Multicenter phase II study of Cabazitaxel in advanced gastroesophageal cancer: Association of HER2 Expression and M2-like Tumor Associated Macrophages with Patient Outcome

Abstract

Purpose:

We examined cabazitaxel, a novel next generation taxoid, in patients with metastatic gastric cancer in a multicenter phase II study.

Experimental Design:

Patients that have progressed on 1 or more prior therapies for locally advanced, unresectable or metastatic disease, were eligible and prior taxane therapy was allowed. Taxane-naïve and pretreated cohorts were analyzed independently for efficacy. The primary endpoint for both cohorts was progression-free survival (PFS) using RECIST 1.1, using a Simon’s two-stage design (10% significance, 80% power) for both cohorts. Comprehensive molecular annotation included whole exome and bulk RNA sequencing.

Results:

Fifty-three patients enrolled in the taxane-naïve cohort (Arm A), and 23 patients in the prior-taxane cohort (Arm B), from January 8, 2013 to April 8, 2015: median age 61.7 years (range 35.5 – 91.8 years), 66% male, 66% Caucasian. The most common adverse events included neutropenia (17% Arm A, 39% Arm B), fatigue/muscle weakness (13%), and hematuria (12%). In Arm A, the 3-month PFS rate was 28% (95%CI 17–42%), and did not meet the pre-specified efficacy target. The 3-month PFS rate in Arm B was 35% (95% CI 16–57%), and surpassed its efficacy target. HER2 amplification or over-expression was associated with improved disease control (P=0.003), PFS (p=0.04) and overall survival (p=0.002). An M2 macrophage signature was also associated with improved survival (p=0.031).

Conclusions:

Cabazitaxel has modest activity in advanced gastric cancer, including in patients previously treated with taxanes. Her2 amplification/overexpression and M2 high macrophage signature are potential biomarkers for taxane efficacy that warrant further evaluation.

INTRODUCTION

Gastric cancer is a global disease that represents an enormous global health burden, responsible for approximately 700,000 deaths worldwide annually¹. Gastric cancers are often grouped together, even though there are considerable clinical and pathologic differences in disease, distinguished by disease location, histology, and molecular characteristics^{2, 3}. For most patients diagnosed with advanced disease, the prognosis remains dismal with median survival less than 1 year^4–6. By improving our understanding of the heterogeneity of this disease and its clinical consequences, we will improve patient outcomes with improved directed treatment options. Our aim with this phase II study is to examine the efficacy of cabazitaxel, a novel next generation taxoid that has demonstrated efficacy in docetaxel refractory disease^{7, 8}, in metastatic gastric cancer and to examine correlative features associated with patient outcomes.

The addition of docetaxel to cisplatin/fluorouracil (DCF) is approved as a first line standard of care treatment option for patients with advanced gastric cancer based on improved patient survival in a random assignment phase II/III study⁵. However, DCF is associated with significant toxicity and currently reserved for patients with excellent performance status^{9, 10}. Two new modified regimens, modified DCF (mDCF)¹¹ and fluorouracil, leucovorin, oxaliplatin and docetaxel (FLOT)^{12, 13}, have been developed to improve efficacy while reducing toxicity. However, these three drug combinations still are more toxic than FOLFOX, which is now a common standard first line treatment option¹⁴, especially since the establishment of taxane based therapy in the 2^nd line treatment setting¹⁵.

In the second line treatment setting, the response rate with single agent platinum, taxanes, or irinotecan is 5–23% associated with an overall survival of 5.2–8.3 months^{16, 17}. When this study was initiated, the administration of second-line therapy in appropriate patients was established as a standard care option, based on three randomized studies each of which demonstrates a survival advantage with chemotherapy over best supportive care (BSC)^18–20. A meta-analysis of these studies demonstrated a HR for OS of 0.73 (95% CI, 0.58–0.96), and in highly functioning patients (PS 0–1), the HR was 0.57 (0.36–0.91) over best supportive care, suggesting even a greater improvement in survival with chemotherapy in the 2^nd line setting in patients with a preserved performance status.²¹

Cabazitaxel is a new taxane that has demonstrated similar antiproliferative activity to docetaxel in taxane-sensitive pre-clinical models and superior activity to docetaxel in taxane-resistant preclinical models²². The preclinical activity of cabazitaxel was confirmed clinically and cabazitaxel has achieved regulatory approval for docetaxel refractory castrate-resistant prostate cancer⁸. We performed this phase II study to examine the efficacy of cabazitaxel in the second line treatment of both taxane-naïve and taxane-treated metastatic gastric cancer. Correlative studies were performed to understand mechanisms of resistance and markers of efficacy in this disease.

