IGF-1R associates with adverse outcomes after radical radiotherapy for prostate cancer.

BACKGROUND: Activated type 1 insulin-like growth factor receptors (IGF-1Rs) undergo internalisation and nuclear translocation, promoting cell survival. We previously reported that IGF-1R inhibition delays DNA damage repair, sensitising prostate cancer cells to ionising radiation. Here we tested the clinical relevance of these findings.
METHODS: We assessed associations between IGF-1R and clinical outcomes by immunohistochemistry in diagnostic biopsies of 136 men treated with 55-70 Gy external beam radiotherapy for prostate cancer, comparing results with publicly available transcriptional data in surgically treated patients.
RESULTS: Following radiotherapy, overall recurrence-free survival was shorter in patients whose tumours contained high total, cytoplasmic and internalised (nuclear/cytoplasmic) IGF-1R. High total IGF-1R associated with high primary Gleason grade and risk of metastasis, and cytoplasmic and internalised IGF-1R with biochemical recurrence, which includes patients experiencing local recurrence within the radiation field indicating radioresistance. In multivariate analysis, cytoplasmic, internalised and total IGF-1R were independently associated with risk of overall recurrence, and cytoplasmic IGF-1R was an independent predictor of biochemical recurrence post radiotherapy. Insulin-like growth factor receptors expression did not associate with biochemical recurrence after radical prostatectomy.
CONCLUSIONS: These data reveal increased risk of post-radiotherapy recurrence in men whose prostate cancers contain high levels of total or cytoplasmic IGF-1R.

Insulin-like growth factors (IGFs) bind to type 1 IGF receptors (IGF-1Rs) on the surface of most cells, activating signalling effectors, including AKT, to promote normal embryonic and post-natal development (Baker ). In tumours, IGF-1R signalling mediates proliferation, invasion and cell survival via protection from apoptosis (Chitnis ). Activated IGF-1Rs undergo internalisation, and are then degraded or recycled back to the plasma membrane (Goh and Sorkin, 2013). We and others reported that some activated internalised IGF-1Rs translocate to the nucleus of human tumour cells, influencing gene expression (Aleksic ; Sehat ; Sarfstein ; Warsito ).

The IGF axis is recognised as mediating resistance to anticancer therapies (King ). The first report linking IGF-1R with radioresistance came from a study of breast cancer patients, where IGF-1R overexpression was associated with early recurrence within the irradiated site (Turner ). High IGF-1R also associates with radioresistance in cervical cancer (Lloret ), and we reported that IGF-1R is overexpressed in HPV-negative head and neck cancers that are characterised by resistance to chemo-radiation (Dale ). Preclinical data support a link with radioresistance: we and others showed that IGF-1R targeting enhances chemo- and radiosensitivity in a range of tumour models in vitro and in vivo, independent of apoptosis induction (Macaulay ; Tezuka ; Cosaceanu ; Isebaert ; Riesterer ). Subsequently, we reported that IGF-1R depletion or inhibition enhances the sensitivity of prostate cancer cells to ionising radiation (IR). This effect was associated with delayed resolution of IR-induced DNA double-strand breaks (DSBs) and inhibition of DSB repair by both non-homologous end-joining and homologous recombination (Turney ; Chitnis ). The aim of the current study was to investigate whether these findings have clinical relevance, by assessing IGF-1R expression in prostate cancers of men treated with radical radiotherapy. Our major findings are first that patients whose tumours express high total IGF-1R experience adverse outcomes, including increased risk of overall and metastatic relapse. Second, high internalised IGF-1R associates significantly with increased risk of biochemical relapse. This group includes patients experiencing recurrence within the irradiated field, supporting a link with clinical radioresistance.

Materials and methods

Clinical cases

Clinical data were extracted from records of patients treated with external beam radiotherapy in Oxford from 2000 to 2005, including age, date of diagnosis, presenting PSA, clinical tumour (cT) stage, Gleason grade and Gleason Sum score. Using follow-up data, including serial PSA monitoring, isotope bone scans, CT scans, MRI and PET-CT scans, patients were assigned to one of three groups: long-term remission, metastatic relapse, or biochemical relapse. The latter was defined using the ASTRO-Phoenix Consensus criteria (Roach ) as +2 ng ml−1 PSA rise above the post-radiotherapy nadir, without evidence of metastatic disease. This study was approved by the National Research Ethics Service (07/H0606/120).

