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PLOS ONE logoLink to PLOS ONE
. 2023 Jun 27;18(6):e0287608. doi: 10.1371/journal.pone.0287608

PAK1 copy number in breast cancer—Associations with proliferation and molecular subtypes

Anette H Skjervold 1,*, Marit Valla 1,2, Borgny Ytterhus 1, Anna M Bofin 1
Editor: Elingarami Sauli3
PMCID: PMC10298784  PMID: 37368917

Abstract

Introduction

P21-activated kinase 1 (PAK1) is known to be overexpressed in several human tumour types, including breast cancer (BC). It is located on chromosome 11 (11q13.5-q14.1) and plays a significant role in proliferation in BC. In this study we aimed to assess PAK1 gene copy number (CN) in primary breast tumours and their corresponding lymph node metastases, and associations between PAK1 CN and proliferation status, molecular subtype, and prognosis. In addition, we aimed to study associations between CNs of PAK1 and CCND1. Both genes are located on the long arm of chromosome 11 (11q13).

Methods

Fluorescence in situ hybridization for PAK1 and Chromosome enumeration probe (CEP)11 were used on tissue microarray sections from a series of 512 BC cases. Copy numbers were estimated by counting the number of fluorescent signals for PAK1 and CEP11 in 20 tumour cell nuclei. Pearson’s x2 test was performed to assess associations between PAK1 CN and tumour features, and between PAK1 and CCND1 CNs. Cumulative risk of death from BC and hazard ratios were estimated in analysis of prognosis.

Results

We found mean PAK1 CN ≥4<6 in 26 (5.1%) tumours, and CN ≥ 6 in 22 (4.3%) tumours. The proportion of cases with copy number increase (mean CN ≥4) was highest among HER2 type and Luminal B (HER2-) tumours. We found an association between PAK1 CN increase, and high proliferation, and high histological grade, but not prognosis. Of cases with PAK1 CN ≥ 6, 30% also had CCND1 CN ≥ 6.

Conclusions

PAK1 copy number increase is associated with high proliferation and high histological grade, but not with prognosis. PAK1 CN increase was most frequent in the HER2 type and Luminal B (HER2-) subtype. PAK1 CN increase is associated with CN increase of CCND1.

Introduction

P21-activated kinases (PAK) are a family of serine/threonine protein kinases comprising six isoforms (PAK1–6). They are overexpressed in several human tumours, such as breast cancer (BC), colon cancer and lung cancer, and in neurofibromatosis [1]. The six PAK isoforms are subdivided in PAK1-3 (group I) and PAK4-6 (group II) [2, 3]. PAKs play a significant role in proliferation, cytoskeletal dynamics, and cell survival [1, 4]. Their roles in these cell processes make them potential therapeutic targets. More is known of the functions of PAK1 and PAK4, than of the other isoforms [5, 6].

PAK1 is located on chromosome 11 (q13.5-q14.1). Amplification of PAK1 and high PAK1 protein levels are found in several human cancers, including BC [79], and are linked to aggressive tumour types, chemotherapy resistance and poor prognosis [4, 1014]. In 2000, Mira et al. first discovered that PAK1 had an important role in proliferation in BC cell lines [15]. Since then, PAK1 has been found to be involved in many stages of the BC process and is known to regulate several signaling pathways. [4, 1621]. PAK1 amplification has recently been found to be significantly associated with reduced relapse-free survival of ER-positive BC patients [19]. PAK1 is localized in the same chromosomal region as CCND1, 11q13 [22, 23]. Cyclin D1 (CCND1) has been found to be overexpressed in breast cancer, and PAK1 is shown to regulate the expression of CCND1 in BC [8, 23].

In this study we aimed to assess PAK1 gene copy number (CN) in a well-characterized series of primary BCs and their corresponding axillary lymph node metastases. We studied associations between PAK1 CN and proliferation, molecular subtypes, and prognosis. In addition, we examined associations between CN of CCND1, assessed in an earlier study by our group [24], and PAK1 CN.

Materials and methods

Study population

A population-based survey for the early detection of BC was conducted in the county of Nord-Trøndelag, Norway, between 1956 and 1959. The study included 25,727 women born 1886–1928 [25]. These women were followed for BC occurrence, through linkage with data from the Cancer Registry of Norway. During the follow-up years, between 1961 and 2008, 1379 new BCs were registered. Of these, 909 cases were included in the study population and were first reclassified into molecular subtypes in a previous published by our group in 2013 (Table 1) [26]. All patients were followed from time of diagnosis until death or December 31st, 2015.

Table 1. Reclassification of breast cancers into molecular subtypes [26].