METHODS

Study Design

This was a multicenter phase II study to examine the efficacy and safety of cabazitaxel monotherapy in advanced gastric or gastroesophageal junction adenocarcinoma (NCT01757171 on clinicaltrials.gov). Patients must have histologically confirmed gastric or gastroesophageal junction adenocarcinoma, or distal esophageal adenocarcinoma that was unresectable or metastatic, and must have received at least one and no more than two prior cytotoxic therapy regimens for incurable disease. Patients must have evaluable disease by RECIST 1.1 criteria, and must have an adequate performance status of ECOG 0–2, 18 years or older, and with adequate baseline end organ function, including WBC ≥ 3000/ml and ANC > 1000/ml, Hgb > 7.5 g/dL (without transfusion within 7 days), Plt > 75,000/ml (without transfusion within 7 days), AST/ALT ≤ 2.5 × upper limit of normal (ULN), TB ≤ 1.5 × ULN, and creatinine < 2.0 g/dl. Patients were excluded if they had brain metastases with active neurological dysfunction, pregnancy, chemotherapy within 3 weeks, radiotherapy within 2 weeks, or nitrosoureas/ mitomycin C within 6 weeks of enrolling onto the study.

The protocol was approved by institutional review boards at each site. The study was conducted in accordance with principles of the Declaration of Helsinki and International Good Clinical Practice Guidelines. All patients provided written informed consent.

Treatment

Cabazitaxel was administered on an outpatient basis initially intravenously at a dose of 25 mg/m² every 3 weeks, but after initial grade 4 neutropenia was observed in 4 out of the first 5 patients, all subsequent patients received cabazitaxel 20 mg/m². Premedications included antihistamine (dexchlorpheniramine 5 mg or diphenhydramine 25 mg, or equivalent), a corticosteroid (dexamethasone 8 mg or equivalent), and H2 antagonist (ranitidine 50 mg or equivalent). Antiemetic prophylaxis was recommended as per institutional guidelines and hematopoietic growth factor support was permitted at the discretion of the treating physician. Dose escalation to 25 mg/m² was permitted provided no grade 3–4 toxicity was experienced in the first 2 cycles of therapy. Toxicity assessments occurred prior to every cabazitaxel dose according to CTCAE 4.0. Patients underwent radiographic imaging, preferentially contrast enhanced CT scans of the chest, abdomen, and pelvis within 4 weeks of starting therapy, and again every 2 cycles (eg. 6 weeks) for the first 6 months and then every 9 weeks thereafter. Patients were followed for survival every 3 months for 6 months after removal from study.

Biostatistics

Because the efficacy of cabazitaxel may be different in a taxane-naïve versus taxane previously-treated patient populations, patients were assigned to one of two independent cohorts (Arm A: taxane-naïve and Arm B: taxane-pre-treated), and each cohort was analyzed independently. Patients were considered evaluable if they met eligibility requirements, initiated therapy and not removed for non-compliance or withdrawal (unrelated to disease progression or clinical deterioration) within the first 3 months. The primary endpoint for both cohorts was progression-free survival (PFS), as measured from the start of the treatment to the date of either documentation of disease progression or death. We defined progression of disease as per RECIST 1.1 criteria²³. All patients will be observed for a minimum of 3 months. Both arms utilized the Simon’s two-stage design²⁴.

Based on a study totaling 154 patients with advanced gastric cancer previously treated with cisplatin/fluorouracil (eg. taxane-naïve), the median time to disease progression or death with docetaxel monotherapy is 2.6 months¹⁶. We therefore estimated the 3-month PFS rate for the taxane-naïve arm (ARM A) to be 40%, with the null hypothesis having a 3-month PFS rate of ≤ 25%. If 8 or more patients out of the first 25 evaluable patients were progression-free after 3 months of follow-up, ongoing accrual proceeded to the target sample size of 52 patients. The treatment would be declared effective and worthy of further testing if 17 or more patients were progression-free after 3 months of follow-up. For Arm B, null hypothesis of the 3-month PFS rate of patients pre-treated with taxanes to be 5%, and the 3-month PFS rate of interest to be 25%. If 2 or more patients out of the first 6 evaluable patients were progression-free after 3 months of follow-up, accrual would proceed to the target sample size of 23 patients. The treatment would be declared effective and worthy of further testing if 3 or more patients were progression-free after 3 months of follow-up. For both arms, this two-stage design yielded 80% power at 10% significance level.

Secondary endpoints:

Secondary endpoints include response rate (RR) and overall survival (OS). Median PFS and OS was estimated using Kaplan-Meier methodology. Greenwood’s formula was used to calculate 95% confidence intervals for the Kaplan-Meier estimates. Exploratory analyses of 3-month PFS and response rate, stratified by gastric cancer subtype (diffuse, non-diffuse proximal, non-diffuse distal)³, was also performed to identify the primary subtypes of interest for future study. Based on an estimated sample size of 25 patients in each disease subtype, 95% confidence intervals for the difference in 3-month PFS between the subtypes could be constructed to be within ±24% of the true difference in 3-month PFS between subtypes (assuming ≥15% 3-month PFS differences between subtypes).

