M O L E C U L A R O N C O L O G Y 1 0 ( 2 0 1 6 ) 1 4 3 0 e1 4 3 6
available at www.sciencedirect.com
ScienceDirect www.elsevier.com/locate/molonc
Concordance between HER-2 status determined by qPCR in Fine Needle Aspiration Cytology (FNAC) samples compared with IHC and FISH in Core Needle Biopsy (CNB) or surgical specimens in breast cancer patients Claudia Rodrigueza,1, Voichita Suciub,1, Audrey Poteriec,1, Ludovic Lacroixb,d, Isabelle Mirand, Amelie Boichardb,d, Suzette Delalogee, Jacqueline Deneuvef, Sandy Azoulayb, Marie-Christine Mathieub, Alexander Valentb, Stefan Michielsc,g,1, Monica Arnedose,h,1, Philippe Vielhb,d,*,1 a
Oncologia Medica, Centro Universitario Contra el Cancer, Universidad Autonoma de Nuevo Leon, Mexico Departement de Biologie et Pathologie Medicales, Gustave Roussy Cancer Campus, Villejuif, France c Service de Biostatistique et d’Epidemiologie, Gustave Roussy Cancer Campus, Villejuif, France d Laboratoire de Recherche Translationnelle et Centre de Ressources Biologiques, AMMICA, INSERM US23/CNRS UMS3655, Gustave Roussy Cancer Campus, France e Departement de Medecine, Gustave Roussy Cancer Campus, Villejuif, France f Service des Operations de Recherche Clinique, Gustave Roussy Cancer Campus, Villejuif, France g INSERM U1018, CESP, Universite Paris-Sud, Universite Paris-Saclay, Villejuif, France h INSERM U981 et Universite Paris-Sud, Universite Paris-Saclay, Villejuif, France b
A R T I C L E
I N F O
A B S T R A C T
Article history:
Determining the status of HER2-neu amplification and overexpression in breast cancer is
Received 10 March 2016
crucial for prognosis but mostly for treatment purposes. Standard techniques include the
Received in revised form
determination of IHC in combination with in situ hybridization techniques to confirm a
13 June 2016
HER2-neu amplification in case of IHC2þ using either a core-needle biopsy or a surgical
Accepted 20 July 2016
specimen. qPCR has been also demonstrated to be able to determine HER2 status, mostly
Available online 5 August 2016
in core biopsies or in surgical specimens. Fine-needle aspiration is a reliable, quicker and less invasive technique that is widely used for diagnosis of invasive breast cancer. In
Keywords:
this study, we assessed the performance of qPCR in invasive breast carcinomas to deter-
Breast cancer
mine HER2-neu status by using fine-needle aspiration samples and comparing to standard
Fine needle aspiration
IHC and FISH. From a total of 154 samples from patients who had nodular breast lesions
Cytology
and attended the 1-day-stop clinic at the Gustave Roussy from March 2013 to October
HER2
2014, qPCR was able to determine the HER2 status in a mean of 3.7 days (SD 3.1). The overall
qPCR
concordance with standard HER2-testing was very high: 97% (95% CI 0.94 to 0.99); sensitivity was 96% (0.87e1), specificity 98% (0.95e1) and positive and negative predictive values 88% (0.75e1) and 99% (0.98e1), respectively.
partement de Biologie et Pathologie Me dicales, Gustave Roussy Cancer Campus, 114, rue Edouard Vaillant, * Corresponding author. De 94805 Villejuif Cedex, France. Fax: þ331 4211 5263. E-mail address: [email protected] (P. Vielh). 1 These authors contributed equally to the work. http://dx.doi.org/10.1016/j.molonc.2016.07.009 1574-7891/ª 2016 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
M O L E C U L A R O N C O L O G Y 1 0 ( 2 0 1 6 ) 1 4 3 0 e1 4 3 6
1431
In conclusion, our study demonstrates that qPCR performed using fine-needle aspiration samples from a primary tumour is a reliable and fast method to determine HER2/neu status in patients with early breast cancer. ª 2016 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
1.
