Pleomorphic lobular carcinoma in situ (PLCIS) is a lobular neoplasm variant with worrying histologic features including high nuclear grade, necrosis, and increased mitoses, features also seen in high-grade ductal carcinoma in situ (hgDCIS). Optimal management of PLCIS is unclear. Classic lobular carcinoma in situ (cLCIS) is considered an incidental risk factor that does not necessitate additional interventions;¹ however, the literature suggests that PLCIS may be a non-obligate precursor to invasive carcinoma.^2,3 The National Health Service and European Society for Medical Oncology advocate for PLCIS to be treated with complete excision followed by adjuvant hormone therapy and/or radiation, similar to management of hgDCIS.¹ The National Comprehensive Cancer Network, while more conservative, still recommends complete surgical excision of PLCIS with negative margins;¹ however, there is a lack of uniform approach among general surgeons as some approach PLCIS in the same way as they deal with cLCIS.⁴ To explore the biological behavior of PLCIS, which has implications for its malignant potential, we looked at gene expression to compare PLCIS's expression profile to those of cLCIS and hgDCIS, entities which were chosen for comparison due to overlaps in histologic features and surgical recommendations.

There were initially 329 cases of PLCIS and 2603 cases of cLCIS identified within the in-house pathology database in the period of 2015 to 2024. Of these, 192 PLCIS cases and 501 cases of cLCIS cases were from core needle biopsies and excluded. Of the remaining 137 PLCIS cases and 2102 cLCIS cases, 121 PLCIS cases and 2085 cLCIS cases were excluded due to either contemporary or historical invasive carcinoma or concurrent DCIS within the specimen. Of the remaining 17 cLCIS cases, 7 were excluded due to concurrent PLCIS within the specimen. In total, there were 16 cases of PLCIS and 10 cases of cLCIS after implementation of the inclusion and exclusion criteria outlined above, all of whom were included within this study. A large number of qualifying hgDCIS cases were identified after implementation of inclusion and exclusion criteria, from whom 9 cases were chosen to match the number of eligible PLCIS and cLCIS cases. In total, there were 16 cases of PLCIS, 10 cases of cLCIS, and 9 cases of hgDCIS who were included for gene expression analysis.

A total of 3 differentially expressed genes (DEGs) were identified between PLCIS and cLCIS (1 upregulated and 2 downregulated in PLCIS). A total of 63 DEGs were identified between PLCIS and hgDCIS (43 upregulated and 20 downregulated in PLCIS). Volcano plots showing pair-wise comparisons of DEGs between PLCIS against cLCIS and hgDCIS are shown in Figures 2A–B, respectively.

Based on nominal P values, gene set analysis between PLCIS-cLCIS showed that PLCIS was significantly enriched in the E2F transcription factor target gene expression (P = 0.028) while cLCIS had enrichment in the apical junction complex (P = 0.037) set of genes; corresponding FDR values were 6.8% for E2F transcription factors and 76.8% for apical junction complex genes, indicating a high likelihood that the difference in apical junction complex was a false positive. Gene set analysis between PLCIS-hgDCIS showed hgDCIS was enriched for glycolysis pathway (P = 0.030) and interferon-α response (P = 0.017) while no gene sets were enriched in PLCIS; corresponding FDR values were 27.7% for glycolysis and 0.9% for the interferon-α response.

Of note, when the familywise-error rate (FWER) correction method was used to correct the P values of the 4 significant gene sets listed, only the interferon-α response remained significant (P = 0.009) while the E2F transcription factor target gene set had borderline significance (P = 0.068); however, it has been noted that FWER is a conservative correction method.⁹ Heat maps from gene set analysis are shown in Figures 2C–D.

There is a much more distinctive differential gene expression profile between PLCIS and hgDCIS than with cLCIS, suggesting that the differences in biological behavior are more pronounced between hgDCIS and PLCIS. This might be intuitively expected due to a shared lobular phenotype and studies showing a stepwise molecular progression from cLCIS to PLCIS;³ however, it is unexpected to find that only few genes have differential expression between cLCIS and PLCIS which could suggest a closer biological behavior than would be expected based on morphology.