Type 1 IGF receptor immunohistochemistry

Archival diagnostic formalin-fixed paraffin-embedded (FFPE) prostate biopsies were selected for this study. Routinely, left- and right-sided biopsies had been embedded in separate blocks, ⩽6 biopsies per block. For patients with unilateral tumour, the ipsilateral (i.e., cancer-containing) block was selected for study. For cases with bilateral tumour, the block selected was the most representative of Gleason grade. The chosen tumour-containing blocks were used for IGF-1R immunohistochemistry (IHC) as described (Aleksic ). For details of method and scoring see Supplementary Information. In brief, IGF-1R signal in ⩽6 biopsies per case was scored blinded by uro-pathologist CV for intensity (0–3) and percentage positivity (0–4) in the malignant prostatic epithelium. This generated immunoreactive scores (intensity × percentage, range 0–12) for membrane, cytoplasmic and nuclear IGF-1R. Internalised IGF-1R (0–24) was represented by the sum of cytoplasmic and nuclear IGF-1R, and total IGF-1R (0–36) as the sum of membrane, cytoplasmic and nuclear IGF-1R. Where present, benign prostatic epithelium was also scored for IGF-1R content.

Statistical analysis

Clinical data were analysed using t-tests and Wilcoxon matched-pairs signed rank test for non-parametric data. The IGF-1R IHC scores were binarised by the median score and analysed as categorical variables by χ2-test. Overall survival (OS) was defined as the follow-up time from radiotherapy until death. Patients not reported as dead were censored at their last known date alive. Recurrence-free survival (RFS) was defined as time from radiotherapy until any recurrence (biochemical or metastatic). Recurrence-free survival data are displayed using Kaplan–Meier curves, with the associated log-rank (Mantel–Cox) test. Cox proportional hazards models were fitted for IGF-1R for RFS and OS in both univariate and multivariate analyses. IGF1R expression data were extracted from (Taylor ) and analysed for association with risk of biochemical recurrence by Wald test. Statistical analysis was performed using GraphPad Prism v6, STATA 11.2 (Stata Corporation, College Station, TX, USA) and R Statistical programming environment (v3.2.4, R package: survival v2.38-3. R Core Team: Vienna, Austria). All statistical tests were two-sided, and P<0.05 was considered significant.

Results

From a database of ∼800 prostate cancer patients treated with radical radiotherapy from 2000 to 2005, we identified 136 with available FFPE tissue (Table 1), and in whom we could ascertain outcomes of remission (75 cases), metastatic recurrence (17) or biochemical recurrence (44) from imaging and serial PSAs. External beam radiotherapy had been 3D conformal CT planned, and treatment typically involved delivering a 55 Gy dose to the planning target volume in 20 fractions over 4 weeks (Table 1). Assuming an α/β ratio for prostate cancer of 1.8 Gy (Dearnaley ), this dose/fractionation schedule is equivalent to 65.9 Gy in 2 Gy fractions. Radiotherapy was administered to all other patients in fractions of 2 Gy. Of the 136 patients, 57 (42%) had also received adjuvant endocrine therapy, generally for 6–48 months (Table 1). Median follow-up was 7.86 years (range 0.46–12.68 years). To assess the extent to which this cohort is representative, we assessed associations of adverse outcome with the principal prognostic factors: primary Gleason grade, stage, and presenting PSA (Heidenreich ). Univariate analysis confirmed significant associations between risk of overall recurrence and Gleason grade (primary grade 4 vs 3), stage (1 vs 2 or 3) and PSA 10–20 vs <10, although increased risk in patients with PSA ⩾20 did not reach significance (Supplementary Table S1). Adjuvant endocrine therapy has been shown to improve disease-free survival and OS following radiotherapy for localised prostate cancer (Kumar ; Bolla ), but was associated with increased risk of overall recurrence in our series (Supplementary Figure S1A). This likely reflects the practice during 2000–2005 of offering endocrine therapy only to high-risk patients. Indeed, those receiving endocrine therapy had higher-grade tumours (median primary Gleason grade 4 vs 3) and a lower proportion of stage cT1 tumours (15 out of 57, 26% vs 30 out of 79, 38%) compared with patients not offered endocrine therapy.