Introduction
Breast cancer is the most common malignancy among women, corresponding to 25% of all cancers and representing the second cause of cancer death in developed countries (GLOBOCAN, 2012). Breast cancer is a heterogeneous disease categorized into different subtypes based on expression of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor-2 (HER-2), that have an impact both in prognosis and treatment (NCCN, 2014). HER-2 amplification and/or overexpression are present in around 10e25% of invasive breast carcinomas (Slamon et al., 1989). Tumours harbouring a HER2 amplification are more aggressive and present with worse prognosis. The use of targeted therapy against HER2, like the monoclonal antibody trastuzumab (Herceptin, Roche), has markedly improved this poor prognosis revolutionizing the treatment of this breast cancer subtype (Slamon et al., 2001). HER2 status at diagnosis might be crucial for treatment purposes as neo-adjuvant chemotherapy in combination with an anti-HER2 therapy can achieve a pathological complete response (pCR) rate of around 50% (Nagayama et al., 2014) (de Azambuja et al., 2014). It has been previously shown that HER2 status at biopsy is highly concordant with HER2 status at the surgical specimen (Arnedos et al., 2009). There are several techniques to determine HER-2 status at a protein, ribonucleic acid (RNA) and/or deoxyribonucleic acid (DNA) level. It can be assessed on the protein level using immunohistochemistry (IHC) to detect the amount of HER-2 protein expressed on the cancer cell surface. This method relies on a qualitative scoring system which is prone to interobserver variability, so false-positive results are possible (Wolff et al., 2013). Assessment by the DNA level can be done by fluorescence in situ hybridization (FISH), in order to detect amplification of the HER-2/neu gene. Finally, the assessment by RNA level can be done using quantitative real-time polymerase chain reaction (qPCR) which allows determination of gene amplification (Bergqvist et al., 2007). This last method is simple, quick and highly automated, producing numerical results for its interpretation. Nowadays, the American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) recommendations state a combined approach of IHC and/or FISH analysis as the current reference method for the detection of HER-2 amplification in breast cancer specimens (Wolff et al., 2013). One of the reasons for these guidelines are due to the discordance observed in HER2 status in several clinical trials between local and central pathology laboratories (Paik et al., 2002). Several studies have investigated the potential use of
alternative techniques to determine HER2 status in a more efficient, quicker and less expensive way (Penault-Llorca et al., 2009), including the use of qPCR (Beatty et al., 2004; Bergqvist et al., 2007; Jacquemier et al., 2013; Susini et al., 2010). Concordance rates for HER2 status between qPCR and standard methodology (IHC and FISH testing in some cases) ranged from 80 to 98%. Nevertheless, the majority of these comparisons had been performed using formalin-fixed paraffin-embedded blocks from surgical specimen for the qPCR analysis; very few used preoperative diagnostic tissue samples for this analysis, and in this case, mostly were an ultrasound (US)-guided coreneedle biopsy (CNB) (Beatty et al., 2004; Jacquemier et al., 2013; Kulka et al., 2006; Suo et al., 2004; Susini et al., 2010). US-guided fine-needle aspiration cytology (FNAC) is a less invasive and simpler way to obtain tumour tissue sample from suspicious breast lumps (Farshid et al., 2008; Hamill et al., 2002; Pagani et al., 2011; Uzan et al., 2009). It provides breast cancer diagnosis in 80%e90% of the cases, with high sensitivity (65%e99%) and specificity (96e100%); a predictive value of 99% and false negatives rates from 0 to 20%. The expression of hormonal receptors using FNAC samples has been analysed in a multinational study in Europe with a great variability of results, probably because of the different types of specimen preparations that were used (Domanski et al., 2013; Marinsek et al., 2013). We had previously reported that FNAC is a good source to obtain good quality mRNA from suspicious breast lumps in order to perform molecular analysis including determination of ER and HER2 status by PCR in confirmed malignant lesions (Uzan et al., 2009). The purpose of this study was to compare the preoperative determination of HER-2/neu status from breast cancer patients from primary tumour samples obtained by US-guided FNAC using qPCR, with HER2 status performed from either a CNB or from the surgical specimen using standard IHC and/or FISH analysis in order to determine their concordance.
2.
Materials and methods
2.1.
