Frequent PIK3CA-activating mutations in hidradenoma papilliferums

    Frequent PIK3CA Activating Mutations in Hidradenoma Papilliferums Jau-Yu Liau, Jui Lan, Jin-Bon Hong, Jia-Huei Tsai, Kuan-Tin Kuo, Chia-Yu Chu, Yi-Shuan Sheen, Wen-Chang Huang PII: DOI: Reference:

S0046-8177(16)30063-6 doi: 10.1016/j.humpath.2016.04.014 YHUPA 3892

To appear in:

Human Pathology

Received date: Revised date: Accepted date:

4 February 2016 5 April 2016 21 April 2016

Please cite this article as: Liau Jau-Yu, Lan Jui, Hong Jin-Bon, Tsai Jia-Huei, Kuo Kuan-Tin, Chu Chia-Yu, Sheen Yi-Shuan, Huang Wen-Chang, Frequent PIK3CA Activating Mutations in Hidradenoma Papilliferums, Human Pathology (2016), doi: 10.1016/j.humpath.2016.04.014

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ACCEPTED MANUSCRIPT Frequent

PIK3CA

Activating

Mutations

in

Hidradenoma

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Papilliferums

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Running Title: PIK3CA mutations in hidradenoma papilliferums

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Jau-Yu Liau,1,2 Jui Lan,3 Jin-Bon Hong,4 Jia-Huei Tsai,1,2 Kuan-Tin Kuo,1 Chia-Yu

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Pathology, National Taiwan University Hospital and National

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Department of

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Chu,4 Yi-Shuan Sheen,4 and Wen-Chang Huang5

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Taiwan University College of Medicine, and 2Graduate Institute of Pathology,

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National Taiwan University College of Medicine, Taipei, Taiwan. Department of 3Pathology, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung, Taiwan Department of 4Dermatology, National Taiwan University Hospital and National Taiwan University College of Medicine, Taipei, Taiwan Department of 5Pathology, Wan Fang Hospital, Taipei Medical University, Taipei, Taiwan

Correspondence: Wen-Chang Huang, Department of Pathology, Wan Fang Hospital,

ACCEPTED MANUSCRIPT Taipei Medical University, No.111, Sec. 3, Xinglong Rd., Wenshan Dist., Taipei 11696, Taiwan. Phone: 886-2-29307930 Ext: 1483; Fax: 886-2-86621139;

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E-mail: [email protected]

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Disclosure/conflict of interest: The authors declare no conflict of interest.

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Funding source: None

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anogenital mammary-like glands

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Keywords: hidradenoma papilliferum, PIK3CA, AKT1, intraductal papilloma,

ACCEPTED MANUSCRIPT Abstract

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Hidradenoma papilliferum (HP) is a benign epithelial tumor most commonly

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seen in the vulva. It is proposed to be derived from the anogenital mammary-like

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glands and is histologically very similar to the mammary intraductal papilloma (IP). Approximately 60% of mammary IPs have activating mutations in either PIK3CA or

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AKT1 gene, with each gene accounting for 30% of cases. In this study, we screened the mutation statuses of PIK3CA, AKT1, RAS, and BRAF in 30 HPs. The results showed that activating mutations in either PIK3CA or AKT1 gene were identified in

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20 tumors (67%); 19 tumors had PIK3CA mutations (63%, 13 in exon 20 and 6 in

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exon 9), and 1 had an AKT1 E17K mutation (3%). BRAF V600E mutation was found

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in a HP that also had a PIK3CA H1047R mutation. No RAS mutation was found. The

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mutation status was not correlated with the degree of epithelial cell hyperplasia. We conclude that although there might be site-related variations in the mutation frequencies of PIK3CA and AKT1 genes, HP is histologically and also genetically very similar to the mammary IP, suggesting that HP can be viewed as the extramammary counterpart of mammary IP.

ACCEPTED MANUSCRIPT 1. INTRODUCTION

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Hidradenoma papilliferum (HP) is an uncommon benign epithelial tumor

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showing apocrine differentiation. HP is most commonly seen in the labia majora or

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labia minora [1]. Less commonly, it may occur in the clitoris, perianal area, and perineum. Clinically, HP presents as a solitary asymptomatic nodule or cyst-like

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lesion. Almost all reported cases occur in women, usually young or middle aged. It was thought to originate from cutaneous sweat glands, but later suggested to be derived from ectopic breast tissue or the so-called anogenital mammary-like glands

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(AGMLGs) [2].