Table 1

Demographic data

Median (range)	Patients, N (%)
Age
69 (55–79)	136
PSA
13.0 (1.3–414)
Primary Gleason grade
3	74
4	61
5	1
cT stage
cT1	45
cT2	58
cT3a	28
Radiotherapy
55 Gy in 20 fractions	120
64 Gy in 32 fractions	6
68 Gy in 34 fractions	1
70 Gy in 35 fractions	9
Adjuvant endocrine therapy
Goserelin plus bicalutamide	57
Outcome
Remission	75
Relapse	61
Metastatic recurrence	17
Biochemical recurrence	44
Death	26
Cancer IGF-1R
Positive	109 (80.1)
Membrane	77 (56.6)
Cytoplasm	86 (63.2)
Nuclear	37 (27.2)
Internalised	96 (70.6)
Membrane only	13 (9.6)
Negative	27 (19.9)

Abbreviations: cT stage=clinical tumour stage at presentation; IGF-1R=type 1 insulin-like growth factor receptor; PSA=prostate-specific antigen.

Demographic data of patients treated with radical radiotherapy for prostate cancer. Adjuvant endocrine therapy was administered concurrently and for median 6 months post radiotherapy; only 4 patients had <6 months endocrine therapy (range 2–48 months). Table also shows number (%) of tumours containing detectable IGF-1R overall and in each subcellular compartment.

We then evaluated the IGF-1R content of the prostate biopsies, detecting IGF-1R in 109 out of 136 (80.1%) of the cancers with variable subcellular localisation in the plasma membrane, cytoplasm and nuclei (Table 2; Figure 1A). All but 8 biopsies contained benign prostatic epithelium, of which 90 out of 128 (70.3%) contained detectable IGF-1R. Compared with benign glands from the same biopsies, IGF-1R expression was higher in the malignant prostatic epithelium (P<0.001; Figure 1B), confirming our previous report of IGF-1R overexpression in primary prostate cancer (Hellawell ). The expression of IGF-1R did not associate with cT stage or presenting PSA, but higher-grade tumours (primary Gleason grade 4–5) contained significantly more IGF-1R than lower-grade tumours (primary Gleason grade 3, P=0.004; Table 2).

Table 2

Association of IGF-1R with clinical factors

	Total IGF-1R
	IGF-1R⩽8	IGF-1R>8	P-value
Clinical stage
cT1	31	14	0.260
cT2	37	21
cT3	14	14
Primary Gleason grade
Gleason 3	53	19	0.004
Gleason ⩾4	31	32
PSA
0–10	32	14	0.289
10–20	28	19
>20	19	17

Abbreviations: cT stage=clinical tumour stage at presentation; IGF-1R=type 1 insulin-like growth factor receptor; PSA=prostate-specific antigen.

χ2-test was used to test for differences in stage, Gleason score and PSA between high and low IGF-1R groups. There was significant association between total IGF-1R and Gleason grade, with higher-grade tumours (primary Gleason grade ⩾4) containing more IGF-1R than tumours with primary Gleason grade 3. There were no significant associations between cT stage or Gleason grade and IGF-1R scores in the plasma membrane, cytoplasm or nucleus, or with internalised (cytoplasmic plus nuclear) IGF-1R.

