University of Veterinary Medicine Hannover Small Animal Clinic
Comparison of fresh frozen vs. formalin-fixed, paraffin-embedded specimens and the expression profiling of 16 target genes in neoplastic and non-neoplastic
canine mammary tissues using a multiplex branched-DNA assay
THESIS
Submitted in partial fulfilment of the requirements for the degree
DOCTOR OF PHILOSOPHY
(PhD)
awarded by the University of Veterinary Medicine Hannover by
Florenza Lüder Ripoli
Santa Maria- RS, Brazil
Hannover, Germany 2016
Supervisor:
Prof. Dr. Ingo Nolte Supervision Group:
Prof. Dr. Ingo Nolte
Prof. Dr. Marion Hewicker-Trautwein Prof. Dr. Hans H. Kreipe
1st Evaluation:
Prof. Dr. Ingo Nolte
Small Animal Clinic, University of Veterinary Medicine Hannover, Hannover, Germany
Prof. Dr. Marion Hewicker-Trautwein
Institute of Pathology, University of Veterinary Medicine Hannover, Hannover, Germany
Prof. Dr. Hans H. Kreipe
Institute of Pathology, Hannover Medical School, Hannover, Germany
2nd Evaluation: Prof. Dr. Johannes Hirschberger
Date of final exam: 27.09.2016
Florenza Lüder Ripoli was funded by CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior), Brazil.
To my family and friends
Parts of the thesis have been published previously in:
Publications:
F. L. Ripoli, A. Mohr, S. C. Hammer, S. Willenbrock, M. Hewicker-Trautwein, S.
Hennecke, H. M. Escobar and I. Nolte. "A Comparison of Fresh Frozen vs. Formalin-Fixed, Paraffin-Embedded Specimens of Canine Mammary Tumors via Branched-DNA Assay". Int.
J. Mol. Sci 17(5), 2016
F. L. Ripoli, S. C. Hammer, A. Mohr, S. Willenbrock, M. Hewicker-Trautwein, B.
Brenig, H. M. Escobar and I. Nolte. "Multiplex Gene Expression Profiling of 16 Target Genes in Neoplastic and Non-neoplastic Canine Mammary Tissues Using Branched-DNA Assay".
Submitted to Int. J. Mol. Sci, 2016
Poster presentations:
F. L. Ripoli, A. Mohr, S. C. Hammer, S. Willenbrock, M. Hewicker-Trautwein, H.
Murua Escobar, I. Nolte. “Influence of the storage time on RNA isolation and expression of relevant genes in canine mammary tumours”. 24. Jahrestagung der Fachgruppe Innere Medizin und klinische Labordiagnostik, January 29 – 30, 2016, Berlin, Germany.
CONTENTS
ABBREVIATIONS ……….I
1 INTRODUCTION ... 1
2 RESULTS ... 4
2.1 A COMPARISON OF FRESH FROZEN VS. FORMALIN-FIXED, PARAFFIN-EMBEDDED SPECIMENS OF CANINE MAMMARY TUMORS VIA BRANCHED-DNAASSAY ... 4
2.2 MULTIPLEX GENE EXPRESSION PROFILING OF 16 TARGET GENES IN NEOPLASTIC AND NON-NEOPLASTIC CANINE MAMMARY TISSUES USING BRANCHED-DNAASSAY ... 17
3 GENERAL DISCUSSION ... 31
4 SUMMARY ... 38
5 ZUSAMMENFASSUNG ... 41
6 REFERENCES ... 44
7 ACKNOWLEDGMENTS ... 51
Abbreviations
ACTB Beta-actin
BC Human Breast cancer
b-DNA assay Branched-DNA assay
BRCA1 BRCA1, DNA repair associated
BRCA2 BRCA2, DNA repair associated
CMT Canine mammary tumor
ER Estrogen receptor
ESS English Springer Spaniels
FF Fresh frozen tissues
FFPE Formalin-fixed, paraffin-embedded tissues
FOXO3 Forkhead box O3
GAPDH Glyceraldehyde 3-phosphate dehydrogenase
GATA4 GATA binding protein 4
GHR Growth hormone receptor
HER2 Erb-b2 receptor tyrosine kinase 2
HKG Housekeeping gene
HMGA1 High mobility group AT-hook 1
HMGA2 High mobility group AT-hook 2
II
HMGB1 High mobility group box 1
HPRT1 Hypoxanthine phosphoribosyltransferase 1
IHC Immunohistochemistry
LOD Limit of detection
MAPK1 Mitogen-activated protein kinase 1
MAPK3 Mitogen-activated protein kinase 3
MCL1 Myeloid cell leukemia 1
mRNA Messenger RNA
MYC V-myc avian myelocytomatosis viral oncogene
homolog
PFDN5 Prefoldin subunit 5
PIK3CA Phosphatidylinositol-4,5-bisphosphate 3- kinase catalytic subunit alpha
PR Progesterone receptor
PRLR Prolactin receptor
PTEN Phosphatase and tensin homolog
qPCR Real-time polymerase chain reaction
RNA Ribonucleic acid
TP53 Tumor protein p53
Introduction
1 1 Introduction
Mammary tumors are the most prevalent neoplasms in female dogs (Dorn et al. 1968, Merlo et al. 2008). Certain breeds are reported to be predisposed to develop canine mammary tumor (CMT) with influence of the geographic location (Moe 2001, Egenvall et al.
2005, Dobson 2013). In a study performed in Germany, the highest relative risk ratio of developing benign mammary tumors was found in Yorkshire Terriers followed by Poodles, Dachshunds and Cocker Spaniels. In malignant mammary tumors, the prevalence was higher in Poodles followed by Dachshunds, Cocker Spaniels and Yorkshire Terriers (von Bomhard 2001).
