PIK3CA mutations in breast cancer

doi:10.21203/rs.3.rs-1500819/v1

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PIK3CA mutations in breast cancer

https://doi.org/10.21203/rs.3.rs-1500819/v1

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Purpose: The phosphatidylinositide-3-kinase (PI3K) pathway regulates intracellular processes in relation to proliferation and apoptosis, which can be activated by somatic PIK3CA mutations. The aim of this work was to analyze the prevalence of PIK3CA mutations in an unselected cohort of patients with early stage breast cancer and their association with survival.

Patients and methods: From a prospective, multicentre cohort of 1,270-breast cancer patients (PiA, Prognostic Assessment in routine application, NCT 01592825) 1,123 tumors were tested for PIK3CA-mutations in three hotspots C775 (H1047R), C763 (E545K) and C760 (E542K) by quantitative PCR. The primary objectives were the prevalence of somatic PIK3CA mutations and the associations between PIK3CA mutations and clinical and histopathological parameters. Secondary objectives were the association of PIK3CA mutations with recurrence free interval (RFI) and overall survival.

Results: The overall PIK3CA mutation rate was 26.7% (300 out of 1,123). PIK3CA mutations were significantly more frequent in tumors with more favorable factors like steroid hormone receptor (HR)-positive and HER2-negative (31.4%), and G1/ G2 tumours (32.8%). We found a significant detrimental prognostic impact of PIK3CA-mutations on RFI for patients with HR-positive BCs who had only aromatase inhibitors as adjuvant therapy (adjusted HR 3.71, 95% CI 1.24-11.11), whereas PIK3CA mutations in HR-negative BCs were insignificantly associated with improved RFI (adjusted HR=0.43; 95% CI 0.10-1.82).

Conclusion: The impaired prognosis for HR-positive BCs and concurrent PIK3CA mutations supports the hypothesis that the activation of PI3K might lead to endocrine resistance. Conversely, patients with HR-negative tumors and PIK3CA mutations might benefit from chemotherapy compared to patients with wildtype PIK3CA gene.

Early breast cancer

PIK3CA

PI3K

somatic mutations

prognosis

Phosphatidylinositol-3-kinase (PI3K) (gene symbol PIK3CA) intracellularly mediates different processes like promoting cell transformation, tumour initiation and proliferation, and resistance to apoptosis. Its activity is stimulated by extracellular growth factors and hormones ¹. The dysregulation of PI3K initiates activity of the serine/threonine kinase AKT in many cancer entities thereby modulating a range of downstream proteins that promote uncontrolled cellular and tumour growth ². Thirty years ago, the PI3K/AKT/mTOR signalling pathway was discovered to be associated with carcinogenesis/oncogenic development ³, as shown by the timeline from Arafeh and Samules ⁴, and to date, PIK3CA-targeted drugs are developed and validated in clinical trials ^{5, 6}.

The lipid-based PI3-kinases phosphorylate the 3-hydroxyl group of phosphatidylinositol (4,5)-bisphosphate (PIP2) to phosphatidylinositol (3,4,5)-trisphosphate (PIP3) followed by the activation of AKT and downstream signalling pathways required for cell growth and survival. PI3K activation is physiologically abrogated by the tumour suppressor phosphatase and tensin-homolog (PTEN) which converts PIP3 back to PIP2. PIP3 peptide levels depend on the competition between PI3K and PTEN. The overactivation of PI3K as well as decreased PTEN expression lead to activated and increased levels of AKT, thus pathologically promoting cell cycle progression ⁶.

There are three classes of PI3Ks according to their primary structures, substrate preferences and regulation: Class I (Ia, Ib), Class II and Class III. Most relevant for cellular regulation are the PI3Ks of class Ia which act as heterodimers of regulatory and catalytic subunits ⁷. The catalytic subunit of the class I PI3-kinase p110α is encoded by the PIK3CA gene with a total genomic size of 86,190 bases in 21 exons and a final transcript of 3,207 bases which encode a protein of 1068 amino acids. The p110α protein has five domains: an adaptor-binding-domain for linking the regulatory subunit, a Ras-binding-domain, a C2-domain for binding PIP2, PIP3, a helical domain and the kinase domain, see https://www.uniprot.org/uniprot/P42336 ⁸.

