
and abemaciclib, but not for palbociclib in the endocrine sensitive setting, we will hypothesize a difference 
of 20% in terms of OS between ribociclib versus palbociclib and between abemaciclib and palbociclib. To 
detect this difference with 80% power with a two-sided alpha error of 0.05, a sample size of 3258 patients 
reaching 977 OS events will be required. The prognostic role of CDK4/6i drug will be weighed for clinically 
and biologically relevant characteristics.89 
Since the number of patients enrolled in the other sub-studies depends on the sample size of the RWD sub-
study, no formal statistical calculation has been performed for 2 to 5 PALMARES-2 sub-studies. 
Statistical methods 
Standard descriptive statistics will be used to describe clinical and biological patients’ characteristics. The 
median and interquartile range (IQR) were reported for continuous variables. Categorical variables were 
summarized as percentages of available data. To evaluate differences, in terms of clinic-pathological 
characteristics, among different CDK4/6i groups, we will use the Kruskal-Wallis Rank Sum test for 

   
       
        
                
 
 
 
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continuous variables and the Chi-square and Fisher exact tests for categorical variables. Unpaired t-test will 
be used to compare numeric variables, at baseline and different timepoints. Classic survival analysis methods 
will be used to evaluate patient survival outcomes and survival curves will be obtained through the Kaplan–
Meier method. Follow-up times will be estimated using the reverse Kaplan–Meier method. Multivariable 
analysis will be performed through Cox regression modeling to estimate the effects of covariates. To 
homogenize patients’ characteristics among treatment groups, additional adjustment techniques will be 
eventually used, such as propensity score matching (PSM) and inverse probability of treatment weighting 
(IPTW). Estimated weights will be then incorporated into a weighted Cox regression model to estimate 
rwPFS. Area Under the ROC Curve (AUC) values of the models will be calculated using the Caret and 
pROC R package and compared using Delong’s test. 
Patients with missing outcome data will be excluded from the analysis. Covariates whose values will be 
missing in less than 20% were imputed by using the median or the mode value (for numerical or categorical 
variables, respectively), while covariates with missing values exceeding 20% will be excluded from the present 
analysis.90 All statistical tests will two-tailed, and a p value (P) <0.05 was considered as significant. Statistical 
analyses will be performed using R software and R Studio (version 4.1.2). 
Image omics and model generation methodology 
Radiomics Methodology 
A team of four radiologists with experience on CT scan/PET images segmentation will identify the Volume 
Of Interest (VOI) through a semi-automated 3D segmentation process performed with Syngo.via software. 
An ad hoc designed segmentation protocol will be applied to homogenize the choice of CT/PET sequence on 
which to perform segmentation and the choice of the lesion to be segmented. Peritumoral area will be 
obtained through automated augmentation techniques (+5 mm, +10 and +15 mm); if the peritumoral area 
obtained crosses blood vessels, gas, bone, bile duct, or exceeded the organ edge, the contour will be 
manually modified to exclude this area. After image pre-processing through different techniques (e.g., gray 
discretization, intensity normalization, and voxel resampling), radiomic features will be extracted from VOIs 
and peritumoral areas using the PyRadiomics library in Python. In order to prevent signature overfitting, 
dimensionality of features is reduced before signature construction, firstly excluding features with high 
intraclass correlation coefficient and significantly different between the two outcome groups as assessed by 
one-way analysis of variance (ANOVA). Least absolute shrinkage and selection operator (LASSO) 
regression and/or Maximum Relevance Minimum Redundancy (MRMR) will be then used for the selection 
of features that will be included in the final model. The cohort will be split in a training (80%) and testing 
cohort (20%). Different standard classifiers, such as Random Forest, Multilayer perceptron, Logistic 

   
       
        
                
 
 
