Droplet-digital pcr reveals frequent mutations in tert promoter region in breast fibroadenomas and phyllodes tumours, irrespective of the presence of med12 mutations

ABSTRACT BACKGROUND Breast fibroadenoma (FA) and phyllodes tumour (PT) often have variations of gene mediator complex subunit 12 (_MED12_) and mutations in the telomerase reverse

transcriptase promoter region (_TERTp_). _TERTp_ mutation is usually tested by Sanger sequencing. In this study, we compared Sanger sequencing and droplet-digital PCR (ddPCR) to measure

_TERTp_ mutations in FA and PT samples. METHODS FA and PT samples were collected from 82 patients who underwent surgery at our institution from 2005 to 2016. _MED12_ mutations for all cases

and _TERTp_ mutations for 17 tumours were detected by Sanger sequencing. ddPCR was performed to analyse _TERTp_ mutation in all cases. RESULTS A total of 75 samples were eligible for

analysis. Sanger sequencing detected _MED12_ mutations in 19/44 FA (42%) and 21/31 PT (68%). Among 17 Sanger sequencing-tested samples, 2/17 (12%) were _TERTp_ mutation-positive. In ddPCR

analyses, a significantly greater percentage of PT (19/31, 61%) was _TERTp_ mutation-positive than was FA (13/44, 30%; _P_ = 0.0046). The mutation positivity of _TERTp_ and _MED12_ did not

correlate, in either FA or PT. CONCLUSIONS ddPCR was more sensitive for detecting _TERTp_ mutation than Sanger sequencing, being able to elucidate tumorigenesis in FA and PT. SIMILAR CONTENT

BEING VIEWED BY OTHERS SYSTEMATIC REVIEW AND FEASIBILITY STUDY ON PRE-ANALYTICAL FACTORS AND GENOMIC ANALYSES ON ARCHIVAL FORMALIN-FIXED PARAFFIN-EMBEDDED BREAST CANCER TISSUE Article Open

access 06 August 2024 GENETIC DIFFERENCES BETWEEN BENIGN PHYLLODES TUMORS AND FIBROADENOMAS REVEALED THROUGH TARGETED NEXT GENERATION SEQUENCING Article 16 March 2021 COMPARATIVE ANALYSIS OF

EGFR MUTATIONS IN CIRCULATING TUMOR DNA AND PRIMARY TUMOR TISSUES FROM LUNG CANCER PATIENTS USING BEAM_ING_ PCR Article Open access 08 January 2025 BACKGROUND Breast fibroepithelial

tumours, which include fibroadenoma (FA) and phyllodes tumour (PT), are characterised by the biphasic proliferation of both epithelial and stromal components.1,2,3,4,5 FA is common benign

tumour that is often observed in young women. It expresses oestrogen receptor (ER)-α in epithelium and ERβ in stromal components, and is known to be hormone dependent.6,7 Small FA (<3 cm)

is usually followed up without resection, and 16–37% of FA cases reportedly regress or completely resolve spontaneously.8,9,10 PT is much less common than FA; it comprises only 2–3% of

fibroepithelial breast tumours and accounts for <1% of all breast tumours. PT is histologically classified as benign, borderline or malignant type. Resection is generally recommended for

PT because it often grows rapidly and can potentially become malignant. Occasionally, PT enlarges to huge sizes that require total mastectomy, and malignant PT has high risks of both local

recurrences and distant metastasis compared with the other types of PT.1,2,3,10,11,12,13,14,15 FA and PT, especially when they remain relatively small, are morphologically so similar that

differentiating them histologically is sometimes difficult. Some studies have suggested that FA could potentially progress to PT, especially FA with monoclonal stromal components; however,

the mechanisms underlying their initiation and progression are unclear.10,11,16,17 Although FA and PT have been genomically analysed,18,19,20,21 little had been known about their genetic

abnormalities. However, since next-generation sequencing (NGS) became widely used in research, several genetical alterations have been revealed in both tumours. The discovery of highly

recurrent Mediator complex subunit 12 (_MED12_) somatic mutations in breast FA was surprising; almost nothing had previously been known about these mutations.22 As for PT, in addition to

