Anargyros Megalios

Bio: Anargyros Megalios is an academic researcher from King's College London. The author has contributed to research in topics: Ductal carcinoma & Breast cancer. The author has an hindex of 2, co-authored 3 publications receiving 10 citations.

Genomic analysis defines clonal relationships of ductal carcinoma in situ and recurrent invasive breast cancer

Abstract: Ductal carcinoma in situ (DCIS) is the most common form of preinvasive breast cancer and, despite treatment, a small fraction (5-10%) of DCIS patients develop subsequent invasive disease. A fundamental biologic question is whether the invasive disease arises from tumor cells in the initial DCIS or represents new unrelated disease. To address this question, we performed genomic analyses on the initial DCIS lesion and paired invasive recurrent tumors in 95 patients together with single-cell DNA sequencing in a subset of cases. Our data show that in 75% of cases the invasive recurrence was clonally related to the initial DCIS, suggesting that tumor cells were not eliminated during the initial treatment. Surprisingly, however, 18% were clonally unrelated to the DCIS, representing new independent lineages and 7% of cases were ambiguous. This knowledge is essential for accurate risk evaluation of DCIS, treatment de-escalation strategies and the identification of predictive biomarkers.

Frequency of pathogenic germline variants in BRCA1, BRCA2, PALB2, CHEK2 and TP53 in ductal carcinoma in situ diagnosed in women under the age of 50 years

Abstract: Ductal carcinoma in situ (DCIS) is a non-obligate precursor of invasive ductal breast cancer, and approximately 20% of screen-detected tumours are pure DCIS. Most risk factors for breast cancer have similar associations with DCIS and IDC; however, there is limited data on the prevalence of the known high and moderate penetrance breast cancer predisposition genes in DCIS and which women with DCIS should be referred for genetic screening. The aim of this study was to assess the frequency of germline variants in BRCA2, BRCA1, CHEK2, PALB2 and TP53 in DCIS in women aged less than 50 years of age. After DNA extraction from the peripheral blood, Access Array technology (Fluidigm) was used to amplify all exons of these five known breast cancer predisposition genes using a custom made targeted sequencing panel in 655 cases of pure DCIS presenting in women under the age of 50 years together with 1611 controls. Case-control analysis revealed an excess of pathogenic variants in BRCA2 (OR = 27.96, 95%CI 6.56–119.26, P = 2.0 × 10−10) and CHEK2 (OR = 8.04, 95%CI 2.93–22.05, P = 9.0 × 10−6), with weaker associations with PALB2 (P = 0.003), BRCA1 (P = 0.007) and TP53 (P = 0.02). For oestrogen receptor (ER)-positive DCIS the frequency of pathogenic variants was 9% under the age of 50 (14% with a family history of breast cancer) and 29% under the age of 40 (42% with a family history of breast cancer). For ER-negative DCIS, the frequency was 9% (16% with a family history of breast cancer) and 8% (11% with a family history of breast cancer) under the ages of 50 and 40, respectively. This study has shown that breast tumourigenesis in women with pathogenic variants in BRCA2, CHEK2, PALB2, BRCA1 and TP53 can involve a DCIS precursor stage and that the focus of genetic testing in DCIS should be on women under the age of 40 with ER-positive DCIS.

Genomic profiling defines variable clonal relatedness between invasive breast cancer and primary ductal carcinoma in situ

Abstract: Pure ductal carcinoma in situ (DCIS) is being diagnosed more frequently through breast screening programmes and is associated with an increased risk of developing invasive breast cancer. We assessed the clonal relatedness of 143 cases of pure DCIS and their subsequent events using a combination of whole exome, targeted and copy number sequencing, supplemented by single cell analysis. Unexpectedly, 18% of all invasive events after DCIS were clonally unrelated to the primary DCIS. Single cell sequencing of selected pairs confirmed our findings. In contrast, synchronous DCIS and invasive disease (n=44) were almost always (93%) clonally related. This challenges the dogma that almost all invasive events after DCIS represent invasive transformation of the initial DCIS and suggests that DCIS could be an independent risk factor for developing invasive disease as well as a precursor lesion. Our findings support a paradigm shift that confirms a more complex role for DCIS than previously recognized, and that the future management of DCIS should take into account both the precursor and risk factor implications of this diagnosis.

