Single Cell Genetic Profiling of Tumors of Breast Cancer Patients Aged 50 Years and Older Reveals Enormous Intratumor Heterogeneity Independent of Individual Prognosis.

Anna-Sophie Liegmann,Jens K Habermann,Thomas Ried,Daniela Hirsch,Wei-Dong Chen,Irianna Torres,Edward Michael Gertz,Christoph Thorns,Timo Gemoll,Achim Rody,Yue Hu,Annette Lischka,Hendrik Alkemade,Kerstin Heselmeyer-Haddad

doi:10.3390/cancers13133366

Abstract

Simple SummaryThe majority (69.7%) of women diagnosed with breast cancer are above the age of 55. The population of older breast cancer patients is growing. Nevertheless, older patients are underrepresented in cancer research. Therefore, our study set the focus on breast cancer patients aged 50 years and older with a median age of 67 years aiming to understand the influence of aneuploidy, genomic instability and inter- and intratumor heterogeneity on disease outcome, being a major obstacle for precise prognostication and successful treatment. We analyzed chromosomal copy number changes, ploidy and specific gene mutations and found an enormous degree of genomic instability and intratumor heterogeneity in our cohort. However, neither the ploidy, the degree of intratumor heterogeneity nor the presence of specific gene mutations was correlated with prognosis. Our findings provide a precise description of the degree of intratumor heterogeneity, genomic instability, and gene mutations in breast cancer patients aged 50 years and older, revealing significant differences between diploid and aneuploid tumors regarding copy number alterations and the extent of genomic instability.Purpose: Older breast cancer patients are underrepresented in cancer research even though the majority (81.4%) of women dying of breast cancer are 55 years and older. Here we study a common phenomenon observed in breast cancer which is a large inter- and intratumor heterogeneity; this poses a tremendous clinical challenge, for example with respect to treatment stratification. To further elucidate genomic instability and tumor heterogeneity in older patients, we analyzed the genetic aberration profiles of 39 breast cancer patients aged 50 years and older (median 67 years) with either short (median 2.4 years) or long survival (median 19 years). The analysis was based on copy number enumeration of eight breast cancer-associated genes using multiplex interphase fluorescence in situ hybridization (miFISH) of single cells, and by targeted next-generation sequencing of 563 cancer-related genes. Results: We detected enormous inter- and intratumor heterogeneity, yet maintenance of common cancer gene mutations and breast cancer specific chromosomal gains and losses. The gain of COX2 was most common (72%), followed by MYC (69%); losses were most prevalent for CDH1 (74%) and TP53 (69%). The degree of intratumor heterogeneity did not correlate with disease outcome. Comparing the miFISH results of diploid with aneuploid tumor samples significant differences were found: aneuploid tumors showed significantly higher average signal numbers, copy number alterations (CNAs) and instability indices. Mutations in PIKC3A were mostly restricted to luminal A tumors. Furthermore, a significant co-occurrence of CNAs of DBC2/MYC, HER2/DBC2 and HER2/TP53 and mutual exclusivity of CNAs of HER2 and PIK3CA mutations and CNAs of CCND1 and PIK3CA mutations were revealed. Conclusion: Our results provide a comprehensive picture of genome instability profiles with a large variety of inter- and intratumor heterogeneity in breast cancer patients aged 50 years and older. In most cases, the distribution of chromosomal aneuploidies was consistent with previous results; however, striking exceptions, such as tumors driven by exclusive loss of chromosomes, were identified.

Highlights

To non-melanoma skin cancer, breast cancer is the most common cancer in women with an estimated 276,480 new cases diagnosed in the United States in 2020 [1]
These most common alterations plus the overall distribution of gains and losses of our multiplex interphase fluorescence in situ hybridization (miFISH) results are consistent with previous results based on comparative genomic hybridization, i.e., the relative gains of chromosome arms or parts of chromosome arms 1q, 8q, 11q, 17q and 20q accompanied by losses of chromosome arms 8p, 16q and 17p [26,27,45] and with results reported in the The Cancer Genome Atlas (TCGA) database [36]
The pattern of copy number alterations (CNAs) of the eight analyzed cancer-related genes and the distribution of gene mutations of the OncoVar panel observed in breast carcinomas of patients aged 50 years and older are similar to an age-unbiased cohort

Summary

Introduction

To non-melanoma skin cancer, breast cancer is the most common cancer in women with an estimated 276,480 new cases diagnosed in the United States in 2020 [1]. One in eight women in the United States will develop breast cancer over an 80-year lifespan. The risk of developing breast cancer depends on a combination of factors, with advancing age playing a major role: the incidence of breast cancer rises dramatically with age [1]. Of all women diagnosed with breast cancer 69.7% are older than 55 years [1]. The majority (81.4%) of women who die of breast cancer are aged 55 years and older with a median age of breast cancer death of 68 years [1]. The rising incidence of cancer with advancing age coupled with an aging population will result in an increase of cancer incidence for patients 65 years and older of 67% between 2010 and 2030 [3].

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