Molecular subtype Classified by
Luminal A ER+ and/or PR+, HER2-, Ki-67<15%
Luminal B (HER2-) ER+ and/or PR+, HER2-, Ki-67≥15%
Luminal B (HER2+) ER+ and/or PR+, HER2+
HER2 type ER-, PR-, HER2+
Basal-like ER-, PR-, HER2-, CK5+ and/or EGFR+
5-negative phenotype ER-, PR-, HER2-, CK5-, EGFR-

*ER = Oestrogen receptor, PR = Progesterone receptor, HER2 = Human epidermal growth factor receptor 2, CK5 = Cytokeratin 5, EGFR = Epidermal growth factor receptor 1

For the present study, we performed fluorescence in situ hybridization (FISH) on tissue specimens from cases mainly diagnosed after 1985 (n = 558). Of these, 46 were excluded due to missing or insufficient tumour tissue (n = 25), or due to unsuccessful FISH (n = 21). Thus, 512 cases were suitable for assessment of PAK1 and chromosome enumeration probe 11 (CEP11) CN in primary tumours (Fig 1). Of the 512 cases, 172 had lymph node metastases, and tissue from lymph node metastases was available for 143 cases. Cases with unsuccessful FISH (n = 9) or insufficient amounts of tumour tissue (n = 11) were excluded. Hence, lymph node metastases from 123 cases were included in the analyses.

Fig 1. Overview of study population and cases included in this study.

Fig 1

Specimen characteristics

The primary tumours were previously reclassified into histological type and grade according to present-day guidelines [2628]. Tissue microarray (TMA) blocks were made using the TissueArrayer Minicore with TMA Designer2 software (Alphelys). Three 1-mm in diameter tissue cylinders were extracted from the periphery of the primary tumour, and from lymph node metastases and transferred to TMA recipient blocks. Using sections from the TMAs, primary tumours were then reclassified into molecular subtypes using immunohistochemistry (IHC) and chromogenic in situ hybridization (CISH) as previously described (Table 1). Briefly, Oestrogen Receptor (ER), Progesterone Receptor (PR), the proliferation marker Ki-67, Cytokeratin 5 (CK5) and Epidermal Growth Factor Receptor 1 (EGFR) were assessed using IHC, and Human Epidermal Growth Factor Receptor 2 (HER2) was assessed using both CISH and IHC [26] (Table 2). In a previous study of CCND1 CN, FISH was used to target CCND1 and CEP11, using Dako Histology FISH Accessory Kit K 579911 probes for CCND1 (3 μL, Empire Genomics) and CEP11 (1 μL, Abbott/VYSIS) [24].

Table 2. Sources and dilutions of primary antibodies used for molecular subtyping [26].

Antibody Clone Manufacturer Concentration of antibody Dilution
ER SP1 Cell Marque 33 mg/ml 1:100
PR 16 Novocastra 360 mg/l 1:400
HER2 CB11 Novocastra 3.9 g/l 1:640
Ki-67 MIB1 Dako 35 mg/l 1:100
CK5 XM26 Novocastra 50 mg/l 1:100
EGFR 2-18C9 Dako Ready to use No dilution

Fluorescence in situ hybridization

For the present study of PAK1 and CEP11 CN, FISH was done using DAKO Histology FISH Accessory Kit K 579911 according to the manufacturer’s instructions. TMA sections were preheated at 60°C for 1–2 h, then de-waxed and rehydrated. The slides were then boiled in a microwave oven for 10 min. in pretreatment solution and washed in DAKO wash buffer (2x3min.) after cooling (15 min.), followed by protein digestion in pepsin solution (37°C, 25 min.). After protein digestion, the slides were washed in DAKO wash buffer (2x3 min.), dehydrated (2 min. in 70%, 85% and 95% ethanol), then air-dried for 15 min. at room temperature.

PAK1 (3 μL, PAK1-20-RE, SpectrumRed fluorochrome Empire Genomics) and CEP11 (3 μL, CEP11 [D11Z19], SpectrumGreen fluorochrome, VYSIS) probes were mixed with hybridizing buffer (9 μL, Empire Genomics) and applied to TMA slides according to the manufacturer’s instructions. Coverslips were then applied to the slides, sealed with DAKO coverslip sealant, and the slides were dried for 20 min. After drying, denaturation was performed at 83°C for 3 min., followed by hybridization at 37°C overnight using DAKO hybridizer. Post-hybridization washes were done in 0.4 X SSC/ 0.3% NP-40 stringent wash buffer at 72°C (2 min.) and 2 X SSC/ 0.1% NP-40 wash buffer at room temperature (1 min.). Slides were then dried at 37°C for 15 min., DAPI II VYSIS (15 μl, no 06J50-001) was applied. The slides were then coverslipped and stored at −20°C.

Scoring and reporting

A fluorescence microscope (Nikon Eclipse 90i) was used for counting PAK1 and CEP11 CN. For each case, all available tissue spots were examined and the number of fluorescent signals for PAK1 and CEP11 were counted in 20 well-preserved, non-overlapping tumour cell nuclei. Mean PAK1 and CEP11 CNs was calculated for tumours and lymph node metastases and were first categorized as <4 and ≥4. In addition, to distinguish between low-level CN gain and high-level gain or gene amplification, we also subdivided CN into three categories: <4; ≥4<6; and ≥6 according to guidelines for categorizing HER2 CNs [29], a strategy which has been used in previous studies of other genes by our group [24, 3032]. The Reporting Recommendations for Tumor Marker Prognostic Studies (REMARK) were followed [33].