The postoperative median survival time of malignant CMT can be shorter than two years depending on the histological stage and histologic grade of the tumor (Betz et al.
2012). Therefore, the histological stage at diagnosis is one of the most important prognostic factors in dogs (Yamagami et al. 1996, Karayannopoulou et al. 2005). However, it is only possible to be assessed in symptomatic patients and is thus not suitable as a mammary tumor predictive parameter. The analysis herein is performed in tumor tissues with the aim to find possible tumor candidate genes. These genes could serve as predictive markers in the future to identify tumor targets in body fluids just like in the human counterpart (Goessl et al.
2002, Hu et al. 2006, Sharma 2009).
The analysis and differentiation of the mammary tumors are the basis for pathogenesis, diagnosis, prognosis, new therapy options and good breed hygiene practice.
Immunohistochemistry (IHC), alongside with conventional histopathology, plays an important role as a diagnostic tool in classifying tumors (Duraiyan et al. 2012, Tavasoly et al. 2013).
Most studies on mammary tumors in dogs are made through IHC and determine biomarkers on the protein level by evaluating estrogen and progesterone receptors (ER and PR), which may assist in the prognosis of patients (Toniti et al. 2009, Cassali et al. 2011, Dolka et al.
2013, Pena et al. 2014). This technique has proven to be suitable when performing large multi-center studies. However, the limitations rely on the semi-quantitative and subjective interpretation of its results (Seidal et al. 2001) and time to execute the method specially when a high number of samples are involved (Kim et al. 2010). To overcome this situation, branched DNA (b-DNA) assays, in combination with the xMAP magnetic beads technology, permit the concomitant expression analysis of several target genes within a single sample (Flagella et al. 2006). The referred technique presents a simpler and more rapid workflow, providing highly sensitive quantitative results when compared to other gene expression techniques such as real time PCR (qPCR) (Knudsen et al. 2008).
Introduction
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B-DNA assays allow the possibility of working with small amounts of RNA and therefore enabling the analysis of valuable formalin-fixed, paraffin-embedded (FFPE) material. Fresh frozen (FF) tissues are ideal for molecular analysis by gene expression measurements considering their RNA is well preserved. However, such tumor sets are not so abundant (Penland et al. 2007) and their collection is mainly restricted to tissue banks and research groups. Alternatively, FFPE tissues represent a unique source of archived biological material (von Ahlfen et al. 2007, Granato et al. 2014) and they are the most widely available source of tissue material for which long-term clinical follow-up data are recorded (Mittempergher et al. 2011). FFPE material offer several advantages such as easy handling and long-term cheap storage (Perlmutter et al. 2004). The chance to assess gene expression profiling proportioned by the mentioned samples enables the performance of many retrospective and multi-center studies, benefiting the correlation of expression profiles with clinical outcomes (Ramaswamy 2004, April et al. 2009).
Bearing in mind that the use of mammary tumor biomarkers in canines is still restricted to research purposes (Queiroga et al. 2011, Hennecke et al. 2015), the need to comprehensively analyze gene profiles in different canine mammary tumor entities to find potential candidate genes which could serve as predictive factors is evident. Still, some genes have already been analyzed in canine mammary tumors with promising results.
Expression of BRCA1 and BRCA2 in CMT was analyzed and it was found to be associated with an increased mammary tumor risk in English Springer Spaniels (ESS) (Rivera et al.
2009). A possible influence of those genes in tumor development in dogs is also suggested in other studies (Ochiai et al. 2001, Nieto et al. 2003, Klopfleisch and Gruber 2009, Enginler et al. 2014). Further targets of the herein analyzed have also been investigated in CMT such as TP53 (Chu et al. 1998, Muto et al. 2000, Lee et al. 2004, Terra et al. 2012); PTEN (Kanae et al. 2006, Qiu et al. 2008, Ressel et al. 2009, Beck et al. 2013, Borge et al. 2015); PFDN5 (Beck et al. 2013, Hennecke et al. 2015); MYC (Borge et al. 2015), HER2 (Hsu et al. 2009, Ressel et al. 2013, Burrai et al. 2015) and MCL1 (Kano et al. 2009) associating their expression patterns with the development of CMT. However, their limitations rely on the small sample number available in each work. The detection of suitable biomarkers could assist in the neoplasms’ early detection and support in providing novel therapeutic approaches. Additionally, the propagation of possible faulty genes to the offspring could be avoided by the breeding hygiene in dogs.
The concomitant analysis of 16 onco- and suppressor-genes (BRCA1; BRCA2;
FOXO3; GATA4; HER2; HMGA1; HMGA2; HMGB1; MAPK1; MAPK3; MCL1; MYC; PFDN5;
PIK3CA; PTEN and TP53) regarded as being involved in mammary neoplasm development
Introduction
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(Li et al. 1997, Sivaraman et al. 1997, Gasco et al. 2002, Antoniou et al. 2003, Ross et al.
2003, O'Driscoll et al. 2004, Aulmann et al. 2006, Li et al. 2006, Fusco and Fedele 2007, Mavaddat et al. 2010, Jiang et al. 2013, Takagi et al. 2014, Sun et al. 2015) using the multiplex b-DNA technology in a large pool of FF and FFPE specimens is herein proposed.