Somatic mutations of the PIK3CA gene in human cancers have been described in recent decades ⁹ with up to 40% in primary breast cancer (http://www.sanger.ac.uk/cosmic) ¹⁰. The most frequent PIK3CA gene mutations are found in the coding sequence inducing a gain –of-function of the PI3K. Three hot spot non-synonymous variants represent 87% of the mutations with known clinical relevance ¹¹ leading to amino acid substitutions: COSMIC 760 in exon 9 (17% incidence) with an E545K mutation, COSMIC 763 in exon 19 (17% incidence) affecting E545 and COSMIC 775 in exon 20 (35% incidence) altering H1047.

The three genomic aberrations are predictive for drug responsiveness, meaning that diagnostic testing can identify patients who might benefit from PI3K-targeted therapy. Recently, the PI3K inhibitor alpelisib was approved by both the FDA and EMA for patients with PIK3CA-mutated, steroid hormone receptor positive and HER2-negative tumours ⁵. The three hotspot mutations described above can be efficiently determined by targeted sequence analysis. Nevertheless, the prognostic and predictive value of the PIK3CA mutation status as a biomarker for early breast cancer is discussed controversially for BC subgroups with respect to hormone receptor and HER2 expression ¹².

In this study, we describe the prevalence of the three most common PIK3CA mutations in subgroups of a breast cancer cohort and its association with clinical, histopathological characteristics and survival data.

Patient and Tumour Characteristics

A prospective study of 1,270 early breast cancer patients from five German certified breast centres (2009 to 2011) was designed in accordance with the REMARK (“Reporting Recommendations for Tumor Marker Prognostic Studies”) criteria ¹³ and registered as the “PiA-study” (Prognostic assessment in routine application, NCT 01592825) using the following inclusion criteria: female patients, aged 18 years or older, invasive, non metastasised BC and no secondary cancer, no limitation in tumour size, lymph node involvement, and grading or receptor status like ER, PgR and HER2. Patients were diagnosed and treated (1,070 with primary surgery, 200 with neoadjuvant chemotherapy, NACT) according to the annually updated German AGO Guidelines (AGO) valid at the respective times https://www.ago-online.de/leitlinien-empfehlungen/leitlinien-empfehlungen/kommission-mamma.

In the current study, we analysed the PIK3CA gene mutation status of 1,123 tumours. The median age of the patients was 60 years at time of diagnosis, with three-quarters of patients being older than 50 years, and two thirds having no lymph node involvement. Considering tumour tissue, three-quarters were well differentiated or intermediate (G1 or G2), and half of the tumours were smaller than 2 cm. The distribution of the patients´ main characteristics, as well as the histopathological parameters of the analysed cohort (n = 1,123), did not significantly differ from the entire PiA cohort (supplementary table S 1). The enrolment of the patients and grouping for the main analysis is shown in Fig. 1.

Endpoints and Statistical Analysis

We selected the prevalence of PIK3CA-mutations and the associations between PIK3CA-mutation status and clinical histopathological parameters as the first objective, evaluated by a binary logistic regression model; the recurrence free interval (RFI) and overall survival (OS) were the second objectives. RFI-related events refer to local invasive recurrence, distant recurrence, and death from breast cancer. OS included death from breast cancer, non-breast cancer causes, and unknown causes¹⁴. Tumour association and survival analysis were only performed for patients with one tumour. The median observation time after diagnosis was 62 months (1-132). The effect of PIK3CA-mutation status was calculated using Kaplan-Meier estimates, differences described by log-rank test and multivariate analysis for selected parameters applying the proportional hazard regression model summarised using the fixed effects model (Review Manager, version 5.3). Recursive partitioning referred to as Classification and Regression Tree (CART) analysis was carried out to examine combinations of clinical and histopathological parameters and to find homogenous risk groups with respect to RFI ¹⁵. To avoid selection bias for any parameters, only significant variables were entered in the CART analysis starting with the most important prognostic parameter resulting from regression model. Patient groups were further recursively divided, considering the best split at each decision point into smaller and more homogenous groups. Unbiased parameter selection was guaranteed by following the best impact parameter (after regression model) ¹⁶.