The frequency of subjects experiencing toxicities was tabulated. Toxicities were assessed and graded according to CTCAE 4.0. Exact 95% confidence intervals around the toxicity proportions were calculated to assess the precision of the obtained estimates.

Correlative Studies

Tissue Acquisition and Processing

A fresh tissue biopsy of the tumor and an adjacent non-tumor sample was requested at baseline prior to study initiation from all participating sites. Matching tumor and adjacent non-tumor fresh biopsies were obtained at baseline from 66 patients (87%). The majority of the baseline tumor biopsies were of the primary tumor (n=63), whereas 1 patient had a liver biopsy and 2 patients had tissue collected from metastatic lymph nodes. We collected an on-treatment biopsy at week 4 (within 72 hours of the 2^nd cabazitaxel treatment) from patients enrolled at Weill Cornell from 21 patients (51% of patients enrolled at Weill Cornell). DNA and RNA was simultaneously extracted from tumor biopsies using the Qiagen Allprep DNA/RNAMicro kit (Cat no: 80284; Qiagen, Hilden, Germany) as previously described^{25, 26}. Briefly, each sample (approximately 3–5mg) was homogenized individually on ice for 30 seconds in RLT lysis buffer (Qiagen^@) using a pre-sterilized homogenizer (Pro250® Pro Scientific). The homogenized lysate was then loaded on Allprep DNA mini spin column (Qiagen®) and isolated by serial washes off the column. The initial flow through was used to extract RNA using the RNeasy MinElute spin column (Qiagen®). After subsequent washing steps with buffers RW1 (Qiagen®), RPE (Qiagen®) and 80% ethanol, RNA was eluted using 14 ul of RNase-free water provided in the kit.

Whole Exome Sequencing

Preprocessing:

Exome capture was performed using NimbleGen (Roche) SeqCap EZ library prep protocol (~62MB, WES) for both tumor and matching normal samples from 66 patients. Sequencing libraries were sequenced on HiSeq2500 as paired-end 100bp with average 78M reads per sample and median exon coverage of 78X. Quality control of tumor and adjacent non-tumor biopsies was performed using FastQC v0.11.5, FastQ Screen v0.11.1, RSeQC v3.0.0, MultiQC v1.6.

Alignment:

low quality reads were filtered using FilterByTile/BBMap v37.90 and aligned to human reference genome GRCh38 (GRCh38.d1.vd1 assembly) using BWA v0.7.17. Duplicate reads were removed using Picard’s v2.6.0 MarkDuplicates, indels were realigned by IndelRealigner and recalibrated by BaseRecalibrator (both of GATK v3.8.1).

Variant calling:

Both germline and somatic single nucleotide variations (sSNVs), small insertions and deletions were all detected using Strelka v2.9. All variants, insertions and deletions were annotated using Variant Effect Predictor v92.1. Tumor purity and ploidy were performed using FACETS v0.5.14. Copy number alterations were evaluated by customized version of Sequenza v2.1.2. Gene fusions were detected by STAR-fusion v1.5.0.

RNA-based support for somatic mutations identified in exome was evaluated by running Strelka v2.9 on RNA-Seq reads aligned to the aforementioned human genome by STAR v2.4.2 with similar duplicates’ marking, realignment and recalibration.

RNA sequencing data analysis

RNA-Seq reads were aligned using Kallisto v0.42.4 to GENCODE v23 transcripts 69. The protein coding transcripts, IGH/K/L and TCR related transcripts were retained and the non-coding RNA transcripts, histones and mitochondria related transcripts being removed resulting in 20,062 protein coding genes. Gene expressions were quantified as transcripts per million (TPM).

Immune Deconvolution:

We applied CIBERSORT to deconvolve the patient’s tumor samples into its constituent immune cell types using patient’s RNAseq expression profiles (quantified as FPKMs). CIBERSORT uses a reference expression matrix of 22 leucocytes (called LM22 signature) to determine the abundance of immune cell types in the tumor microenvironment. Tumor samples were segregated into those with high or low macrophage M2 levels using the cohort specific median macrophage M2 abundance as the threshold. Statistical significance was determined using the Mann-Whitney-Wilcoxon test in R. Fisher’s exact test was applied to test for significant associations between M2 macrophage levels and patient response (i.e., responders versus non-responders with progressive disease). Paired t-test was applied to test for statistical significance between baseline and their matched on-treatment samples. We examined 20 biopsy samples for validation of the M2 signature by immunohistochemistry using the following markers, CD68 (pan-macrophage marker), CD163 (M2 preference), and iNOS (M1 preference). We examined the presence of staining both within the tumor microenvironment and away from the tumor, counting the number of positive cells in 0.785mm². The pathologist was blinded to the deconvolution analysis prior to scoring the macrophages by IHC. Additional macrophage gene signature (MSR1, CSF1R, SIGLEC1, IL10, IL4I1, CD68, MRC1, CD163) was calculated by ssGSEA.