The hypothesis is if the multiplex b-DNA assay is feasible when analyzing canine mammary tumor entities. It is also of interest to determine whether the gene expression between FFPE and FF specimens is comparable when applying the referred technique and whether the different storage times of FFPE samples have any influence on the outcomes. Moreover, considering that there are still no commonly accepted housekeeping genes (HKG) for analysis in canine mammary neoplasms, the opportunity to investigate the performance of three different HKG is taken in the present study. The HKG herein utilized were based on the data normalizers commonly reported in BC (Majidzadeh et al. 2011, Kilic et al. 2014).
Furthermore, it was also aimed to compare the expression patterns of the chosen targets in neoplastic (benign and malignant) and non-neoplastic tissues with the intention of finding new potential tumor markers. This characterization might serve to gain a better understanding of the tumor pathogenesis of different canine mammary tumor types in further studies. Moreover, potential target genes might assist to determine whether certain breeds are at risk of developing mammary tumors and if the castration status plays a role. In addition, potential targets might be identified to contribute to a better therapeutic approach in the future.
Results
4 2 Results
2.1 A Comparison of Fresh Frozen vs. Formalin-Fixed, Paraffin-Embedded Specimens of Canine Mammary Tumors via Branched-DNA Assay
F. L. Ripoli, A. Mohr, S. C. Hammer, S. Willenbrock, M. Hewicker- Trautwein, S. Hennecke, H. Murua Escobar and I. Nolte
International Journal of Molecular Sciences, 2016, 17(5)
Contribution to this study:
Florenza Lüder Ripoli performed all the experiments, data analyses and wrote
the manuscript.
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Abstract:
Mammary neoplasms are the tumors most affecting female dogs and women.
Formalin-fixed, paraffin-embedded (FFPE) tissues are an invaluable source of archived biological material. Fresh frozen (FF) tissue is considered ideal for gene expression analysis.
However, strategies based on FFPE material offer several advantages. Branched-DNA assays permit a reliable and fast workflow when analyzing gene expression. The aim of this study was to assess the comparability of the branched-DNA assay when analyzing certain gene expression patterns between FF and FFPE samples in canine mammary tumors. RNA was isolated from 109 FFPE samples and from 93 FF samples of different canine mammary tissues. Sixteen (16) target genes (TP53; MYC; HMGA1; PIK3CA; MCL1; MAPK3; FOXO3;
PTEN; GATA4; PFDN5; HMGB1; MAPK1; BRCA2; BRCA1; HMGA2; and HER2) were analyzed via branched-DNA assay (b-DNA). ACTB, GAPDH, and HPRT1 were used as data normalizers. Overall, the relative gene expression of the two different origins of samples showed an agreement of 63%. Still, care should be taken, as FFPE specimens showed lower expression of the analyzed targets when compared to FF samples.
The fact that the gene expression in FFPE proved to be lower than in FF specimens is likely to have been caused by the effect of storage time. ACTB had the best performance as a data normalizer.
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2.2 Multiplex Gene Expression Profiling of 16 Target Genes in Neoplastic and Non-neoplastic Canine Mammary Tissues Using Branched-DNA Assay
F. L. Ripoli, S. C. Hammer, A. Mohr, S. Willenbrock, M. Hewicker- Trautwein, B. Brenig, H. Murua Escobar and I. Nolte
Submitted to: International Journal of Molecular Sciences
Contribution to this study:
Florenza Lüder Ripoli performed all the experiments, data analyses and wrote
the manuscript.
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Abstract:
Mammary gland tumors are one of the most common neoplasms in female dogs and certain breeds are prone to develop the disease. The use of biomarkers in canines is still restricted to research purposes. Therefore, the necessity to analyze gene profiles in different mammary entities in large sample sets is evident in order to evaluate the strength of potential markers serving as future prognostic factors. The aim of the present study was to analyze the gene expression of 16 target genes (BRCA1; BRCA2; FOXO3; GATA4; HER2; HMGA1;
HMGA2; HMGB1; MAPK1; MAPK3; MCL1; MYC; PFDN5; PIK3CA; PTEN and TP53) known to be involved in human and canine mammary neoplasm development. Expression was analyzed in 111 fresh frozen (FF) and in 170 formalin-fixed, paraffin-embedded (FFPE) specimens of neoplastic and non-neoplastic canine mammary tissues using a multiplex branched-DNA (b-DNA) assay. TP53, FOXO3, PTEN and PFDN5 expression revealed consistent results with significant low expression in malignant tumors. The possibility of utilizing them as predictive factors as well as them assisting in the choice of an adequate gene therapy may help in the development of new and improved approaches in canine mammary tumors.
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General discussion
31 3 General Discussion
Different risk factors are reported to have certain influence in the development of canine mammary tumor (CMT). The most known are age, breed (genetic predisposition) and hormones (Rivera et al. 2009, Sleeckx et al. 2011). The analysis and differentiation of mammary tumors lead to a better understanding of their behavior. Considering that still no prognostic factors and targeted therapy are routinely used in dogs, the need to comprehensively analyze gene profiles in different canine mammary entities to find potential candidate genes is evident as little is known in this regard to date (Queiroga et al. 2011).
Furthermore, the possibility to identify markers which would assist to better elucidate the pathogenesis of CMT is also an important aspect. In addition to that, potential target genes could be identified which might assist to prospectively determine whether certain breeds are at risk of developing mammary tumors.
The multiplex b-DNA assay, utilized in the present study, offers a quantitative and sensitive analysis of several targets within a single sample. The technique revealed to be a feasible and reliable method for analyzing canine mammary tissues of FF and FFPE samples. This fact was essential for further data analysis. Most studies on mammary tumors in dogs are made through IHC (Cassali et al. 2011, Pena et al. 2014). It is certainly a suitable method when performing large multi-center studies, nonetheless it is laborious and time consuming specially when the objective is to analyze several markers (Kim et al. 2010). Still, this technique is, together with conventional histopathology, an important diagnostic tool to classify tumors (Duraiyan et al. 2012, Tavasoly et al. 2013). Results of IHC can, however, vary depending on the sample fixation type and time, antibody clones and assay interpretation utilized in different studies (Kraus et al. 2012). Although qPCR is considered the method of choice for gene expression analysis (Bernardo et al. 2013, Adamski et al.