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Histologically, HP is very similar, if not identical, to the intraductal papilloma (IP)

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of the breast, demonstrating solid-cystic proliferation of branching papillae and

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tubular glands lined by epithelial cells containing moderate to abundant eosinophilic cytoplasm and a layer of myoepithelial cells. All kinds of epithelial, metaplastic or stromal changes that can be seen in mammary IPs such as decapitation secretion, epithelial hyperplasia, mucinous metaplasia, and stromal sclerosis can be observed in HPs. HP is circumscribed, often surrounded by a fibrous pseudocapsule, and can be removed by simple excision. The pathogenesis of HP is not clear. Previously it has been evaluated for the presence of human papilloma virus, but the virus does not appear to play crucial roles in its pathogenesis [3,4].

ACCEPTED MANUSCRIPT The AGMLG constitutes a ductal-glandular tissue mainly seen in the sulcus

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between the labia majora and labia minora [5], and its anatomical distribution mirrors

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the predilected sites of HP. It has been suggested that AGMLG is an ectopic breast

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tissue representing the distal end of the milk ridges. However, van der Putte believed that the milk line does not exist in humans and that the AGMLG is a normal

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constituent of the anogenital area [5].

Although the histogenesis of AGMLG is disputed, on morphological grounds the AGMLG is very similar to mammary tissue, and many disease entities that are

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suggested to arise in AGMLG, such as HP, fibroepithelial tumors (fibroadenoma and

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phyllodes tumor) and mammary type ductal carcinoma, have morphological

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counterparts in the breast [1]. It is reasonable to speculate that the mechanisms of

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tumorigenesis may be similar in these 2 tissues. Recently, nearly two-thirds of mammary IPs have been shown to harbor activating mutations in PIK3CA, AKT1, or RAS genes [6]. On the basis of their morphological similarities, we speculated that HP may also have mutations in these genes and conducted the present study to test this hypothesis.

ACCEPTED MANUSCRIPT 2. MATERIALS AND METHODS

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2.1 Tumor Samples

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Cases of HP with available formalin-fixed and paraffin-embedded tissue blocks

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were retrieved from the archives of the Department of Pathology of National Taiwan University Hospital. The diagnosis was confirmed through re-examination of the

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histological sections by 3 pathologists (JY Liau, KT Kuo, and WC Huang). Because in IPs of the breast, AKT1 mutations have been shown to be more common in tumors with no to mild epithelial hyperplasia, whereas PIK3CA mutations are more common

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in tumors with moderate to marked epithelial hyperplasia [6], the degree of epithelial

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hyperplasia was recorded. The microscopic tumor size was also measured. This study

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was approved by the Research Ethics Committee of National Taiwan University

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Hospital, and the specimens were anonymous and analyzed in a blind manner. 2.2 Mutation Analyses DNA extraction, amplification, and sequencing were performed as previously described [7]. Briefly, tumoral and non-tumoral tissues were dissected from 10-µm sections that were cut from paraffin blocks. Genomic DNA was extracted using a QIAamp DNA FFPE Tissue Kit (Qiagen, Santa Clarita, CA, USA) according to the manufacturer’s protocol. After polymerase chain reaction amplification and purification, direct sequencing was performed using an automated ABI 3770

ACCEPTED MANUSCRIPT sequencer (Applied BioSystems, Foster City, CA, USA). The primer sequences used

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to amplify the following genes: KRAS (exons 2 and 3), HRAS (exons 2 and 3), NRAS

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(exons 2 and 3), BRAF (exon 15), PIK3CA (exons 9 and 20), and AKT1 (exon 4) are

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listed in Table 1. 2.3 Statistical Analyses

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Data analyses were conducted using SPSS 19 (IBM Corp., Armonk, NY). Comparisons of categorical variables were performed using the Pearson  method or Fisher’s exact test as appropriate. Continuous variables were analyzed using the

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Student’s t test.