RESULTS

From January 8, 2013 through April 8, 2015, 76 evaluable patients were enrolled from 6 different institutions (sTable 1). Patient demographics are provided in Table 1. The median age of the total study population was 61.7 years (range 35.5–91.8 years), which was predominantly male (66%) and Caucasian (66%), and 96% were ECOG 0–1 performance status. There was an equal split of intestinal and diffuse histologies for the entire study population and per study arm. Overall, there were no significant differences in the study population between Arms A and B, in particular with regard to HER2 status, gender, and ethnic distribution (Table 1). Cohort B was however more heavily pretreated, as might be expected (Table 1, and sTable 2). Specifically, whereas 92% (49/53) of patients in cohort A had only one prior chemotherapy (most commonly a platinum doublet, sTable 2), 60% (14/23) of patients in cohort B had received 2 or more prior lines of chemotherapy (p<0.0001).

Table 1.

Patient Demographics.

	Total (N=76) (%)	Arm A (evaluable): Taxane Naïve (N=53)	Arm B (evaluable): Prior Taxane (N=23)	P-value
Age at Diagnosis:	61.67	62.1	57.34	0.151
Median (Range)	(35.46 – 91.76)	(35.46 – 91.76)	(36.89 – 73.45)	0.151
Gender:
Female	26 (34)	20 (38)	6 (26)
Male	50 (66)	33 (62)	17 (74)	0.432
Ethnicity:
Hispanic	9 (12)	5 (9)	4 (17)
Non-Hispanic	50 (66)	36 (68)	14 (61)	0.358
Declined	16 (21)	12 (23)	4 (17)
Unknown	1 (1)	0 (0)	1 (4)
Race
African American	1 (1)	1 (2)	0 (0)
Asian	8 (11)	7 (13)	1 (4)
Caucasian	50 (66)	36 (68)	14 (61)	0.185
Other	15 (20)	7 (13)	8 (35)
Declined	2 (3)	2 (4)	0 (0)
ECOG at Baseline:
0	16 (21)	11 (21)	5 (22)
1	57 (75)	39 (74)	18 (78)	1
2	2 (3)	2 (4)	0 (0)
Not Done	1 (1)	1 (2)	0 (0)
Histological Subtype:
Diffuse	33 (43)	23 (43)	10 (43)	0.932
Intestinal	36 (47)	25 (47)	11 (48)
Mixed	5 (7)	4 (8)	1 (4)
Unknown	2 (3)	1 (2)	1 (4)
Disease Location
Esophagus	14 (18)	12 (23)	2 (9)	0.871
GEJ	25 (33)	16 (30)	9 (39)
Gastric	37 (49)	25 (47)	12 (52)
HER2 Status*:
Negative	57 (75)	42 (79)	15 (65)	0.251
Positive	19 (25)	11 (21)	8 (35)
Prior Treatments
One Prior Rx	58 (76)	49 (92)	9 (39)	< 0.0001
Two or more Prior Rx	18 (24)	4 (8)	14 (61)^

^*HER2 status was based on local lab testing. Positive was considered IHC 3+ or IHC 2+ and FISH positive.

^{^}One patient had 3 prior therapies.

Safety and Toxicity

Table 2 provides the grade 2–4 toxicity that is possibly, probably, or definitely attributable to cabazitaxel (all reported toxicity regardless of attribution in sTable 3). Overall, cabazitaxel was well tolerated, with the main toxicity being myelosuppression in patients who were taxane naïve (Arm A) and who had received prior taxanes (Arm B). Although the rate of neutropenia was moderate (17% for taxane-naïve (Arm A), and 39% for prior taxane (Arm B)), the rate of febrile neutropenia was low in both study Arms, 4% in Arm A and 0% in Arm B. The only other grade 2–4 cabazitaxel associated toxicities that occurred in at least 5% of patients occurred in the taxane naïve (Arm A) study group, with fatigue/muscle weakness observed in 15% (G2 n=5, G3–4, n=3) patients, and hematuria observed in 11% (G2 n=2, G3–4 n=4) patients. None of these toxicities were dose limiting. As a toxicity of interest, grade 2 neuropathy was observed in 5 patients, all in Arm A, with no grade 3–4 neuropathy observed. There were 3 deaths on study, each unrelated to cabazitaxel therapy (sTable 4).

Table 2.