_MED12_,10,11,12,13,23,24,25,26,27 the telomerase reverse transcriptase (_TERT_) promoter has been shown to have repeated mutations in these tumours.28,29,30,31,32,33,34 In 2014, Lim et al.

first found highly frequent _MED12_ exon 2 mutations in FA (58/98, 59%) using exome analysis.22 The _MED12_ gene, located on the X chromosome, encodes MED12 protein, a member of the

multiprotein mediator complex that regulates transcription of all RNA polymerase II-dependent genes.35 Reportedly, up to 60% of FA, 80% of benign and borderline PT and 40% of malignant PT

harbours somatic mutations in exon 2 of the _MED12_ gene,10,11,12,13,22,23,24,25,26,27 which suggests that FA and PT have much more in common in their origin or development than previously

thought. However, the underlying mechanisms, and how this mutation generates or induces the progression of FA and PT, are unknown. Since the discovery of frequent _MED12_ mutations in FA and

PT, next-generation sequencing has been used to search for other gene mutations in these tumours. Many mutations found in PT, such as in _RARA_, _EGFR_, _RB1_ or _TP53_, are very uncommon

compared with _MED12_ mutations,12,21,27,28,29,30 whereas mutations in the _TERT_ promoter region (_TERTp_) are reportedly more frequent: 0–7% in FA, and 27–70.6% in PT.29,30,31,32,33,34

_TERTp_ is considered to be a critical regulatory element for telomerase expression.35,36 Hotspots for mutations in this region in PT and FA are reported to be c.−146 C > T (C250T) and

c.−124 C > T (C228T),29,31,32,33,34 which are concordant with those in other tumours, including central nervous system tumours, thyroid cancers, bladder cancers and skin melanoma.36,37

Previous studies found correlations between _TERTp_ mutations (_TERTp__Mut_) and _MED12_ mutations (_MED12__Mut_),28,29,30,31 which suggests that these mutations interact, especially in the

development of PT. Recently, McEvoy et al. showed that droplet-digital polymerase chain reaction (ddPCR) is very sensitive in detecting _TERTp__Mut_ in melanoma compared with pyrosequencing

or Sanger sequencing (SS).38,39 Digital PCR was developed to yield absolute measures for nucleic acid concentrations by a combination of limiting dilution, end-point PCR and Poisson

statistics.40 ddPCR is a newer, more precise and less subjective assay to quantify DNA amplification, based on water–oil emulsion droplet technology. In ddPCR, a sample is fractionated into

20,000 droplets, and PCR amplification of the template molecules occurs in each individual droplet. ddPCR has also been shown to obtain high levels of partitioning at a low cost.41,42

_TERTp__Mut_ was previously evaluated with ddPCR, but only in melanoma.38,39 We found no reports of studies that evaluated mutations in breast fibroepithelial lesions using ddPCR. In this

study, we used ddPCR to measure _TERTp__Mut_ in formalin-fixed, paraffin-embedded (FFPE) samples of resected FA or PT, and compared the results with those from conventional SS. We also

analysed the relationships among _TERTp__Mut_ and _MED12__Mut_ status and histopathological characteristics in FA and PT. METHODS TISSUE SAMPLES We collected FFPE samples of 54 FA and 31 PT

from 82 patients who underwent surgery at the University of Tokyo Hospital from 2005 to 2016. Three patients had two tumours excised at different times. All samples were diagnosed by two

expert pathologists. FA was classified as intracanalicular type, pericanalicular type, mastopathic type, organoid type,43,44 complex fibroadenoma45 or juvenile fibroadenoma;46 PT was

subclassified according to the WHO classification as benign, borderline or malignant lesions.2 All patients consented to the use of their stored tumour tissue. This study protocol was

approved by the ethics committee at the University of Tokyo Hospital, Tokyo, Japan. DNA EXTRACTION For each specimen, two or three 10-µm FFPE sections were cut from a single representative

block per case. Macrodissection was performed with a scalpel as needed to adjust the tumour content to be visually more than 20%. Microdissection was not performed. DNA was isolated using a

GeneRead DNA FFPE Kit (Qiagen, Hilden, Germany), in accordance with the manufacturer’s instructions. Purified DNA was quantified using a NanoDrop 2000 spectrophotometer (Thermo Scientific,

