Transcriptomic properties of her2+ ductal carcinoma in situ of the breast associate with absence of immune cells

SIMPLE SUMMARY: Tumor-infiltrating lymphocytes (TILs) are likely to play a role in the biological behavior of HER2+ ductal carcinoma in situ (DCIS). To prevent invasiveness, the potential of targeted immune-modulating treatment of HER2+ DCIS has been explored. We identified a 29-gene expression profile that was associated with the density of TILs. These genes included CCND3, DUSP10 and RAP1GAP, which may guide towards more rationalized choices with respect to immune-mediated therapy in HER2+ DCIS, such as targeted vaccine therapy. ABSTRACT: The identification of transcriptomic alterations of HER2+ ductal carcinoma in situ (DCIS) that are associated with the density of tumor-infiltrating lymphocytes (TILs) could contribute to optimizing choices regarding the potential benefit of immune therapy. We compared the gene expression profile of TIL-poor HER2+ DCIS to that of TIL-rich HER2+ DCIS. Tumor cells from 11 TIL-rich and 12 TIL-poor DCIS cases were micro-dissected for RNA isolation. The Ion AmpliSeq Transcriptome Human Gene Expression Kit was used for RNA sequencing. After normalization, a Mann–Whitney rank sum test was used to analyze differentially expressed genes between TIL-poor and TIL-rich HER2+ DCIS. Whole tissue sections were immunostained for validation of protein expression. We identified a 29-gene expression profile that differentiated TIL-rich from TIL-poor HER2+ DCIS. These genes included CCND3, DUSP10 and RAP1GAP, which were previously described in breast cancer and cancer immunity and were more highly expressed in TIL-rich DCIS. Using immunohistochemistry, we found lower protein expression in TIL-rich DCIS. This suggests regulation of protein expression at the posttranslational level. We identified a gene expression profile of HER2+ DCIS cells that was associated with the density of TILs. This classifier may guide towards more rationalized choices regarding immune-mediated therapy in HER2+ DCIS, such as targeted vaccine therapy

Loss of Y-Chromosome during Male Breast Carcinogenesis

Loss of Y-chromosome (LOY) is associated with increased cancer mortality in males. The prevalence of LOY in male breast cancer (BC) is unknown. The aim of this study is to assess the presence and prognostic effect of LOY during male BC progression. We included male BC patients diagnosed between 1989 and 2009 (n = 796). A tissue microarray (TMA) was constructed to perform immunohistochemistry and fluorescent in situ hybridization (FISH), using an X and Y probe. We also performed this FISH on a selected number of patients using whole tissue slides to study LOY during progression from ductal carcinoma in situ (DCIS) to invasive BC. In total, LOY was present in 12.7% (n = 92) of cases, whereby LOY was associated with ER and PR negative tumors (p = 0.017 and p = 0.01). LOY was not associated with the outcome. Using whole slides including invasive BC and adjacent DCIS (n = 22), we detected a concordant LOY status between both components in 17 patients. In conclusion, LOY is an early event in male breast carcinogenesis, which generally starts at the DCIS stage and is associated with ER and PR negative tumors

Transcriptomic Properties of HER2+ Ductal Carcinoma In Situ of the Breast Associate with Absence of Immune Cells

The identification of transcriptomic alterations of HER2+ ductal carcinoma in situ (DCIS) that are associated with the density of tumor-infiltrating lymphocytes (TILs) could contribute to optimizing choices regarding the potential benefit of immune therapy. We compared the gene expression profile of TIL-poor HER2+ DCIS to that of TIL-rich HER2+ DCIS. Tumor cells from 11 TIL-rich and 12 TIL-poor DCIS cases were micro-dissected for RNA isolation. The Ion AmpliSeq Transcriptome Human Gene Expression Kit was used for RNA sequencing. After normalization, a Mann–Whitney rank sum test was used to analyze differentially expressed genes between TIL-poor and TIL-rich HER2+ DCIS. Whole tissue sections were immunostained for validation of protein expression. We identified a 29-gene expression profile that differentiated TIL-rich from TIL-poor HER2+ DCIS. These genes included CCND3, DUSP10 and RAP1GAP, which were previously described in breast cancer and cancer immunity and were more highly expressed in TIL-rich DCIS. Using immunohistochemistry, we found lower protein expression in TIL-rich DCIS. This suggests regulation of protein expression at the posttranslational level. We identified a gene expression profile of HER2+ DCIS cells that was associated with the density of TILs. This classifier may guide towards more rationalized choices regarding immune-mediated therapy in HER2+ DCIS, such as targeted vaccine therapy

Radioactive Seed Versus Wire-Guided Localization for Ductal Carcinoma in Situ of the Breast: Comparable Resection Margins

Background: There are currently two widely used methods for preoperative localization of ductal carcinoma in situ (DCIS) of the breast: wire-guided localization (WGL) and radioactive seed localization (RSL). Several studies compared these localization techniques in small cohorts. Objective: The aim of this study was to compare the surgical resection margin status between RSL and WGL in a large national cohort of patients with DCIS. Patients and Methods: We included patients from the Dutch Pathology Registry who underwent breast-conserving surgery for DCIS by either RSL (n = 1851) or WGL (n = 2187) between 2009 and 2019. Several clinicopathological characteristics were compared between these two groups, including resection margin status and number of re-excisions. Results: Patients undergoing RSL were younger (p = 0.014) and were more often diagnosed with a large DCIS (p = 0.013), high grade DCIS (p < 0.001) and comedonecrosis (p < 0.001) compared with patients undergoing WGL. There was no significant difference in resection margin status between both groups (p = 0.089) and the number of re-excisions (p = 0.429). However, in case of re-excision, patients in the RSL group were more often treated with breast-conserving surgery (p = 0.029). Conclusion: In this large national cohort study of patients with DCIS, we demonstrated that there was no difference in resection margin status between both procedures, or in the number of re-excisions, but patients in the RSL group were more often treated with breast-conserving therapy in case of a re-excision

Correction: Immune response and stromal changes in ductal carcinoma in situ of the breast are subtype dependent.