Grade 2–4 adverse events, possibly, probably, or definitely related to cabazitaxel.

	Taxane Naïve (n=53) Grade, Frequency			Prior Taxane (n=23) Grade, Frequency
	G2	G3–4	G3–4%	G2	G3–4	G3–4%
Hematologic Toxicity
Anemia	4	2	4%	4	0	0%
Leukopenia	1	7	13%	4	5	22%
Neutropenia	5	9	17%	2	9	39%
Febrile/Neutropenia	0	2	4%	0	0	0%
Lymphopenia	0	0	0	0	1	4%
Thrombocytopenia	3	0	0	1	1	4%
Non-Heme Toxicity
Pulmonary Embolism/Thrombus	0	1*	2%	0	1	4%
Hypotension	0	0	0	1	0	0%
Sepsis	0	1	2%	0	0	0%
Fall/Dehydration	0	1	2%	0	1	4%
Dizziness	0	0	0	1	0	0%
Nausea	2	0	0	2	0	0
Anorexia	1	0	0	0	0	0
Abdominal pain	0	0	0	1	1	4%
Diarrhea	0	1	2%	0	0	0
Colitis	1	0	0	0	0	0
Arthralgia/ Muscle pain	1	0	0	0	1	4%
Fatigue/ Muscle weakness	5	3	6%	5	0	0
Edema	1	0	0	0	0	0
Headache	1	0	0	0	0	0
Eye Dryness	1	0	0	0	0	0
Dyspnea/ Pneumonitis	0	2	4%	0	0	0
Increased Creatinine	0	2	4%	1	0	0
Hematuria	2	4	8%	2	0	0
Proteinuria	1	0	0	0	0	0
Dysuria	0	0	0	1	0	0
Hypernatremia	0	1	2%	0	0	0
Hyponatremia	0	0	0	0	1	4%
Neuropathy	5	0	0	0	0	0
Hemorrhage	2	0	0	0	0	0
Allergic reaction	0	0	0	0	1	4%
Infusion reaction	1	0	0	0	0	0

Efficacy

For each arm, the primary study endpoint is the rate of 3-month progression free survival. For patients in Arm A (Taxane-naïve cohort), the first stage of the Simon’s 2 stage design required 8 or more patients out of the first 25 evaluable patients to be progression-free at 3 months to proceed to the target sample size of 52 evaluable patients. This criterion was met, with 9 patients without disease progression at 3 months of follow up. The study continued to enroll 53 evaluable patients, with a final total of 15 patients (28%, 95%CI 17%−42%) who were progression free at 3 months. The Simon 2 stage criteria required 17 or greater patients to be progression-free at 3 months to achieve a target 40% progression free 3-month survival rate for this to be an encouraging outcome, and thus the stage 2 criteria for efficacy was not met.

For patients in Arm B (prior taxane treatment), the first stage of the Simon’s 2 stage design required 1 patient in the first 6 evaluable patients to be progression free at 3 months for the study to proceed to the 2^nd stage. In the first 6 evaluable patients, 3 were without progression and the study completed enrollment of 23 evaluable patients, with a final of 7 patients (30%, 95% CI 13%−53%) who were progression-free at 3 months, meeting our pre-specified efficacy target.

The response rate table for both Arm A (taxane naïve) and B (prior taxane therapy) is provided in sTable 4 and Figure 1, and the progression free and overall survival Kaplan Meier curve is provided in Figure 1. The overall response rate (ORR) for cabazitaxel in taxane treatment naïve patients (PR + CR) is 11.32% (95%CI 4%−23%), whereas the ORR for cabazitaxel in patients who have received prior taxane therapy is 13.0% (95%CI 3%−34%). One patient in Arm A achieved a complete radiographic response. The disease control rate (SD+PR+CR) in Arm A is 32% (95%CI 20%−46%), and the disease control rate for Arm B is 35% (16%−57%). Notably, both ORR and the disease control rate are numerically similar in the two arms. The progression free survival (PFS) curve demonstrates modest activity of cabazitaxel regardless of prior taxane use. The median PFS is 2.17 mo (95%CI 1.31–2.6 mo) for Arm A (taxane naïve), and 1.31 mo (95% CI 1.15 – 3.98 mo) for Arm B (prior taxane therapy). There was no difference in 3-month PFS according to disease subtypes (p=0.76). The median OS was 6.83 mo (95% CI 4.76–10.3 mo) for Arm A, and 5.98 mo (95%CI 2.43 – NR) for Arm B (Figure 1).

Figure 1.

Efficacy of Cabazitaxel in previously treated gastric and GEJ adenocarcinoma. Panel A: Progression free survival and overall response rate (partial response and complete response) with cabazitaxel in taxane-naïve (arm A) and taxane-pretreated (arm B) patients. Panel B: Overall survival.