2014, Pabinger et al. 2014), the method presents major limitations such as the necessity of previous purification of the nucleic acid, need of target gene pre-amplification and high amounts of RNA (Perlmutter et al. 2004). The RNA retrieved from the FFPE samples herein were considerable low in comparison to the ones obtained from FF, limiting qPCR analyses due to the small amounts of RNA. Furthermore, limited number of targets analyzed per time, a more complex and longer work flow with a consequent higher probability of pipetting errors and lower sensitivity are still disadvantages of qPCR when compared to b-DNA assays (Urdea et al. 1991, Soutschek et al. 2004). The b-DNA method in combination with the xMAP technology allow rapid and accurate analysis (Knudsen et al. 2008) of up to 100 unique targets within a single sample (Flagella et al. 2006), thereby reducing hands-on time and
General discussion
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costs. This fact was decisive in choosing the method in this study as 24 tumor associated target genes were concomitantly analyzed of which 16 were used for the present study.
Moreover, the simple workflow enables the analysis of a large pool of samples which leads to an increase of the statistical power and reliability of the results.
To analyze if both FF and FFPE specimens are suitable for the multiplex bDNA assay, a comparison of the origins of samples was performed. The analysis showed an agreement of 63% between FF and FFPE specimens. Previous studies regarding the comparison of FF vs. FFPE revealed a correlation of almost 100%. However, those studies usually analyzed matched pairs of both origin of samples and the storage time was shorter (Knudsen et al. 2008, Webster et al. 2015), reasons which might explain the 37% of significant differences between FF and FFPE specimens in the present study. The choice of non-matched samples herein was deliberately as the aim was to resemble what is normally seen in the routine. The achievement of FF samples is restricted to tissue banks and research groups (Penland et al. 2007) and FFPE samples are widely available (Mittempergher et al. 2011) as the collection procedures happen as a result of the sampling normally sent to pathology laboratories for diagnostic reasons.
As the measured normalized expression in FFPE samples revealed to be lower than in FF specimens, a comparison of the raw data between different storage times of FFPE and FF was performed to investigate if the storage time had major influence on the expression results. For FF samples, no significant difference was found. The FFPE samples were divided into three groups: 1. Storage time of 16 and 17 years, n = 74; 2. Storage time of 18, 19, and 20 years, n = 61; 3. Storage time of 21, 22, and 23 years, n = 30. A clear higher expression of target genes in the short storage time group with a decrease of the expression in the groups with longer storage time was revealed for FFPE specimens. This outcome shows that an overtime effect plays a role in detecting the gene expression. Resembling to that, a study from 2004 showed that fragmentation of RNA continues to occur over the storage time for human FFPE specimens (Cronin et al. 2004), which could be a reason for the lower expression seen in the present study. Nam and colleagues reported similar results when analyzing samples via qPCR where the target gene had its amplification decreased as the storage time of FFPE specimens increased (Nam et al. 2014). Their study analyzed specimens dated from 2003 to 2011 (three to 11 years storage time). In the present study, it would be ideal to directly compare the storage time of FF and FFPE samples to check if other variables with exception for the time have also influenced in the lower expression of FFPE when compared to FF specimens. It was unfortunately not possible to be performed in this work due to no presence of FF and FFPE samples with the same storage time. The
General discussion
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matched samples as well the short storage time are two relevant aspects to be considered which might explain the 37% of discrepancy between other studies (Scicchitano et al. 2006, Oberli et al. 2008) in contrast to the present work. The multiplex bDNA method revealed to be feasible when willing to analyze FFPE specimens. However, the results should be carefully interpreted.
With regard to the HKG herein utilized, their selection was based on data normalizers commonly reported in human literature regarding breast cancer (Majidzadeh et al. 2011, Kilic et al. 2014). HKG are commonly selected to correct expression data due to the variability between samples when analyzing the results of gene expression experiments (de Kok et al.
2005). As little is known about housekeepers for gene expression analysis in canine mammary tissue, the opportunity to separately analyze the performance of the HKG was taken in the present study. Therefore, housekeepers were separated and data were normalized against each one individually. 27.4 % of the genes within the histological groups showed differences when data were normalized against ACTB. For GAPDH, 50.9 % presented difference, whereas for HPRT1 47.0 % revealed difference. De Kok and team showed that the best HKGs as data normalizers for human breast cancer were, when only taking into consideration the ones herein utilized, HPRT1 followed by ACTB and GAPDH (de Kok et al. 2005). Considering that the expression of HKG varies (Kunth et al. 1994, Zhong and Simons 1999, Mane et al. 2008) and leads to misinterpretation of the results, the average of the three was chosen to normalize the data. This procedure is recommended nowadays to avoid bias analysis (Lyng et al. 2008). The results herein might serve as basis for further studies to characterize the expression patterns of HKG in canine mammary tissues.
To elucidate the role of tumor-associated genes in canine mammary tissue, 16 target genes (BRCA1; BRCA2; FOXO3; GATA4; HER2; HMGA1; HMGA2; HMGB1; MAPK1;
MAPK3; MCL1; MYC; PFDN5; PIK3CA; PTEN and TP53) were analyzed given their known importance in the development of mammary neoplasms (Li et al. 1997, Sivaraman et al.