Statistical analyses

Pearson’s chi square test was used to compare tumour characteristics across categories of PAK1 mean CN. Cumulative incidence of death from breast cancer was estimated, and Gray’s test was used to compare equality between cumulative incidence curves. Cox proportional hazard analyses were used to estimate hazard ratios (HR) of breast cancer death with 95% confidence intervals (CI). The analyses were adjusted for age (≤ 49, 50–59, 60–64, 65–69, 70–74, ≥ 75), stage (I–IV), histological grade (1–3), and Ki67 status (</≥ 15%). Adjustments were made for each variable separately, and for age, grade, and stage combined. No clear violations of proportionality were observed in log minus-log plots. All statistical tests were two-sided and statistical significance was assessed at the 5% level. We used Stata 16 (Stata corp., College station, TX, USA) in the statistical analyses.

Ethics statement

This study was granted approval including dispensation from the general requirement of informed consent, by the Regional Committee for Medical and Health Research Ethics, Midt-Norge (REK 836/2009). All methods were carried out in accordance with relevant guidelines and regulations (The Declaration of Helsinki and national regulations (ACT 2008-06-20 no. 44: Act on medical and health research (the Health Research Act)).

Results

Patient and tumour characteristics for the 512 patients included in the present study are given in Table 3. The mean age at diagnosis was 75.4 years (range 41–96) and the mean follow-up after diagnosis was 9.1 years (SD = 7.2). At end of follow-up, 35.4% of patients had died from BC and 54.3% had died from other causes.

Table 3. Patient and tumour characteristics according to PAK1 copy number.