Correlative studies

We have previously demonstrated that microtubule bundling identified on on-treatment biopsies is associated with response to therapy²⁷. The underlying purpose of the correlative studies in this report was to identify any potential association of genetic aberrations or tumor microenvironment signatures with cabazitaxel activity. We examined baseline tumor tissue for genomic alterations associated with taxane efficacy. Given that the efficacy of cabazitaxel was similar regardless of prior taxane administration, arms A and B were collapsed together for the correlative analyses.

Genomic Alterations and Cabazitaxel Efficacy

Whole exome sequencing of tumor biopsies (mean coverage 83x) and matched adjacent non-tumor tissues (mean coverage 64x) identified various somatic DNA alterations, including SNVs (single nucleotide variants), small indels and copy number alterations (Figure 2). Tumor cellularity fraction (purity) ranged significantly from 18 to 86 percentages according to tumor purity estimation from WES, and was categorized into high and low tumor purity using a 45% cutoff. The majority of somatic alterations were missense mutations and chromosomal rearrangements. Tumors had very complex karyotypes with multiple amplifications and deletions. Recurrent somatic mutations were observed in TP53 (26/47 cases). Clinically actionable alterations included mutations in BRAF V600E (sample 100_36), amplifications in EGFR (10/47) and amplifications in ERBBR2 (8/47) genes. Other alterations in RTK signaling pathway included AXL gene fusions, mutations in MET, ALK and FLT3 genes. One patient had a KRAS Q61H mutation (sample 100_41) which would predict for resistance to a broad spectrum of receptor tyrosine kinase inhibitors. Several patients also had deletions of MTOR and STK11 suppressor genes, amplifications and mutations in PI3CA associated with PI3K/mTOR signaling pathway (Figure 2A).

Figure 2.

Landscape of somatic mutations identified from whole exome sequencing for patients receiving cabazitaxel and Impact of HER2 status on patient outcomes. Panel A) Alterations plot of integrated genomic data for pre-treatment samples from patients including somatic mutations, high amplifications (>=2), deep deletions and fusions. Each column represents one sample and each line corresponds to one gene. Genes were grouped into major signaling pathways. Top panel represents sample annotation: treatment arm, purity group and RECIST status. Alternative treatments options potentially associated with genetic alterations are mentioned on the right (sensitive - blue and resistant – red). Panel B) Left: Comparison ERBB2 expression level between HER-2 negative (red) and HER-2 positive (green) samples. HER2 status was defined by FISH or IHC. Right: Percentage of samples with experimental HER-2 status (positive or negative) in non-responders and responders groups. Panel C) Time to treatment failure analysis between HER-2 positive and negative samples. Panel D) Survival analysis between HER-2 positive and negative samples.

No correlation was observed between mutation status and the treatment arm. Tumor mutation burden was also not associated with response to therapy (sFigure 2). We investigated the correlation between genetic alterations and response (PR or SD) and found that ERBB2 amplification was significantly more prevalent in responders to cabazitaxel (p=0.003)(Figure 2B). Key genes in the ERBB2/RAS/MAPK pathway demonstrate enrichment of alterations in cabazitaxel responding patients (sFigure 3), further highlighting the significance of this signaling pathway in this cohort of patients. Patients with HER2 positive status (according to both IHC/FISH as assessed on the diagnostic sample) had significantly higher ERBB2 expression by RNASeq analysis in comparison with HER-2 negative samples. Furthermore, we observed a significant enrichment of HER2 positive samples in combined group of patients with disease control (SD and PR) in comparison with non-responders (Figure 2B). In addition, patients with HER2 positive tumors had better progression free (p=0.04) and overall survival (p=0.002) (Figure 2C and 2D).

Tumor Immune Microenvironment and Cabazitaxel Efficacy

Deconvolution analysis was available in 54 patients in both groups (18 patients with a PR or SD, and 36 patients with disease progression as their best response), and revealed an enrichment of an M2 macrophage signature in a cohort of patients that was not associated with other immune infiltration (sFigure 1). We confirmed M2 tumor associated macrophage enrichment by immunohistochemistry, with more than 65% of tumors (of a cohort of 26 patients) demonstrating enrichment of M2 (CD68+, CD163+ staining macrophages) within the tumor versus the periphery. The M2 signature score above the median was associated with improved disease control (PR + SD) compared to M2 score below the median (Figure 3C, p= 0.042). Tumors with high M2 signature also had an improved progression free survival (Figure 3D, p=0.031). Ten of these patients had matching on-treatment biopsies, and in 8 out of 10 matching biopsies, the M2 signature declined (sFigure 4). Using bulk RNA sequencing, we also examined the correlation of the M2 macrophage gene signature after accounting for the relative value of macrophages in each sample, and found a similar association with improved survival (Figure 4, p = 0.009). Her2 status and macrophage M2 signature both showed significant association with patient survival by cox-regression analysis (p<0.005).

