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Quantitative Real Time PCR assessment of hormonal receptors and HER2status on fine-needle aspiration pre-operatory specimens from a prospectivelyaccrued cohort of women with suspect breast malignant lesions

Anna Garuti, Ilaria Rocco, Gabriella Cirmena, Maurizio Chiaramondia,Paola Baccini, Massimo Calabrese, Claudia Palermo, Daniele Friedman,Gabriele Zoppoli, Alberto Ballestrero

PII: S0090-8258(13)01342-5DOI: doi: 10.1016/j.ygyno.2013.11.020Reference: YGYNO 975271

To appear in: Gynecologic Oncology

Received date: 21 August 2013Accepted date: 14 November 2013

Please cite this article as: Garuti Anna, Rocco Ilaria, Cirmena Gabriella, ChiaramondiaMaurizio, Baccini Paola, Calabrese Massimo, Palermo Claudia, Friedman Daniele, Zop-poli Gabriele, Ballestrero Alberto, Quantitative Real Time PCR assessment of hormonalreceptors and HER2 status on fine-needle aspiration pre-operatory specimens from aprospectively accrued cohort of women with suspect breast malignant lesions, Gyneco-logic Oncology (2013), doi: 10.1016/j.ygyno.2013.11.020

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http://dx.doi.org/10.1016/j.ygyno.2013.11.020

http://dx.doi.org/10.1016/j.ygyno.2013.11.020

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Title: Quantitative Real Time PCR assessment of hormonal receptors and HER2 status on

fine-needle aspiration pre-operatory specimens from a prospectively accrued cohort of

women with suspect breast malignant lesions

Authors: Anna Garuti1,*, Ilaria Rocco1,*, Gabriella Cirmena1, Maurizio Chiaramondia2, Paola

Baccini3, Massimo Calabrese4, Claudia Palermo1, Daniele Friedman5, Gabriele Zoppoli1,#, and

Alberto Ballestrero1

Affiliations:

1Department of Internal Medicine, Istituto di Ricerca a Carattere Clinico e Scientifico (IRCCS),

Azienda Ospedaliera Universitaria (AOU) San Martino Istituto Nazionale Tumori (IST), Genoa, Italy

2Division of Pathology, Busto Arsizio Hospital, Busto Arsizio (Varese), Italy

3Department of Surgical Sciences and Integrated Diagnostics, University of Genoa and Anatomic

Pathology Service, IRCCS AOU San Martino IST, Genoa, Italy

4Department of Radiology, IRCCS AOU San Martino IST, Genoa, Italy

5Breast Surgery Unit, Department of Surgery, University of Genoa and IRCCS AOU San Martino

IST, Genoa, Italy

*These authors contributed equally to the present study

Running title: Expression of hormonal receptors and HER2 on FNA in breast cancer

#To whom correspondence and re-prints should be sent:

Dr. Gabriele Zoppoli - Department of Internal Medicine and IRCCS AOU San Martino IST - Viale

Benedetto XV 6, 16132 Genoa Italy - E-mail: [email protected] - Tel. +390103537968

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Abstract:

Objectives: Reliable assessment of estrogen, progesterone (ER and PR), and HER2 receptors

status are essential in breast cancer (BC) treatment. Immunohistochemical methods are limited by

intra- and inter-laboratory variability. Furthermore, current methods are not the ideal approach for

reproducing the biological continuum of ER, PR, and HER2 receptor levels, due to their intrinsic,

semi-quantitative nature, relying in part on subjective interpretation.

Methods: In the present study, we tested a molecular approach to define ER, PR, and HER2 status

in fine-needle-aspirate (FNA) samples from patients with early BC. We performed flow cytometry

analysis on 88 FNA specimens from suspect BC patients to determine cellularity. We used

quantitative real time PCR (QRT-PCR) to assess ER, PR, HER2 status, and qPCR for HER2 gene

copy number (GCN).

Results: ER and PR mRNA levels showed a highly significant correlation with IHC data on surgical

samples. qPCR showed greater accuracy than IHC in defining HER2 status. QRT-PCR defined

better than IHC the continuous spectrum of the expression of the assessed receptors. Moreover,

PCR analysis demonstrated a strict correlation between HER2 status and higher levels of its

transcript, correctly stratifying HER2+ and HER2- patients. Finally, there was a strongly significant

agreement between HER2 GCN assessed on FNA specimens by qPCR and FISH data obtained

on pathological tissue specimens.

Conclusions: The present results support a comprehensive approach to determine ER, PR, and

HER2 status by PCR (QRT-PCR and qPCR) in FNA specimens, with high relevance for

therapeutic strategies like neoadjuvant treatment.

Keywords:

Breast cancer, fine needle aspiration, PCR, hormonal receptors, HER2

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Introduction:

Estrogen (ER), progesterone (PR) and HER2 receptors are key factors in determining breast

cancer sensitivity to endocrine and anti-HER2 treatments. As a consequence, accurate and

reliable assessment of ER, PR and HER2 status is of paramount relevance for clinical

management.