ACCEPTED MANUSCRIPT 3. RESULTS

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3.1 Clinicopathological Features

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In total, 32 HP were retrieved and analyzed. All patients were female ranging in

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age from 29 to 67 years (mean: 44; median: 43) at the time of excision. To our knowledge, most, if not all of the patients were Taiwanese. The majority of cases (29)

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occurred in the vulva. Three cases occurred in the perianal region. All lesions were solitary and presented with asymptomatic papulonodules or painful tumors when the surface was eroded. Tumor excision was performed in all patients. Histologically, 12

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tumors showed no or mild epithelial hyperplasia, and 20 showed prominent

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hyperplasia. Peritumoral stroma was included in 22 tumors. AGMLG was identified

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in 13 and absent in 9. Representative histological images are shown in Figure 1.

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3.2 Mutation Analyses

After discarding 2 tumors in which DNA quality was poor and PCR was unsuccessful, 30 HPs were included as the study cohort. The results of mutation analysis are shown in Table 2 and representative sequencing chromatographies are shown in Figure 1. PIK3CA activating mutations were identified in 19 tumors (63%). Thirteen occurred in exon 20 (12 with the H1047R mutation and 1 with the H1047L mutation), and 6 occurred in exon 9 (3 with the E542K mutation and 3 with the E545K mutation). One tumor harbored an AKT1 E17K mutation (Figure 1D). No

ACCEPTED MANUSCRIPT PIK3CA mutations were identified in the non-tumor parts of 5 PIK3CA-mutated

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tumors with available tissue.

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All mutations in the PIK3CA and AKT1 genes were mutually exclusive; therefore,

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two-thirds (20 of 30) of the HPs had a mutation in either gene. In addition, one tumor with a PIK3CA H1047R mutation also had a BRAF V600E mutation (Figure 1A). No

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mutation was found in RAS genes. All mutations (PIK3CA, AKT1, and BRAF) identified in this study have been previously shown to be activating and are the most common variants recorded in the COSMIC database [8-12].

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3.3 Molecular-Histological Correlations

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Of the 30 successfully analyzed tumors, 11 showed no or mild epithelial

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hyperplasia, whereas 19 showed prominent hyperplasia. PIK3CA mutations were

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found in 64% (7 of 11) and 63% (12 of 19) of the former and latter subgroups, respectively. The AKT1-mutated tumor also exhibited prominent hyperplasia. Of the 13 tumors in which AGMLG could be seen, 8 had PIK3CA mutations (62%), whereas 5 of the 9 tumors (56%) without AGMLG had PIK3CA (4 tumors) or AKT1 (1 tumor) mutations. The presence of a PIK3CA or AKT1 mutation was not correlated with the presence or absence of AGMLG (P = 1.000, Fisher’s exact test). Eight tumors did not have sufficient peritumoral stroma for evaluation of the presence of absence of AGMLG. Interestingly, this subset had the highest PIK3CA mutation

ACCEPTED MANUSCRIPT rate: mutation was found in 7 of the 8 tumors (88%).

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Regarding tumor size, although the mean tumor size of those with mutations

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were slightly larger than those without mutations (6.20 mm vs. 5.30 mm), the

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presence of a PIK3CA or AKT1 mutation was not associated with the tumor size (P = 0.418). Notably, although statistically non-significant, the 8 tumors in which the

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presence or absence of AGMLG could not be determined tended to be larger than the 22 tumors in which the presence of AGMLG could be evaluated (7.50 mm vs. 5.32

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mm, P = 0.162).

ACCEPTED MANUSCRIPT 4. Discussion

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In this study, we investigated the mutation statuses of PIK3CA, AKT1, RAS, and

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BRAF genes in a series of 30 HPs on the basis that HPs and mammary IPs show

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similar histological features. We demonstrated that activating mutations in either PIK3CA or AKT1 gene were present in two-thirds of HPs, thus providing a strong

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molecular link between these 2 entities.

Phosphoinositide 3-kinase (PI3K) is a heterodimeric lipid kinase composed of the catalytic subunit (p110α, PIK3CA) and the regulatory subunit (p85, PIK3R1) [13].

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The PI3K/AKT pathway is implicated in cell proliferation, protein translation, cell

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metabolism, and anti-apoptosis. Approximately 30% of in situ and invasive breast

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carcinomas have activating mutations in PIK3CA (26%) or AKT1 (4%) gene [14].