All statistical tests were two-sided, and a p-value below 0.05 was considered to be significant. Statistical analyses were carried out using SPSS 25 (IMB, Armonk, NY, USA).

DNA extraction and Hotspot Mutation assay

Fresh frozen tissue (FF) (n = 813) and formalin-fixed, paraffin-embedded tumour material (FFPE) (n = 310) of the tumours were used for DNA isolation. The FF-tissue was dismembrated in liquid nitrogen, and powder was used for DNA extraction using the QIAamp DNA Mini Kit (Cat. No. 51304; Qiagen, Hilden, Germany). For isolation of DNA from FFPE specimens, 3–5 adjacent unstained tumour slices (5 µm) were processed using QIAamp DNA FFPE Tissue kit (Cat. No. 56404; Qiagen, Hilden, Germany). All preparations were performed according to the manufacturer’s instructions. Quality and concentration of the extracted DNA were assessed with a Tecan Infinite PRO® 200 (Tecan, Männedorf, Switzerland). A standard amount of 50 ng DNA was subjected to mutation analysis.

With respect to mutation status we focused on the three most common hotspot mutations COSMIC C775 (H1047R), C763 (E545K) and C760 (E542K). TaqMan® Mutation Detection and reference assays were performed in duplicates (Life Technologies, Carlsbad, CA, USA) and were used for quantitative PCR (qPCR) with the StepOne Plus® Real-Time PCR System (Life Technologies) for 40 cycles with 60°C amplification temperature.

PIK3CA Mutation Prevalence and Association with Clinical and Histopathological Parameters

In this cohort study, 88% of the patients were tested for single nucleotide substitutions at three hotspot positions in the PIK3CA gene, and we found a mutation prevalence of 26.7% (n = 300 of 1,123) at these positions. The mutation rates for the three hot spot sites were 58% (n = 174) for COSMIC C775 (H1047R), 28% (n = 85) for C763 (E545K) and 14% (n = 43) for C760 (E542K). Co-occurrence of mutations at C775 and C763 were found in two tumours, although PIK3CA mutations were significantly more frequent in well (G1, OR = 3.13, 95% CI 1.970–4.986, in relation to G3) or intermediately differentiated (G2, OR = 2.14, 95% CI 1.478–3.085) tumours. Tumours with PIK3CA mutations were more likely to be steroid hormone receptor-positive compared to HR-negative tumours (OR 3.38, 95% CI 2.103–5.438), and in HER2-negative than in HER2-positive tumours (OR 2.25, 95% CI 1.451–3.501). Considering immunohistochemistry (IHC) types, PIK3CA mutations were most prevalent (n = 259 of 824, 31.4%) in HR-positive, HER2-negative tumours (OR = 4.13, 95% CI 1.753–9.712). Only 11.7% of TNBC tumours were mutated in PIK3CA (15 of 128). There was no significant association with age, tumour size and nodal status. The characteristics of patients and tumours according to PIK3CA mutation status are reported in Table 1.

Tab. 1 PIK3CA-mutation prevalence (%) in selected clinical and histopathological groups