Current tests to assess the status of these receptors have some limitations that give rise to

concern in the clinical decision-making setting. Two key points are relevant.

The first problem involves correct patient classification that can alter the predictive value of the

test. In fact, there is considerable intra- and interlaboratory variability in the diagnostic methods

for both the hormone receptor by immunohistochemical (IHC) and HER2 by fluorescence-in-situ-

hybridization (FISH) determination [1, 2].

For example, variability is high in low ER expressing tumors [3] and may potentially interfere with

the correct allocation of patients within the clinically relevant grey zone of incomplete endocrine

responsiveness [4]. Equivocal results may also be obtained through FISH HER2 testing in specific

cases, for example when the values are in the intermediate range, or with polisomy of

chromosome 17 [2].

Secondly, current methods are not the ideal way to reproduce the biological continuum of hormone

and HER2 receptor levels due to their intrinsic, semi-quantitative nature, which depends on the

partially subjective interpretation of the IHC and FISH specimens by the pathologist. The

importance of the quantitative assessment of hormone receptors and HER2 is highlighted by

several clinical trials. Indeed, the quantitative levels of these receptors correlate with patient

outcome after chemo-endocrine or anti-HER2 treatment [5, 6].

These results point out the possible clinical relevance of the quantitative levels of hormone and

HER2 receptor expression rather than the use of an absolute cut-off criterion.

On the basis of the good correlation between the amount of hormone receptors and Her-2 protein

and their transcripts [7], nucleic acid-based molecular analysis may be a useful approach to

overcome the above mentioned limits.

Microarray gene expression profile is a reliable method for the quantitative assessment of ER, PR

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and HER2 mRNA levels in several types of tissue samples, including whole sections, core

biopsies, and fine-needle aspiration biopsies [8, 9].

Quantitative polymerase chain reaction (PCR) is easy, fast, independent of specific reagents like

antibodies, and it has presently reached a high level of sensitivity and accuracy. All these factors

contribute in supporting its use as a reliable method for accurate measurement of gene transcripts.

Therefore, it is now considered the most appropriate method for validating microarray results and

would appear to be a suitable technique to replace microarray technology in diagnostic medical

applications [10].

In the present study, we suggest a comprehensive approach to the definition of ER, PR and HER2

status by both a Real Time quantitative Reverse Transcription PCR (QRT-PCR) method to

measure the mRNA levels of the three receptors and a Real Time quantitative PCR (qPCR)

method to detect the HER2 gene copy number. The tissue samples used for the measurements

were collected by fine needle aspiration (FNA), which is an easy sampling method. FNA allows the

quick collection of cancer cells from fresh tumor tissues and the immediate analysis of intra-cellular

nucleic acids, thus avoiding any bias due to tissue preserving techniques.

Materials and methods

Patients and samples: This study involved 88 prospectively enrolled cases of women with

suspect breast lesions (BI-RADS category 4 or 5) referred to the Breast Diagnostic Center at

IRCCS AOU San Martino IST, Genoa, Italy.

A single experienced cytopathologist performed all FNA procedures using a 23-gauge needle and

a 13 MHz transducer under image-guidance using stereotactic ultrasound.

On the basis of the study design, we performed cytological analysis to determine cell composition,

cytometric evaluation of leukocyte contamination, and quantitative PCR determination of ER, PR

and HER2 status of all FNA samples. Sixty patients confirmed to bear a malignant lesion

(cytological category C5) were included in the present study.

The first 10 patients underwent a single FNA, while the subsequent 50 patients underwent two

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FNA procedures, the first for the conventional cytological diagnosis and the other for the planned

study analyses. Samples for molecular analysis from 10 of the 50 patients who underwent double

aspiration were collected into ATL buffer for total DNA extraction (QIAGEN, Valencia, CA). A small

portion of each aspiration sample (1:10 of the total) was used for cytometric cellular analyses and

the remaining cell suspension was collected into a vial containing 0.5 mL of RNAlater solution

(Ambion, Austin, TX, USA), kept at room temperature for no more than 1 hour and stored at -80°C

until total RNA extraction.

Cell composition of the FNA samples was determined by cell counts of the Diff-Quik-stained slides

using light microscopy at high magnification (X400). In a representative area of the slide, the first

300 cells were classified as tumor cells, leukocytes or stromal cells. The results were recorded as

percentages.

Leukocyte contamination was also analyzed by flow cytometry by evaluating two cell surface

antigens, i.e., CD19 and CD45, which are typically expressed in leukocytes.

Ethics committee approval was granted (n°6/2010 director: Dr. Luigi Francesco Meloni).