Wilmington, DE, USA), with 0.5–14.0 µg of DNA recovered per section. SANGER SEQUENCING TO DETECT MED12 MUTATION FA and PT were analysed for mutations in exon 2 of _MED12_ by SS for all

samples. Exon 2 was amplified with the following primers: (exon 2 forward) 5′-AACTAAACGCCGCTTTCCTG-3′, (exon 2 reverse) 5′-TTCCTTCAGCCTGGCAGAG-3′10,47 (Supplementary Table S1). The PCR

products were purified using agarose gel electrophoresis, labelled with Big Dye Terminator (Applied Biosystems, Foster City, CA, USA) with bidirectional primers and subjected to 3130 × l

Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) in accordance with standard protocols. The Catalogue of Somatic Mutations in Cancer (COSMIC) database48 was used to identify

already-known somatic mutations and mutation types. SANGER SEQUENCING FOR DETECTING TERTP MUTATIONS _TERTp_ mutations (C228T and C250T) were analysed in FA and PT with the same extracted DNA

as used for the _MED12__Mut_ analysis. We performed SS on 17 samples, which had been surgically resected in 2015 and 2016. _TERTp_ was amplified with the following primers: (promoter

forward) 5′-AGCGCTGCCTGAAACTCG-3′, (promoter reverse) 5′-CCTGCCCCTTCACCTTCCAG-3′31,34 (Supplementary Table S1). Q-Solution (Qiagen) was added to the reaction to amplify _TERTp_ by PCR, in

accordance with previous reports.38,39 The SS method was the same as that for the _MED12__Mut_ analysis described above. DDPCR FOR DETECTING TERTP MUTATIONS Analysis of _TERTp_ mutations

(C228T and C250T) with ddPCR was performed for all of the samples, using a previously described ddPCR method.38,39 Primers were the same as those used in SS (Supplementary Table S1). Two

probes were used to detect the mutations: one (the mutant probe) was designated as “/56-FAM/CCC + C + T + T + CCGG/3IABkFQ/” to detect both C228T and C250T mutations (as both mutations

result in the same sequencing string), and the other (the wild probe) was designated as “/5HEX/CCCC + C + T + CCGG/3IABkFQ/,” to recognise the C228 locus (Supplementary Fig. S1). The probes

were custom-synthesised by Integrated DNA Technologies (Coralville, IA, USA). PCR reactions were performed in 20-mL reactions that contained 10 µL of Bio-Rad 2× ddPCR Supermix for Probes (no

dUTP) (Bio-Rad, Hercules, CA, USA), 250 nmol/L probe, 900 nmol/L primers, 10–290 ng of DNA and water. Reaction mixtures were partitioned into emulsions of ~20,000 droplets in oil using a

QX200 Droplet Generator (Bio-Rad). The droplets were then transferred to a 96-well PCR plate, heat-sealed and placed in a thermal cycler (Bio-Rad PX1). Droplets were generated and analysed

using the QX200 Droplet Digital PCR System (Bio-Rad). The thermal cycling conditions were 1 cycle at 95 °C (2.5 °C/s ramp) for 10 min, 40 cycles at 94 °C (2.5 °C/s ramp) for 30 s and at 57 

°C followed by 98 °C (2.5 °C/s ramp) for 10 min. Samples were held at 4 °C until further processing. After PCR, the PCR plates were loaded on a Bio-Rad QX200 droplet reader, and ddPCR

absolute quantifications of mutant and wild-type alleles were estimated by modelling them as a Poisson distribution, using Bio-Rad QuantaSoft version 1.6.6 software. Thresholds were defined

based on signals from empty droplets, wild-type DNA controls and mutant-positive controls, as described in the Droplet Digital Application Guide (Bio-Rad). STATISTICAL ANALYSIS All analyses

were performed using JMP Pro statistical software (ver. 14.1.0, SAS Institute, Tokyo, Japan). Fisher’s exact test or chi-squared test was used to analyse categorical data. Proportions were

compared by two-sample tests. The _t_ test, Mann–Whitney _U_ test or Kruskal–Wallis test was used to analyse continuous variables. _P_ < 0.05 was considered significant. RESULTS Among 85

FFPE tissue samples, 75 (44 FA and 31 PT) were eligible for analysis. Ten samples, including five FA samples with insufficient extracted DNA and five FA samples in which the _MED12_ amplicon

could not be amplified to perform SS, were excluded. We successfully performed ddPCR to detect _TERTp_ mutations in all PT samples and SS for _TERTp_ regions in all but one PT sample.

