Clinicopathological features of breast cancer progression: From DCIS to invasive ductal carcinoma

doi:10.21203/rs.3.rs-3410901/v1

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Clinicopathological features of breast cancer progression: From DCIS to invasive ductal carcinoma

https://doi.org/10.21203/rs.3.rs-3410901/v1

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Purpose

Ductal carcinoma in situ (DCIS) is regarded as non-invasive precursor lesion of invasive ductal carcinoma (IDC) but the molecular mechanisms and tumorigenesis behind DCIS progression remain to be elucidated. IDC sometimes present with a synchronous in-situ component (IDC-DCIS). The current study investigated the clinicopathological features that could predict DCIS progression and trace the origin of IDC.

Methods

Total 501 breast ductal carcinoma patients who underwent surgery as a first-line treatment between 2019 to 2022 from Sir Run Run Shaw Hospital were retrospectively reviewed. The clinical outcomes in different molecular subtypes and nuclear grade were evaluated.

Results

DCIS was significantly associated with a positive CK5/6 expression (P < 0.001), which was observed especially in HER2 overexpression subtype (P = 0.0027). Compared to IDC-DCIS, EGFR expression was significantly higher in IDC (P < 0.001) in triple-negative subtype (P < 0.001). In IDC-DCIS, co-expression of several biomarkers was observed in the DCIS component and IDC component. High grade DCIS component was significantly associated with HER2 and high Ki-67 (P < 0.001). Compared to pure DCIS, the in-situ component of IDC-DCIS was associated with high Ki-67 (P = 0.004), negative EGFR (P = 0.003), positive CK5/6 (P < 0.001) and high grade (P = 0.004).

Conclusion

Molecular subtypes, nuclear grade, and expressions of EGFR and CK5/6 resulted in different clinicopathological profiles in DCIS, IDC-DCIS and IDC. Presence of in-situ component is a marker of reduced aggressiveness and also supported that DCIS is the precursor lesion. Overall, our study traced the origin of IDC and propounded that HER2 targeted therapies could be of potential use in DCIS clinical trials.

DCIS

IDC

clinicopathology

molecular subtype

Mammary ductal carcinoma is the most prevalent type of invasive breast carcinoma, accounting for 70–80%, and is divided into invasive ductal carcinoma (IDC) and ductal carcinoma in situ (DCIS). DCIS is defined as mammary carcinoma that is still confined within the ductal basement membrane whereas IDC invades the ducts and exists in the stroma (Sgroi 2021). DCIS is regarded as a non-invasive precursor of IDC and is associated with a 10-fold risk of developing into breast carcinoma (Rebbeck et al. 2022). Approximately 20–40% of untreated DCIS will progress to IDC (Thennavan et al. 2022). However, the transition from DCIS to IDC, the related-pathways and biomarkers involved remain to be elucidated.

IDC commonly presents alone (pure IDC) or with a co-existing DCIS component (IDC-DCIS). The clinical significance and prognosis of IDC-DCIS has not been defined conclusively, and treatment is similar as pure IDC since clinical management depend entirely on the pathological and molecular characteristics of the invasive component of IDC-DCIS (Kole et al. 2019; Chen et al. 2019). Previous studies have demonstrated that IDC-DCIS tumors are associated with favorable clinical characteristics and reduced biological aggressiveness such as smaller tumor size, lower tumor nuclear grade, less lymph node involvement and lower Ki-67 expression compared to IDC alone (Dieterich et al. 2014). There is a high concordance of receptor expression between the DCIS component and adjacent IDC component in IDC-DCIS (Doebar et al. 2016), postulating that IDC may arise from a preexisting DCIS lesion and indicate that IDC-DCIS represents a disease entity biologically distinct from pure IDC (Iakovlev et al. 2008; Yu et al. 2011). Identifying changes between them will be helpful for finding associated instances of breast cancer progression.