Total study population Mean PAK1 copy number, three categories Mean PAK1 copy number, two categories
<4 ≥4 to <6 ≥6 p value (χ2) <4 ≥4 p value (χ2)
N (%) 512 464 (90.6) 26 (5.1) 22 (4.3) 464 (90.6) 48 (9.4)
Mean age at diagnosis, years (SD) 75.4(41–96) (8.2) 75.5 (8.1) 75.2 (7.3) 74.3 (10.0) 75.5 (8.1) 74.8 (8.6)
Mean follow-up, years (SD) 9.1 (7.2) 9.0 (7.0) 9.6 (6.5) 9.0 (7.5) 9.0 (7.0) 9.3 (6.9)
Deaths from breast cancer (%) 181 (35.4) 161 (34.7) 9 (34.6) 11 (50.0) 161 (34.7) 20 (41.7)
Deaths from other causes (%) 278 (54.3) 255 (55.0) 15 (57.7) 8 (36.4) 255 (55.0) 23 (47.9)
Histological grade (%)
I 56 (10.9) 55 (11.9) 0 (0) 1 (4.6) 0.082 55 (11.9) 1(2.1) 0.037
II 287 (56.1) 262 (56.5) 12 (46.2) 13 (59.1) 262 (56.5) 25 (52.1)
III 169 (33.0) 147 (31.7) 14 (53.9) 8 (36.4) 147 (31.7) 22 (45.8)
Lymph node metastasis (%)
Yes 172 (33.6) 153 (33.0) 13 (50.0) 6 (27.3) 0.272 153 (33.0) 19 (39.6) 0.360
No 228 (44.5) 209 (45.0) 9 (34.6) 10 (45.5) 209 (45.0) 19 (39.6)
Unknown histology 112 (21.9) 102 (22.0) 4 (15.4) 6 (27.3) 102 (22.0) 10 (20.8)
Tumor size (%)
≤2 cm 245 (47.9) 217 (46.8) 16 (61.5) 12 (54.6) 0.327 217 (46.8) 28 (58.3) 0.516
>2 cm, ≤ 5 cm 95 (18.6) 88 (19.0) 4 (15.4) 3 (13.6) 88 (19.0) 7 (14.6)
>5 cm 10 (2.0) 9 (1.9) 1 (3.9) 0 (0) 9 (1.9) 1(2.1)
Uncertain, but >2 cm 63 (12.3) 60 (12.9) 3 (11.5) 0 (0) 60 (12.9) 3 (6.3)
Uncertain 99 (19.3) 90 (19.4) 2 (7.7) 7 (31.8) 90 (19.4) 9 (18.8)
Stage (%)
I 242 (47.3) 221 (47.6) 9 (34.6) 12 (54.6) 0.027 221 (47.6) 21 (43.8) 0.117
II 218 (42.6) 198 (42.7) 14 (53.9) 6 (27.3) 198 (42.7) 20 (41.7)
III 27 (5.3) 22 (4.7) 3 (11.5) 2 (9.1) 22 (4.7) 5 (10.4)
IV 23 (4.5) 22 (4.7) 0 (0) 1 (4.6) 22 (4.7) 1 (2.1)
Unknown 2 (0.4) 1 (0.2) 0 (0) 1 (4.6) 1 (0.2) 1 (2.1)
Molecular subtype (%)
Luminal A 272 (53.1) 251 (54.1) 11 (42.3) 10 (45.5) 0.649 251 (54.1) 21 (43.8) 0.375
Luminal B (HER2-) 121 (23.6) 105 (22.6) 8 (30.8) 8 (36.4) 105 (22.6) 16 (33.3)
Luminal B (HER2+) 42 (8.2) 39 (8.4) 1 (3.9) 2 (9.1) 39 (8.4) 3 (6.3)
HER2 type 27 (5.3) 23 (5.0) 3 (11.5) 1 (4.6) 23 (5.0) 4 (8.3)
5NP 11 (2.2) 11 (2.4) 0 (0) 0 (0) 11 (2.4) 0 (0)
BP 39 (7.6) 35 (7.5) 3 (11.5) 1 (4.6) 35 (7.5) 4 (8.3)
Histological type (%)
Invasive carcinoma NOS 353 (69.0) 318 (68.5) 19 (73.1) 16 (72.7) 0.593 318 (68.5) 35 (69.0) 0.273
Lobular carcinoma 66 (12.9) 61 (13.2) 2 (7.7) 3 (13.6) 61 (13.2) 5 (10.4)
Tubular carcinoma 1 (0.2) 1 (0.2) 0 (0) 0 (0) 1 (0.2) 0 (0)
Mucinous carcinoma 24 (4.7) 23 (5.0) 1 (3.9) 0 (0) 23 (5.0) 1 (2.1)
Medullary carcinoma 14 (2.7) 10 (2.2) 3 (11.5) 1 (4.6) 10 (2.2) 4 (8.3)
Papillary carcinoma 25 (4.9) 23 (5.0) 1 (3.9) 1 (4.6) 23 (5.0) 2 (4.2)
Metaplastic 8 (1.6) 8 (1.7) 0 (0) 0 (0) 8 (1.7) 0 (0)
Other 21 (4.1) 20 (4.3) 0 (0) 1 (4.6) 20 (4.3) 1 (2.1)
Ki67 high/low (%)
Ki67 <15% 308 (60.2) 286 (61.6) 12 (46.2) 10 (45.5) 0.104 286 (61.6) 22 (45.8) 0.033
Ki67 ≥15% 204 (39.8) 178 (38.4) 14 (53.9) 12 (54.6) 178 (38.4) 26 (54.2)
Mitoses/10 HPF, median (IQR p25, p75) 5 (1, 12) 5 (1, 11) 9 (3,20) 6 (2, 12) 5 (1, 12) 8 (2.5, 16.5)
Mitoses/10 HPF, quartiles (%)
≤1 136 (26.6) 128 (27.6) 6 (23.1) 2 (9.1) 0.025 128 (27.6) 8 (16.7) 0.162
>1 ≤5 133 (26.0) 123 (26.5) 1 (3.9) 9 (40.9) 123 (26.5) 10 (20.8)
>5 ≤12 123 (24.0) 107 (23.1) 9 (34.6) 7 (31.8) 107 (23.1) 16 (33.3)
>12 120 (23.4) 106 (22.8) 10 (38.5) 4 (18.2) 106 (22.8) 14 (29.2)

Abbreviations: SD = standard deviation, HER2 = human epidermal growth factor receptor 2, 5NP = 5 negative phenotype, BP = basal phenotype, HPF = high power fields

PAK1 and CEP11 copy number, and histological grade and proliferation

PAK1 CN ≥4 was found in 48 (9.4%) tumours (Table 3, Fig 2). Of these, 26 (5.1%) cases had mean CN ≥4<6, and 22 (4.3%) had mean CN ≥6. While 147/464 (31.7%) cases with CN <4 were grade 3, 22/48 (45.8%) cases with CN ≥4 were grade 3 (p = 0.037). We found no significant associations between PAK1 CN increase and high histological grade using three categories of mean PAK1 CN (Table 3).

Fig 2. Fluorescence in situ hybridization using probes for CEP11 (fluorochrome SpectrumGreen) and PAK1 (fluorochrome SpectrumRed).

Fig 2

Fig 2 showing 2–3 copies of CEP11 and 6–8 copies of PAK1 in each tumour cell nucleus.