PATIENT CHARACTERISTICS Histological classifications of FA, histological grades of PT, ages and tumour sizes of each case are listed in Table 1. Among 44 FA, 18 samples were typed as

intracanalicular, 6 as pericanalicular, 8 as mastopathic, 7 as organoid, 4 as complex and 1 as juvenile fibroadenoma. Among 31 PT samples, 17 were classified as benign, 9 as borderline and 5

as malignant. The FA ranged in size from 9 to 125 mm, and the PT ranged from 23 to 130 mm. The Mann–Whitney _U_ test showed that patients with PT were significantly older (_P_ = 0.006) and

had larger tumours (_P_ < 0.001) than those with FA. MED12 MUTATIONS IN FA AND PT A significantly higher percentage of PT (21/31, 68%) was _MED12__Mut+_ than was FA (19/44, 42%, _P_ = 

0.035, two-sample test of proportions). Among FA, the intracanalicular type showed _MED12_ mutations more frequently than the other types (_P_ = 0.046, chi-squared test). The frequency of

_MED12_ mutations did not significantly differ among histological grades of PT (_P_ = 0.81, chi-squared test; Supplementary Table S2). Table 2 shows the types and locations of _MED12_

mutations. All of the missense mutations have already been reported in COSMIC,47 but many deletion and deletion/insertion patterns were newly discovered in this study (shown in red in Table 

2). TERTP MUTATIONS IN FA AND PT In this study, the only mutation site for _TERTp_ was C228T; we saw no case of C250T. In 16 cases examined for _TERTp__Mut+_ by both SS and ddPCR, SS found

that 12.5% (2/16) were _TERTp__Mut+_, whereas ddPCR found 37.5% (6/16; _P_ = 0.037, two-sample test of proportions). Both cases determined as _TERTp__Mut+_ by SS were PT; no FA case carrying

_TERTp_ mutations was found (Fig. 1 and Supplementary Table S3). ddPCR analyses of all tumours found that 42.7% (32/75) were _TERTp__Mut+_, which was significantly more frequent in PT

(19/31; 61.3%) than in FA (13/44; 29.5%; _P_ = 0.009, Fisher’s exact test, Fig. 1 and Table 3). The two samples that were found to be _TERTp__Mut+_ by SS (16a and 16i) were strongly positive

in ddPCR analysis, whereas those with discrepant results between SS and ddPCR (e.g. 16b and 16k) were weakly positive by ddPCR (Fig. 2). Correlations between _TERTp-_mutation status and

clinicopathological variables in FA and PT are shown in Table 3. Among FA, _TERTp__Mut+_ tumours were significantly larger than _TERTp__Mut–_ ones (_P_ = 0.015, Mann–Whitney _U_ test), but

this was not the case in PT (_P_ = 1.0). None of the other variables, such as patients’ age, FA classification, PT grade and _MED12-_mutation status, differed significantly between

_TERTp__Mut+_ and _TERTp__Mut−_ tumours. FRACTIONAL ABUNDANCE OF FAS AND PTS WITH TERTP MUTATION A total of 70 FA and PT samples with _TERTp_ mutation were detected by ddPCR at a fractional

abundance from 0.7% to 92% (Supplementary Table S4). PT had significantly higher fractional abundance of _TERTp_ mutation than FA: the mean fractional abundance in FA was 20% (range:

1.0–89%), while that in PT was 40% (range: 0.7–92%) (_P_ = 0.049, Mann–Whitney _U_ test). Among the cases analysed by both SS and ddPCR, fractional abundance was relatively high in the two

concordant cases (28.8% and 52.3%; Supplementary Table S5). Those cases that were both _MED12-_mutation-positive with SS and _TERTp-_mutation-positive with ddPCR did not show high fractional

abundance compared with the others (_P_ = 0.067, _t_ test; Supplementary Table S4). CONCORDANCE OF MED12 AND TERTP MUTATIONS BETWEEN METACHRONOUS MULTIPLE TUMOURS FROM THE SAME INDIVIDUALS