Figure 1

Type 1 IGF receptor associates with adverse outcomes in patients treated with radical radiotherapy for prostate cancer. (A) Examples of IGF-1R IHC in diagnostic biopsies of prostate cancer from patients treated with radiotherapy. (a) Gleason 3+4=7 cancer, IGF-1R score membrane 0, cytoplasm 12, nuclear 9; (b) Gleason 4+3=7 cancer, IGF-1R score membrane 6, cytoplasm 3, nuclear 0; (c) Gleason 3+3=6 cancer containing no detectable IGF-1R; (d) Gleason 4+5=9 cancer, IGF-1R score membrane 0, cytoplasm 12, nuclear 0. Scale bar, 20 μm. (B) Total IGF-1R score (bars, mean±s.e.m.) in benign and malignant areas (***P=0.001 by Wilcoxon matched-pairs signed rank test). (C–E) Kaplan–Meier plots with log-rank (Mantel–Cox) tests to assess association of total, cytoplasmic (Cyto) and internalised (Intern) IGF-1R with (C) all recurrences, (D) metastatic recurrence and (E) biochemical recurrence.

Using Kaplan–Meier and univariate analyses, we tested for associations between clinical outcomes and IGF-1R expression and subcellular localisation. The risk of overall recurrence was significantly greater in men whose prostate cancers contained high total IGF-1R. There were also significant associations between overall recurrence and cytoplasmic or internalised (nuclear plus cytoplasmic) IGF-1R (Table 3; Figure 1C). Analysis of membrane and nuclear IGF-1R separately showed no significant association with outcome (Table 3). Of the 136 patients, 17 (12.5%) developed scan-confirmed metastatic disease, which associated significantly with high total IGF-1R (Table 3; Figure 1D). We also assessed associations with OS, as 26 out of 136 (19%) of the cohort had died, finding borderline association between risk of death and high total IGF-1R (P=0.059; Table 3). Neither cytoplasmic nor internalised IGF-1Rs were associated with metastatic disease or OS (Table 3; Supplementary Figure 1B).

Table 3

Univariate analysis

RFS: overall recurrence	Comparison	Relapse/total	HR	95% CI	P-value
Membrane IGF-1R	⩽1	27/66	Baseline		0.291
	>1	34/70	1.31	0.79–2.18
Cytoplasmic IGF-1R	⩽4	33/89	Baseline		0.001
	>4	28/47	2.32	1.39–3.87
Internalised IGF-1R	⩽6	35/89	Baseline		0.010
	>6	26/47	1.97	1.17–3.29
Nuclear IGF-1R	0	42/99	Baseline		0.625
	⩾1	19/37	1.14	0.67–1.97
Total IGF-1R	⩽8	32/85	Baseline		0.007
	>8	29/51	2.02	1.22–3.36
RFS: biochemical recurrence
Cytoplasmic IGF-1R	⩽4	23/89	Baseline		0.002
	>4	21/47	2.62	1.44–4.79
Internalised IGF-1R	⩽6	26/89	Baseline		0.034
	>6	18/47	1.94	1.05–3.58
Total IGF-1R	⩽8	25/85	Baseline		0.059
	>8	19/51	1.79	0.98–3.27
RFS: metastatic recurrence
Cytoplasmic IGF-1R	⩽4	10/89	Baseline		0.284
	>4	7/47	1.70	0.64–4.48
Internalised IGF-1R	⩽6	9/89	Baseline		0.147
	>6	8/47	2.03	0.78–5.26
Total IGF-1R	⩽8	7/85	Baseline		0.040
	>8	10/51	2.76	1.05–7.26
Overall survival	Comparison	Death/total	HR	95% CI	P-value
Cytoplasmic IGF-1R	⩽4	18/89	Baseline		0.827
	>4	8/47	0.91	0.39–2.11
Internalised IGF-1R	⩽6	16/89	Baseline		0.448
	>6	10/47	1.36	0.61–3.04
Total IGF-1R	⩽8	12/85	Baseline		0.059
	>8	14/51	2.12	0.97–4.61

Abbreviations: CI=confidence interval; HR=hazard ratio; IGF-1R=type 1 insulin-like growth factor receptor; RFS=recurrence-free survival.

Univariate analysis of associations between IGF-1R and RFS and overall survival post-radiotherapy. Table shows IGF-1R immunoreactive scores, number experiencing recurrence or death/total number, HR for recurrence with 95% CI.