Abstract P4-07-04: Genomic analysis of paired DCIS and subsequent recurrence to assess clonal relatedness in screen detected DCIS

Abstract: In order to understand the clonal relatedness of DCIS and subsequent recurrence, 40 patients with primary DCIS that developed a subsequent ipsilateral event (28 invasive and 12 pure DCIS) and 17 with a subsequent contralateral event, were identified within the Sloane project, a prospective national cohort study of DCIS embedded in the UK Breast Screening Programme. Median follow up was 5.4 years. All cases were reviewed by a breast histopathologist and macro-dissected to separate the DCIS or invasive disease from the normal tissue. DNA was extracted using the AllPrep DNA/RNA FFPE Kit (Qiagen). Somatic copy number aberrations were assessed using the HumanCytoSNP-12 BeadChip Kit (Illumina) and ASCAT. Targeted sequencing of a custom panel of 120 breast cancer-associated genes was performed using the SureSelect XT low input Target Enrichment System (Agilent Technologies). Prior to undertaking clonal related analysis, the relatedness of the samples was confirmed by cryptic relatedness analysis in PLINK v1.07. Clonal relatedness was assessed using two methods. The first used copy number data, available for all samples, where each breakpoint was compared between the pairs of tumours from the same individual and a concordance score calculated based on the presence of shared and unique breakpoints. The second used targeted sequencing, available on 23 paired ipsilateral cases and 7 contralateral paired recurrences, that was run through the previously described Clonality package (Biometrics. 2018). The results of the clonal relatedness analysis are summarized in below. There were 2 cases where mutation data showed clear evidence of clonal relatedness but copy number did not, suggesting that the copy number approach may underestimate relatedness. There was also a proportion of cases where the pairs shared only one mutation and it was not possible to ascertain conclusively whether they were related, particularly when the shared mutation was a common driver mutation in PIK3CA that could have arisen by chance in unrelated samples. Of the 28 primary DCIS that developed ipsilateral invasive recurrence, 8 cases (29%) showed no evidence of clonal relatedness by one or other method. Of these 3 recurred in the original tumour bed, 3 in a different quadrant and in 2 the site of recurrence was unknown. Of the 12 primary DCIS that developed an ipsilateral DCIS recurrence 92% showed evidence of clonal relatedness and also recurred earlier than the invasive recurrences, mean time to recurrence 2.9 years compared to 4.15 years for recurrent invasive disease (P=0.04, Student’s t-test). The mutations that were most commonly shared between the primary and recurrence were PIK3CA and TP53, followed by KMT2C, CAD and DNAH2. As expected, the majority of the contralateral cases were unrelated. Of the 28 cases of DCIS that recurred ipsilaterally with invasive disease 16 had synchronous DCIS at the time of detection of the invasive disease and in 12 cases we were able to analyse the two synchronous components separately. In all cases the synchronous recurrent DCIS and invasive components showed evidence of clonal relatedness. This study shows synchronous DCIS and IDC are clonally related as is DCIS that recurs as DCIS in the majority of cases. However, 29% of DCIS that recurred as invasive disease showed no evidence of clonal relatedness. It remains to be seen whether exome sequencing of the unrelated lesions would reveal evidence of relatedness or whether they are truly new primaries. Citation Format: Vandna Shah, Anargyros Megalios, Rana Shami, Mathini Sridharan, Carolina Salinas de Souza, Tapsi Kumar, Karen Clements, Andrew Futreal, Sarah Pinder, Alastair Thompson, Elinor Jane Sawyer, Sloane Steering Committee, CRUK Grand Challenge PRECISION Team. Genomic analysis of paired DCIS and subsequent recurrence to assess clonal relatedness in screen detected DCIS [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-07-04.

Abstract P1-22-05: Identifying predictors of invasive recurrence based on molecular profiles of DCIS lesions

Abstract: Introduction Ductal carcinoma in situ (DCIS) is a non-obligate precursor of invasive breast cancer. Patients with DCIS are routinely treated by breast-conserving surgery often supplemented by radiotherapy, although many will never develop invasive disease. To date, no robust predictors of invasive breast cancer recurrence following DCIS have been identified. In our efforts to find such predictors, we performed gene expression, copy number and mutation analysis on two large DCIS cohorts with long-term follow-up. Methods Two nested case control series were analyzed, where cases are defined as DCIS with a subsequent invasive breast cancer and controls remained disease free during follow up. Cases and controls were matched on age and on follow up duration and were derived from two nation-wide cohort studies. The Sloane cohort is a prospective breast screening cohort from the UK, median follow up is 6 years (range 1-10). The Dutch cohort is population-based and had a median follow up of 13 years (range 2-23). We performed copy number analysis using CytoSNP array or low pass whole genome sequencing (lpWGS) on 310 controls and 196 cases, and RNA-seq on 295 controls and 206 cases. Results First analyses on the copy number data suggest that cases are genetically more aberrant with multiple regions of amplification compared to controls (p < 0.05). RNA-seq was used to classify DCIS into the PAM50 subtypes which did not appear to be predictive of recurrence. Initial RNA-seq analysis did not show consistent gene expression differences between cases and controls in the Sloane or Dutch cohorts, possibly explained by differences in clinical characteristics of the cohorts. A new computational method has been developed accounting for the differences in follow-up times, results will be presented at SABCS. Targeted sequencing revealed that the most common mutations were in PIK3CA and TP53, but there was no association with recurrence. Conclusion Only small molecular differences were identified between DCIS that recurs as invasive breast cancer and DCIS that remains disease-free. Currently, we are seeking to identify reproducible differences by a combined analysis of two population-based cohorts in a time dependent fashion. These will be presented at the SABCS. This work was supported by Cancer Research UK and by KWF Dutch Cancer Society (ref.C38317/A24043) Citation Format: Tycho Bismeijer, Ahmed A Ahmed, Michael Sheinman, Maria Roman-Escorza, Vandna Shah, Jeffrey R Marks, Lorraine M King, Anargyros Megalios, Lindy L Visser, Marlous Hoogstraat, Helen R Davies, Tapsi Kumar, Deborah Collyar, Hilary Stobart, Nicholas N Navin, Andrew Futreal, Serena Nik-Zainal, Shelley Hwang, Esther H Lips, Alastair Thompson, Lodewyk FA Wessels, Elinor J Sawyer, Jelle Wesseling, Grand Challenge PRECISION Consortium. Identifying predictors of invasive recurrence based on molecular profiles of DCIS lesions [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-22-05.