Patients and samples
Data was collected retrospectively from patients who had nodular breast lesions and attended the 1-day-stop clinic at the Gustave Roussy Cancer Campus from March 2013 to October 2014 and who had undergone a diagnostic FNAC and subsequent CNB and/or surgical tumour excision. USguided FNAC was performed in all patients. FNAC was performed as part of standard diagnosis in our one-stop clinic for patients with suspicious lumps. FNA samples (1 or 2
1432
M O L E C U L A R O N C O L O G Y 1 0 ( 2 0 1 6 ) 1 4 3 0 e1 4 3 6
passes) were obtained with a 23-gauge needle and were harvested by 3 cytopathologists. Cytological examination for diagnosis of adenocarcinoma was performed as per standards procedures. A portion of each aspiration sample was used for preparing cytological slides, and the rest of it was collected into a vial containing 350 mL of the lysis solution ALT buffer (Qiagen, Courtaboeuf, France) to allow for HER-2/neu status testing inside the HERBIN (Herceptin biomarker investigation) clinical trial aiming at identifying biomarkers of sensitivity and resistance after two cycles of 3-weekly pre-operative trastuzumab in HER2-amplified tumours (https://clinicaltrials.gov; NCT01834950). All patients provided their written consent for use of the samples for research purposes. Information regarding final diagnosis and complete histology on tissue sections after surgery was collected prospectively. Cases were eligible if HER2 status was available for FNAC and CNB and/or surgical excision specimens. If a patient had both HER2 testing performed from both the surgery and CNB, we used the result of the surgical specimen.
2.2.
DNA extraction and qPCR
In FNAC samples HER-2 status was determined by qPCR. FNAC samples collected in ATL buffer were stored at 4 C and were extracted within one day using QIAamp DNA Mini Kit according to manufacturer recommendation for fresh cells extraction. HER2 status was determined by qPCR adapted to FNAC based on our previous study (Jacquemier et al., 2013). Briefly, a set of ten primer pairs were used to determine HER2 status including 2 pairs targeting HER2 (exons 8 and 26), 5 pairs targeting chromosome 17 polysomy detection (TAOK1 and UTP6, MRM1 and MKS1 in sub-centromeric region and SSTR2, noted as CEN17) completed by 3 pairs as diploid control targeting genomic region on chromosomes 2, 4 and 5 (TSN, LAP3 and ADAMTS, respectively). PCR reactions were carried out in final volume of 15 ml using FastSYBRgreen Master Mix (Life technologies, Darmstadt, Germany) with 5 ng of genomic DNA. Each PCR were run in duplicate. Fluorescence was recorded and cycles to threshold (CT) are calculated using ViiA7 RUO Software (Life Technologies). During inclusion time-frame, the FNAC samples were prospectively analysed each week, by batch from 1 to 10 samples according to recruitments. Specific controls were included in each batch of analysis, consisting in normal lymph nodes as 2 copies (normal) control, SKBR-3 breast cancer cell lines, as amplified and no template control. Copy number calculation for each target was based on DCt method (DCt ¼ DCt_Sple(Ct_TSNeCt_Target)/DCt_2CopiesNormalReference(Ct_TSNeCt_Target)). The median of copy number values obtained for HER2 probes or CEN17 probes or diploid control probes was retain for calculation of ratio. Cycle threshold (Ct) values above 35 were defined as without amplification. Results are expressed as HER2/CEN17 ratio calculated with the median copy number of the 5 genes used for chromosome 17 control of polysomy and the median HER2 copy number. Cases with more than 3 copies of HER2 and a HER2/CEN17 ratio >1.5 were considered amplified. Cycle threshold (Ct) values above 35 were defined as without amplification. Results are expressed as HER2/CEN17 ratio calculated with the median copy number of the 5 genes used for chromosome 17
control of polysomy and the median HER2 copy number. Cases with more than 3 copies of HER2 and a HER2/CEN17 ratio >1.5 were considered amplified.
2.3.
Routine determination of ERBB2 status
Gold-standard HER-2 status was determined only in FFPE samples from CNB and/or surgical excision specimens by IHC using the 4B5 monoclonal antibody (Ventana Medical Systems Inc., Tucson, AZ) on a BenchMark XT system (Ventana Medical Systems Inc.). HER2 expression was scored from 0 to 3þ according to the American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) guideline recommendations (Wolff et al., 2013). In each case, benign glands were used as internal negative controls for specificity and an invasive breast cancer case with known HER2 overexpression was used as an external positive control. FISH analysis was performed only in cases with a IHC score 2þ to determine the presence of HER-2 amplification, using direct-labelled probes (DAKO) specific for HER2. Sections (4 mm thick) were deparaffinised in xylene and rehydrated through a graded series of ethanol solutions. We followed the procedure as recommended by manufacturers and slides were viewed under a fluorescent microscope equipped with multi-bandpass filters to visualise colours simultaneously. Cases with IHC score 3þ or FISH showing amplification were considered HER-2 positive as per ASCO/CAP recommendations. All other cases were considered HER-2 negative.
