In the past three decades, the detection rate of ductal carcinoma in situ of the breast has dramatically increased due to breast screening programs. As a consequence, about 20% of all breast cancer cases are detected in this early in situ stage. Some ductal carcinoma in situ cases will progress to invasive breast cancer, while other cases are likely to have an indolent biological behavior. The presence of tumor-infiltrating lymphocytes is seen as a promising prognostic and predictive marker in invasive breast cancer, mainly in HER2-positive and triple-negative subtypes. Here, we summarize the current understanding regarding immune infiltrates in invasive breast cancer and highlight recent observations regarding the presence and potential clinical significance of such immune infiltrates in patients with ductal carcinoma in situ. The presence of tumor-infiltrating lymphocytes, their numbers, composition, and potential relationship with genomic status will be discussed. Finally, we propose that a combination of genetic and immune markers may better stratify ductal carcinoma in situ subtypes with respect to tumor evolution.

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Intratumour heterogeneity fuels carcinogenesis and allows circumventing specific targeted therapies. HER2 gene amplification is associated with poor outcome in invasive breast cancer. Heterogeneous HER2 amplification has been described in 5–41% of breast cancers. Here, we investigated the genetic differences between HER2‐positive and HER2‐negative admixed breast cancer components. We performed an in‐depth analysis to explore the potential heterogeneity in the somatic mutational landscape of each individual tumour component. Formalin‐fixed, paraffin‐embedded breast cancer tissue of ten patients with at least one HER2‐negative and at least one HER2‐positive component was microdissected. Targeted next‐generation sequencing was performed using a customized 53‐gene panel. Somatic mutations and copy number variations were analysed. Overall, the tumours showed a heterogeneous distribution of 12 deletions, 9 insertions, 32 missense variants and 7 nonsense variants in 26 different genes, which are (likely) pathogenic. Three splice site alterations were identified. One patient had an EGFR copy number gain restricted to a HER2‐negative in situ component, resulting in EGFR protein overexpression. Two patients had FGFR1 copy number gains in at least one tumour component. Two patients had an 8q24 gain in at least one tumour component, resulting in a copy number increase in MYC and PVT1. One patient had a CCND1 copy number gain restricted to a HER2‐negative tumour component. No common alternative drivers were identified in the HER2‐negative tumour components. This series of 10 breast cancers with heterogeneous HER2 gene amplification illustrates that HER2 positivity is not an unconditional prerequisite for the maintenance of tumour growth. Many other molecular aberrations are likely to act as alternative or collaborative drivers. This study demonstrates that breast carcinogenesis is a dynamically evolving process characterized by a versatile somatic mutational profile, of which some genetic aberrations will be crucial for cancer progression, and others will be mere ‘passenger’ molecular anomalies.

Ductal carcinoma in situ (DCIS) of the breast is a nonobligate precursor of invasive breast cancer, accounting for 20 % of screen‐detected breast cancers. Little is known about the natural progression of DCIS because most patients undergo surgery upon diagnosis. Many DCIS patients are likely being overtreated, as it is believed that only around 50 % of DCIS will progress to invasive carcinoma. Robust prognostic markers for progression to invasive carcinoma are lacking. In the past, studies have investigated women who developed a recurrence after breast‐conserving surgery (BCS) and compared them with those who did not. However, where there is no recurrence, the patient has probably been adequately treated. The present narrative review advocates a new research strategy, wherein only those patients with a recurrence are studied. Approximately half of the recurrences are invasive cancers, and half are DCIS. So‐called “recurrences” are probably most often the result of residual disease. The new approach allows us to ask: why did some residual DCIS evolve to invasive cancers and others not? This novel strategy compares the group of patients that developed in situ recurrence with the group of patients that developed invasive recurrence after BCS. The differences between these groups could then be used to develop a robust risk stratification tool. This tool should estimate the risk of synchronous and metachronous invasive carcinoma when DCIS is diagnosed in a biopsy. Identification of DCIS patients at low risk for developing invasive carcinoma will individualize future therapy and prevent overtreatment.

PIK3CA is one of the most frequently mutated genes in invasive breast cancer (IBC). These mutations are generally associated with hyper-activation of the phosphatidylinositol 3-kinase signaling pathway, which involves increased phosphorylation of AKT (p-AKT). This pathway is negatively regulated by the tumor suppressor PTEN. Data are limited regarding the variant allele frequency (VAF) of PIK3CA, PTEN and p-AKT expression during various stages of breast carcinogenesis. Therefore, the aim of this study was to gain insight into PIK3CA VAF and associated PTEN and p-AKT expression during the progression from ductal carcinoma in situ (DCIS) to IBC. We isolated DNA from DCIS tissue, synchronous IBC and metastasis when present. These samples were pre-screened for PIK3CA hotspot mutations using the SNaPshot assay and, if positive, validated and quantified by digital PCR. PTEN and p-AKT expression was evaluated by immunohistochemistry using the Histo-score (H-score). Differences in PIK3CA VAF, PTEN and p-AKT H-scores between DCIS and IBC were analyzed. PIK3CA mutations were detected in 17 out of 73 DCIS samples, 16 out of 73 IBC samples and 3 out of 23 lymph node metastasis. We detected a significantly higher VAF of PIK3CA in the DCIS component compared to the adjacent IBC component (P = 0.007). The expression of PTEN was significantly higher in DCIS compared to the IBC component in cases with a wild-type (WT) PIK3CA status (P = 0.007), while it remained similar in both components when PIK3CA was mutated. There was no difference in p-AKT expression between DCIS and the IBC component. In conclusion, our data suggest that PIK3CA mutations could be essential specifically in early stages of breast carcinogenesis. In addition, these mutations do not co-occur with PTEN expression during DCIS progression to IBC in the majority of patients. These results may contribute to further unraveling the process of breast carcinogenesis, and this could aid in the development of patient-specific treatment.