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Furthermore, several benign proliferative breast lesions, such as IP, usual ductal hyperplasia, columnar cell lesion, and radial scar harbor mutations in these 2 genes in up to one-half to two-thirds of cases [6,15-17], indicating that the activation of this pathway plays crucial roles in mammary tumorigenesis. In addition to HP, a wide variety of benign and malignant tumors, such as lactating adenoma, fibroadenoma, and mammary ductal carcinoma, are also supposed to be derived from the AGMLG. The presence of similar genetic alterations in HPs and mammary IPs may lead to a suggestion that AGMLG is a type of ectopic breast

ACCEPTED MANUSCRIPT tissue. However, because the breast is supposed to be a modified apocrine gland, we

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cannot exclude the possibility that activation of the PI3K/AKT pathway via mutation

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is a shared and common feature in the tumorigenesis of cutaneous sweat glands and

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all of their derivatives. Nevertheless, we are accord with others that the most effective approach to study AGMLG-derived tumors is to compare them with their mammary

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counterparts [1]. We also speculate that mutations found in mammary tumors, such as the recently reported MED12 mutations in mammary fibroepithelial tumors [18,19], may also be present in their extramammary counterparts.

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Although HPs and mammary IPs showed a similar overall mutation rate in

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PIK3CA and AKT1 genes, the distribution of the mutations appeared to be quite

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different between them. In mammary IPs, PIK3CA and AKT1 mutations are equally

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common [6]. In addition, in tumors showing absent to mild hyperplasia, AKT1 mutations are more common than PIK3CA mutations, whereas the reverse is seen in IPs with prominent epithelial hyperplasia. By contrast, in our study, 19 of 20 mutations identified (95%) were PIK3CA mutations, and AKT1 mutation was found in only one tumor. The mutations were also not correlated with the degree of epithelial hyperplasia. In fact, the AKT1-mutated tumor exhibited prominent hyperplasia histologically. We speculate that there might be potential site- and/or ethnicity-related variations in the genes involved in the pathogenesis of HPs and mammary IPs.

ACCEPTED MANUSCRIPT In our study, one tumor exhibited both BRAF V600E and PIK3CA H1047R

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mutations. No BRAF mutation was found in the previous study of mammary papillary

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neoplasms, but 2 of 89 papillary tumors had RAS mutations (HRAS G12D and NRAS

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Q61H) [6]. The first tumor was an IP exhibiting atypical ductal hyperplasia, and the second was an invasive papillary carcinoma which also had multiple PIK3CA exon 13

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mutations. Similarly, a small subset (approximately 5%) of extramammary Paget disease cases have concurrent PIK3CA/AKT1 and RAS/BRAF mutations, suggesting that activating mutations in both pathways are not mutually exclusive and may

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provide additive growth advantages [20]. We also considered the possibility that this

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BRAF-mutated tumor might be a misclassified syringocystadenoma papilliferum

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(SCAP), because BRAF V600E mutation was recently found in approximately 50% of

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nevus sebaceus-unrelated SCAPs [21], and some authors have suggested that HP and SCAP are closely related tumors. However, this BRAF-mutated tumor exhibited typical histological features of HP, with no atypical proliferative changes or features suggesting SCAP. Therefore, the significance of this combination in HP is undetermined. Nevertheless, our data indicate that HP and SCAP are genetically distinct tumors. Tumors derived from the sweat glands exhibit diverse histomorphologies. The underlying genetic changes are increasingly deciphered and most of them harbor

ACCEPTED MANUSCRIPT distinct molecular alterations. For example, BRAF/RAS mutations are commonly

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present in SCAPs whereas the CRTC1-MAML2 gene fusion is identified in 50% of

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cutaneous hidradenomas [21,22]. We also analyzed the PIK3CA/AKT1 status in small

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numbers of eccrine poromas and apocrine cystadenomas but none had mutations (data not shown). Further studies are necessary to identify the genetic changes in these

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tumors.

In our series, PIK3CA and AKT1 mutations were not correlated with the presence or absence of AGMLG or tumor size. Interestingly, tumors lacking sufficient

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peritumoral stroma to determine the presence of AGMLG had the highest mutation

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rate and tended to be larger. We speculate that tumors harboring mutations might be

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larger and have a greater compression effect, enabling them to have fibrous

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pseudocapsules that permit the clinicians to perform a more conservative surgery with narrow margins.

In summary, we demonstrate that, like the mammary IPs, activating mutations in PIK3CA or AKT1 gene are present in the majority of HPs. Our data suggest that they can essentially be regarded as the same tumor on the basis of their shared histomorphologies and molecular alterations. We also speculate that other tumors derived from the AGMLG will possess the same or closely related genetic changes as their mammary counterparts do.