Determination of hormonal-receptors and HER2 status in tumor specimens: Hormonal

receptor and HER2 status for all study patients was determined by IHC on formalin-fixed, paraffin-

embedded tumor samples that were examined after surgical removal. Hormonal status was

identified by monoclonal antibodies 1D5 (M7047, Dako, Glostrup, Denmark) and 1A6 (M3569

Dako, DK-2600 Glostrup Denmark), respectively for ER and PR. Samples showing nuclear staining

in at least 10% of cancer cells were defined as positive for ER or PR receptors. The Allred score,

which is a composite of the percentage of cells that stained and the intensity of their staining, was

also determined for all patients. The percentage of stained cells is classified from 0 through 5,

while the intensity of cell staining is rated as 1, 2 or 3 [11]. DAKO HercepTest kit (polyclonal

antibody DA485, K5206, Dako DK-2600 Glostrup Denmark), a Food and Drug Administration

(FDA) approved specific reagent, was used to determine the amplification status of HER2. Tumors

with a score of 1+, 2+ or 3+ were confirmed by FISH (PathVision kit, Vysis AutoVysion Downers

Grove, IL, USA). A positive HER2 test was defined according to ASCO guidelines [2].

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RNA isolation, cDNA and QRT-PCR: RNA was extracted from 50 of the 60 FNA samples that

were taken, using the RNAeasy Micro Kit (Qiagen, Valencia, CA) according to the manufacturer’s

instructions.The amount and quality of RNA were assessed using ExperionTM RNA HighSens

Chips (Bio-Rad, Hercules CA). Total RNA was reverse transcribed using random hexamers in a

final volume of 50µl as previously described [12]. The expression levels of the target genes, ER,

PR and HER2, were measured in each sample by QRT-PCR amplification performed with TaqMan

Gene Expression Assays products in an ABI PRISM 7900 HT Fast Real Time PCR System

(Applied Biosystem Monza, Italy). RPLP0 was used as the internal control gene. Primers and

probes were obtained from Applied Biosystems (Applied Biosystems, Monza, Italy). All

measurements were performed in triplicate. The following QRT-PCR run protocol was used:

activation Taq program (50°C for 2 min and 95°C for 10 min), amplification and quantification

program repeated 50 times (95°C for 15 sec, 60°C for 1 min with a single fluorescence

measurement). In the 6 cases with low extraction yield, RNA was linearly amplified with a

PreAmplified Kit (Applied Biosystems, Monza, Italy) as previously described [13]. QRT-PCR

reaction was carried out in a final volume of 20µl starting from 2µl of cDNA.

DNA isolation and qPCR: Genomic DNA was extracted from 10 of the 60 FNA samples that were

taken, using the DNeasy Blood & Tissue Kit (Qiagen, Valencia, CA) according to the manufactures

instructions. The amount and quality of DNA were assessed with NanoDrop ND-1000 (Celbio, MI,

Italy). Then, 5ng of DNA were used for qPCR amplification with TaqMan system (ABI PRISM 7900

HT Fast Real Time PCR System, Applied Biosystems Monza, Italy) for HER2 and APP,

respectively as the target and internal control genes; the reaction was performed in a final volume

of 20 µl. HER2 and APP primers and probes were derived from the literature [14]. All

measurements were performed in triplicate. The following qPCR run protocol was used: activation

Taq program (50°C for 2 min and 95°C for 10 min), amplification and quantification program

repeated 40 times (95°C for 15 sec, 60°C for 1 min with a single fluorescence measurement).

In each experiment, DNA extracted from MCF7 and SKBR-3 breast cancer cell lines was included

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as the control for non-amplified and amplified HER2 genes, respectively.

The DNA samples from thirty-five healthy volunteers were used to calculate the cut-off values for

amplification (range 0.91-1.48) [15]. Values below 1.5 were considered as non-amplified, those

greater than 2.0 were considered as amplified, and those greater than 5.0 were scored as high-

level amplification.

Data Analysis: We used the comparative cycle threshold (Ct) method to determine both the

amount of mRNA encoded by each target gene and the HER2 gene copy number [16]. In

particular, the changes in the target gene expression levels measured in tumor samples as

compared to normal mammary tissue were calculated according to the 2-ΔΔCt method using the

following formula:

ΔΔCt = (Ct,Target gene - Ct,control gene) breast tumor - (Ct,Target gene - Ct,control gene) normal

breast.

RPLP0 was the internal control gene. HER2 gene copy number was also measured from qPCR

data according to the previous formula where HER2 is the target gene and APP is the internal

control gene. Human Mammary Gland RNA (Clontech laboratories Inc) and Human genomic DNA

(Clontech laboratories Inc) were used as calibrators for gene expression assay and gene copy

number determination, respectively. Cut-off values discriminating negative from positive ER, PR

and HER2 mRNA measurements were determined by receiver operating characteristic (ROC)

analysis. Correlations between ER and PR mRNA expression and IHC hormonal-receptor status

and between HER2 mRNA expression and IHC/FISH HER2 status were calculated by Spearman’s

rank correlation coefficient.