Pathological features of 11,337 patients with primary ductal carcinoma in situ (DCIS) and subsequent events: results from the UK Sloane Project

Abstract: The Sloane audit compares screen-detected ductal carcinoma in situ (DCIS) pathology with subsequent management and outcomes. This was a national, prospective cohort study of DCIS diagnosed during 2003–2012. Among 11,337 patients, 7204 (64%) had high-grade DCIS. Over time, the proportion of high-grade disease increased (from 60 to 65%), low-grade DCIS decreased (from 10 to 6%) and mean size increased (from 21.4 to 24.1 mm). Mastectomy was more common for high-grade (36%) than for low-grade DCIS (15%). Few (6%) patients treated with breast-conserving surgery (BCS) had a surgical margin <1 mm. Of the 9191 women diagnosed in England (median follow-up 9.4 years), 7% developed DCIS or invasive malignancy in the ipsilateral and 5% in the contralateral breast. The commonest ipsilateral event was invasive carcinoma (n = 413), median time 62 months, followed by DCIS (n = 225), at median 37 months. Radiotherapy (RT) was most protective against recurrence for high-grade DCIS (3.2% for high-grade DCIS with RT compared to 6.9% without, compared with 2.3 and 3.0%, respectively, for low/intermediate-grade DCIS). Ipsilateral DCIS events lessened after 5 years, while the risk of ipsilateral invasive cancer remained consistent to beyond 10 years. DCIS pathology informs patient management and highlights the need for prolonged follow-up of screen-detected DCIS.

Genomic analysis defines clonal relationships of ductal carcinoma in situ and recurrent invasive breast cancer

Abstract: Ductal carcinoma in situ (DCIS) is the most common form of preinvasive breast cancer and, despite treatment, a small fraction (5-10%) of DCIS patients develop subsequent invasive disease. A fundamental biologic question is whether the invasive disease arises from tumor cells in the initial DCIS or represents new unrelated disease. To address this question, we performed genomic analyses on the initial DCIS lesion and paired invasive recurrent tumors in 95 patients together with single-cell DNA sequencing in a subset of cases. Our data show that in 75% of cases the invasive recurrence was clonally related to the initial DCIS, suggesting that tumor cells were not eliminated during the initial treatment. Surprisingly, however, 18% were clonally unrelated to the DCIS, representing new independent lineages and 7% of cases were ambiguous. This knowledge is essential for accurate risk evaluation of DCIS, treatment de-escalation strategies and the identification of predictive biomarkers.

Prevalence of germline pathogenic variants in 22 cancer susceptibility genes in Swedish pediatric cancer patients

Abstract: Up to 10% of pediatric cancer patients harbor pathogenic germline variants in one or more cancer susceptibility genes. A recent study from the US reported pathogenic variants in 22 out of 60 analyzed autosomal dominant cancer susceptibility genes, implicating 8.5% of pediatric cancer patients. Here we aimed to assess the prevalence of germline pathogenic variants in these 22 genes in a population-based Swedish cohort and to compare the results to those described in other populations. We found pathogenic variants in 10 of the 22 genes covering 3.8% of these patients. The prevalence of TP53 mutations was significantly lower than described in previous studies, which can largely be attributed to differences in tumor diagnosis distributions across the three cohorts. Matched family history for relatives allowed assessment of familial cancer incidence, however, no significant difference in cancer incidence was found in families of children carrying pathogenic variants compared to those who did not.