Gene set analysis can give a more nuanced insight into biological behavior. Although the apical junction gene set represents adherents and tight junctions which are characteristically lost in cancer, it is difficult to draw any conclusions from this finding given the high probability that it is a false positive result.^6,10 Because of this, the primary difference between cLCIS and PLCIS is upregulation of E2F transcription factor targets in PLCIS. E2F transcription factors are primarily associated with cellular proliferation due to their regulation of DNA replication and checkpoint control genes and are frequently upregulated in cancer.^11–13 Increased proliferation due to upregulation of the E2F transcription factor targets could explain the characteristic necrosis which is one of the distinguishing features between cLCIS and PLCIS. Previous studies have linked increased E2F target gene expression in breast cancer with worse prognosis, including decreased survival rates and heightened mortality risk.¹⁴

Between PLCIS and hgDCIS, the primary DEGs sets were upregulation of glycolysis genes and interferon-α response genes in hgDCIS. Aerobic glycolysis is a key process in cancer cells; not only is it responsible for the majority of energy production in cancer, but it also influences other aspects of tumor development and progression, such as immune suppression and modulation of the tumor microenvironment.^15,16 Although the differences in glycolysis gene set expression had a relatively high chance of being a false positive finding at 27.7%, previous studies exploring gene expression between invasive lobular carcinoma (ILC) and invasive ductal carcinoma (IDC) similarly found an increased expression of glycolysis-related genes in IDC relative to ILC, which does provide some reassurance that this could be a real finding;¹⁷ however, upregulation of glycolysis in the fully malignant IDC does also limit the inferences which can be made about malignant progression of in-situ lesions, as this could instead just be a biological feature associated with the ductal phenotype. The exact role of interferon-α in cancer biology is still not entirely known. Interferon-α has classically been associated with antitumoral properties, being a driver of apoptosis and modulator of the immune microenvironment;^18,19 however, interferon-α has been shown to have procancer effects on cells, such as the ability to activate the NF-κB pathway, promote angiogenesis, and induce cell survival.^20–22

Importantly, there is a noticeable lack of differences in TGF-β, hedgehog, notch, Wnt/β-catenin, or PI3K-AKT-mTOR signaling pathway which are key signaling pathways in breast cancer biology.^23,24 The significance of this finding is hard to determine, especially since mutational profiles are not available for the samples used in this study. Given that gain-of-function mutations in PIK3CA are ubiquitous throughout breast neoplasms, including both DCIS and LCIS, and are an early event implicated in tumor initiation, it is possible that a high prevalence of activating PIK3CA mutations in the study samples could be upregulating the PI3K pathways at comparable rates.^25,26 It is interesting to note, however, that a gene expression study between IDC and ILC found lower mTOR activity and increased AKT and PI3K activity in ILC, which is suggestive of a unique mTOR regulation pathway in ILC.¹⁷ The absence of such findings in our study could be due either to absence of this regulation pathway in the in-situ lesions or because such a granular pathway might not be represented in the gene set collection used in analyses and the resultant counterintuitive changes in mTOR, AKT, and PI3K expression could be confounding the analysis of the broader PI3K-AKT-mTOR pathway; a separate ILC-IDC gene expression study using GSEA analysis with the same Hallmark gene set collection used in this study likewise did not find any differences in the PI3K-AKT-mTOR signaling pathway which supports the latter interpretation.²⁷ The lack of gene-gene interaction analysis has been a criticism of GSEA and could explain why it may not be able to discern such a specific interaction.²⁸

It is important to note that although gene set analysis can provide vague insights into biological behavior, the main purpose of GSEA is the generation of hypotheses and so it is difficult to use these findings to extrapolate firm conclusions about biological behavior.⁹ Additional nuances can be introduced by placing the findings of this study within the context of similar lobular and ductal breast cancer gene expression studies, but even this exploration is limited due to the lack of literature analyzing gene expression in specifically pleomorphic lobular variants. Furthermore, this study was limited by a relatively small sample size due to stringent inclusion/exclusion criteria as well as a relatively low number of genes analyzed, which are all factors likely contributing to the highly variable FDRs.²⁹ Findings with low FDR, primarily the differences in E2F transcription factor and interferon-α gene expressions, are expected to be relatively stable while the reliability of other findings are questionable and could be further resolved by increasing the sample size.

While this study provides a general hierarchy of disease severity and disease overlap, it does highlight the challenges associated with using gene expression data to reach conclusions about biological behavior or clinical significance. As such, larger cohort studies interrogating long-term clinical outcomes of PLCIS are needed to determine prognosis and optimal management.

PLCIS gene expression profile had greater overlap with cLCIS than with hgDCIS. Gene set analysis showed increased expression of E2F transcription factor targets in PLCIS relative to cLCIS and increased expression of glycolysis genes and interferon-α genes in hgDCIS as compared to PLCIS. These findings suggest that PLCIS behaves more similarly to cLCIS than hgDCIS and may be more aggressive than cLCIS, but not as aggressive as hgDCIS. Due to limitations translating gene expression studies to clinical outcomes, large cohort studies evaluating long-term clinical outcomes of PLCIS are still needed to definitively determine prognosis and optimal management.