Results

Cellular composition of FNA samples: Samples of cells collected by echo-guided FNA from 60

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consecutively screened women with suspect malignant nodules were analyzed. The first ten

samples were obtained from a single FNA that was split into two parts for diagnostic cytology and

study analyses, respectively. However, the number of cells that were obtained from these samples

was too small to allow us to perform all the planned cellular and molecular analyses, i.e., cytology,

flow cytometry and PCR. Therefore, the subsequent 50 patients underwent two FNAs, the second

of which was entirely dedicated to the planned analyses. The percentage of tumor cells in these 50

samples, as evaluated by cytological examination, was equal to or greater than 80% (mean

91.51%, median 97.76 % range 80-99.8 %), which suffices for molecular evaluation [17]. The

mean value of leukocyte contamination of the sample, as determined by flow cytometry, was =

1.98% (range 0.3-14.3).

RNA and DNA total extraction: RNA and DNA extractions were performed on 40 and 10 of the

50 dedicated FNA samples, respectively. The total amount of RNA we extracted from 34 out of 40

samples was equal to or greater than 100ng (mean 1021ng, range 108-4035ng), which was

enough for the direct QRT-PCR measurement of ER, PR and HER2 gene expression. Total RNA

yield in the other 6 samples was below 100ng, mean value 28ng (range 5-91ng), so these samples

underwent a pre-amplification procedure before PCR analysis. DNA extraction performed on the

10 FNA samples dedicated to HER2 gene copy number determination yielded enough high quality

DNA to allow qPCR analysis to be carried out. In fact, the mean value of total DNA extraction per

sample was 510ng (range 218-1719ng).

RNA pre-amplification: RNA pre-amplification was carried out on the 6 cases that had a total

RNA yield below 100ng. It is noteworthy that this procedure can be performed in the range from 1

to 250ng of extracted RNA [18, 19]. The pre-amplification procedure was successful in all cases

and allowed us to obtain an RNA yield that was suitable for QRT-PCR analysis. In particular, we

obtained more than 400ng of amplified RNA for each case, which proved to be sufficient to perform

not only the planned PCR analysis of the three target genes but also multiple gene assays, such

as low density array. Therefore, the RNA pre-amplification technique allowed us to perform

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molecular analysis on all FNA samples.

Evaluation of ER and PR status by molecular analysis: ER and PR mRNA expression levels

were measured in all 40 FNA samples by QRT-PCR. The results represent the quantitative

measurement of gene expression in tumor cells relative to the normal breast tissue used as a

calibrator. In each case, QRT-PCR expression data were compared to the matched IHC data

obtained from surgically removed primary tumors.

When mRNA expression data are listed as a monotonic series there is a ROC analysis-determined

cut-off value that discriminates negative subgroups from positive ones (Fig 1a and 1b). In fact,

there was perfect agreement (kappa test = 1) between IHC and QRT-PCR in classifying patients

into negative and positive groups. Therefore, routine IHC and FNA QRT-PCR carried out on

patients enrolled in this study performed just as well in the dichotomous allocation of patients with

regards to ER and PR.

According to IHC analysis, ER and PR data can be considered both as a continuous variable,

when gathered as a percentage of staining cells, or as a discrete variable, when gathered

according to Allred’s score. When considered as continuous variables, IHC data show a highly

significant correlation with ER and PR mRNA levels. In fact, the Spearman correlation coefficients

were 0.85 (95%CI 0.73-0.92; p<0.0001) and 0.88 (95%CI 0.77-0.93; p<0.0001), for ER and PR,

respectively. However, by considering the distribution of the mRNA values within each

homogeneous percentage class as defined by IHC, it is noteworthy that within each class there

was a wide range of values which reached a maximum of 70- and 8-fold for ER and PR,

respectively. This also implies some overlapping of mRNA values when ranked according to the

percentage of IHC positive cells (Fig 2a and 2b). For instance, in the group with 50% or higher

positive staining for ER, that conventionally defines patients who are highly responsive to

endocrine therapy, the quantity values were scattered over a 100-fold wide range. These data

suggest that in terms of clinical behavior, apparently IHC homogeneous patients could encompass

various degrees of hormonal dependency.

Similar observations can be made if one considers ER and PR IHC data as discrete variables

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according to Allred’s score that integrates the percentage of positive-staining tumor cells with

staining intensity. Despite the high correlation between the mRNA levels and Allred’s score,

correlation coefficients of 0.82 (95%CI 0.68-0.90; p < .0001) and 0.90 (95%CI 0.82-0.95,

p<0.0001) for ER and PR, respectively, there is a wide distribution of the mRNA values within each

homogeneous Allred’s class (Fig 3a and 3b).

Although derived from a small patient group, the present data suggest that quantitative molecular

analysis may be more accurate than semi quantitative IHC to describe the continuous spectrum of

variation of ER and PR into tumor tissue.

