Janani Jayanthan

Metastatic progression of breast cancer: insights from 50 years of autopsies

There remain no clear guidelines for the optimal management of patients with metastatic breast cancer. To better understand its natural history, we undertook a detailed examination of 197 autopsies performed on women who died of breast cancer. We reviewed clinical, treatment and pathological aspects of all cases and, additionally, pathological features and biomarker expression (ER, PgR, HER2, EGFR, p53, Ki67, c‐Kit, CK AE1/AE3) were assessed in detail for the primary tumour and matched metastases for 55 of the cases. Genomes of the primary tumour and multiple metastases were analysed by array‐based comparative genomic hybridization for six cases##. 945 metastatic deposits were identified, with a median of four/patient. The most common organs involved were lung/pleura (80%), bone (74%), liver (71%) and non‐axillary lymph nodes (55%). Major findings included: (a) patients with CNS metastases were more likely to have bone metastases (p < 0.013); (b) younger age was associated with metastasis to the liver (≤ 49 years; p < 0.001) and to gynaecological organs (≤ 49 years; p = 0.001); (c) surgical excision of the primary tumour was associated with metastasis to the liver (p = 0.002); and (d) ER and PgR showed down‐regulation during progression in a non‐random manner, particularly in lung/pleura (ER; p < 0.001), liver and bone metastases. Genomic analysis revealed DNA copy number variation between the primary tumour and metastases (e.g. amplification of 2q11.2–q12.1 and 10q22.2–q22.3) but little variation between metastases from the same patient. In summary, the association of CNS and bone metastases, liver and gynaecological metastases in young women and the risk of liver metastases following surgery have important implications for the management of patients with breast cancer. Clonal heterogeneity of the primary tumour is important in developing metastatic propensity and the change in tumour phenotype during progression/colonization highlights the importance of sampling metastatic disease for optimal treatment strategies.

Molecular evidence for progression of microglandular adenosis (MGA) to invasive carcinoma.

Microglandular adenosis (MGA) is an uncommon, benign breast lesion that is characterized by a proliferation of small uniform, round glands lined by a single layer of epithelial cells around open lumina with haphazard infiltrative growth in fibrous and fatty breast tissue. Although MGA usually has an indolent course, there is morphologic evidence that MGA can be a precursor for the development of intraductal and invasive ductal carcinoma. To investigate the possibility of such a transition, we studied 17 cases of MGA or atypical MGA some of which had given rise to carcinoma in situ (CIS) and/or invasive ductal carcinoma using the reticulin stain, immunohistochemistry (S-100, p63, Ki-67, and p53), and a molecular approach involving microdissection and high-resolution comparative genomic hybridization and MYC chromogenic in situ hybridization. MGA and carcinomas arising from MGA were typically negative for p63 and positive for S-100 and Ki-67 and occasionally positive for p53. High-resolution comparative genomic hybridization identified recurrent gains and losses in MGA (2q+, 5q−, 8q+, and 14q−) and atypical MGA (1q+, 5q−, 8q+, 14q−, and 15q−). Some examples of MGA and carcinomas arising from MGA harbored few gross chromosomal abnormalities whereas others had considerable genetic instability with widespread aberrations affecting numerous chromosomal arms. Such widespread genetic changes, together with recurrent loss of 5q and gain of 8q were reminiscent of those reported specifically for basal-like, estrogen receptor-negative, and BRCA1-associated breast tumors. Concordant genetic alterations were identified between MGA, atypical MGA, and higher risk lesions (CIS and invasive ductal carcinoma) and in some cases there was an accumulation of genetic alterations as cases “progressed” from MGA to atypical MGA, CIS, and invasive ductal carcinoma. The molecular data suggests that MGA, atypical MGA, and carcinoma arising in MGA in a single case were clonally related. This result implicates MGA as a nonobligate precursor for the development of intraductal and invasive ductal carcinoma.

Evaluating the repair of DNA derived from formalin-fixed paraffin-embedded tissues prior to genomic profiling by SNP-CGH analysis

Pathology archives contain vast resources of clinical material in the form of formalin-fixed paraffin-embedded (FFPE) tissue samples. Owing to the methods of tissue fixation and storage, the integrity of DNA and RNA available from FFPE tissue is compromized, which means obtaining informative data regarding epigenetic, genomic, and expression alterations can be challenging. Here, we have investigated the utility of repairing damaged DNA derived from FFPE tumors prior to single-nucleotide polymorphism (SNP) arrays for whole-genome DNA copy number analysis. DNA was extracted from FFPE samples spanning five decades, involving tumor material obtained from surgical specimens and postmortems. Various aspects of the protocol were assessed, including the method of DNA extraction, the role of Quality Control quantitative PCR (qPCR) in predicting sample success, and the effect of DNA restoration on assay performance, data quality, and the prediction of copy number aberrations (CNAs). DNA that had undergone the repair process yielded higher SNP call rates, reduced log R ratio variance, and improved calling of CNAs compared with matched FFPE DNA not subjected to repair. Reproducible mapping of genomic break points and detection of focal CNAs representing high-level gains and homozygous deletions (HD) were possible, even on autopsy material obtained in 1974. For example, DNA amplifications at the ERBB2 and EGFR gene loci and a HD mapping to 13q14.2 were validated using immunohistochemistry, in situ hybridization, and qPCR. The power of SNP arrays lies in the detection of allele-specific aberrations; however, this aspect of the analysis remains challenging, particularly in the distinction between loss of heterozygosity (LOH) and copy neutral LOH. In summary, attempting to repair DNA that is damaged during fixation and storage may be a useful pretreatment step for genomic studies of large archival FFPE cohorts with long-term follow-up or for understanding rare cancer types, where fresh frozen material is scarce.