In this study, three patients had metachronous multiple tumours. All three cases harboured the same _MED12_ mutation in the primary and secondary lesions (Table 4). However, only one case,

of malignant PT, was _TERTp__Mut+_ in both primary and secondary tumours, whereas the other two cases carried this mutation only in the second tumour. DISCUSSION In this study, we describe a

method to detect _TERT_ promoter mutations in FA and PT using ddPCR. _TERT_ promoter mutation has been reported to be one of the most prevalent mutations in PT other than _MED12_ mutation.

ddPCR not only succeeded in detecting _TERT_ promoter mutations, but also revealed this mutation to be much more frequent among breast fibroepithelial tumours, especially in FA, than was

previously known. The advantages of ddPCR are its high sensitivity, quantitative measurement and low running cost. As ddPCR can detect mutations that have few patterns of single-base

substitution, it is entirely appropriate for _TERTp_ mutations, because only two hotspots have been reported in this region.29,31,32,33,34 ddPCR can distinguish mutations with a very low

frequency of allele variants that SS would not have detected. Here, we show the potential high sensitivity of ddPCR in detecting _TERTp__Mut+_ FA and PT, as in a previous report on melanoma

by McEvoy et al.39 Around the mutation sites of C228T and C250T, _TERTp_ region contains guanine and cytosine in high frequency. Such GC-rich regions affect the generation of secondary

products during PCR, and as a consequence, inhibit DNA polymerases from copying these sites correctly. Besides, the two mutation hotspots, C228T and C250T, locate close to each other. These

inconvenient factors reduce the sensitivity in sequencing analyses, including NGS. Such disadvantageous potentials remain challenging in ddPCR research; however, Corless BC et al.49 showed

excellent sensitivity and specificity in detecting _TERTp_ mutations by ddPCR. According to them, in ddPCR analysis of mutations in GC-rich regions, shorter amplicon length provides better

detection sensitivity. They mentioned that DNA in FFPE samples is shortly fragmented due to the chemical reaction between formalin and nucleic acid, which helps primers bind to the target

regions and generate short amplicons, leading to the efficient PCR reaction.49 Although FFPE samples are often considered to be unfavourable in sequencing analysis due to the low quality of

nucleic acid conditions, they might be even advantageous in ddPCR analysis for _TERTp_-mutation detection. There have been a few reports comparing the detection of genetic mutations with SS

and digital PCR, including one from McEvoy et al.39 and another from Sho et al.50 McEvoy et al. examined mutations in _BRAF_, _NRAS_ and _TERTp_ in melanoma. According to them, the frequency

of mutation detection in SS was 20% (8/40) in _BRAF_, 0% (0/40) in _NRAS_ and 12.5% (5/40) in _TERTp_, whereas that with ddPCR was 55% (22/40) in _BRAF_, 10% (41/40) in _NRAS_ and 37.5%

(15/40) in _TERTp_.39 Sho et al. performed SS on resected specimens of the pancreas, and digital PCR on preoperative endoscopic ultrasound–fine-needle aspiration specimens. All 22 cases

positive for mutation by SS were also determined to be mutation-positive by digital PCR, and digital PCR detected five more mutations. This report demonstrates the utility of digital PCR in

cytology samples, which have a much lower tumour burden than surgical specimens.50 In these reports, digital PCR determined as mutation-positive for all cases in which SS detected the

mutation, and this result is consistent with ours. In this study, we used SS instead of NGS to detect _TERTp_ mutations in some cases. NGS’s deep sequencing is well known to be more

sensitive than Sanger sequencing, and it would be interesting to know what the results of NGS analysis would be in these samples. However, conducting NGS targeting only the _TERT_ promoter

region is too costly. In addition, because one of our future goals is to apply our method to clinical practice, we judged it impractical to perform additional NGS in this study from the

beginning of the research. With our _TERTp__Mut_ analysis by ddPCR, the frequency of this mutation was shown with histological grade, from FA (29%) and benign PT (59%) to borderline PT (67%,