Figure 3.

Macrophage characteristics and association with patient outcomes when treated with cabazitaxel. The photomicrographs of gastric adenocarcinoma presented in Panel A demonstrate the variability of the overall macrophage (CD68+ cells) and M2-specific macrophage (CD163+ cells) infiltrate within tumor stroma from different patients. In general, there appears to be enrichment of an M2 macrophage signature within the tumor microenvironment, which is quantified in panel B, where over 65% of samples had evidence of an M2 tumor associated macrophage signature. M2 enrichment in the tumor stroma appears to be associated with improved progression free survival. Panel C provides the proportion and distribution of M2 immune signature when separating high and low M2 macrophages by the median. Panel D provides the estimated progression free survival probability according to M2 high and low, (p=0.031).

Figure 4. Bulk RNA Seq M2 signature and association with HER2.

In panel A, we correlate the M2 macrophage signature using bulk RNA sequencing after accounting for the macrophage infiltrate, and still find a significant association with patient survival. Panel B identifies no correlation between the M2 signature and HER2 expression, suggesting that these are two independent factors associated with outcome in patients treated with cabazitaxel.

Discussion

We completed a multicenter phase II study of cabazitaxel in the 2^nd or 3^rd line treatment setting for patients with advanced unresectable, or metastatic gastric and gastroesophageal junction adenocarcinoma. The study included patients who had received prior taxane therapy as well as taxane treatment naïve patients. We demonstrate that cabazitaxel can be administered safely in this patient population, without significant toxicity. We reduced the cabazitaxel dose from the standard dose at the time (25 mg/m²) down to 20 mg/m², primarily due to increased neutropenia observed in 4 of the first 5 patients enrolled. Since that time, cabazitaxel 20 mg/m² was proven to be non-inferior compared with cabazitaxel 25 mg/m² in prostate cancer, and with significantly less neutropenia (grade 3–4 neutropenia rate 41.8% at 20mg/m² compared with 73.3% at cabazitaxel 25 mg/m²).²⁸ Cabazitaxel single agent therapy was associated with modest antitumor activity, regardless of the previous use of taxanes. The modest antitumor activity in taxane naïve patients did not meet the pre-specified efficacy threshold, whereas in the prior taxane treated group, there was encouraging activity with a 3-month progression free survival rate of 30% and a disease control rate of 35%, meeting the pre-specified efficacy cutoff. This activity is greater than would be expected in this setting, especially in a more heavily pre-treated patient population, and would suggest further drug development of cabazitaxel in a taxane refractory patient population.

This study was initiated prior to the approval of ramucirumab, which is now a standard option in the second line setting, either alone²⁹, or with paclitaxel¹⁵. The Rainbow study compared the efficacy of paclitaxel and ramucirumab to paclitaxel alone in patients who received a platinum/fluoropyrimidine first line combination, with or without an anthracycline¹⁵, and specifically excluded patients who received prior taxane therapy in the first line setting. The median progression free survival for the paclitaxel alone arm was 2.9 months (95%CI 2.8–3.0 months), which appears to be at least equivalent and possibly better than the efficacy of cabazitaxel in Arm A (taxane-naïve) of this study in which cabazitaxel demonstrated median PFS of 2.4 months (95%CI 1.31–2.6 months). Alternatively, there are no available data of the efficacy of paclitaxel and ramucirumab in patients who received prior first line taxane therapy. However, in the Couger-02 study, the response rate to docetaxel in the 2^nd line setting was 7% and the median time to disease progression was 2.6 months, which compares favorably to cabazitaxel in the taxane-prior treatment cohort¹⁸. In a more comparable 3^rd line treatment setting, trifluridine/tipiracil is associated with a response rate of 4% and irinotecan 6.8%, which also compares favorably to the efficacy of cabazitaxel in this treatment setting^{30, 31}. Given that cohort B is significantly more heavily pre-treated, it is perhaps even more compelling that cabazitaxel had some efficacy in this cohort, demonstrating a disease control rate of 35%, and notably similar to the activity in taxane naïve patients.