PAK1 CN ≥4 was associated with high Ki-67 (≥15%). Of cases with PAK1 CN <4, 178/464 (38.4%) had Ki-67 ≥15%, compared to 26/48 (54.2%) among those with PAK1 CN ≥4 (p = 0.033). No association between PAK1 CN increase and Ki-67 status was found when PAK1 CN was subdivided into three categories. The median mitotic count was higher in cases with mean PAK1 CN ≥4, compared to cases with mean CN <4 (8 mitoses/10 high power fields [HPF] and 5 mitoses/10 HPF, respectively). The proportion of cases with mitotic counts in the upper quartile was also higher for cases with mean PAK1 CN ≥4, compared to those with mean CN <4 (106/464 [22.8%] and 14/48 (29.2%), respectively (p = 0.162)) (Table 3). Only seven cases showed CEP11 CN increase. Five of these were in cases with PAK1 CN <4. Of the 26 cases with PAK1 CN ≥4<6, only two were accompanied by CEP11 CN increase (≥4<6). Of the 22 cases with PAK1 CN ≥6, none had concurrent CN increase of CEP11.

PAK1 copy number and molecular subtypes

Copy number increase of PAK1 was found in all molecular subtypes, except the 5-negative phenotype (5NP). The highest proportion of cases with PAK1 CN ≥4 was found in the HER2 type, followed by Luminal B (HER2-). Of a total of 27 cases of the HER2 type, four (14.7%) had PAK1 CN ≥4, one of which (3.7%) had PAK1 CN ≥6. In Luminal B (HER2-), 16/121 (13.2%) had PAK1 CN ≥4, and of these, 8/121 (6.6%) had PAK1 CN ≥6. Among Luminal B (HER2+) cases, 3/42 (7.1%) showed PAK1 CN ≥4 (Table 3).

PAK1 and prognosis

The cumulative risk of death from BC during the first 5 years after diagnosis was 20.3% (95%CI 16.9–24.2) for cases with mean PAK1 CN <4, 23.1% (95% CI 11.1–44.3) for cases with CN ≥4<6, and 18.2% (95% CI 7.2–41.5) for cases with CN ≥6 (Fig 3, Table 4). During the first 10 years after diagnosis, the cumulative risk of death from BC was 30.1% (95% CI 26.1–34.5) for cases with mean PAK1 CN <4, 26.9% (95% CI 13.9–48.3) for cases with CN ≥4<6, and 40.9% (95% CI 23.8–63.9) for cases with CN ≥6. In the Cox regression analyses using mean PAK1 CN <4 as the reference, no significantdifference was observed in the rate of death from breast cancer for cases with PAK1 CN increase (HR 1.4 [95% CI 0.8–2.7]) for cases with mean PAK1 copy number ≥6). Fourteen of the 123 cases for which lymph node metastases were available had PAK1 CN ≥4 in the primary tumour. Of these, 8 also had PAK1 CN ≥4 in the corresponding lymph node metastasis. Of the five cases with PAK1 CN ≥6 in the primary tumour, 3 also had PAK1 CN ≥6 in the corresponding lymph node metastasis (Table 5).

Fig 3. Cumulative incidence of death from breast cancer according to mean PAK1 copy number in primary breast cancer tumours.

Fig 3

Cumulative incidence curves show no significant association between PAK1 copy number and risk of death. A) Mean PAK1 copy number <4, ≥4<6 and ≥6. p = 0.39. B) Mean PAK1 copy number <4 and ≥4. p = 0.42.

Table 4. Absolute and relative risk of death from breast cancer according to mean PAK1 copy number/tumour cell nucleus in primary tumours.

Mean PAK1 copy number
<4 ≥4<6 ≥6
Cumulative risk after 5 years (%) (95% CI) 20.3(16.9–24.2) 23.1 (11.1–44.3) 18.2 (7.2–41.5)
Cumulative risk after 10 years (%) (95% CI) 30.1 (26.1–34.5) 26.9 (13.9–48.3) 40.9 (23.8–63.9)
HR unadjusted (95% CI) 1.0 0.9 (0.5–1.8) 1.4 (0.8–2.7)
HR adjusted for age (95% CI) 1.0 0.9 (0.5–1.8) 1.5 (0.8–2.7)
HR adjusted for stage (95% CI) 1.0 0.8 (0.4–1.6) 1.7 (0.9–3.2)
HR adjusted for grade (95% CI) 1.0 0.8 (0.4–1.6) 1.4 (0.8–2.6)
HR adjusted for Ki-67 (95% CI) 1.0 0.8 (0.4–1.7) 1.3 (0.7–2.3)
HR adjusted for age, stage, and grade (95% CI) 1.0 0.8 (0.4–1.5) 1.7 (0.9–3.2)

Abbreviations: HR = Hazard ratio, CI = confidence interval

Table 5. PAK1 copy number in primary tumours and corresponding axillary lymph node metastases.