An epithelial to mesenchymal transition programme does not usually drive the phenotype of invasive lobular carcinomas

Epithelial to mesenchymal transition (EMT) is a cellular phenotype switching phenomenon which occurs during normal development and is proposed to promote tumour cell invasive capabilities during tumour progression. Invasive lobular carcinoma (ILC) is a histological special type of breast cancer with a peculiar aetiology – the tumour cells display an invasive growth pattern, with detached, single cells or single files of cells, and a canonical feature is the loss of E‐cadherin expression. These characteristics are indicative of an EMT or at the very least that they represent some plasticity between phenotypes. While some gene expression profiling data support this view, the tumour cells remain epithelial and limited immunohistochemistry data suggest that EMT markers may not feature prominently in ILC. We assessed the expression of a panel of EMT markers (fibronectin, vimentin, N‐cadherin, smooth muscle actin, osteonectin, Snail, Twist) in 148 ILCs and performed a meta‐analysis of publically available molecular data from 154 ILCs. Three out of 148 (2%) ILCs demonstrated an early and coordinated alteration of multiple EMT markers (down‐regulation of E‐cadherin, nuclear TWIST, and up‐regulation of vimentin, osteonectin, and smooth muscle actin). However, the data overall do not support a role for EMT in defining the phenotypic peculiarities of the majority of ILCs. Copyright

The use of the Illumina FFPE Restoration Protocol to obtain suitable quality DNA for SNP-based CGH– a pilot study

The use of formalin fixed paraffin embedded (FFPE) tissues for DNA analysis has been met with great difficulties due to the degradative effect this fixation, processing and storage has on DNA quality. The lack of a suitable protocol to enhance the quality of such degraded DNA has been a great hindrance to our ability to make full use of clinically annotated FFPE cancer tissue banks. In this report we have begun to investigate the effectiveness and limitations to Illumina’s recent platform for the restoration of DNA derived from archival FFPE tissues. With the exception of select fresh frozen and blood samples, all DNA samples were extracted from FFPE tissues and restored according to Illumina’s protocol. The quality of the FFPE extracted DNA was then assessed by Illumina’s PCR-based quality control assay (QC-PCR) and the resultant DNA was subsequently run on Illumina’s SNP-based CGH chips. Chip call rates were largely used in order to determine the quality of a particular array. Overall, the chip data were highly reproducible as determined by comparing several technical replicate samples. FFPE-extracted and restored DNA performed well in comparison to DNA extracted from fresh frozen tumor and blood from the same patients, meeting the minimum standard for continuation of this platform. It should also be noted that the quality of the chip data was not appreciably enhanced by the use of sodium thiocyanate during the extraction of DNA from FFPE tissues. Moreover, chip quality was significantly lower, with regards to call rates and ‘quality’ of b allele frequency plots, when the recommended Roche DNA extraction kit was used instead of the Qiagen DNA extraction kit. Of great importance, we found that the QC-PCR provided an accurate prediction of chip quality as determined by comparing chip call rates with PCR signals derived from proprietary primer sets (rho= 0.6242, p<0.0001). The selection of restored DNA for future studies will be guided by the results from the QC-PCR assay. These preliminary data demonstrate the promise of Illumina’s DNA restoration protocol for FFPE extracted DNA from tissues older than three decades. Further studies are required to determine the full potential of this method for SNP-based CGH analysis of large FFPE tumor banks.

Tumor heterogeneity in a follicular carcinoma of thyroid: a study by comparative genomic hybridization.

We report a follicular carcinoma of thyroid that showed a range of histologic appearances, with microfollicular, macrofollicular/pseudopapillary, oncocytic, and poorly differentiated areas. We used comparative genomic hybridization to detect the major DNA copy number changes in each component, in order to study the inter-relationships among them. All showed gains in 11q and 17q, suggesting that these were early events in the development of the tumor, and these were the only changes in the follicular component. The other components each showed additional gains and losses, some unique to one component. The oncocytic component showed most changes, including loss on 16q in the region of the E-cadherin gene. This was associated with reduced intensity of immunostaining for E-cadherin specifically in that component. No mutations in the E-cadherin gene were detected in this component. The demonstration that some DNA copy number changes are consistent across each component suggests that they are all clonally related. The additional chromosomal and immunohistochemical heterogeneity across the macrofollicular/pseudopapillary, oncocytic, and poorly differentiated components would be consistent with the emergence of subclones, possibly as part of tumor progression.