1997, Gasco et al. 2002, Antoniou et al. 2003, Ross et al. 2003, O'Driscoll et al. 2004, Aulmann et al. 2006, Li et al. 2006, Fusco and Fedele 2007, Mavaddat et al. 2010, Jiang et al. 2013, Takagi et al. 2014, Sun et al. 2015). The analysis of estrogen and progesterone receptors (ER and PR) via IHC is important in order to evaluate BC (Hammond et al. 2010).
Considering that the expression at mRNA levels of them is not characterized in different histopathological subgroups of CMT yet, the opportunity to concomitantly analyze those receptors with the genes herein described was taken. However, as the data belong to a
General discussion
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parallel project of the present group (Mohr et al. 2016), the outcomes are not described and discussed herein.
Of the 16 onco- and suppressor-genes, significant reduction in the expression of the tumor suppressors TP53, FOXO3, PTEN and PFDN5 in malignant tumors were found in the present study. These results are in line with the literature described in humans (Li et al. 1997, Gasco et al. 2002, Accili and Arden 2004) and in dogs (Muto et al. 2000, Borge et al. 2015, Hennecke et al. 2015), underlining their role in cellular growth. To the best of knowledge, that is the first time the gene FOXO3 has been analyzed on mRNA level in dogs. Hence, these genes could represent potential prognostic markers in CMT. TP53, FOXO3 and PTEN have already been checked for target therapy in human BC with promising results (Bertheau et al.
2013, Guo et al. 2015, Smit et al. 2016). Therefore, they may also help in therapeutic approaches in CMT.
TP53 demonstrated to be more highly expressed in non-neoplastic tissue herein.
Furthermore, the target gene showed higher expression in benign than in malignant tumors in FFPE tissue samples. The TP53 is known to be a suppressor gene (Gasco et al. 2002) and its inactivation is a frequent event in tumorigenesis (Rivlin et al. 2011). Interestingly, reports of TP53 on protein levels in CMT have detected stronger staining in malignant tumors (Lee et al. 2004, Terra et al. 2012, Koltai and Vajdovich 2014) and its higher expression was associated with poor prognosis in canine mammary tumors (Lee et al. 2004). The reason for that might be related to a point mutation region of the gene encoding the p53 protein reported to occur in CMT, leading to the inactivation of the protein (Veldhoen et al. 1999). Its expression can be either normal, under- or overexpressed. The results presented herein are interesting considering little is known about the role of TP53 in CMT on mRNA levels.
Therefore, the findings of the present study might serve as basis for further analyses of TP53 on mRNA levels to better understand its interaction in CMT.
A study from 2013 showed that a high expression level of FOXO3 was significantly correlated with long-term survival of women suffering from breast cancer, indicating that FOXO3 expression is a favorable prognostic marker (Jiang et al. 2013). The results of the present study corroborate what has been reported in humans, demonstrating its higher expression in non-neoplastic tissues (Jiang et al. 2013). Moreover, the target gene showed to be higher expressed in benign tumors when compared to malignant tumors in FF and FFPE specimens. Even though no studies describing the behavior of this gene in CMT have been performed, the results herein confirm what has been described in humans and might, therefore, be also applicable for dogs as a prognostic marker in different malignant mammary tumors.
General discussion
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PTEN is known as a tumor suppressor gene either (Li et al. 1997) and the role of its loss has been largely investigated in human breast carcinomas (Chen et al. 1998, Bose et al.
2002, Li et al. 2015). Known for its importance in humans, the role of PTEN has also been examined in CMT and its behavior in dogs resembles what has been seen in BC, associating PTEN low expression with malignancy (Kanae et al. 2006, Qiu et al. 2008, Ressel et al.
2009, Beck et al. 2013). The present study revealed higher expression of the target gene in non-neoplastic tissues and benign tumors in comparison to malignant tumors in FFPE samples. Surprisingly, it did not show any significant differences in FF tissue samples. The explanation for that might be the smaller number of samples of benign and malignant tumors of FF (n= 33 for benign tumors; n= 60 for malignant tumors) when compared to FFPE (n= 47 for benign tumors; n= 84 for malignant tumors), decreasing the statistical power. Based in the outcomes herein, the potential of PTEN as a predictive marker in CMT might be considered and further studies are necessary to better characterize its interaction in CMT.
Hennecke and colleagues (2015) suggested PFDN5 as a potential tumor marker in CMT. Another study demonstrated that PFDN5 was recurrently deleted in CMT (Beck et al.
2013). In humans, few reports mention the role of the gene in breast cancer. However, it has been shown to be a tumor suppressor candidate in leukemia and tongue cancer (Fujioka et al. 2001). The results herein revealed higher expression of the referred gene in non- neoplastic tissue as well as in benign when compared to malignant tumors in FF and FFPE samples. The outcomes are, therefore, in line with literature reports corroborating its tumor marker potential in CMT.
Further studies at mRNA level are worth carrying out to deepen existing knowledge concerning the role of the four previously described genes in CMT. Besides assessing their pathway in CMT, it would be interesting to also check the combination / interaction of them with each other as well as with other genes. By that, novel therapies would be useful to manage canine mammary tumors.
The genes MAPK3, HMGB1, HMGA1 and MCL1 demonstrated to be consistently lower expressed in malignant tumors in FF and FFPE specimens in this study. PIK3CA showed to be lower expressed in malignant tumors solely in FF samples. Their expression revealed not to be in line to what is normally reported in humans as they are known to be higher expressed in BC (Sivaraman et al. 1997, O'Driscoll et al. 2004, Levine et al. 2005, Fusco and Fedele 2007, Sun et al. 2015), stimulating, therefore, cellular growth. Therefore, a hypothesis is raised whether those genes play a different role in canine mammary tumors.