Mean PAK1 copy number in primary tumours (%)
Mean PAK1 copy number in lymph node metastases (%) <4 ≥4<6 ≥6 Total
<4
≥4<6
≥6
Total
103 (94.5) 6 (66.7) 0 109
5 (4.6) 3 (33.3) 2 (40) 10
1 (0.9) 0 3 (60) 4
109 9 5 123
Mean PAK1 copy number in primary tumours (%)
Mean PAK1 copy number in lymph node metastases (%) <4 ≥4 Total
<4
≥4
Total
103 (94.5) 6 (42.9) 109
6 (5.5) 8 (57.1) 14
109 14 123

PAK1 and CCND1

Among the 512 cases included in this study, CCND1 CN status was available for 504 cases [24]. A total of 84/504 cases showed CCND1 CN ≥4 and 40 of these had ≥6 copies of CCND1/nucleus (Table 6). Of the 22 patients with PAK1 CN ≥6, 12 (54.6%) cases also had CCND1 CN ≥6. Of the 48 cases with PAK1 CN ≥4, 30 (62.5%) cases also had CCND1 CN ≥4. However, 54 cases had CCND1 CN ≥4 without a corresponding increase in PAK1 CN and 18 cases showed CN increase ≥4 for PAK1 without CN increase of CCND1 (Table 6).

Table 6. PAK1 and CCND1 copy numbers in primary tumours.

Mean PAK1 CN in primary tumours (%)
Mean CCND1 CN <4 ≥4<6 ≥6 Total
<4 402 (88.2) 11 (42.3) 7 (31.8) 420 p<0.001
≥4<6 31 (6.8) 10 (38.5) 3 (13.6) 44
≥6 23 (5.0) 5 (19.2) 12 (54.6) 40
Total 456 26 22 504
Mean PAK1 CN in primary tumours (%)
Mean CCND1 CN <4 ≥4 Total
<4 402 (88.2) 18 (37.5) 420 p<0.001
≥4 54 (11.8) 30 (62.5) 84
Total 456 48 504

We found no significant difference in the cumulative risk of death from BC between cases with CN ≥4 of PAK1 alone, CN ≥4 CCND1 alone, and cases with CN ≥4 for both PAK1 and CCND1 combined (Fig 4). Similarly, The Cox regression analysis using combined PAK1 CN <4 and CCND CN <4 as the reference value, showed no significant difference in the rate of death from BC between the three groups of patients with copy number increase (Table 7).

Fig 4. Cumulative incidence of death from breast cancer according to copy numbers of PAK1 and CCND1, and co-amplification of PAK1 and CCND1.

Fig 4

Cumulative incidence curves show no significant association between PAK1 copy number, CCND1 copy number, and co-amplification of PAK1 and CCND1, and risk of death. p = 0,81.

Table 7. Relative risk of death from breast cancer according to copy numbers of PAK1 and CCND1, and co-amplification of PAK1 and CCND1.

Copy number of PAK1 and CCND1 Hazard ratio
HR CI p-value
PAK1 CN<4 & CCND1 CN<4 (reference value) 1.0 0.872
PAK1 CN≥4 & CCND1 CN<4 1.3 0.6–2.6
PAK1 CN<4 & CCND1 CN≥4 0.9 0.6–1.5
PAK1 CN≥4& CCND1 CN≥4 1.1 0.6–2.0

Hazard ratio = HR, Confidence interval = CI

Discussion

In this study of 512 primary BC tumours, we found PAK1 CN ≥4 in 48 (9.4%) cases, of which 22 cases showed high grade CN increase of PAK1 CN ≥6. We found an association between PAK1 CN ≥4, and high Ki-67 (≥15%) and high histological grade. The highest proportion of cases with increased CN of PAK1 (≥4) was found in the HER2 type and Luminal B (HER2-) breast cancer subtype. Concurrent CN increase (≥4) of PAK1 and CCND1 was observed in 30 cases. Of the 123 cases with available lymph node metastases, only three cases had PAK1 CN ≥6 in both the primary tumour and the corresponding lymph node metastases.

The cohort of Norwegian BC patients from which the cases of this study are derived is well-described, with mean follow-up of nine years. Since recurrence and death from BC may occur many years after the primary diagnosis, long-term follow-up is important in studies of prognostic markers. While recurrence data was unavailable to us, long-term survival data is complete, enabling us to assess the influence of biomarkers on prognosis. Histological typing and grading of all cases in this cohort were revised by experienced pathologists according to current guidelines. All biomarkers were stained at the same laboratory, and the same antibodies, cut-off levels and algorithm for molecular subtyping were used for all cases in the cohort [26].

In this study we used FISH on TMAs. TMAs provide the opportunity to efficiently study biomarkers in a large number of samples simultaneously under similar laboratory conditions at a relatively low cost. FISH is a method available in most laboratories, contrary to multigene assays. It enables us to assess the morphology of the section and ensure that only invasive tumour cell nuclei are assessed. Despite this, FISH applied to tissue sections may lead to an underestimation of CN compared to analysis of whole nuclei, due to nuclear truncation [34]. This would be of particular importance in cases with low CN increase. Preanalytical conditions will have varied considering that the cases included in the present study were diagnosed over decades. This could have affected the cases suitable for FISH analysis. However, few cases were discarded due to unsuccessful FISH. There are no established guidelines for cut-off levels in the assessment of PAK1 CN. We chose to follow HER2 guidelines for categorizing CN, as in previous studies by our group [24, 2932]. While we also registered CN of CEP 11, we did not calculate the ratio between CNs of PAK1 and CEP11 as this would have masked the true gene CN increase. Furthermore, we found that CEP11 CN increase was observed in only seven cases, of which only two were accompanied by CN increase of PAK1.