HER2 status definition by molecular analysis: In the present series of 50 patients, 44 were

HER2 negative according to IHC (score 0 in 28, 1+ in 12 and 2+ in 4), while only 6 patients were

HER2 positive (score 3+). FISH analysis was performed on all patients whose IHC scores were 1+

or more and confirmed IHC results. The amplification status of the HER2 gene can be molecularly

assessed by both mRNA level measurement and DNA gene copy number. HER2 mRNA

expression in the 40 patients evaluated by QRT-PCR shows a cut-off value, as determined by

ROC analysis, that discriminates negative subgroups from positive ones (Fig 1c). In fact the

molecular test was able to cluster these patients into two definitely distinct groups, corresponding

to amplified and non-amplified cases (Fig 4a). The difference between these two groups is highly

significant (p = 0.001) and very robust. As a matter of fact, positive patients had a median value of

84.6, which was more than 25 times the median value of negative patients. In the HER2 negative

patient group, no significant differences were observed between the three IHC score groups that

define the negative status (Fig 4b).

The HER2 gene was found to be amplified in two of the 10 patients evaluated by qPCR, 2.39 and

7.99 gene copies respectively, whereas it was not amplified in the remaining patients. These

results were in agreement with the IHC/FISH and mRNA analysis (Table 1), performed on surgical

samples as a control. Thus, the arranged use of the two molecular tests, copy number and gene

expression analysis, could be a robust way to correctly define the HER2 status in virtually all breast

cancer patients.

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Discussion

FNA is an easy sampling method that allows for the collection of enriched cancer cells and the

direct analysis of nucleic acids, thereby avoiding any bias due to tissue preserving techniques.

Currently existing methods allow for extraction of high-quality total RNA, which is suitable for cDNA

microarray or qPCR analysis from more than 70% of FNA samples [9, 20].

In the present study, we report how FNA, performed by a trained cytopathologist, allowed us to

collect a percentage of tumor cells > 80% in all the analyzed patients, with a total RNA or DNA

yield > 100ng in the majority of cases. Six patients in our series had an RNA yield below 100ng.

However, this number of inadequate samples is likely overestimated because of the initial study

design that foresaw the use of part of the same FNA sample for both cytometric cellular analyses,

and the consequent low quantity of material suitable for RNA extraction without additional, time-

and money-consuming pre-amplification steps. These six patients underwent molecular analysis

after linear RNA amplification, developed in collaboration with Applied Biosystems Inc [13].

In the present study, we tested a molecular approach for defining simultaneously hormonal

receptors and HER2 status in FNA specimens from patients with suspect early breast cancer. This

approach is not new, since other Authors demonstrated the feasibility and valuable results of HER2

and/or hormone receptor status determination on diagnostic samples, either by PCR [21, 22] or

IHC-based methods [23]. However, our results expand such analysis to include ER and PR

determination by QRT-PCR. The results we obtained demonstrate that QRT-PCR performs

optimally in the dichotomous allocation of patients with respect to both ER and PR status in FNA

specimens. Of interest, unlike conventional IHC, QRT-PCR was able to discriminate between

positive and negative samples without discordant results even in spite of the small available

material (Fig 1a and 1b). Using a QRT-PCR approach, Uzan et al. [20] also reported a low

discordance rate of roughly 10% in ER status determination. These results were obtained in two

small patient groups. However, similar results were obtained [8] by using cDNA microarray in a

larger data set, confirming the validity of nucleic acid determination in pre-operatory samples.

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In order to reach high performance levels, QRT-PCR methods applied to FNA samples have to

fulfill several conditions. In particular, a trained cytopathologist must be available to perform FNA,

to carry out the microscopic evaluation of the collected cells, and to follow material collection

protocols correctly. Ideally, such as in our experience, a single cytopathologist should be the

referent for such studies to avoid inter-operator variability. Furthermore, mRNA transcript

assessment by QRT-PCR requires a suitable reference gene and an adequate calibrator tissue

[16]. We chose RPLP0 as a reference gene for data normalization, since we found it to be stably

expressed in both breast carcinomas and in normal breast tissue (our unpublished observations

and [24]). The reliability of hormonal receptors and HER2 status definition by standard immune

staining-based methods and FISH techniques is suboptimal in terms of both intra- and inter-

laboratory reproducibility, as well as for true quantitative assessment of protein expression [25].

These limitations raise some concerns in clinical practice due to the critical role of these

parameters in determining treatment strategies, especially in the neoadjuvant setting. Therefore,

accurate pre-operatory testing of hormonal receptors and HER2 status with a reliable method such

as PCR is of high clinical relevance [4]. The use of technique is an attractive and potentially cost-

effective way to overcome the limits of current, standard analytical methods. Moreover, while FNA

is an easier and more common sampling methodology than core-biopsy procedures, it is scarcely

amenable to FISH analysis.

Several authors have proposed the use of cDNA microarray technology for the molecular

characterization of breast cancer samples derived from FNA [8, 9, 26, 27]. In particular, in a large

series of patients [8], Gong et al. showed that the DNA microarray technique is able to reliably

determine ER and HER2 status and can provide better quantification than currently available,

semi-quantitative methods (like IHC and FISH) thus leading to an increase in the predictive value

of the test with regards to the possible clinical benefits. However, microarray technologies appears

less cost-effective than PCR for the determination of single genes, show strong inter-operator and

inter-platform variability, are not widely available, and require dedicated bioinformatical personnel,

thus not appearing as an easily applicable methodology for routine diagnostics. On the contrary,

PCR is easily standardizable, fast, and does not depend on costly and difficult-to-handle reagents.

