Primary tumor somatic mutations in the blood of women with ductal carcinoma in situ of the breast
Authors: S. Di Cosimo, V. Appierto, M. Silvestri, E. Ortolan, L. De Cecco, S. Veneroni, G. Pruneri, A. Vingiani, A. Belfiore, G. Scaperrotta, S. Folli, M.G. Daidone
Journal: Annals of Oncology (2020)

In their review on the clinical validity of liquid biopsy, Siravegna et al. report on ongoing observational/interventional trials based on circulating tumor DNA (ctDNA) in different clinical settings.1Siravegna G. Mussolin B. Venesio T. et al.How liquid biopsies can change clinical practice in oncology.Ann Oncol. 2019; 30: 1580-1590Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar The increased sensitivity of available technologies has markedly improved the feasibility of ctDNA, currently detectable in early stage cancer and exploitable for tracking minimal residual disease. Hence, it is likely to assume that ctDNA could be informative even when invasive cancer has not been diagnosed, yet could occur in the future. In this context, diagnosis and treatment of ductal carcinoma in situ (DCIS), the incidence of which has sharply increased in the last years, have sparked debate over a molecular characterization to assist personalized treatment in cases at risk of invasive recurrence.2Siegel R.L. Miller K.D. Jemal A. Cancer statistics, 2018.CA Cancer J Clin. 2018; 68: 7-30Crossref PubMed Scopus (5029) Google Scholar,3Morrow M. Katz S.J. Addressing overtreatment in DCIS: what should physicians do now?.J Natl Cancer Inst. 2015; 107: djv290Crossref PubMed Scopus (16) Google Scholar Here, we report our preliminary findings demonstrating that ctDNA is detectable at the time of initial diagnosis in patients with DCIS by using a pragmatic approach to blood tracing by digital droplet PCR (ddPCR) the somatic mutations detected by targeted-gene sequencing in the primary tumors. Our study was carried out on 36 primary tumor and matched plasma samples from consenting patients, obtained from the bio-bank linked to our institutional breast cancer (BC) registry. Nine DCIS were not evaluable due to low DNA integrity number or coverage; mutational analysis was carried out on the remaining 27 formalin-fixed paraffin-embedded samples by 50 cancer-related gene sequencing (Ion AmpliSeq™ Hot Spot Cancer Panel), or by 409 cancer-related gene sequencing (IonAmpliSeq™ Comprehensive Cancer Panel) in the wild-type cases with tissue DNA still available. Among the DCIS with adequate DNA for mutational analysis, 13 resulted wild type, and 14 had at least one mutation, with ≥2 mutations in seven cases (Table 1), all validated by ddPCR. PIK3CA was the most commonly mutated gene, followed by TP53. Customized ddPCR assays detected mutations with a variant allele frequency (VAF) ≥0.001% in pre-surgery blood from 10/14 (71%) patients harboring mutated DCIS, with variable levels among patients (median VAF = 0.14%; range 0.001%–5%) and detection rate unrelated to clinicopathological features. Our series also included three recurrent patients with evaluable DNA from primary DCIS, resected ipsilateral breast tumor recurrence (IBTR), and matched plasma pairs. Patient 24 had no mutations in all surgical specimens, while patient 1 had mutated PIK3CA in each surgical specimen (i.e. primary DCIS, non-invasive IBTR, and subsequent triple-negative BC), and patient 6 had an IBTR with DCIS and invasive breast cancer which harbored the same TP53 mutation of initial DCIS. The corresponding plasma samples revealed that primary DCIS mutations in PIK3CA and TP53 were present at time of recurrence.Table 1DCIS patients with and without evaluable tissue and plasma mutational profilePatient numberAge (years)DetectionSurgeryHistologyGradeSize (mm)HRaHR status classified as positive when either estrogen receptor or progesterone receptor were ≥1%, or negative when both ER and PR were <1%. statusHER2Ki-67 (%)Adjuvant therapyMutational profileTissueVAF (%)PlasmaVAF (%)DCIS patients with evaluable tissue and plasma mutational profile 128ClinicalMastectomyComedoIII12+1+NENonePIK3CA N345K8PIK3CA N345K0.04 243ClinicalMastectomyComedoIII30+2+25NonePIK3CA E542Q33PIK3CA E542Q0.12 PIK3CA H1047R32 KIT V532I49 364ClinicalMastectomyNon comedoII30NENENENonePIK3CA H1047R9PIK3CA H1047R0.11 460ScreeningBCSNon comedoI6+03NonePIK3CA E542K34PIK3CA E542K0.14 547ScreeningMastectomyComedoII35+3+35NoneTP53 T163C11TP53 T163C0.9 ATM V410A62 644ScreeningBCSComedoIII7+2+30TAMTP53 R282W23TP53 R282W5 