Tamoxifen is an established hormonal therapy used in ER positive BC. Five years of tamoxifen therapy nearly halves the risk of BC recurrence among ER positive patients [35]. Phosphorylation of ER by PAK1 may induce tamoxifen-resistance in ER positive tumours and tamoxifen itself may also increase nuclear PAK1 and PAK1 kinase activity [14, 23]. Patients with PAK1 amplification have reduced benefit from tamoxifen and PAK1 CN may therefore be a predictor of tamoxifen resistance [23]. Thus, PAK1-inhibitors may be useful in ER positive tumours, to improve the effect of tamoxifen in these cases [36].

Both PAK1 and CCND1 encode proteins shown to activate ER [23, 36]. Both are located on 11q13 and are thought to be frequently co-amplified. In this study, of the 504 patients analyzed for both CCND1 and PAK1, 84 cases had CN ≥4 for CCND1 and 48 with PAK1 CN ≥4. A total of 30 (62.5%) cases had CN increase of both genes. These results are in accordance with the findings of others [23]. In the present study, co-amplification of PAK1 and CCND1 was not associated with prognosis.

The proportion of cases with increased PAK1 CN in this study was lower compared to the results of previous studies [7, 8]. However, the mean age at diagnosis in our study was 75.4 years, which is high compared to other studies and higher than the mean age for diagnosis of breast cancer in Norway which is 62 years of age [37]. Fumagalli et al found CN increase in 11% of cases in a selected series of ER+, metastatic breast cancer cases. In our series of cases, PAK1 CN increase was found among Luminal B HER2- and the HER2 type [38]. High proliferation rate and poor prognosis are found to be associated in BC [39, 40], and the prognostic effect of proliferation has been shown to vary with age, exerting a greater effect on prognossis among younger BC patients [41]. This may, in part, explain the discrepant results compared to other studies of PAK1 and further studies including a wider age range are warranted. Furthermore, the choice of method may also have contributed to these results. Tissue microarrays include only small tissue cylinders from the tumour and may not be representative of the whole tumour, particularly in cases with tumour heterogeneity [42, 43]. In the TMAs used in our study, tissue cylinders were extracted from the tumour periphery and are therefore not necessarily representative of other areas of the tumour. However, we considered the tumour periphery to be the region of greatest interest in the tumour given its greater proliferative activity [44] and its proximity to surrounding breast tissue. Furthermore, selecting tissue for TMA form the same region of all tumours contributes to a certain standardization of the material examined in the study.

Despite associations between PAK1 CN increase and high histological grade and high proliferation, we failed to demonstrate a statistically significant association between increased PAK1 CN and prognosis. It would be interesting to study prognosis according to PAK1 CN for each of the molecular subtypes separately. However, in the present study the number of cases in some of the molecular subtypes was too low to warrant further analyses of subgroups. The numbers of cases showing PAK1 CN increase in primary tumours only, lymph node metastases only, or both were too low to give reliable prognostic information. The frequency of PAK1 CN change in this study was lower than the expression of established biomarkers, such as ER, PR and HER2 in BC. However, in an era of personalized medicine, its known influence on the effect of tamoxifen in BC makes it an interesting biomarker and potential target for treatment.

Conclusion

PAK1 CN increase is found in all molecular subtypes, except the 5-negative phenotype (5NP), and most frequently in the HER2 and Luminal B (HER2-) subtypes. It is associated with aggressive tumour characteristics such as high histological grade and high Ki-67 protein expression, but not with prognosis. It is co-amplified with CCND1 in a proportion of cases. Few cases showed PAK1 CN increase in both the primary tumour and the corresponding lymph node metastases.

Acknowledgments

The authors thank the Department of Pathology, St. Olav´s Hospital, Trondheim University Hospital for making the diagnostic archives available for the study, and the Cancer Registry of Norway for supplying the patient data.

Data Availability

The datasets generated in the current study are not publicly available due to ethical and legal restrictions imposed by General Data Protection Regulations (GDPR), National health research legislation and the conditions for approval by the Regional Committee for Medical and Health Research Ethics, Midt-Norge (REK 836/2009), but may be available from the corresponding author on reasonable request and/or the Institutional Research Officer, Department of Clinical and Molecular Medicine, Faculty of Medicine, NTNU at postmottak@mh.ntnu.no.