2.4.
Statistical analysis
Descriptive statistics were used to describe the study population. Sensibility, specificity, likelihood ratios and their confidence interval, were estimated. In this analysis, following the ASCO/CAP recommendations, IHC and FISH tests were considered the reference method and qPCR the test assessment. An exploratory analysis using ROC curves was performed to analyse the association between the number of copies of ERBB2 and HER2 status, and also between the ratio of the number of copies of ERBB2 and control CEN17 and HER2 status.
3.
Results
A total of 156 samples from 151 patients were available. Five patients were diagnosed with two primary tumours. We excluded one sample due to analytical failure for the qPCR test, leaving 155 samples from 150 patients for the final analysis. All samples had sufficient cells for the qPCR analysis. The mean age of the population was 58.2 years (þ/12.09). Histologically, most of the tumours were invasive ductal carcinoma in 84%, invasive lobular carcinoma in 10% and the remaining 6% other invasive tumour types. Histological grade was grade 2 in 46%, and grade 3 in 39% of the tumours. ER was positive in 85%, and PR positive in 77% of the cases. The most frequent clinical stage was IIA with 40.4%, followed by IA with 37.2%, IIB with 17.3% and IIIA with 5.1%. Tumours and samples characteristics are summarized in Table 1.
1433
M O L E C U L A R O N C O L O G Y 1 0 ( 2 0 1 6 ) 1 4 3 0 e1 4 3 6
Table 1 e Tumours and samples characteristics. Number of specimens N ¼ 155 (%) Origin of the sample Surgery Biopsy Surgery and biopsy Unknown Progesterone receptor Negative Positive Estrogen receptor Negative Positive Histology grade 1 2 3 Unknown Histology type Ductal Lobular Papillary Apocrine Mucinous Ki 67 (%) Mean (SD) Median Minemax N
61 (39) 87 (56) 6 (4) 1 (
available at www.sciencedirect.com
ScienceDirect www.elsevier.com/locate/molonc
Concordance between HER-2 status determined by qPCR in Fine Needle Aspiration Cytology (FNAC) samples compared with IHC and FISH in Core Needle Biopsy (CNB) or surgical specimens in breast cancer patients Claudia Rodrigueza,1, Voichita Suciub,1, Audrey Poteriec,1, Ludovic Lacroixb,d, Isabelle Mirand, Amelie Boichardb,d, Suzette Delalogee, Jacqueline Deneuvef, Sandy Azoulayb, Marie-Christine Mathieub, Alexander Valentb, Stefan Michielsc,g,1, Monica Arnedose,h,1, Philippe Vielhb,d,*,1 a
Oncologia Medica, Centro Universitario Contra el Cancer, Universidad Autonoma de Nuevo Leon, Mexico Departement de Biologie et Pathologie Medicales, Gustave Roussy Cancer Campus, Villejuif, France c Service de Biostatistique et d’Epidemiologie, Gustave Roussy Cancer Campus, Villejuif, France d Laboratoire de Recherche Translationnelle et Centre de Ressources Biologiques, AMMICA, INSERM US23/CNRS UMS3655, Gustave Roussy Cancer Campus, France e Departement de Medecine, Gustave Roussy Cancer Campus, Villejuif, France f Service des Operations de Recherche Clinique, Gustave Roussy Cancer Campus, Villejuif, France g INSERM U1018, CESP, Universite Paris-Sud, Universite Paris-Saclay, Villejuif, France h INSERM U981 et Universite Paris-Sud, Universite Paris-Saclay, Villejuif, France b
A R T I C L E
I N F O
A B S T R A C T
Article history:
Determining the status of HER2-neu amplification and overexpression in breast cancer is
Received 10 March 2016
crucial for prognosis but mostly for treatment purposes. Standard techniques include the
Received in revised form
determination of IHC in combination with in situ hybridization techniques to confirm a
13 June 2016
HER2-neu amplification in case of IHC2þ using either a core-needle biopsy or a surgical
Accepted 20 July 2016
specimen. qPCR has been also demonstrated to be able to determine HER2 status, mostly
Available online 5 August 2016
in core biopsies or in surgical specimens. Fine-needle aspiration is a reliable, quicker and less invasive technique that is widely used for diagnosis of invasive breast cancer. In
Keywords:
this study, we assessed the performance of qPCR in invasive breast carcinomas to deter-
Breast cancer
mine HER2-neu status by using fine-needle aspiration samples and comparing to standard
Fine needle aspiration
IHC and FISH. From a total of 154 samples from patients who had nodular breast lesions
Cytology
and attended the 1-day-stop clinic at the Gustave Roussy from March 2013 to October
HER2
2014, qPCR was able to determine the HER2 status in a mean of 3.7 days (SD 3.1). The overall
qPCR
concordance with standard HER2-testing was very high: 97% (95% CI 0.94 to 0.99); sensitivity was 96% (0.87e1), specificity 98% (0.95e1) and positive and negative predictive values 88% (0.75e1) and 99% (0.98e1), respectively.