There have been several reports evaluating prognostic factors associated with improved outcome in the second line setting^32–34. The Regard and Rainbow investigators pooled the trial data together to examine for prognostic factors associated with efficacy in 2^nd line therapy³³, and found several clinical factors associated with a worse survival (eg. presence of primary tumor, poor/unknown tumor differentiation, ECOG 1, peritoneal metastases). These studies did not report on the association of HER2 status and taxane sensitivity³³. In a separate large retrospective analysis of markers associated with patient outcome in the 2^nd line setting, HER2 status was not found to be prognostic in a more heterogeneous patient population³². There have been very few reports of specific tumor-intrinsic predictive factors for taxane-based therapy. We identified a very diverse mutational landscape in our patient population, with prevalent mutations in TP53, RHOA, and RTK/RAS signaling, consistent with previous reports³⁵. We identified HER2 amplification/over expression as significantly associated with improved survival in this patient population. Previous retrospective reports in breast cancer suggested that HER2 positive breast cancer may also be associated a better response to taxanes^36–38. However, there were no reports of the impact of HER2 status in breast cancer patients treated with taxane monotherapy. In the context of taxane and anthracycline based therapy, response was associated with the co-expression of topoisomerase II alpha³⁶. HER2 positive breast cancer is also associated with reduced expression of the microtubule associated protein, Tau, which may also be associated with taxane efficacy in the context of ER positive disease^{36, 39}. No data evaluating the sensitivity of HER2 positive gastric cancer to taxanes in gastric cancer could be identified, and as such, this novel finding should be further explored.

We also identified a subset of gastric cancers that were enriched in an M2-like tumor associated macrophage signature that was associated with improved survival, independent of HER2 signaling. An M2-like macrophage signature has been reported in gastric cancer^{40, 41}, and co-culturing of undifferentiated macrophages with gastric cancer cell lines can induce M2 macrophage polarization⁴⁰. Detailed spatial analysis identifies that CD68+CD163+ macrophages was the M2 subtype that was in closest proximity to gastric tumor cells⁴¹. This is consistent with our finding that the dominant M2 subtype in our cohort was CD68+, CD163+, and that we specifically had very few CD206+ M2 macrophages.

Tumor associated macrophages are a diverse population of cells with important clinical implications with regard to tumor aggressiveness and immunosurveillance⁴². M2-like macrophage tumor infiltration has been associated with poor prognosis in several cancers, including glioma⁴³, renal cell carcinoma⁴⁴, and cholangiocarcinoma⁴⁵. In gastric cancer, the presence of M2-like macrophages is associated with increasing stage and resultant worse prognosis⁴⁰. Further, in tumor xenograft models, the presence of M2 macrophages was associated with a more aggressive cancer phenotype and worse survival⁴⁰. However, our data suggest that the M2 enriched cohort has improved survival in the context of cabazitaxel treatment. Notably, taxane treatment at low doses has been demonstrated to reduce the M2-like differentiation of macrophages via the Toll-like receptor 4 (TLR4)⁴⁶. Further, taxane therapy was associated with reprogramming tumor-associated M2-like macrophages to an M1 (proinflammatory, antitumor) profile in preclinical models⁴⁷. Consistent with these preclinical studies, we found that the M2-tumor associated macrophage signature in our biopsy samples was significantly diminished in cabazitaxel treated patients with matched baseline and on-treatment biopsies. It is also notable that tumor associated macrophages may facilitate resistance to anti-VEGF therapy⁴⁸. This may explain the synergy between paclitaxel and ramucirumab in the 2^nd line treatment for advanced gastric cancer. Taken together, our data suggest the enriched M2 tumor-associated macrophage signature warrants further evaluation as a novel predictive marker for taxane sensitivity in this patient population.

Mechanisms of resistance to cytotoxic therapy are multifactorial and are likely to include both tumor intrinsic genomic alterations and aspects of the tumor microenvironment. We have previously demonstrated that microtubule engagement within the tumor is an important prerequisite for taxane efficacy²⁷. Here, our data suggests that both HER2 amplification/over-expression and enrichment of M2 macrophages in the tumor immune microenvironment may also play a role in taxane sensitivity. These analyses are limited by the small sample size, but supported by the orthogonal analyses performed. The interaction between molecular alterations in the tumor and the microenvironment remains an area of active investigation. Cabazitaxel has modest activity in the 2^nd line therapy in gastric cancer. It is noteworthy that there appears to be activity in patients previously treated with taxanes, particularly considering that this group was more heavily pretreated. This finding warrants further evaluation given the limited sample size. Further studies on the role of the M2-like tumor associated macrophage signature are also underway.

Translational Relevance:

Our study demonstrates that cabazitaxel, a novel 3^rd generation taxane, has modest anti-tumor activity in both taxane-naïve and taxane-pre-treated patients with advanced gastroesophageal cancer in the 2^nd/3^rd line treatment setting. A comprehensive evaluation of the genomic alterations of the tumor and the tumor microenvironment was undertaken to identify tissue specific characteristics associated with cabazitaxel efficacy. Two independent potential biomarkers for cabazitaxel efficacy in advanced gastroesophageal cancer were identified. We identified HER2 amplification/over-expression and an M2 tumor associated macrophage signature as potential biomarkers associated with improved survival in patients with metastatic gastric cancer treated with cabazitaxel.