All	1,123	300	(26.7%)
Age at time of diagnosis
≤ 50	293	68	(23.2%)	1
> 50	830	232	(28.0%)	1.284	0.941–1.751	0.115
Histological Type
ductal	905	241	(26.5%)	2.132	0.993–4.578	0.058
lobular	163	51	(31.3%)	2.675	1.179–6.071	0.019
others	55	8	(14.5%)	1
Tumour size at time of diagnosis
< 2 cm	575	172	(23.0%)	1.4	1.073–1.828	0.013
≥ 2 cm	548	128	(18.9%)	1
Nodal status at time of diagnosis
Node-negative	688	180	(26.2%)	1
Node-positive	435	120	(27.6%)	1.075	0.820–1.407	0.599
Tumour differentiation
G1	154	57	(37.0%)	3.134	1.970–4.986	< 0.000
G2	703	201	(28.6%)	2.135	1.478–3.085	< 0.000
G3	266	42	(15.8%)	1
ER status
ER positive (≥ 1%)	919	278	(30.3%)	3.588	2.255–5.708	< 0.00
ER negative (< 1%)	204	22	(10.8%)	1
PgR status
PgR positive (≥ 1%)	776	245	(31.6%)	2.45	1.769–3.392	< 0.000
PgR negative (< 1%)	347	55	(15.9%)	1
HR status
HR positive	935	279	(29.8%)	3.382	2.103–5.438	< 0.000
HR negative	188	21	(11.2%)	1
HER2 Status
HER2 negative	952	274	(28.8%)	2.254	1.451–3.501	< 0.000
HER2 positive	171	26	(15.2%)	1
IHC-types
HR positive and HER2 negative	824	259	(31.4%)	4.126	1.753–9.712	0.001
HER2 positive and HR positive	111	20	(18.0%)	1.978	0.748–5.231	0.169
HER2 positive and HR negative	60	6	(10.0%)	1
TNBC	128	15	(11.7%)	1.195	0.439–3.250	0.728
CI, confidence interval; HR, hazard ratio bold: significant in prognostic parameters

Association with survival

Overall, we did not observe any significant association of the presence of PIK3CA mutations with RFI (event-free at 5 yrs 90.9% for mutated, 89.9% for wildtype; adjusted HR 1.19, 95% CI 0.752–1.894, Fig. 2A) and OS (alive at 5 yrs 88.2% for mutated, 87.2% for wildtype; adjusted HR 1.08, 95% CI 0.714–1.638, Fig. 2B), neither in univariate analysis nor after adjustment (Table 2, summary for the entire cohort and subgroups is presented in supplementary Table S 2 A, B).

Tab. 2 Univaraite and multivariate analysis of RFI and OS for all patients with regard to selected parameters

Next, we studied the subgroups with the highest frequency of PIK3CA mutations. A part of the hormone receptor-positive and HER2-negative patient group, was treated with aromatase inhibitors (AIs) in monotherapy (n = 220). We detected in a multivariate analysis a significant 3.7 times higher occurrence of RFI-influencing events for patients harbouring a PIK3CA mutation (n = 70) compared to patients with PIK3CA-wildtype (n = 150), (95% CI 1.240-11.112) (Fig. 2C). Regarding OS, PIK3CA mutations were merely associated with a trend towards impaired performance (Fig. 2D). In contrast, no correlation between PIK3CA mutation status and RFI or OS was observed for luminal patients who had been treated with tamoxifen in monotherapy (fig. S1 C, D)).

Considering all patients with a positive steroid hormone receptor status (irrespective of HER2 status, n = 935) 8.5% of the patients with PIK3CA-mutations suffered events within 5 years of RFI analysis as compared to 6.2% with PIK3CA-wildtype (adjusted HR = 1.64, 95% CI 0.958–2.807, p = 0.071). Overall survival probability at 5 years was 88.1% and 90.5%, respectively (adjusted HR = 1.37, 95% CI 0.867–2.152). We found numerically more RFI events at 5yrs in patients with hormone receptor-positive, HER2-negative and PIK3CA mutated tumours than in patients with PIK3CA-wildtype tumours (7.9% and 6.0%, resp., supplementary Fig. S 1 A). Notably, more patients died if their tumours were PIK3CA mutated in this group (11.4% and 8.5%, resp., supplementary Fig. S1 B). However, the effect was not significant, neither in univariate, nor in multivariate analyses.