partement de Biologie et Pathologie Me dicales, Gustave Roussy Cancer Campus, 114, rue Edouard Vaillant, * Corresponding author. De 94805 Villejuif Cedex, France. Fax: þ331 4211 5263. E-mail address: [email protected] (P. Vielh). 1 These authors contributed equally to the work. http://dx.doi.org/10.1016/j.molonc.2016.07.009 1574-7891/ª 2016 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
M O L E C U L A R O N C O L O G Y 1 0 ( 2 0 1 6 ) 1 4 3 0 e1 4 3 6
1431
In conclusion, our study demonstrates that qPCR performed using fine-needle aspiration samples from a primary tumour is a reliable and fast method to determine HER2/neu status in patients with early breast cancer. ª 2016 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
1.
Introduction
Breast cancer is the most common malignancy among women, corresponding to 25% of all cancers and representing the second cause of cancer death in developed countries (GLOBOCAN, 2012). Breast cancer is a heterogeneous disease categorized into different subtypes based on expression of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor-2 (HER-2), that have an impact both in prognosis and treatment (NCCN, 2014). HER-2 amplification and/or overexpression are present in around 10e25% of invasive breast carcinomas (Slamon et al., 1989). Tumours harbouring a HER2 amplification are more aggressive and present with worse prognosis. The use of targeted therapy against HER2, like the monoclonal antibody trastuzumab (Herceptin, Roche), has markedly improved this poor prognosis revolutionizing the treatment of this breast cancer subtype (Slamon et al., 2001). HER2 status at diagnosis might be crucial for treatment purposes as neo-adjuvant chemotherapy in combination with an anti-HER2 therapy can achieve a pathological complete response (pCR) rate of around 50% (Nagayama et al., 2014) (de Azambuja et al., 2014). It has been previously shown that HER2 status at biopsy is highly concordant with HER2 status at the surgical specimen (Arnedos et al., 2009). There are several techniques to determine HER-2 status at a protein, ribonucleic acid (RNA) and/or deoxyribonucleic acid (DNA) level. It can be assessed on the protein level using immunohistochemistry (IHC) to detect the amount of HER-2 protein expressed on the cancer cell surface. This method relies on a qualitative scoring system which is prone to interobserver variability, so false-positive results are possible (Wolff et al., 2013). Assessment by the DNA level can be done by fluorescence in situ hybridization (FISH), in order to detect amplification of the HER-2/neu gene. Finally, the assessment by RNA level can be done using quantitative real-time polymerase chain reaction (qPCR) which allows determination of gene amplification (Bergqvist et al., 2007). This last method is simple, quick and highly automated, producing numerical results for its interpretation. Nowadays, the American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) recommendations state a combined approach of IHC and/or FISH analysis as the current reference method for the detection of HER-2 amplification in breast cancer specimens (Wolff et al., 2013). One of the reasons for these guidelines are due to the discordance observed in HER2 status in several clinical trials between local and central pathology laboratories (Paik et al., 2002). Several studies have investigated the potential use of