ACCEPTED MANUSCRIPT References

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[1] Kazakov DV, Spagnolo DV, Kacerovska D, Michal M. Lesions of anogenital

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mammary-like glands: an update. Adv Anat Pathol 2011;18:1-28.

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[2] van der Putte SC. Mammary-like glands of the vulva and their disorders. Int J Gynecol Pathol 1994;13:150-60.

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[3] Kazakov DV, Mikyskova I, Kutzner H, et al. Hidradenoma papilliferum with oxyphilic metaplasia: a clinicopathological study of 18 cases, including detection of human papillomavirus. Am J Dermatopathol 2005;27:102-10.

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[4] Kazakov DV, Nemcova J, Mikyskova I, Belousova IE, Vazmitel M, Michal M.

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Human papillomavirus in lesions of anogenital mammary-like glands. Int J Gynecol

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Pathol 2007;26:475-80.

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[5] van der Putte SC. Anogenital "sweat" glands. Histology and pathology of a gland that may mimic mammary glands. Am J Dermatopathol 1991;13:557-67. [6] Troxell ML, Levine J, Beadling C, et al. High prevalence of PIK3CA/AKT pathway mutations in papillary neoplasms of the breast. Mod Pathol 2010;23:27-37. [7] Liau JY, Tsai JH, Yuan RH, Chang CN, Lee HJ, Jeng YM. Morphological subclassification of intrahepatic cholangiocarcinoma: etiological, clinicopathological, and molecular features. Mod Pathol 2014;27:1163-73. [8] Zhao L, Vogt PK. Class I PI3K in oncogenic cellular transformation. Oncogene

ACCEPTED MANUSCRIPT 2008;27:5486-96.

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[9] Vogt PK, Kang S, Elsliger MA, Gymnopoulos M. Cancer-specific mutations in

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phosphatidylinositol 3-kinase. Trends Biochem Sci 2007;32:342-9.

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[10] Gymnopoulos M, Elsliger MA, Vogt PK. Rare cancer-specific mutations in PIK3CA show gain of function. Proc Natl Acad Sci U S A 2007;104:5569-74.

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[11] Carpten JD, Faber AL, Horn C, et al. A transforming mutation in the pleckstrin homology domain of AKT1 in cancer. Nature 2007;448:439-44. [12] Davies H, Bignell GR, Cox C, et al. Mutations of the BRAF gene in human

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cancer. Nature 2002;417:949-54.

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[13] Engelman JA. Targeting PI3K signalling in cancer: opportunities, challenges and

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limitations. Nat Rev Cancer 2009;9:550-62.

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[14] Troxell ML. PIK3CA/AKT1 mutations in breast carcinoma: a comprehensive review of experimental and clinical studies. J Clin Exp Pathol 2012;S1:002. [15] Troxell ML, Brunner AL, Neff T, et al. Phosphatidylinositol-3-kinase pathway mutations are common in breast columnar cell lesions. Mod Pathol 2012;25:930-7. [16] Wolters KL, Ang D, Warrick A, Beadling C, Corless CL, Troxell ML. Frequent PIK3CA mutations in radial scars. Diagn Mol Pathol 2013;22:210-4. [17] Ang DC, Warrick AL, Shilling A, Beadling C, Corless CL, Troxell ML. Frequent phosphatidylinositol-3-kinase mutations in proliferative breast lesions. Mod Pathol

ACCEPTED MANUSCRIPT 2014;27:740-50.

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[18] Lim WK, Ong CK, Tan J, et al. Exome sequencing identifies highly recurrent

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MED12 somatic mutations in breast fibroadenoma. Nat Genet 2014;46:877-80.

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[19] Yoshida M, Sekine S, Ogawa R, et al. Frequent MED12 mutations in phyllodes tumours of the breast. Br J Cancer 2015;112:1703-8.

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[20] Kang Z, Xu F, Zhang QA, et al. Oncogenic mutations in extramammary Paget's disease and their clinical relevance. Int J Cancer 2013;132:824-31. [21] Shen AS, Peterhof E, Kind P, et al. Activating mutations in the

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RAS/mitogen-activated protein kinase signaling pathway in sporadic trichoblastoma

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and syringocystadenoma papilliferum. Hum Pathol 2015;46:272-6.