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Furthermore, it has presently reached high levels of sensitivity and accuracy, which strongly

support its use in clinical practice.

In our experience, although ER and PR mRNA levels had a highly significant correlation with IHC

data, QRT-PCR was able to detect wider expression ranges for both genes than IHC (either

expressed as a percentage of stained cells or as Allred’s score, see Fig 2a and 2b). These data

suggest that QRT-PCR describes the continuous spectrum of these receptors more accurately

than IHC. Also, the cost of commercially available QRT-PCR primers has been steadily declining

over the last few years, so much so that it is often comparable with, when not smaller than the

price of IHC-grade antibodies. Hence, when the correlation of transcripts and their protein products

is very high, and detecting post-translational protein modifications is not the purpose of an

experiment, we believe the use of QRT-PCR as performed in our study is actually cost-effective

compared to traditional immune-based cyto- or histochemistry.

Specific quantitative assessment of ER and PR expression is of potentially great clinical value for

several reasons. It can improve the accuracy of hormonal status definition by reducing the negative

effect of the intra- and inter-laboratory variability observed in IHC methods [1, 28]. Furthermore, the

QRT-PCR approach might allow us to define a more reliable cut-off to predict endocrine response

in the patients within the “grey zone” of incomplete endocrine responsiveness [4, 29]. Finally, more

precise measurement of hormone receptor quantitative levels may also improve their prognostic

and predictive value, as suggested by recent results from the central evaluation of randomized

clinical trials that established a significant relationship between IHC values and patient outcome [5,

30].

Concerning HER2 status assessment, our data support the usefulness of the molecular test. In our

patient series, QRT-PCR analysis correctly identified the four HER2 positive patients, whose

mRNA levels were on average 25 times higher than HER2 negative patients. Furthermore, the

gene copy number measured on FNA specimens was consistent with the mRNA and FISH data

obtained on pathological tissue samples in the ten additional patients who underwent qPCR

analysis (see Table1). Of course, these results require validation in larger patient cohorts.

However, they represent the proof of principle for a double test approach combining both DNA

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copy number and mRNA transcript quantification. This approach may in part overcome the present

limitations of IHC and FISH tests, in particular by resolving the issue of equivocal results due to

both borderline FISH results and polysomy 17 (Garuti A. et al., manuscript in preparation).

In conclusion, the present results support a comprehensive PCR-based approach to define

hormone receptor and HER2 status in pre-operatory FNA specimens. To our knowledge, the

present work is one of the few prospectively enrolled FNA sets tested by PCR for both hormone

receptors and HER2 status. This approach has strong clinical implications in the neoadjuvant

treatment setting. Furthermore, our results point toward a high agreement with conventional

analyses performed on surgical tissues, and may even be superior in specific settings, by

combining qPCR with QRT-PCR for HER2 status determination. Nonetheless, we are aware that

our results are preliminary and require validation in larger patient cohorts.

Abbreviations:

estrogen (ER),

progesterone (PR),

immunohistochemical (IHC)

fine-needle aspirates (FNA)

Real Time quantitative Reverse Transcription PCR (QRT-PCR)

Real Time quantitative PCR (qPCR)

fluorescence in situ hybridization (FISH)

cycle threshold (Ct)

Acknowledgments:

This study was mainly supported by grants from the Associazione Italiana per la Lotta alle

Leucemie e ai Linfomi (AIL), from Compagnia del San Paolo and Banca CARIGE. GZ is supported

by an AIRC “My First AIRC Grant” no. MFAG10570. We wish to thank Dr. P. Ciotti, Dr. P. Mandich

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(Department of Neuroscience, Ophthalmology and Genetics, Section of Medical Genetics,

University of Genoa, Genoa, Italy), Dr. F. Murelli (Breast Surgery Unit, Department of Surgery,

University of Genoa and IRCCS AOU San Martino IST, Genoa, Italy), Dr. E. Carminati

(Department of Internal Medicine, Istituto di Ricerca a Carattere Clinico e Scientifico (IRCCS),

Azienda Ospedaliera Universitaria (AOU) San Martino Istituto Nazionale Tumori (IST), Genoa,

Italy).

Authors’ Contributions:

AG, IL,, GC, CP and participated in the study design, performed the analyses, and carried out the

experiments on RNA and DNA; PB, DF and FM supplied patients, performed FNA and evaluated

the IHC and FISH analysis; MC performed ultrasound image-guidance; GZ and EC analyzed data;

PM, GZ and AB wrote the manuscript. All authors have read and approved the final manuscript.