alternative techniques to determine HER2 status in a more efficient, quicker and less expensive way (Penault-Llorca et al., 2009), including the use of qPCR (Beatty et al., 2004; Bergqvist et al., 2007; Jacquemier et al., 2013; Susini et al., 2010). Concordance rates for HER2 status between qPCR and standard methodology (IHC and FISH testing in some cases) ranged from 80 to 98%. Nevertheless, the majority of these comparisons had been performed using formalin-fixed paraffin-embedded blocks from surgical specimen for the qPCR analysis; very few used preoperative diagnostic tissue samples for this analysis, and in this case, mostly were an ultrasound (US)-guided coreneedle biopsy (CNB) (Beatty et al., 2004; Jacquemier et al., 2013; Kulka et al., 2006; Suo et al., 2004; Susini et al., 2010). US-guided fine-needle aspiration cytology (FNAC) is a less invasive and simpler way to obtain tumour tissue sample from suspicious breast lumps (Farshid et al., 2008; Hamill et al., 2002; Pagani et al., 2011; Uzan et al., 2009). It provides breast cancer diagnosis in 80%e90% of the cases, with high sensitivity (65%e99%) and specificity (96e100%); a predictive value of 99% and false negatives rates from 0 to 20%. The expression of hormonal receptors using FNAC samples has been analysed in a multinational study in Europe with a great variability of results, probably because of the different types of specimen preparations that were used (Domanski et al., 2013; Marinsek et al., 2013). We had previously reported that FNAC is a good source to obtain good quality mRNA from suspicious breast lumps in order to perform molecular analysis including determination of ER and HER2 status by PCR in confirmed malignant lesions (Uzan et al., 2009). The purpose of this study was to compare the preoperative determination of HER-2/neu status from breast cancer patients from primary tumour samples obtained by US-guided FNAC using qPCR, with HER2 status performed from either a CNB or from the surgical specimen using standard IHC and/or FISH analysis in order to determine their concordance.
2.
Materials and methods
2.1.
Patients and samples
Data was collected retrospectively from patients who had nodular breast lesions and attended the 1-day-stop clinic at the Gustave Roussy Cancer Campus from March 2013 to October 2014 and who had undergone a diagnostic FNAC and subsequent CNB and/or surgical tumour excision. USguided FNAC was performed in all patients. FNAC was performed as part of standard diagnosis in our one-stop clinic for patients with suspicious lumps. FNA samples (1 or 2
1432
M O L E C U L A R O N C O L O G Y 1 0 ( 2 0 1 6 ) 1 4 3 0 e1 4 3 6
passes) were obtained with a 23-gauge needle and were harvested by 3 cytopathologists. Cytological examination for diagnosis of adenocarcinoma was performed as per standards procedures. A portion of each aspiration sample was used for preparing cytological slides, and the rest of it was collected into a vial containing 350 mL of the lysis solution ALT buffer (Qiagen, Courtaboeuf, France) to allow for HER-2/neu status testing inside the HERBIN (Herceptin biomarker investigation) clinical trial aiming at identifying biomarkers of sensitivity and resistance after two cycles of 3-weekly pre-operative trastuzumab in HER2-amplified tumours (https://clinicaltrials.gov; NCT01834950). All patients provided their written consent for use of the samples for research purposes. Information regarding final diagnosis and complete histology on tissue sections after surgery was collected prospectively. Cases were eligible if HER2 status was available for FNAC and CNB and/or surgical excision specimens. If a patient had both HER2 testing performed from both the surgery and CNB, we used the result of the surgical specimen.
2.2.