Intriguingly, despite the well characterized and important role of HER2, BRCA1 and BRCA2 in breast cancer (Petrucelli et al. 1993, Welcsh and King 2001, Yarden 2001, Ross et
General discussion
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al. 2003, Murphy and Modi 2009, Sana and Irshad 2012, Figueroa-Magalhaes et al. 2014, McCarthy and Armstrong 2014), the present work demonstrated lower expression of HER2 in malignant tumors and no conclusive results for BRCA1 and BRCA2. BRCA1 and BRCA2 have already demonstrated to be associated with development of CMT in dogs in other studies (Nieto et al. 2003, Enginler et al. 2014). The explanation for the results of those genes herein might be related to the extremely low expression of BRCA1 and BRCA2 in FF and FFPE samples in all diagnostic groups. Therefore, no significant differences could be observed as the expression levels were constant and practically the same in neoplastic and non-neoplastic tissues.
In conclusion, the multiplex b-DNA assay revealed to be a feasible and reliable method for analyzing canine mammary tissues of FF and FFPE samples in the present study. This technique is more simple, rapid and accurate than qPCR (Knudsen et al. 2008), thereby reducing hands-on time and costs. Moreover, several specimens are possible to be analyzed at the same time and the technique permits to detect up to 100 target genes within a simple sample (Flagella et al. 2006). In this work, a considerably larger number of samples was analyzed when compared to other studies performed in this field so far, permitting, consequently, an increase of the statistical power and reliability of the results. The principal differences between the present work when compared to others are with regard to the non- matched samples and longer storage time herein utilized.
Still, care should be taken when analyzing FFPE specimens considering they presented a generally lower expression depending on the storage period of the analyzed target genes when compared to FF samples. It would certainly be ideal to always utilize RNA from FF samples if available for gene expression analyses based on the findings of the present study. However, several FFPE samples are worth being analyzed considering their scientific value as unique samples of certain clinical case or disease but results should be taken with caution.
As limitations of the present study, aspects such as the non-matched samples of FF and FFPE (despite deliberately) as well as not same time of storage do not allowing direct comparisons of the samples are to be considered. It is also important to mention that, even though the FF and FFPE tissues were diagnostically confirmed in the pathology, it is difficult to isolate RNA only of the tumor parts and therefore the samples might also contain traces of other materials such as adipose or healthy mammary tissues. Another important point to mention is with regard to the method herein utilized. The majority of the target genes were possible to be detected with the multiplex bDNA assay, however no direct comparison with
General discussion
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other techniques such as IHC or qPCR was performed. Therefore, no statements can be affirmed regarding the comparison of the three methods when assessing the targets analyzed in this study.
The suppressor genes TP53, FOXO3, PTEN and PFDN5 are considered as potential markers for predicting canine mammary tumors. The results herein are in line with the literature in humans (Li et al. 1997, Fujioka et al. 2001, Gasco et al. 2002, Accili and Arden 2004) and in dogs with exception of FOXO3 (Muto et al. 2000, Borge et al. 2015, Hennecke et al. 2015), revealing low expression of these genes in malignant tumors. This suggests their role in cellular growth. Further investigations are needed to prove if the analyzed genes alone play a role in general tumor progression or if the combination of each other or with other genes confers an important predisposition to the disease with further development of mammary tumors.
The findings herein contribute to further investigations about CMT by leading to a better differentiation and characterization of them. These two last aspects lead to a better understanding of the diseases’ course. As already described, certain breeds are at genetic risk of developing CMT. Thereby, the outcomes herein might relevantly contribute to deepen consecrated studies which have elucidated the predisposition of certain breeds to develop CMT (von Bomhard 2001, Rivera et al. 2009, Dobson 2013) as well as to guide further studies. A review revealed that the possibilities of early neutering as well as of age at castration to reduce the risk of mammary neoplasia were not consistent enough for a concrete conclusion (Beauvais et al. 2012). Therefore, the findings herein may also help to prospectively assess whether the castration status plays a role in CMT development.
As next step, it would be interesting to analyze dog blood samples to identify those markers herein described with the aim to early detect CMT cases. This identification might also prevent genetic predisposed animals from breeding with the consequent avoidance of propagation of defective genes to their offspring. In the UK, for example, women who are at genetic risk of having a BC faulty gene due to family inheritance are offered screening for BRCA1, BRCA2 and TP53 (Gadzicki et al. 2011). Therefore, just like the human counterpart, the direct analysis of blood or other body fluids to detect markers involved with tumor development would lead to a better approach of affected patients as well as permitting the possibility of an early therapy.
Summary
38 4 Summary
Comparison of fresh frozen and formalin-fixed, paraffin-embedded canine mammary tissues and the gene expression analysis of 16 target genes via multiplex branched-DNA assay
Florenza Lüder Ripoli
Mammary gland tumors are one of the most common neoplasms in female dogs and certain breeds are prone to develop the disease. The use of markers in human BC has been widely studied and some genes are routinely used as predictive, diagnostic and prognostic factors as well as for treatment reasons. The use of biomarkers in CMT is, however, still restricted to research purposes only. Therefore, gene profiles in different canine mammary entities were analyzed in the present study in order to evaluate the strength of potential markers serving as future prognostic and predictive factors.