Biochemical recurrence was experienced by 44 (32%) patients, comparable with reported biochemical failure rates of 26–40% for low/intermediate risk disease and up to 70% for high-risk disease following radical external beam radiotherapy (Grossfeld ; Morgan ; Gabriele ; Zargar ). Biochemical recurrence showed borderline association with total IGF-1R (P=0.059 by univariate analysis, Table 3; P=0.055 by Kaplan–Meier analysis, Supplementary Figure S1C). Analysing IGF-1R scores by subcellular compartment (Table 3; Figure 1E), we identified significant associations between biochemical recurrence and IGF-1R in the cytoplasmic (P=0.002 by univariate analysis and 0.001 by Kaplan–Meier) or internalised (nuclear plus cytoplasmic; P=0.034 and 0.031, respectively) compartments. Patients experiencing biochemical recurrence include those with occult metastases and patients experiencing local recurrence within the radiation field. It is not possible to differentiate these two outcomes with certainty, because routine prostate biopsies are not performed in this situation (Morgan ), and none of these patients underwent salvage prostatectomy. However, it is estimated that ⩾30% of post-irradiation biochemical recurrences are due to local recurrence, which is an indicator of clinical radioresistance (Bolla ). In an attempt to further test associations of IGF-1R with local recurrence, we repeated the Kaplan–Meier analyses after excluding patients (n=5) who experienced scan-confirmed metastatic recurrence within 2 years, and were therefore likely to have had occult metastases at the time of radiotherapy. This had no effect on the significance of associations identified between risk of biochemical recurrence and high cytoplasmic (P=0.001, unchanged), internalised (P=0.030 with 5 cases excluded and P=0.031 for all 136 cases) or total IGF-1R (P=0.055, unchanged; Supplementary Figure 1D).

Using multivariate analysis, tumour stage was independently associated with overall and biochemical recurrence (Table 4), as is predictable (Heidenreich ), although this effect was apparent only when comparing clinical stage cT2 (not cT3) with cT1. Importantly, cytoplasmic, internalised and total IGF-1Rs were independent predictors of overall recurrence. Furthermore, cytoplasmic IGF-1R and stage (cT1 vs 2) were the only factors independently associated with the risk of biochemical recurrence (Table 4; Supplementary Table S2). In this cohort, primary Gleason grade (3 vs ⩾4) was not independently associated with overall or biochemical recurrence, but was the only factor independently predictive of metastatic recurrence (Table 4; Supplementary Table S2).

Table 4

Multivariate analysis

	Comparison	HR	95% CI	P-value
RFS: all recurrence
Cytoplasmic IGF-1R	⩽4	Baseline		0.007
	>4	2.05	1.21–3.45
Stage	cT1	Baseline
	cT2	2.61	1.33–5.11	0.005
	cT3	1.84	0.81–4.19	0.145
Primary Gleason grade	3	Baseline		0.108
	⩾4	1.56	0.91–2.68
RFS: all recurrence
Internalised IGF-1R	⩽6			0.036
	>6	1.75	1.04–2.97
Stage	cT1	Baseline
	cT2	2.76	1.40–5.42	0.003
	cT3	1.90	0.72–4.34	0.130
Primary Gleason grade	3	Baseline		0.155
	⩾4	1.49	0.86–2.56
RFS: all recurrence
Total IGF-1R	⩽8	Baseline		0.037
	>8	1.77	1.04–3.02
Stage	cT1	Baseline
	cT2	2.88	1.47–5.65	0.002
	cT3	1.87	0.82–4.26	0.136
Primary Gleason grade	3	Baseline		0.315
	⩾4	1.33	0.76–2.34
RFS: biochemical recurrence
Cytoplasmic IGF-1R	⩽4	Baseline		0.009
	>4	2.29	1.23–4.26
Stage	cT1	Baseline
	cT2	2.62	1.19–5.74	0.016
	cT3	2.01	0.76–5.32	0.160
Primary Gleason grade	3	Baseline
	⩾4	1.12	0.59–2.13	0.729

Abbreviations: CI=confidence interval; HR=hazard ratio; IGF-1R=type 1 insulin-like growth factor receptor; RFS=recurrence-free survival.

Multivariate analysis to identify factors showing independent association with clinical outcomes post radiotherapy.