Table 3). Nault et al. analysed _TERTp_ mutations in cirrhotic dysplastic nodules, which are premalignant lesions of hepatocellular carcinoma (HCC), and in early and progressed HCC. They

reported a strong relationship between _TERTp_ mutations and hepatocarcinogenesis: the mutations were identified in 6% of low-grade dysplastic nodules, 19% of high-grade nodules, 61% of

early HCC and 42% of small and progressed HCC.51 Our results in breast FA and PT were quite similar to their results in HCC, in that the frequency of _TERTp_ mutations increased as tumours

went from benign to malignant, which implies a sequential development by some FA into PT. Interestingly, our study showed a positive correlation between FA size and frequency of _TERTp_

mutations (_P_ = 0.015, Table 3). Some FA, especially large ones, may be genetically similar to benign PT, although morphologically distinguishable from them. The relationship between

_MED12_ and _TERTp_ mutations varies in previous reports.29,30,31,33,34 Piscuoglio et al.,29 Pareja et al.30 and Liu et al.34 reported that just 50% and slightly over of PT were

_MED12__Mut+_ (13/25, 5/9 and 3/6), whereas Yoshida et al.31 and Garcia-Dios et al.33 reported that almost all of them were simultaneously _MED12__Mut+_ (29/30 and 12/13, respectively). Only

one study found _TERTp_ mutations in FA:31 4 cases out of 58 (7%) and all of the _TERTp__Mut+_ FA also harboured _MED12_ mutations. Our study found no significant correlation between

_MED12_ and _TERTp_ mutations, in either FA or PT. Although _MED12_ mutations are most frequently observed in intracanalicular-type FA,10,11,21,23,24,28 _TERTp_ mutations detected in our

study had no statistical relationship with FA classification or PT grade (Table 3). These two mutations might independently affect the genesis or development of FA and PT. Among the 72

patients in our study, three experienced recurrences (Table 4). When we compared the first resected tumour with later ones, two of them, an FA and a benign PT, showed _TERTp__Mut_ status

change from negative to positive. Considering that all cases harboured the same pattern of _MED12_ mutations within each case, the secondary resected tumours seemed to be genetically

identical to the primary tumours, suggesting that they were truly recurrent tumours. Garcia-Dios et al. also reported evidence of recurrent PT that acquired _TERTp_ mutations,33 but our

result indicates that _TERTp_ mutations can be acquired during tumour growth or recurrence in both FA and PT. We detected _TERTp_ mutations at a much higher rate in FA than were seen in

previous studies,29,31,33 but some might doubt the accuracy of the FA diagnoses. In the current study, two expert pathologists diagnosed all of the tumours. The distinction between FA and PT

is often challenging, and in fact, we had eight cases, all eventually diagnosed as benign PT, on which the pathologists did not initially agree whether they were intracanalicular-type FA or

benign PT (data not shown). _TERTp_ mutations were found in five of these controversial tumours (63%), which is similar to the mutation rate of PT as a whole (61%), but much higher than

that of FA (30%, Table 3). We consider that these controversial cases were credibly distinguished from other FA, and would be similarly diagnosed by most pathologists. Although we found

ddPCR to be more sensitive than SS for _TERTp_ mutations, technical challenges remain. Sufficient quality and quantity of DNA is essential for detecting low-frequency mutations; therefore,

small tumours or hyalinised FA may be inadequate for mutation analysis with ddPCR. Another challenge to successful analysis is artefacts from formalin-fixed samples when using sensitive

molecular assays causing false-positive calls.52,53 We set thresholds based on signals from empty droplets, wild-type DNA and mutant-positive controls, and determined samples as negative

when they showed fewer than ten mutation calls, to decrease the possibility of error due to false positives. In other words, detecting very low-frequency mutations with fewer than ten calls

is highly challenging. Additional studies are required to resolve these problems and improve accuracy. To examine the influence of FFPE-derived artefacts of DNA in ddPCR analysis, it would

be more robust to prepare an FFPE-derived negative and a positive control for both C228T and C250T mutation-positive controls. We set distilled water as a negative control, and used DNA

collected from a cell line as a positive control, so the DNA of these controls did not derive from FFPE. However, in our positive control, only C228T positivity was confirmed; thus, it did

not show what type of dot plot would be obtained by ddPCR when C250T had a mutation in our study, although this was depicted in previous reports. These matters can be considered limitations