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[22] Winnes M, Mölne L, Suurküla M, et al. Frequent fusion of the CRTC1 and

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MAML2 genes in clear cell variants of cutaneous hidradenomas. Genes Chromosomes Cancer 2007;46:559-63.

ACCEPTED MANUSCRIPT Figure Legend

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Figure 1. A hidradenoma papilliferum with concomitant PIK3CA exon 20 H1047R

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(lower panel) and BRAF exon 15 V600E (upper panel) mutations (A). A hidradenoma

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papilliferum with a PIK3CA exon 20 H1047L mutation (B). A hidradenoma papilliferum with a PIK3CA exon 9 E542K mutation. AGMLG is present adjacent to

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the tumor (asterisk and the inset). (C). A hidradenoma papilliferum with an AKT1 exon 4 E17K mutation (arrowhead, GAG>AAG) (D). Histological images of A, B, C, and D: hematoxylin and eosin stain; original magnification: A, B, and D: X100; C:

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X40.

ACCEPTED MANUSCRIPT Table 1 Primer sequences Gene

Forward

Reverse

GTGTGACATGTTCTAATATAGTCA

GAATGGTCCTGCACCAGTA

KRAS exon 3

CCAGACTGTGTTTCTCCCTT

GGCAAATACACAAAGAAAGCCC

HRAS exon 2

CAGGAGACCCTGTAGGAGG

GCCAGGCTCACCTCTATAGT

HRAS exon 3

GTCCTCCTGCAGGATTCCTA

TTCACCTGTACTGGTGGATG

NRAS exon 2

GGTGTGAAATGACTGAGTAC

GGGCCTCACCTCTATGGTG

NRAS exon 3

GGTGAAACCTGTTTGTTGGA

GCTCCTAGTACCTGTAGAGGT

BRAF exon 15

TCATAATGCTTGCTCTGATAGGA

GGCCAAAAATTTAATCAGTGGA

PIK3CA exon 9

TTTTCTGTAAATCATCTGTGAATCC

PIK3CA exon 20

ATCATTTGCTCCAAACTGACCA

AKT1 exon 4

TAAGAAACAGCTCCCGTACC

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KRAS exon 2

TCTCCATTTTAGCACTTACCTGTGA

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TTGTGTGGAAGATCCAATCCAT ACAGAGAAGTTGTTGAGG

ACCEPTED MANUSCRIPT Table 2 Results of histological features and mutation analysis Sample

Prominent

Microscopic

PIK3CA

PIK3CA

AKT1

BRAF

hyperplasia

tumor size (mm)

exon 9

exon 20

exon 4

exon 15

WT

WT

AGMLG present

absent

4

WT

H1047R

2

present

absent

6

WT

WT

WT

WT

3

n/a

present

6

WT

H1047R

WT

WT

4

present

present

8

WT

H1047R

WT

WT

5

present

absent

5

WT

H1047R

WT

WT

6

n/a

present

6

WT

WT

WT

7

present

present

4

WT

WT

WT

WT

8

absent

present

4

WT

WT

WT

WT

9

present

absent

10

present

absent

11

absent

present

12

present

present

13

present

present

14

present

present

15

absent

16

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6

WT

WT

WT

WT

3

WT

H1047R

WT

WT

3

WT

WT

WT

WT

6

WT

WT

WT

WT

3

WT

H1047R

WT

WT

6

E542K

WT

WT

WT

present

12

WT

H1047R

WT

WT

n/a

present

15

E545K

WT

WT

WT

17

present

absent

3

E542K

WT

WT

WT

18

absent

present

8

WT

WT

WT

WT

19

present

present

5

WT

H1047R

WT

V600E

20

absent

present

6

WT

WT

E17K

WT

21

absent

present

6

WT

H1047R

WT

WT

n/a

absent

5

E542K

WT

WT

WT

n/a

absent

12

WT

H1047R

WT

WT

n/a

absent

5

WT

WT

WT

WT

25

absent

present

3

WT

H1047L

WT

WT

26

absent

absent

5

E542K

WT

WT

WT

27

absent

present

4

WT

WT

WT

WT

28

n/a

present

5

E542K

WT

WT

WT

29

present

absent

7

WT

WT

WT

WT

30

n/a

present

6

WT

H1047R

WT

WT

23 24

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AC

22

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H1047R

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Abbreviations: n/a: not available; WT: wild type

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ACCEPTED MANUSCRIPT

Figure 1