Finally, we utilised publically available data to test for associations between IGF-1R and outcome in men with prostate cancer treated by radical prostatectomy. Excluding 37 patients who had received previous radiotherapy, the cohort of Taylor ) included 103 patients with microarray data on IGF1R expression, and clinical data on biochemical recurrence. In this surgical data set there was no significant association between IGF1R expression and biochemical recurrence (Supplementary Figure S1E). However, as noted previously, we found only borderline association of biochemical recurrence and total IGF-1R in the radiotherapy cohort (Table 3; Supplementary Figure S1C), and we are comparing semi-quantitative IHC scores with microarray analysis of IGF1R mRNA. Therefore, it will be important to assess the prognostic significance of IGF-1R at the protein level in a surgically treated cohort, to enable a more robust comparison.

Discussion

The major findings of our study are the identification of significant associations between total IGF-1R and risk of overall and metastatic recurrence, and between cytoplasmic or internalised IGF-1R and biochemical recurrence, following radical radiotherapy but not surgery for prostate cancer. These associations were revealed by univariate and Kaplan–Meier analyses; multivariate analysis also indicated that cytoplasmic IGF-1R is an independent predictor of biochemical recurrence. Most of the patients in our series were treated pre-2006 and received radiotherapy at 55 Gy in 20 fractions, equivalent to 65.9 Gy in 2 Gy fractions. Although this seems a modest dose by current standards, this dose/fractionation schedule was widely used in the United Kingdom during the era in which these patients were treated. It remains to be seen whether IGF-1R retains predictive significance in patients treated at 74 Gy in 37 fractions as is current practice, or the biologically equivalent hypo-fractionated dose of 60 Gy in 3 Gy fractions (Dearnaley ). Forty-two percent of patients in our series had received adjuvant endocrine therapy. This is relevant, given known crosstalk between IGF-1R, androgen receptor signalling and the DNA damage response, and reports that anti-androgen therapy enhances prostate cancer radiosensitivity in preclinical models and clinically (Pandini ; Kumar ; Bolla ; Goodwin ; Polkinghorn and Zelefsky, 2013; Zelefsky ). In the current study, we found no evidence of better outcomes in the group receiving endocrine therapy, likely because this had been offered only to high-risk patients. It is possible that the use of adjuvant endocrine therapy in a subset of patients could have influenced the associations we found between IGF-1R and clinical outcomes.

In probing the relevance of IGF-1R subcellular localisation, we noted that the association of cytoplasmic or internalised IGF-1R with biochemical recurrence was stronger than for total IGF-1R. Type 1 IGF receptor internalisation is required for prolonged AKT activation (Romanelli ), and for IGF-1R nuclear translocation, which contributes to IGF axis activity by promoting gene expression (Aleksic ; Sehat ; Sarfstein ; Warsito ). The lack of significant association with nuclear IGF-1R alone, and the stronger association of cytoplasmic IGF-1R than internalised (nuclear plus cytoplasmic) IGF-1R with overall and biochemical recurrence (Table 3) suggest that cytoplasmic IGF-1R is the major contributor here. Supporting this view, cytoplasmic IGF-1R was an independent predictor of biochemical recurrence in multivariate analysis (Table 4). Therefore, it is plausible that the association of biochemical recurrence with cytoplasmic IGF-1R could reflect increased IGF-1R activation in radioresistant tumours. This would be consistent with data we reported in cell lines, where suppression of IGF-1R activity enhanced radiosensitivity (Turney ; Chitnis ). In patients with PSA-detected localised prostate cancer, radiotherapy and surgery are reported to lead to comparable outcomes at median 10 years’ follow-up (Hamdy ). Several predictive biomarkers have been identified to predict outcomes post irradiation for prostate cancer (Wilkins ), but a role for IGF-1R has not previously been highlighted in this context. Our data suggest that patients whose prostate cancers contain high cytoplasmic or high total IGF-1R should be offered surgery or dose-escalated radiotherapy, hypotheses that warrant prospective evaluation. Taken together with preclinical data (Isebaert ; Turney ; Chitnis ), these results also support evaluation of IGF axis inhibition as a novel route to radiosensitisation of prostate cancers that express high total or cytoplasmic IGF-1R.