In the hormone receptor-negative group (irrespective of HER2 status), patients with PIK3CA mutated tumours (n = 21 of 188, 11.2%) showed numerically fewer RFI-events (3 of 21 versus 43 of 167) consistent with a better disease specific outcome (84.4% versus 72.9%; adjusted HR = 0.49, 95% CI 0.152–1.597; supplementary Fig. S 1 C). Similarly, in TNBC, 84% patients were without any BC-related recurrences after five years if they were PIK3CA-mutated compared to only 71.5% for those with wildtype PIK3CA (adjusted HR = 0.43, 95% CI 0.103–1.822, Fig. 2E). These observations were similar after exclusion of patients without adequate (neo)adjuvant therapy. For the HER2-positive group, we did not observe any significant impact of PIK3CA mutations on RFI or OS (supplementary Fig. 1G, H). Interestingly, patients with HR-negative tumours who were either of the TNBC or HER2-positive subtypes experienced a better overall survival if a PIK3CA mutation was detected (Fig. 2F, supplementary Fig. S 1 F, H). The different impact of PIK3CA mutation status on RFI in relation to steroid hormone receptor- and HER2 expression is visualized in the corresponding forest plot (supplementary Fig. S 2).

In order to identify homogenous risk groups with regards to PIK3CA mutation status, we used a recursive partitioning procedure (Fig. 3). In node-negative, HR-positive and undifferentiated (G3) tumours, patients with a PIK3CA-mutation (n = 14) had a worse 5 year-RFI (70.5%) than those with wildtype PIK3CA (5 year RFI 96.4%, HR = 11.92; 95% CI 1.724–82.461, p = 0.012). In contrast, in HR-negative, larger tumours (≥ 2cm), patients with PIK3CA mutations (n = 14) had a better 5yrs-RFI probability (85.7%) than those with the wildtype (5 yrs RFI 66.1%, HR = 2.75; 95% CI 0.657–11.527). The survival differences are substantial and are relevant for 18.9% of all patients.

Association of PIK3CA mutation status to pathologic complete response (pCR) rates

The PIK3CA mutation status was available for 120 tumour samples of TNBC patients receiving NACT, 20 of them with a PIK3CA mutation. Due to the small numbers, only a trend indicating that patients with wildtype tumours reached pCR more often than patients harbouring mutations was observed. No subgroup analysis was possible.

In this study we present the largest analysis of PIK3CA oncogenic mutations to date, using an unselected routine cohort of early stage breast cancer patients (n = 1,123). The aim of our study was to evaluate the prevalence of PIK3CA mutations and their associations with clinical and histopathological parameters as well as clinical outcome. Since the landmark study of Samuels et al. ⁹, it has been known that presence of somatic PIK3CA mutations promote cancer progression also for breast cancer. Most previous publications have reported on heterogeneous sample sets including mixed sets of early stage and metastatic breast cancer patients from retrospective studies. Instead, our findings provide new knowledge about the realistic frequency of PIK3CA mutations overall and in subgroups, as well as their correlation with recurrence free interval and overall survival.

We detected an overall somatic mutation rate of 26.7% (300 of 1,123 samples) when testing the three most common hot spots C775 (H1047R), C763 (E545K), and C760 (E542K) (https://www.mycancergenome.org/), which represent 87% of all mutations currently known in the PIK3CA gene ¹¹. Of interest, the highest frequencies (> 30%) of PIK3CA gene mutations were detected in tumours with more favourable characteristics (G1, G2, ER-positive, PgR-positive, luminal-like), which is in agreement with most other studies and available data in the COSMIC database ¹⁷. In tumours with high risk biology (HER2-positive, TNBC) we found the lowest rate of PIK3CA-mutations (15.2% and 11.4), which is also consistent with published data ¹¹.

These findings provoke the question why PIK3CA mutations are more frequently detected in ER-positive disease. The current state of research postulates that PIK3CA-mutation-dependent activation of AKT phosphorylates and activates ER leading to transcriptional activity of ER in an oestradiol-independent manner and consecutively to preferential growth of ER-positive cancer ^{18, 19}. Thus, mutated PI3K likely promote ER-positive cancer growth and may explain the overrepresentation of PIK3CA-mutated tumours in luminal breast cancer. In addition, PIK3CA-mutations were detected even in small tumours as well as in non-invasive precursor lesions, like DCIS ²⁰, and consequently, as an early event in breast cancer development, PIK3CA-mutations can then be observed often in ER-positive and well differentiated tumours; fast growing ER-negative and undifferentiated tumours, however, may be derived from different precursor cells and independent of activating PIK3CA-mutations.