Legends:

Table 1: HER2 status in tissue samples as determined by IHC/FISH, gene expression and gene

copy-number in FNA samples. In the copy number analysis, values smaller than 1.5 were

considered as non-amplified, whereas those greater than 2.0 were considered as amplified. HER2

expression was performed on FFPE samples and HER2 gene was considered over-expressed

when the mRNA value was greater than 2.5.

Figure 1: Rank ordered mRNA expression levels of ER (a), PR (b) and HER2 (c). Each sample is

identified as negative (green column) or positive (red column) according to IHC for ER and PR

status and according to IHC and FISH for HER2 status. Horizontal dotted lines indicate the

threshold values as defined by ROC curves that discriminate patient subgroups optimally: ER =

0.11; PR = 0.28; HER2=30.

* This data is out of scale, the real value is 138.1

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Figure 2: ER (a) and PR (b) mRNA level distribution according to percentage of positive cells as

determined by IHC. * This data is out of scale, the real value is 138.1.

Figure 3: ER (a) and PR (b) mRNA level distribution according to Allred’s classes. * This data is

out of scale, the real value is 138.1.

Figure 4: Box and whiskers plot showing the polarization of mRNA measurements between HER2

negative and positive samples. Analysis was performed according to dichotomic (a) or IHC (b)

groups. Rectangles=IQR (25th and 75th percentiles). Horizontal line within rectangle=median.

Outer boundary brackets=2·5th and 97·5th percentiles.

Conflicts of interest:

None declared.

Bibliography:

[1] Rudiger T, Hofler H, Kreipe HH, Nizze H, Pfeifer U, Stein H, et al. Quality assurance in

immunohistochemistry: results of an interlaboratory trial involving 172 pathologists. The American

Journal of Surgical Pathology. 2002;26:873-82.

[2] Wolff AC, Hammond ME, Schwartz JN, Hagerty KL, Allred DC, Cote RJ, et al. American Society

of Clinical Oncology/College of American Pathologists guideline recommendations for human

epidermal growth factor receptor 2 testing in breast cancer. Archives of Pathology & Laboratory

ACC

EPTE

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17

Medicine. 2007;131:18-43.

[3] Rhodes A, Jasani B, Barnes DM, Bobrow LG, Miller KD. Reliability of Immunohistochemical

demonstration of oestrogen receptors in routine practice: interlaboratory variance in the sensitivity

of detection and evaluation of scoring systems. Journal of Clinical Pathology. 2000;53:125-30.

[4] Goldhirsch A, Wood WC, Gelber RD, Coates AS, Thurlimann B, Senn HJ. Progress and

promise: highlights of the international expert consensus on the primary therapy of early breast

cancer 2007. Annals of Oncology. 2007;18:1133-44.

[5] Dowsett M, Allred C, Knox J, Quinn E, Salter J, Wale C, et al. Relationship between quantitative

estrogen and progesterone receptor expression and human epidermal growth factor receptor 2

(HER-2) status with recurrence in the Arimidex, Tamoxifen, Alone or in Combination trial. Journal

of Clinical Oncology. 2008;26:1059-65.

[6] Krop IE, Winer EP. Ten years of HER2-directed therapy: still questions after all these years.

Breast Cancer Research and Treatment. 2009;113:207-9.

[7] Brown PO, Botstein D. Exploring the new world of the genome with DNA microarrays. Nature

Genetics. 1999;21:33-7.

[8] Gong Y, Yan K, Lin F, Anderson K, Sotiriou C, Andre F, et al. Determination of oestrogen-

receptor status and ERBB2 status of breast carcinoma: a gene-expression profiling study. The

Lancet Oncology. 2007;8:203-11.

[9] Symmans WF, Ayers M, Clark EA, Stec J, Hess KR, Sneige N, et al. Total RNA yield and

microarray gene expression profiles from fine-needle aspiration biopsy and core-needle biopsy

samples of breast carcinoma. Cancer. 2003;97:2960-71.

[10] Perez-Diez A, Morgun A, Shulzhenko N. Microarrays for cancer diagnosis and classification.

Advances in Experimental Medicine and Biology. 2007;593:74-85.

[11] Harvey JM, Clark GM, Osborne CK, Allred DC. Estrogen receptor status by

immunohistochemistry is superior to the ligand-binding assay for predicting response to adjuvant

endocrine therapy in breast cancer. Journal of Clinical Oncology. 1999;17:1474-81.

[12] Ballestrero A, Coviello DA, Garuti A, Nencioni A, Fama A, Rocco I, et al. Reverse-

transcriptase polymerase chain reaction of the maspin gene in the detection of bone marrow breast

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

18

carcinoma cell contamination. Cancer. 2001;92:2030-5.

[13] Ciotti P, Garuti A, Ballestrero A, Cirmena G, Chiaramondia M, Baccini P, et al. Reliability and

reproducibility of a RNA preamplification method for low-density array analysis from formalin-fixed

paraffin-embedded breast cancer samples. Diagnostic Molecular Pathology. 2009;18:112-8.