For this study, a branched-DNA assay was utilized to analyze the gene expression in FF and FFPE samples of canine mammary tissues. The b-DNA assay permits to analyze specimens with small amounts of RNA (and therefore suitable to analyze valuable FFPE specimens) as well as several target genes within a single sample. The reasons for the study were: (i) to analyze if valuable FFPE specimens, which represent a unique source of archived biological material, could be measured with the branched DNA assay; (ii) to discuss the advantages offered by the referred method when analyzing gene expression in comparison to techniques such as IHC and qPCR. Therefore, it was of interest to determine whether the gene expression between FFPE and FF specimens is comparable when applying the referred technique and whether the different storage times have any influence on the outcomes. Moreover, the opportunity to evaluate the performance of three different housekeepers in mammary canine tissues was taken.
Overall, the technique revealed to be feasible to analyze FF and FFPE samples of canine mammary tissues. The relative gene expression of the two different origins of samples showed an agreement of 63% when normalizing the data to the average of the three HKG. Still, care should be taken as FFPE specimens showed, in general, lower expression of the analyzed targets when compared to FF samples.
It was hypothesized that this finding is caused by the effect of storage time. The subsequent comparison among three different storage times of FFPE revealed a clearly higher expression of target genes in the short storage time group with a decrease in the expression in the other groups over time. Regarding the different HKG, data normalized against ACTB
Summary
39
was found to present the lowest significant difference between the FF and FFPE samples when comparing the histological groups followed by HPRT1 and GAPDH.
Bearing in mind that the technique showed to be feasible for the analysis of FF and FFPE samples in canine mammary tissues, it enabled us to analyze the gene expressions of different target genes in neoplastic (benign and malignant) and non-neoplastic tissues with the intention of finding new potential tumor markers. 16 onco- and suppressor-genes (BRCA1; BRCA2; FOXO3; GATA4; HER2; HMGA1; HMGA2; HMGB1; MAPK1; MAPK3;
MCL1; MYC; PFDN5; PIK3CA; PTEN and TP53) regarded as being involved in neoplasm development were analyzed in the present study using a multiplex b-DNA assay. As hormone receptors also play an important role in human BC, the opportunity was taken to concomitantly analyze the gene expressions of ER, PR, PRLR and GHR herein. The results and discussion about them are, however, described in another work.
Considering that an agreement of 63% was revealed between FF and FFPE, the samples were analyzed separately.
The significant reduction in the expression of the tumor suppressors TP53, FOXO3, PTEN and PFDN5 in malignant tumors found in the present study confirms previous reports, underlining their role in cellular growth. This is the first study which analyzed the gene expression of FOXO3 in CMT. Hence, these genes could represent potential markers for CMT. On the contrary to what is normally reported in humans, the genes HMGA1, HMGB1, MCL1, MAPK3 and PIK3CA, which are genes known to stimulate cellular growth, revealed to be consistently lower expressed in malignant tumor. Therefore, a hypothesis is raised whether those genes play a different role in canine mammary tumor.
The multiplex b-DNA technology is certainly a method which enables the analysis of several samples which present small amounts of RNA of FF and FFPE tissue. Still, care should be taken considering that FFPE specimens generally present a lower expression of the analyzed target genes when compared to FF samples, fact which is related to the storage time. In this work, a considerably larger number of samples was analyzed when compared to other studies performed in this field so far, permitting, consequently, a more reliable outcome.
Based on the outcomes of the present work, the suppressor genes TP53, FOXO3, PTEN and PFDN5 are considered as potential markers for the behavior of canine mammary tumors. The comparison of the expression patterns of the candidate genes in neoplastic (benign and malignant) and non-neoplastic tissues herein described might serve to gain a better understanding of the tumor pathogenesis of different canine mammary tumor subtypes in further studies. Moreover, potential target genes might assist to determine whether certain breeds are prone to develop CMT. The identification of suitable target genes offers the
Summary
40
prerequisite to search for novel therapeutic approaches in the future, just like in the human counterpart.
Zusammenfassung
41 5 Zusammenfassung
Vergleich von kryokonservierten und Formalin-fixierten, Paraffin-eingebetteten Mammagewebe des Hundes und die Expressions-Analyse von 16 Target-Genen mittels Multiplex Branched-DNA Assay
Florenza Lüder Ripoli
Mammatumoren sind die häufigste Neoplasie der Hündin. Dabei kommen sie bei bestimmten Rassen gehäuft vor. Die Anwendung von Tumormarkern bei Brustkrebs ist bereits intensiv untersucht worden und manche Gene werden beim Menschen bereits routinemäßig als prädiktive, diagnostische und prognostische Marker oder für Therapie- Optionen genutzt. Der Einsatz von Tumormarkern bei kaninen Mammatumoren ist jedoch zurzeit nur für Forschungszwecke von Bedeutung. Dementsprechend werden in der vorliegenden Studie Genprofile in verschiedenen Mammatumorentitäten des Hundes untersucht, um potenzielle prognostische oder prädiktive Marker zu finden.
Für die vorliegende Studie wird zur Genexpressionsanalyse aus Mammagewebe des Hundes ein Branched DNA Assay für kryokonservierte und FFPE-Proben verwendet. Das Branched DNA Assay ermöglicht die Analyse von Proben mit geringer Menge an RNA (dementsprechend passend für die Untersuchung von wertvollen FFPE-Proben), sowie die gleichzeitige Analyse von mehreren Target-Genen in einer Probe. Gründe für die vorliegende Untersuchung waren: (i) festzustellen, ob FFPE-Proben, die im Rahmen von histopathologischen Untersuchungen als archiviertes Material einen großen Probenpool darstellen, für zukünftige Genexpressions-Analysen mit dieser Methode gemessen werden können; (ii) die Vorteile der genutzten Methode mit anderen Methoden, wie IHC oder qPCR, zu diskutieren. Deshalb wurde untersucht, ob die gemessene Genexpression zwischen kryokonservierten und FFPE-Proben mit dem b-DNA-Assay vergleichbar ist, und ob die verschiedenen Lagerungszeiten der Proben einen Einfluss auf die Genexpression haben.