Prognostic and predictive implications

The second objective of our study was the prognostic impact of PIK3CA mutations, and we did not find any association with recurrence free interval (RFI) or overall survival (OS) within the entire cohort of 1,123 patients. We choose RFI as endpoint since we wanted to analyse the clean disease-related impact of PIK3CA-mutations. We tested an unselected and rather large cohort of early breast cancer patients, thus we assume that our data provide a realistic view, demonstrating a lack of a general impact of PIK3CA mutations on the course of disease in breast cancer. This is in contrast to the published meta-analyses and single studies on PIK3CA mutations that present conflicting results on its association to prognosis; studies found an association to better survival (e.g. Dumont et al., Pang et al. ^{21, 22}) as well as to inferior survival (Sobhani et al., Fan et al.) ^{12, 23}. These divergent results might presumably result from the heterogeneity of the populations that were studied with regard to sample size, subgroups, and type of treatment, so, selection bias cannot be excluded (for review see ²⁴).

The published results are also inconclusive with regard to a potential predictive impact of PIK3CA mutations. However, in our study we found a significant predictive value of PIK3CA mutations in luminal breast cancer by observing more disease-related events in patients with PIK3CA mutations. Most importantly, there was an association to the type of endocrine therapy: We found a significant impact of PIK3CA-mutations on the effect of adjuvant aromatase inhibitors, but no impact on the effect of adjuvant tamoxifen. This observation may be explained by PI3K-triggered estradiol-independent activation of the ER that can be observed in estradiol-deprived situations but might be blocked by ER-modulation by tamoxifen as postulated by Campbell and colleagues ¹⁸. This differential therapy response is also described for advanced BC by Ramirez-Ardila et al.²⁵.

It has to be acknowledged that these relations are complex and other mechanisms are involved. For example, recent findings suggest that PI3K pathway alterations might be associated with the composition of the tumour microenvironment in luminal breast cancer, including the attraction of CD8-positive T-cells ²⁶. Our observations are fully in line with the data of Stemke-Hale and colleagues who also did not find an association between PIK3CA mutations and the effect of adjuvant tamoxifen ²⁷. However, data are again heterogeneous; some authors described resistance to tamoxifen ^{28, 29}, whilst others found significantly improved endocrine sensitivity to tamoxifen if PIK3CA-mutations were detected ³⁰. In summary, with our observations from this cohort study we would suggest to consider tamoxifen as preferred adjuvant endocrine therapy if PIK3CA-mutations are detected.

In patients with HER2 positive breast cancer we found no impact of PIK3CA mutations on RFI. Similarly, a well described pooled analysis of five prospective clinical trials showed no significant impact of PIK3CA mutation on the course of disease in patients with adequately treated HER2 positive BC, although the PIK3CA mutated group had a significantly lower pCR rate ³¹. In an uniformly treated early-stage HER2-positive Danish cohort, the PIK3CA alterations predicted a significantly worse OS (adjusted HR 2.14), but had no significant impact on invasive disease free survival (iDFS) presumably due to the small sample size³².

An exploratory analysis of the CLEOPATRA trial identified a subgroup of HER2 positive PIK3CA-mutated patients who were resistant to anti-HER2-therapy with trastuzumab and pertuzumab (worse OS if mutated, HR multivariate 1.48, p = 0.0025) ³³. Contradictory results may be explained by the continued activation of PI3K and an inhibitory effect on HER2 signalling ³⁴. Thus, in patients with HER2-positive tumours the impact of PIK3CA-mutations is not clear; at least the effects are small and not significant.