Correction to: Modern Pathology https://doi.org/10.1038/s41379-020-0553-9 There are some errors in the P values reported in this paper. [...

Immune response and stromal changes in ductal carcinoma in situ of the breast are subtype dependent.

Ductal carcinoma in situ (DCIS) associated stromal changes and influx of immune cells might be mediators of progression to invasive breast cancer. We studied the interaction between DCIS-associated stromal changes, and immune cell distribution and composition in a well-characterized patient cohort. We included 472 patients with DCIS. The presence of stromal changes, signs of regression, and DCIS-associated immune cell position were determined on hematoxylin and eosin-stained slides. Immune cell composition was characterized by immunohistochemistry (CD4, CD8, CD20, CD68, and FOXP3). The number of intraductal immune cells was quantified per mm. The interaction between stromal changes, signs of DCIS regression, immune cell composition and location was explored. Stromal changes and signs of DCIS regression were identified in 30 and 7% of the patients, respectively. Intraductal immune cells mainly comprised CD68+ macrophages and CD8+ T cells. Patients with stromal changes had significantly less influx of immune cells within the duct. DCIS regression was associated with an increased number of intraductal FOXP3+ T cells. The highest number of intraductal CD8+ T cells was seen in the ER+ HER2+ subtype. We suggest that DCIS-associated stromal changes prevent the interaction between immune cells and DCIS cells. However, in case of DCIS regression, we surmise a direct interaction between DCIS cells and immune cells, in particular FOXP3+ cells. Furthermore, the increased number of intraductal CD8+ T cells in the ER+ HER2+ DCIS subtype suggests a subtype-specific immune response, which is likely to play a role in the distinct biological behavior of different DCIS subtypes

Ductal carcinoma in situ of the breast: immune cell composition according to subtype.

Ductal carcinoma in situ of the breast includes several subtypes with a divergent biological behavior. Data regarding the composition of ductal carcinoma in situ-associated immune cells and their potential role in progression is limited. We studied ductal carcinoma in situ-associated immune response by characterizing immune cell subsets according to ductal carcinoma in situ subtypes. Ductal carcinoma in situ-associated tumor infiltrating lymphocyte (TIL) density was evaluated based on hematoxylin and eosin (H&E)-stained sections from 473 patients. Cases were subtyped based on ER, PR, and HER2. Patients were categorized as TIL-high or low. Ductal carcinoma in situ-associated immune cells of TIL-high cases were immunostained on whole slides with CD4, CD8, CD20, CD68, FOXP3, and PD-L1 (SP142 and SP263). In total, 131/473 patients (28.0%) were considered as TIL-high. The percentage of TIL-high cases was significantly higher in HER2+ and triple-negative ductal carcinoma in situ (P < 0.0001). Overall, no statistical difference in immune cell composition according to subtypes was found. However, individual subtype comparison showed that ER+ HER2+ cases had a significantly higher proportion of CD8+ T cells compared with triple-negative cases (P = 0.047). In TIL-high cases, PD-L1-SP142 expression on tumor cells was associated with subtype (P = 0.037); the lowest number of positive cases was observed in the HER2+ subtype (independent of ER). However, in TIL-high ductal carcinoma in situ, PD-L1 expression by both clones was limited. In conclusion, high numbers of TILs are predominantly observed in HER+ and triple negative ductal carcinoma in situ. The ER+ HER2+ subtype seems to attract a higher proportion of CD8+ T cells compared with the triple negative subtype. Among TIL-high cases, the HER2+ subgroup had the lowest PD-L1-SP142 expression on tumor cells. This suggests a more pronounced antitumor immunity in HER2+ ductal carcinoma in situ, which could play a role in its biological behavior

Stromal Changes are Associated with High P4HA2 Expression in Ductal Carcinoma in Situ of the Breast.

Ductal carcinoma in situ (DCIS) of the breast is able to induce stromal changes, which likely reflect the crosstalk between DCIS and its microenvironment. These changes harbor prognostic information, although the interobserver variability of scoring stromal changes is moderate. A more robust evaluation of the DCIS-associated stroma is therefore needed. The aim of this study was to characterize P4HA2 expression, which is involved in collagen biosynthesis, in DCIS and to assess whether P4HA2 expression enables a more robust evaluation of the DCIS-associated stroma compared to histomorphology. This study included 410 patients with DCIS. Stromal changes were scored on hematoxylin/eosin-stained whole slides. P4HA2 expression in DCIS-associated stroma was assessed by whole slide immunohistochemistry. One hundred DCIS lesions were evaluated by seven pathologists to study the interobserver variability in the assessment of stromal changes and stromal P4HA2 expression. High P4HA2 expression in stromal fibroblasts was present in 14.1% of the patients. High P4HA2 expression was associated with the presence of periductal stromal changes (P = 0.004). The interobserver variability was similar for the assessment of stromal changes and the percentage of P4HA2-positive fibroblasts. Although we demonstrated a significant association between high P4HA2 expression in fibroblasts and the morphological presence of stromal changes, it seems unlikely that P4HA2 expression can be used as an alternative for the histopathological evaluation of the DCIS-associated stroma

Somatic mutations and copy number variations in breast cancers with heterogeneous HER2 amplification.

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