of this work. Another limitation is the primers that we used in this study, which were the same as those reported by Yoshida et al.31 and Liu et al.34 The nucleotide sequences of these

primers differ from those in the study by McEvoy et al.,39 albeit by only a few bases. As there is no previous report describing the performance of ddPCR analysis using our primers, it may

have been necessary to verify the findings of the study by using the primers that McEvoy et al. used for validation. Extracted tumour DNA in the present study contained mixtures of that from

epithelium, stroma or somewhat normal mammary cells and lymphocytes surrounding the tumour. Earlier studies found mutations for _TERTp_ and _MED12_ only in PT stromal components,29,31,32 so

strictly speaking, DNA extraction from only the stromal components through microdissection would be necessary to identify _TERTp_ mutations in FA and PT stroma, and to measure the allele

frequency by ddPCR. Extracting DNA and measuring _TERTp_ mutations from only the tumour’s stromal components may solve the problem of false positivity and -negativity more precisely, thus

enhancing studies of relationships of _TERTp__Mut_ allelic frequency with tumour growth rate and malignancy in FA and PT. Further study with targeted tumour cells is warranted. In

conclusion, we have presented the first assessment by ddPCR of _TERTp_ mutations in FFPE breast FA and PT, and detected these mutations at a higher rate than previously reported. Our new

findings reconfirm the genomic similarity of FA to PT and may help elucidate the biology of these tumours. We have shown ddPCR to be a robust method of detecting _TERTp_ mutations, which

suggest a wider clinical potential for this technology. A large-scale study is needed to determine whether _TERTp_ mutation detection by ddPCR can be predictive, and has prognostic value for

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Res._ 21, 3552–3560 (2015). Article  CAS  PubMed  Google Scholar  Download references ACKNOWLEDGEMENTS We thank Marla Brunker, from Edanz Group (www.edanzediting.com/ac), for editing a draft

of this paper. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Department of Breast and Endocrine Surgery, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan Kazutaka Otsuji, 

Masahiko Tanabe & Yasuyuki Seto * Department of Next-Generation Pathology Information and Networking, Faculty of Medicine, The University of Tokyo, Tokyo, Japan Takeshi Sasaki *

Department of Gastrointestinal Surgery, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan Yasuyuki Seto Authors * Kazutaka Otsuji View author publications You can also

search for this author inPubMed Google Scholar * Takeshi Sasaki View author publications You can also search for this author inPubMed Google Scholar * Masahiko Tanabe View author

publications You can also search for this author inPubMed Google Scholar * Yasuyuki Seto View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS

K.O. and T.S. conceived the study. T.S., M.T. and Y.S. directed the study and supervised the research. K.O. collected tumour specimens. K.O. and T.S. confirmed the histopathological findings

and interpreted the clinical data. K.O. performed Sanger sequencing and droplet-digital PCR analyses. K.O. wrote the paper, with assistance and final approval of all authors. CORRESPONDING

AUTHOR Correspondence to Takeshi Sasaki. ETHICS DECLARATIONS ETHICS APPROVAL AND CONSENT TO PARTICIPATE This study protocol was approved by the ethics committee at the University of Tokyo

Hospital, Tokyo, Japan. Written informed consent was obtained from all patients. The study was performed in accordance with the Declaration of Helsinki. CONSENT TO PUBLISH Not applicable.

DATA AVAILABILITY All data supporting the study are available on request. No proprietary materials, except patient tissues, were used. COMPETING INTERESTS The authors declare no competing

interests. FUNDING INFORMATION This study was supported by “Project for the Advanced Genome-Based Medicine” from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

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http://creativecommons.org/licenses/by/4.0/. Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Otsuji, K., Sasaki, T., Tanabe, M. _et al._ Droplet-digital PCR reveals frequent

mutations in _TERT_ promoter region in breast fibroadenomas and phyllodes tumours, irrespective of the presence of _MED12_ mutations. _Br J Cancer_ 124, 466–473 (2021).

https://doi.org/10.1038/s41416-020-01109-8 Download citation * Received: 23 December 2019 * Revised: 10 September 2020 * Accepted: 16 September 2020 * Published: 13 October 2020 * Issue

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