 
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Regression, Support Vector Machine, CatBoost, AdaBoost, XGBoost, will be trained and evaluated for this 
task.91,92 
Pathomics Methodology 
Pathology slides will be digitalized with Leica Scanscope AT2 and stored as “.svs” files to obtain Whole 
Slide Images (WSIs). Hematoxilin and Eosin (HE) slides will be digitalized for pathomics purposes. WSIs 
will be analysed through Slideflow, a Python package that provides a unified Application Programming 
Interface for building and testing models for histopathology. After color normalization procedures, 
quantitative pathomics features will be extracted. Model creation and performance evaluation will follow the 
same methodology discussed in the “Radiomics” workflow.93 
Multimodal integration 
We will use several architectures, including logistic regression, classification and regression trees, random 
forest, multi-layer perceptron, gradient boosted trees (XGBoost), gradient boosting machines (LightGBM) 
and attentive tabular networks TabNet and we will compare their performance to heuristically decide on the 
best model choice for follow-up experiments. Our modelling experiments will include different combinations 
of input embeddings, with concatenation of each data stream permutation to produce fusion embeddings and 
train models using single-modality or multi-modality of input combinations. All models will be trained 
multiple times with multiple different data splits to repeat the experiments and compute average metrics and 
SDs. All defined models will be trained and tested to evaluate the advantage of multimodal predictive 
systems, as compared to single modality ones for the pre-established classification prediction. To assess 
model performance, different metrics will be used, including AUC, Sensitivity and Specificity, which will be 
calculated on the training set through 10-fold cross-validation and on the test set, which will be obtained by 
randomly splitting the entire cohort in a 80:20 way. The hyperparameter combinations of individual models, 
such as maximum depth of the trees, number of estimators and learning rate, will be selected within each 
training loop using a fivefold cross-validated grid search on the training set.94,95 
Since these complex models are considered as "black boxes", since they make decisions without clear 
reasoning, explainability techniques will be adopted to provide insights into how models make predictions, 
guaranteeing human interpretability. In particular, SHapley Additive exPlanations (SHAP) techniques will be 
used for both global and local explanations.96 
Biological analyses methodology 
Genomic analyses 
A next generation sequencing (NGS) panel (Oncomine Comprehensive Assay Plus) will be used to perform 
analysis of various single-gene variants, such as single nucleotide variations (SNVs), indels, fusions, splice 

   
       
        
                
 
 
 
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variants, and copy number variations (CNVs), including both copy number gains and losses, across ~500 
genes. The panel will include, but will not be limited to, genes involved in DNA repair systems (e.g. BRCA1, 
BRCA2, PALB2, CHEK2, ATM), PIK3CA pathway (e.g. PIK3CA, AKT1, PTEN), estrogen receptor (e.g. 
ESR1). It also allows the quantitative evaluation of tumor mutational burden (TMB) and the analysis of the 
microsatellite instability (MSI) status.97 
Transcriptomic analyses 
After cellular RNA extraction, RNA quality check and preparation of libraries, RNA will be sequenced with 
NovaSeq 6000 System. Counts will be analyzed to dissect differential gene expression both at single gene 
level – through differential gene expression analysis (i.e, Deseq2 method) – and at pathway level – through 
Gene Set Enrichment Analysis (GSEA), Over Representation Analyses (ORA) and Gene Set Variation 
Analysis (GSVA).(30,31) The pathway-level analysis will be carried out using the Bioconductor packages 
fgsea, gsva, and clusterprofiler, taking advantage of HALLMARK, KEGG, and REACTOME gene sets 
available from GSEA Molecular Signatures Database (http://www.gsea-msigdb.org/gsea/msigdb/collections. 
jsp). Intrinsic subtypes will be assessed through Absolute Intrinsic Molecular Subtyping (AIMS) 
algorithm.98–100 
Study duration 
Patient enrollment and data collection will start upon approval of the study protocol by the competent ethics 
committee. All consecutive HR+/HER2- aBC patients treated with first-line ET plus CDK 4/6 inhibitors as 
per clinical practice in the time interval between January 2016 and December 2030 will be enrolled. The 
collection and the update of clinical data, medical images and biological samples will continue for an 
expected duration of 10 years from the enrollment of the last patient. The clinical data, medical images and 
data obtained from the biological samples will be retained for further 10 years after the end of the study, 
while the biological samples will be returned to the pathology departments of the Center of origin at the end 
of the analyses foreseen in the protocol. 
Ethical considerations 
This study will be conducted according to the Declaration of Helsinki and in compliance with ethical 
principles regarding human research. 
Based on its observational nature, and in accordance with the Italian Ministerial Decree of 30th November 
2021, the present study was approved by the Ethics Committee of the Sponsor Institution (Fondazione IRCCS 
Istituto Nazionale dei Tumori), and it was subsequently adopted by all the participating centers. Based on 

   
       
        
                
 
 
 
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Article 110bis of the Italian Privacy Code (Legislative Decree of 30 June 2003, n.196 updated 01 May 2024), 
the privacy impact assessment of the study is performed at the level of the Sponsor center and published on 
Institutional website, therefore no additional assessment by the other centers participating in the study is 
required. 
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