The presence of PIK3CA mutations may have a favourable impact in early TNBC, suggested by a 2.3-fold improved RFI and a 3-fold OS, which is in line with Mosele and Takeshita, even though they worked with samples from advanced BC ³⁵ analysing cell-free DNA ³⁶.

To the best of our knowledge, this is the first study using CART in order to find out if combinations of variables could predict the risk of an RFI event. For nearly one fifth of the patients the PIK3CA gene modifications seem to have a significant prognostic impact depending on the HR- status of the tumour. Overall, using the CART algorithms (see Fig. 3) for 121 of 1,123 patients, the gene modification predicts a worse prognosis. Patients with a PIK3CA mutated tumour (n = 20) had lower pCR rates than wildtype tumours. This observation is similar to other studies ³¹.

Our real world data from a multicentre cohort adds exploratory, but valuable information, as our patients were enrolled in the daily clinical routine.

The real overall somatic mutation rate of PIK3CA is 26.7% when testing the three most common hot spots H1047R, E545K, and E542K in a representative cohort of patients with early breast cancer. We did not find an impact of PIK3CA-mutation on RFI in general. As clinical relevant result, we demonstrated resistance of early breast cancer with somatic PIK3CA mutation to adjuvant aromatase inhibitor therapy, suggesting tamoxifen as preferred therapy in these patients. Though only exploratory, this observation is in line with previous observations in metastatic disease. More functional studies are needed to understand the interactions/crosstalk between the activated PI3K signalling pathway and tumorigenesis.

Although this study should be considered exploratory, it has significant strengths like the huge sample size with PIK3CA gene modification status of a prospective, representative cohort, strictly accompanied so that each early breast cancer patient could be enrolled. Therapy adherence to national guidelines was respected. As in most studies, we have analysed only the three most frequent PIK3CA “hot spot” mutations, so a small underrepresentation of the total number of mutations may be possible.

Funding Sources

This work was supported by Wilhelm Roux Program of the Medical Faculty, Martin Luther University Halle Wittenberg (FKZ 25/36) and German Federal Ministry of Education and Research (e:Med FKZ: 031A429).

Competing Interest

The authors have no relevant financial or non-financial interests to disclose.

Acknowledgements

We thank all patients participating the PiA-study and the study nurses Katrin Losse (St. Elisabeth and St. Barbara Halle), Birgit Mauder (Fürth), Andrea Meyer-Kühn (Hildesheim) for excellent documentation.

Author contributions

EJK and MV designed the PiA study; KR, CT, and MV conceived the PIK3CA analysis; KR and MV wrote the manuscript; CU, VK, TL, SP, JJ, MP, CF, FKB, EW, JB, EJK contributed substantial input to the conception and acquisition of the work; KR, KT and SK and carried out DNA mutations experiments, EJK is responsible for statistical evaluation; KR, MV, CT performed data interpretation; all authors revised the manuscript critically

Availability of Data and Materials

The data generated in this study are available within the article and its supplementary data files. Raw data were generated and processed from the authors and are available on request to the corresponding author.

Ethical approval

This study was performed in line with the principles of the Declaration of Helsinki. The study protocol was approved by the Ethical Board of the Medical Faculty of the Martin Luther University Halle Wittenberg. All patients signed a written informed consent

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Editorial decision: Major Revisions Needed
12 Apr, 2022
Reviews received at journal
31 Mar, 2022
Reviewers invited by journal
31 Mar, 2022
Editor invited by journal
30 Mar, 2022
Editor assigned by journal
30 Mar, 2022
First submitted to journal
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PIK3CA mutations in breast cancer

Status:

Version 1

Abstract

Figures

Introduction

Material And Methods

Patient and Tumour Characteristics

Endpoints and Statistical Analysis

DNA extraction and Hotspot Mutation assay

Results

PIK3CA Mutation Prevalence and Association with Clinical and Histopathological Parameters

Association with survival

Association of PIK3CA mutation status to pathologic complete response (pCR) rates

Discussion

Prognostic and predictive implications

Conclusion

Strength & Limitations

Declarations

References

Supplementary Files

Status:

Version 1