Mixed ductal-lobular carcinomas: evidence for progression from ductal to lobular morphology

Mixed ductal–lobular carcinomas (MDLs) show both ductal and lobular morphology, and constitute an archetypal example of intratumoural morphological heterogeneity. The mechanisms underlying the coexistence of these different morphological entities are poorly understood, although theories include that these components either represent ‘collision’ of independent tumours or evolve from a common ancestor. We performed comprehensive clinicopathological analysis of a cohort of 82 MDLs, and found that: (1) MDLs more frequently coexist with ductal carcinoma in situ (DCIS) than with lobular carcinoma in situ (LCIS); (2) the E‐cadherin–catenin complex was normal in the ductal component in 77.6% of tumours; and (3) in the lobular component, E‐cadherin was almost always aberrantly located in the cytoplasm, in contrast to invasive lobular carcinoma (ILC), where E‐cadherin is typically absent. Comparative genomic hybridization and multiregion whole exome sequencing of four representative cases revealed that all morphologically distinct components within an individual case were clonally related. The mutations identified varied between cases; those associated with a common clonal ancestry included BRCA2, TBX3, and TP53, whereas those associated with clonal divergence included CDH1 and ESR1. Together, these data support a model in which separate morphological components of MDLs arise from a common ancestor, and lobular morphology can arise via a ductal pathway of tumour progression. In MDLs that present with LCIS and DCIS, the clonal divergence probably occurs early, and is frequently associated with complete loss of E‐cadherin expression, as in ILC, whereas, in the majority of MDLs, which present with DCIS but not LCIS, direct clonal divergence from the ductal to the lobular phenotype occurs late in tumour evolution, and is associated with aberrant expression of E‐cadherin. The mechanisms driving the phenotypic change may involve E‐cadherin–catenin complex deregulation, but are yet to be fully elucidated, as there is significant intertumoural heterogeneity, and each case may have a unique molecular mechanism.

Abstract P1-07-08: Mixed ductal-lobular carcinomas of the breast: Abrogated cell adhesion in the clonal evolution from ductal to lobular morphology

Mixed ductal-lobular carcinomas (MDL) display both ductal and lobular morphology, and are a clear example of intratumour morphological heterogeneity. The evolution of MDL carcinomas is not well understood. There is a paucity of data surrounding the genetic origin of the different morphological compartments and it remains to be seen whether the coincident presentation of these distinct morphological entities represents two independent tumours that have collided (so called 9collision tumours9), or whether they arise from a common clone. We propose that clonal progression during the evolution of these tumours is associated with a change in phenotype. To address this, a cohort of 82 MDLs was studied for clinical, morphological and molecular features. Key findings include: i) MDLs more frequently co-exist with ductal carcinoma in situ (DCIS) than lobular carcinoma in situ (LCIS); ii) the E-cadherin-catenin complex was recurrently normal in the ductal component but aberrantly localised in the lobular component of the same tumour; iii) E-cadherin deregulation in the lobular component was almost always aberrantly located to the cytoplasm, conversely classic ILCs are typically completely negative for this molecule; iv) epithelial to mesenchymal transition marker expression was not associated with E-cadherin deregulation. Comparative Genomic Hybridsation (CGH) and exome sequencing was performed to investigate clonal relationships between the different intratumour morphologies and identify mechanisms underlying the change in phenotype. Our analysis revealed that i) all morphological components within a case are clonally related; ii) divergence of the morphological components may occur early during tumour evolution (where both DCIS and LCIS are present) or later during tumour progression (cases with only DCIS detectible); and iii) mutations were identified in genes such as CDH1 and ESR1 , and other breast cancer driver genes. Together, these data strongly support the concept that the disparate morphological components of these mixed tumours are clonally related, and are not the result of a collision event. Furthermore, we show that lobular morphology can arise via a 9ductal9 pathway of tumour progression. The mechanisms driving the change in phenotype are yet to be fully elucidated, but there is significant intertumour heterogeneity and each case may utilise a unique molecular mechanism. Citation Format: McCart Reed AE, Kutasovic JR, Nones K, da Silva L, Melville L, Jayanthan J, Vargas AC, Reid LE, Saunus JM, Cummings MM, Porter A, Evans E, Waddell N, Lakhani SR, Simpson PT. Mixed ductal-lobular carcinomas of the breast: Abrogated cell adhesion in the clonal evolution from ductal to lobular morphology [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-07-08.

The role of epithelial to mesenchymal plasticity in driving the invasive capacity of 'lobular-like' tumour cell differentiation in breast cancer

15th International Biennial Congress of the METASTASIS RESEARCH SOCIETY Heidelberg, Germany, June 28th–July 1st, 2014 Springer Science+Business Media Dordrecht 2015