DNA extraction and qPCR
In FNAC samples HER-2 status was determined by qPCR. FNAC samples collected in ATL buffer were stored at 4 C and were extracted within one day using QIAamp DNA Mini Kit according to manufacturer recommendation for fresh cells extraction. HER2 status was determined by qPCR adapted to FNAC based on our previous study (Jacquemier et al., 2013). Briefly, a set of ten primer pairs were used to determine HER2 status including 2 pairs targeting HER2 (exons 8 and 26), 5 pairs targeting chromosome 17 polysomy detection (TAOK1 and UTP6, MRM1 and MKS1 in sub-centromeric region and SSTR2, noted as CEN17) completed by 3 pairs as diploid control targeting genomic region on chromosomes 2, 4 and 5 (TSN, LAP3 and ADAMTS, respectively). PCR reactions were carried out in final volume of 15 ml using FastSYBRgreen Master Mix (Life technologies, Darmstadt, Germany) with 5 ng of genomic DNA. Each PCR were run in duplicate. Fluorescence was recorded and cycles to threshold (CT) are calculated using ViiA7 RUO Software (Life Technologies). During inclusion time-frame, the FNAC samples were prospectively analysed each week, by batch from 1 to 10 samples according to recruitments. Specific controls were included in each batch of analysis, consisting in normal lymph nodes as 2 copies (normal) control, SKBR-3 breast cancer cell lines, as amplified and no template control. Copy number calculation for each target was based on DCt method (DCt ¼ DCt_Sple(Ct_TSNeCt_Target)/DCt_2CopiesNormalReference(Ct_TSNeCt_Target)). The median of copy number values obtained for HER2 probes or CEN17 probes or diploid control probes was retain for calculation of ratio. Cycle threshold (Ct) values above 35 were defined as without amplification. Results are expressed as HER2/CEN17 ratio calculated with the median copy number of the 5 genes used for chromosome 17 control of polysomy and the median HER2 copy number. Cases with more than 3 copies of HER2 and a HER2/CEN17 ratio >1.5 were considered amplified. Cycle threshold (Ct) values above 35 were defined as without amplification. Results are expressed as HER2/CEN17 ratio calculated with the median copy number of the 5 genes used for chromosome 17
control of polysomy and the median HER2 copy number. Cases with more than 3 copies of HER2 and a HER2/CEN17 ratio >1.5 were considered amplified.
2.3.
Routine determination of ERBB2 status
Gold-standard HER-2 status was determined only in FFPE samples from CNB and/or surgical excision specimens by IHC using the 4B5 monoclonal antibody (Ventana Medical Systems Inc., Tucson, AZ) on a BenchMark XT system (Ventana Medical Systems Inc.). HER2 expression was scored from 0 to 3þ according to the American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) guideline recommendations (Wolff et al., 2013). In each case, benign glands were used as internal negative controls for specificity and an invasive breast cancer case with known HER2 overexpression was used as an external positive control. FISH analysis was performed only in cases with a IHC score 2þ to determine the presence of HER-2 amplification, using direct-labelled probes (DAKO) specific for HER2. Sections (4 mm thick) were deparaffinised in xylene and rehydrated through a graded series of ethanol solutions. We followed the procedure as recommended by manufacturers and slides were viewed under a fluorescent microscope equipped with multi-bandpass filters to visualise colours simultaneously. Cases with IHC score 3þ or FISH showing amplification were considered HER-2 positive as per ASCO/CAP recommendations. All other cases were considered HER-2 negative.
2.4.
Statistical analysis
Descriptive statistics were used to describe the study population. Sensibility, specificity, likelihood ratios and their confidence interval, were estimated. In this analysis, following the ASCO/CAP recommendations, IHC and FISH tests were considered the reference method and qPCR the test assessment. An exploratory analysis using ROC curves was performed to analyse the association between the number of copies of ERBB2 and HER2 status, and also between the ratio of the number of copies of ERBB2 and control CEN17 and HER2 status.
3.
Results
A total of 156 samples from 151 patients were available. Five patients were diagnosed with two primary tumours. We excluded one sample due to analytical failure for the qPCR test, leaving 155 samples from 150 patients for the final analysis. All samples had sufficient cells for the qPCR analysis. The mean age of the population was 58.2 years (þ/12.09). Histologically, most of the tumours were invasive ductal carcinoma in 84%, invasive lobular carcinoma in 10% and the remaining 6% other invasive tumour types. Histological grade was grade 2 in 46%, and grade 3 in 39% of the tumours. ER was positive in 85%, and PR positive in 77% of the cases. The most frequent clinical stage was IIA with 40.4%, followed by IA with 37.2%, IIB with 17.3% and IIIA with 5.1%. Tumours and samples characteristics are summarized in Table 1.
1433
M O L E C U L A R O N C O L O G Y 1 0 ( 2 0 1 6 ) 1 4 3 0 e1 4 3 6
Table 1 e Tumours and samples characteristics. Number of specimens N ¼ 155 (%) Origin of the sample Surgery Biopsy Surgery and biopsy Unknown Progesterone receptor Negative Positive Estrogen receptor Negative Positive Histology grade 1 2 3 Unknown Histology type Ductal Lobular Papillary Apocrine Mucinous Ki 67 (%) Mean (SD) Median Minemax N
61 (39) 87 (56) 6 (4) 1 (