[14] Lehmann U, Glockner S, Kleeberger W, von Wasielewski HF, Kreipe H. Detection of gene

amplification in archival breast cancer specimens by laser-assisted microdissection and

quantitative real-time polymerase chain reaction. The American Journal of Pathology.

2000;156:1855-64.

[15] Konigshoff M, Wilhelm J, Bohle RM, Pingoud A, Hahn M. HER-2/neu gene copy number

quantified by real-time PCR: comparison of gene amplification, heterozygosity, and

immunohistochemical status in breast cancer tissue. Clinical Chemistry. 2003;49:219-29.

[16] Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time

quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods (San Diego, Calif). 2001;25:402-8.

[17] Wang Y, Klijn JG, Zhang Y, Sieuwerts AM, Look MP, Yang F, et al. Gene-expression profiles

to predict distant metastasis of lymph-node-negative primary breast cancer. Lancet. 2005;365:671-

9.

[18] Li J, Smyth P, Cahill S, Denning K, Flavin R, Aherne S, et al. Improved RNA quality and

TaqMan Pre-amplification method (PreAmp) to enhance expression analysis from formalin fixed

paraffin embedded (FFPE) materials. BMC Biotechnology. 2008;8:10.

[19] Mengual L, Burset M, Marin-Aguilera M, Ribal MJ, Alcaraz A. Multiplex preamplification of

specific cDNA targets prior to gene expression analysis by TaqMan Arrays. BMC Research Notes.

2008;1:21.

[20] Uzan C, Andre F, Scott V, Laurent I, Azria E, Suciu V, et al. Fine-needle aspiration for nucleic

acid-ased molecular analyses in breast cancer. Cancer. 2009;117:32-9.

[21] Susini T, Bussani C, Marini G, Nori J, Olivieri S, Molino C, et al. Preoperative assessment of

HER-2/neu status in breast carcinoma: the role of quantitative real-time PCR on core-biopsy

specimens. Gynecologic Oncology. 2010;116:234-9.

[22] Konofaos P, Kontzoglou K, Parakeva P, Kittas C, Margari N, Giaxnaki E, et al. The role of

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

19

ThinPrep cytology in the investigation of ki-67 index, p53 and HER-2 detection in fine-needle

aspirates of breast tumors. Journal of BUON : official journal of the Balkan Union of Oncology.

2013;18:352-8.

[23] Tamaki K, Sasano H, Ishida T, Miyashita M, Takeda M, Amari M, et al. Comparison of core

needle biopsy (CNB) and surgical specimens for accurate preoperative evaluation of ER, PgR and

HER2 status of breast cancer patients. Cancer science. 2010;101:2074-9.

[24] Lyng MB, Laenkholm AV, Pallisgaard N, Ditzel HJ. Identification of genes for normalization of

real-time RT-PCR data in breast carcinomas. BMC Cancer. 2008;8:20.

[25] Hayes DF. Quantitative measures of oestrogen receptor and ERBB2 expression. The Lancet

Oncology. 2007;8:184-5.

[26] Assersohn L, Gangi L, Zhao Y, Dowsett M, Simon R, Powles TJ, et al. The feasibility of using

fine needle aspiration from primary breast cancers for cDNA microarray analyses. Clinical Cancer

Research. 2002;8:794-801.

[27] Sotiriou C, Powles TJ, Dowsett M, Jazaeri AA, Feldman AL, Assersohn L, et al. Gene

expression profiles derived from fine needle aspiration correlate with response to systemic

chemotherapy in breast cancer. Breast Cancer Research. 2002;4:R3.

[28] Roche PC, Suman VJ, Jenkins RB, Davidson NE, Martino S, Kaufman PA, et al. Concordance

between local and central laboratory HER2 testing in the breast intergroup trial N9831. Journal of

the National Cancer Institute. 2002;94:855-7.

[29] Increasing steroid hormone receptors expression defines breast cancer subtypes non

responsive to preoperative chemotherapy. Breast Cancer Research and Treatment. 2009;116:359-

69.

[30] Chemoendocrine compared with endocrine adjuvant therapies for node-negative breast

cancer: predictive value of centrally reviewed expression of estrogen and progesterone receptors--

International Breast Cancer Study Group. Journal of Clinical Oncology. 2008;26:1404-10.

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Figure 1

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Figure 2

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Figure 3

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Figure 4

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Table 1

Patient IHC / FISH HER2 expression

(mRNA)

HER2 gene (copy

number)

1 neg / neg neg (0.68) non amplified (0.91)



4 pos / pos pos (31.37) amplified (7.99)




8 pos / pos pos (6.76) amplified (2.39)



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Highlights:

Hormone receptors and Her2 are essential biomarkers in breast cancer; which we

assessed using PCR in fine needle aspiration samples

By combining flow cytometry and PCR we obtain extremely accurate results with extremely

small samples using an inexpensive technique

Our approach is extremely feasible and has high clinical relevance, especially in breast

cancer neoadjuvant treatment

35 Quantitative Real Time FNAC

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