IDH2 R149Q19 736ClinicalMastectomyComedoII25+020NoneTP53 R282Q27TP53 R282Q0.1 PIK3CA H1047R28 852ScreeningMastectomyNon comedoIII30+1+<5NoneBRAFV600E34BRAFV600E0.1 PIK3CA E542Q29 936ScreeningMastectomyNon comedoII11+025NoneNTRK3 R793Ter15NTRK3 R793Ter0.34 1052ClinicalBCSNon comedoII10−0NERTKRAS G12V5KRAS G12V0.013 1173ScreeningBCSNon comedoIII25−3+15RTPIK3CA E545K34PIK3CA E545K0 GNAS R201C32 1272ScreeningMastectomyComedoIII22−1+NENoneERBB2 V777L18ERBB2 V777L0 KRAS G12D18 1350ScreeningBCSComedoII12+1+25NoneAKT1 E17K6AKT1 E17K0 1436ClinicalMastectomyNon comedoII22+1+12NoneGATA3 P409fs22GATA3 P409fs0 1548ScreeningBCSNon comedoII22+2+10TAMWild type 1646ScreeningBCSNon comedoIII6+1+NETAM+RTWild type 1751ScreeningBCSNon comedoII & III7+2+1+NETAMWild type 1854ScreeningBCSNon comedoII30+1+15TAMWild type 1950ScreeningMastectomyNon comedoII50+2+5NoneWild type 2079ClinicalMastectomyNon comedoI & II23−1+<5NoneWild type 2153ScreeningBCSNon comedoI2+022NoneWild type 2258ClinicalMastectomyComedoII & III35+015NoneWild type 2378ScreeningMastectomyNon comedoII34+1+14NoneWild type 2443ScreeningBCSNon comedoI5NENENENoneWild type 2542ScreeningMastectomyComedoIII6+2+20NoneWild type 2673ClinicalBCSNon comedoII28+1+14TAM+RTWild type 2765ScreeningBCSComedoIII21+1+20TAM+RTWild typeDCIS patients not evaluable for tissue and plasma mutational profile 2871ScreeningBCSComedoII & III6+1+35TAMLow coverage 2974ScreeningBCSNon comedoI27+2+4RTLow coverage 3047ScreeningMastectomyNon comedoII45+3+15NoneLow coverage 3145ScreeningBCSNon comedoIII7+3+NETAM+RTLow DIN 3255ScreeningBCSNon comedoII & III15+2+10TAM+RTLow DIN 3358ScreeningBCSComedoIII40+3+NETAM+RTLow DIN 3446ScreeningBCSNon comedoI15+2+<5NoneLow DIN 3569ScreeningBCSNon comedoII2+1+NETAMLow DIN 3643ScreeningBCSNon comedoIII15+3+NETAMLow DINMutations assayed in plasma were those with the highest VAF (patients 6 and 8) or for which a sensitive digital droplet PCR assay was already available (patients 2, 5, and 7). Primary tumor tissue mutations were not tested in plasma when plasma DNA was not available (patients 11 and 12).BCS, breast conserving surgery; DCIS, ductal carcinoma in situ; DIN, DNA integrity number; ER, estrogen receptor; HR, hormone receptor; NE, not evaluated; PR, progesterone receptor; TAM, tamoxifen; VAF, variant allele frequency.a HR status classified as positive when either estrogen receptor or progesterone receptor were ≥1%, or negative when both ER and PR were <1%. Open table in a new tab Mutations assayed in plasma were those with the highest VAF (patients 6 and 8) or for which a sensitive digital droplet PCR assay was already available (patients 2, 5, and 7). Primary tumor tissue mutations were not tested in plasma when plasma DNA was not available (patients 11 and 12). BCS, breast conserving surgery; DCIS, ductal carcinoma in situ; DIN, DNA integrity number; ER, estrogen receptor; HR, hormone receptor; NE, not evaluated; PR, progesterone receptor; TAM, tamoxifen; VAF, variant allele frequency. These findings extend the ctDNA value to additional clinical scenarios, and represent, to our knowledge, the first report showing that primary DCIS mutations can be found in plasma samples similarly to invasive BCs, possibly at lower VAF. The value of ctDNA as a biomarker in DCIS, even from screening-detected cases, represents an avenue for further investigations.4Pantel K. Alix-Panabières C. Liquid biopsy and minimal residual disease -latest advances and implications for cure.Nat Rev Clin Oncol. 2019; 16: 409-424Crossref PubMed Scopus (248) Google Scholar The challenge to move forward will be to analyze ctDNA in patients with newly diagnosed DCIS for stratification and longitudinal monitoring.5Garcia-Murillas I. Chopra N. Comino-Méndez I. et al.Assessment of molecular relapse detection in early-stage breast cancer.JAMA Oncol. 2019; https://doi.org/10.1001/jamaoncol.2019.1838Crossref PubMed Scopus (63) Google Scholar SDC and MGD are recipients of the Fondazione Associazione Italiana Ricerca contro il Cancro (Fondazione AIRC) Investigator Grant projects numbers 20774 and 16900, respectively. This work was supported by the Italian Ministry of Health to VA. No grant number is applicable, funds obtained through a law that allows tax-payers to allocate to research the 5 × 1000 share of their payments. The funder had no role in the design of the study, in the collection, analyses, or interpretation of data, in the writing of the manuscript, or in the decision to publish the results.