Zusätzlich wurden die Ergebnisse von drei unterschiedlichen Housekeeping-Genen bewertet.
Insgesamt zeigte die angewendete Methode sowohl für kryokonservierte, als auch für FFPE-Proben weitgehend verlässliche Ergebnisse im Mammagewebe des Hundes. Die relative Genexpression der beiden Probenarten zeigte, unter Verwendung des Mittelwertes von drei Housekeeping-Genen, eine Übereinstimmung von 63%. Hierbei muss beachtet werden, dass FFPE-Proben allgemein eine niedrigere Expression zeigten als die kryokonservierten Proben. Als eine Ursache dafür wurde die Lagerungszeit vermutet. Der daraufhin durchgeführte Vergleich von drei verschiedenen Lagerungszeiten von FFPE- Proben ergab, dass eine eindeutig höhere Expression der Target-Gene in der Gruppe der
Zusammenfassung
42
Proben mit kürzerer Lagerungszeit vorlag, während die Expression in den anderen beiden Gruppen mit der Zeit abnahm. Im Bezug auf die eingesetzten Housekeeping-Gene stellte sich heraus, dass ACTB für die Normalisierung der Daten am wenigsten Unterschiede zwischen kryokonservierten und FFPE-Proben im Vergleich der Genexpression innerhalb der histologischen Gruppen zeigte, gefolgt von HPRT1 und GAPDH.
Die Erkenntnis, dass sowohl kryokonservierte, als auch FFPE-Proben von kaninem Mammagewebe verlässliche Ergebnisse liefern, war ein wichtiger Befund für die Analyse verschiedener Target-Gen-Expressionen in neoplastischem (gutartig und bösartig) und nicht- neoplastischem Mammagewebe mit dem Ziel potentielle Tumormarker zu detektieren. 16 Onko- und Tumorsuppressorgene (BRCA1; BRCA2; FOXO3; GATA4; HER2; HMGA1;
HMGA2; HMGB1; MAPK1; MAPK3; MCL1; MYC; PFDN5; PIK3CA; PTEN und TP53), welche bei der Entwicklung von Neoplasien der Mamma eine Rolle spielen, wurden in der vorliegenden Studie mittels Multiplex Branched DNA Assay untersucht. Da auch Hormonrezeptoren eine wichtige Bedeutung bei Brustkrebs beim Menschen besitzen, wurde die Expression von ER, PR, PRLR und GHR in der vorliegenden Studie zeitgleich mitgemessen. Die Ergebnisse und deren Diskussion waren aber Teil einer weiteren Studie.
Aufgrund der Tatsache, dass lediglich eine Übereinstimmung von 63% im ersten Teil der Studie vorhanden war, wurden kryokonservierten und FFPE-Proben getrennt voneinander analysiert.
Die in der vorliegenden Studie festgestellte signifikante Abnahme der Expression von den Tumorsuppressor-Genen TP53, FOXO3, PTEN und PFDN5 in malignen Tumoren bestätigt Ergebnisse früherer Studien und unterstreicht die Bedeutung der genannten Gene beim Zellwachstum. Dabei wurde die FOXO3-Expression in der vorliegenden Studie erstmalig im Zusammenhang mit kaninen Mammatumoren gemessen. Dementsprechend haben diese vier Gene das Potenzial als Tumormarker bei Mammatumoren des Hundes genutzt zu werden. Im Gegenteil zu dem was beim Menschen festgestellt wurde, wiesen die Gene HMGA1, HMGB1, MCL1, MAPK3 und PIK3CA, welche das Zellwachstum stimulieren sollen, eine durchweg niedrigere Expression in malignen Mammatumoren auf. Ensprechend könnten sie in Mammatumoren des Hundes eine andere Aufgabe einnehmen, als beim Menschen.
Die Multiplex-Branched-DNA-Technologie stellt definitiv eine Methode dar, welche die Analyse von mehreren Proben mit einer geringen Menge an Startmaterial von kryokonservierten und FFPE-Proben ermöglicht. Dennoch muss beachtet werden, dass FFPE-Proben im Allgemeinen eine niedrigere Expression der analysierten Target-Gene aufweisen, als aus kryokonservierten Proben. Dabei spielt die Lagerungszeit eine Rolle. In der vorliegenden Studie wurde, im Vergleich mit anderen Studien, eine größere Zahl an Proben analysiert wodurch entsprechend eine verlässlichere Aussage zu erwarten ist.
Zusammenfassung
43
Basierend auf den Ergebnissen der vorliegenden Studie, können die Tumorsuppressor-Gene TP53, FOXO3, PTEN und PFDN5 als potenzielle Marker für das Verhalten von Mammatumoren beim Hund angesehen werden. Der Vergleich der Expressionsmuster der Target-Gene in neoplastischem (benigne und maligne Tumoren) und nicht-neoplastischem Gewebe könnte für das bessere Verständnis der Tumorpathogenese in verschiedenen kaninen Mammatumor-Untergruppen in zukünftigen Studien von Bedeutung sein. Zusätzlich könnten die potenziellen Target-Gene Grundlagen sein herauszufinden, warum verschiedene Rassen eine Prädisposition für Mammatumoren haben. Die Identifikation von geeigneten Target-Genen bietet auch die Voraussetzung in der Zukunft nach gentherapeutischen Ansätzen zu forschen.
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