Funding Statement

The research leading to these results received funding from The Liaison Committee between the Central Norway Regional Health Authority and the Norwegian University of Science and Technology (NTNU), The Joint Research Committee between St. Olav’s Hospital and the Faculty of Medicine and Health Sciences, NTNU (FFU), and the Department of Clinical and Molecular Medicine, NTNU. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Decision Letter 0

Elingarami Sauli

11 Apr 2023

PONE-D-23-02665PAK1 copy number in breast cancer - Associations with proliferation and molecular subtypesPLOS ONE

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Reviewer #1: Dear authors,

I read with interest the article which analyzes the associations between PAK1 CN and proliferation status, molecular subtype, and prognosis in addition to the correlation between CNs of PAK1 and CCND1.

I also think that the molecular targets related to the biological aggressiveness of the tumors could constitute a pharmacological target in the personalization of the treatments.

I recommend this article.

The manuscript clear, relevant for the field and presented in a well-structured manner.

The cited references mostly recent publications (within the last 5 years) are relevant.

The manuscript scientifically sound and is the experimental design appropriate to test the hypothesis.

The manuscript’s results reproducible based on the details given in the methods section (to be implemented).

The figures/tables/images/schemes are appropriate and they properly show the data (easy to interpret and understand).

The statistical analysis or data acquired are appropriate.

The conclusions are consistent with the evidence and arguments presented the ethics statements and data availability statements to ensure they are adequate.

The results interpreted appropriately and are significant. All conclusions are justified and supported by the results and the hypotheses carefully identified. The article written in an appropriate way and the data and analyses presented appropriately.

Reviewer #2: The manuscript was well written, the methodology was sound and the results were highlighted systematically.

However, very few issues need address.

1. Line 8 (page 4): Patients were diagnosed, however it was not stated if patients were categorized into treatment options. We assumed molecular classification played role in treatment options and this will be important in the effect of PAK 1 and Tamoxifen as mentioned in the discussion.

2. Table 3 which should highlight the association of PAK 1 CN with Ki67 and Histological grade is missing

3. Kindly check results for line 15 and 16 on page 7 to make sure that comparisons are made with common denominator

4. Line 2 of page 7 described described age demography, with inconsistent gap, while table 3 assumed to highlight this data is conspicuously missing.

5 The discussions on PAK1 CN and CCND1 was scanty.

Reviewer #3: The manuscript titled PAK1 copy number in breast cancer - Associations with proliferation and molecular subtypes is relevant and corroborated knowledge already known.

Abstract

Aims - change to background or introduction. Define all abbreviations first before use.

Method -This was over summarised. More explanation needed.

Result- Add a comment on how the association was found.

Conclusion- Too long. Let it answer the research question

Body of manuscript

Introduction: adequate. Define all abbreviations before use

Material & methods: Adequate and sound. Check the fish protocol and edit appropriately- Make it flow for ease of understanding.

Result: Adequate but need revisions. 'No clear association....' was used several times. It is unclear what it means.

Pg 7 Line 18-25 and 8:1-3 - revise for clarity; HER2- or HER2 superscript - Stick to one

There is repetition of the entire results in the tables- This makes tables/figures redundant. Just mention important findings in-text.

Table 3 missing

Table 5 and 6 confusing. I will suggest the authors stick to the classification stated in the methodology for copy numbers

Discussion: Generally inadequate. Line 4-9: Repetition of result; not necessary. Line 12-19: This was not given attention in the result.

No discussion on comparison of primary tumor with corresponding lymph node metastasis

Why was the TMA taken from tutor periphery?

Conclusion: Fair.

References: Adequate

General: The manuscript needs grammar and spelling check and discussion should be tailored to the results.

**********

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Reviewer #1: Yes: Paolo Orsaria, MD, PhD;

Reviewer #2: No

Reviewer #3: No

**********

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PLoS One. 2023 Jun 27;18(6):e0287608. doi: 10.1371/journal.pone.0287608.r002

Author response to Decision Letter 0


5 Jun 2023

The authors would like to thank both the editor and reviewers for their comprehensive and informative assessment of our manuscript.

Our responses to their comments and suggestions are uploaded as a separate file in this revised submission.

Attachment

Submitted filename: Response to Reviewers.pdf

Decision Letter 1

Elingarami Sauli

8 Jun 2023

PAK1 copy number in breast cancer - Associations with proliferation and molecular subtypes

PONE-D-23-02665R1

Dear Dr. Anette,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Elingarami Sauli, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Acceptance letter

Elingarami Sauli

19 Jun 2023

PONE-D-23-02665R1

PAK1 copy number in breast cancer - Associations with proliferation and molecular subtypes

Dear Dr. Skjervold:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Elingarami Sauli

Academic Editor

PLOS ONE

Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    Attachment

    Submitted filename: Response to Reviewers.pdf

    Data Availability Statement

    The datasets generated in the current study are not publicly available due to ethical and legal restrictions imposed by General Data Protection Regulations (GDPR), National health research legislation and the conditions for approval by the Regional Committee for Medical and Health Research Ethics, Midt-Norge (REK 836/2009), but may be available from the corresponding author on reasonable request and/or the Institutional Research Officer, Department of Clinical and Molecular Medicine, Faculty of Medicine, NTNU at postmottak@mh.ntnu.no.


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