DCIS as well as IDC are comprised of heterogeneous phenotypes, with differences in pathologic indexes such as estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor-2 (HER2) status which are important prognostic markers as well as predictors of therapy response in breast carcinoma, and may contemplate important pathways through which DCIS lesions may progress towards invasion (Clark at al. 2011; Wan et al. 2018). However, the pathologic and molecular differences among the different entities of ductal related carcinoma (including DCIS, IDC-DCIS, and pure IDC) are poorly understood as different expressions of ER, PR and HER2 have been reported among DCIS, IDC-DCIS and IDC (Liu et al. 2020). Based on gene expression analysis, breast cancer is classified into 5 different molecular subtypes: luminal A, luminal B/HER2-negative, luminal B/HER2-positive, HER2-overexpression and triple-negative subtypes, which are correlated with ER, PR, HER2 and Ki-67 index (Goldhirsch et al. 2013). Different breast cancer subtypes have been known to exhibit different clinicopathologic characteristics (Goh et al. 2019). Interestingly, subtype-specific progression paths have been speculated since tumors of the different subtypes are found among both DCIS and IDC (Lesurf et al. 2016; Allred et el. 2008), thus suggesting that DCIS, IDC-DCIS and IDC may have different clinicopathological outcomes in different molecular subtypes. Relatively little is known regarding the clinicopathological characteristics of different types of breast ductal carcinomas (DCIS, IDC-DCIS, IDC) according to molecular subtypes. Very few studies assessed molecular phenotypes or biomarker expression to the clinical outcomes (Clark et al. 2011).

Different pathways have been speculated in the development of high grade and non-high grade carcinomas (Steinman et al. 2007). However, the differences in grade between the different subtypes of DCIS, IDC-DCIS and IDC are controversial. In IDC-DCIS, the grade of the accompanying DCIS component is a significant prognostic factor. Moreover, additional markers such as epidermal growth factor receptor (EGFR), cytokeratin 5/6 (CK5/6) and p63 are warranted to devise more individualized therapy. Identifying any alteration in these molecular markers involved in DCIS transition to invasive disease as well as their potential correlation in terms of different subtypes and grade among DCIS, IDC-DCIS and IDC would be of utmost clinical significance and modernize management.

In this study, we explored the differences in pathological characteristics and clinical outcomes among pure DCIS, IDC-DCIS and pure IDC to gain an increased understanding of the tumor properties that drive disease progression. We further classify the different disease entities into molecular subtypes and grade-wise to pinpoint any changes in potential molecular markers for prognostic and therapeutic purposes. In IDC-DCIS, we compared the in-situ component to the invasive component to identify any molecular changes, and we also assessed the prognostic value and implication of different pathological grade of the accompanying in-situ component. We also assess the correlation among the biomarkers in the different groups and their relationship with clinical outcomes.

Patients

Between January 1, 2019, and April 30, 2022, a total of 501 eligible patients who underwent surgery as a first-line treatment due to breast ductal carcinoma at Sir Run Run Shaw Hospital were retrospectively reviewed. All database used in the present study was approved by the Ethics Committee of the Sir Run Run Shaw Hospital, affiliated with Zhejiang University and informed consent was obtained from all individual participants included in the study. The inclusion criteria were as follows: (1) female patients newly diagnosed with breast cancer; (2) complete tumor resection with axillary or sentinel lymph node dissection. The exclusion criteria were as follows: (1) preoperative chemotherapy, radiotherapy, or immunotherapy; (2) non-ductal carcinoma, such as lobular, papillary, mucinous tumor; (3) a history of other malignancies; (4) incomplete clinical and immunohistochemical data. A flowchart of this retrospective study is shown in Fig. 1.

Data collection

The following variables were collected: age, type of tumor, tumor size, tumor histological grading, lymph node involvement, tumor marker profile (ER, PR, HER2, Ki-67, E-cadherin, p120, EGFR, CK5/6, p63). According to the type of tumor, the population was divided into three groups, including 107 cases of pure DCIS, 253 cases of pure IDC, and 141 cases of IDC with in-situ component (IDC-DCIS). In the group of IDC-DCIS, the clinical index and IHC results of the two components (IDC and DCIS) were separately recorded.

The immunohistochemical results were evaluated independently by professional pathologists. ER and PR assays were considered positive if there are at least 1% tumor nuclei stained and negative if < 1% of tumor cell nuclei were immunoreactive (Hammond et al. 2010; Untch et al. 2013). HER2 positivity was considered as score 3 + by IHC or FISH positive. For E-cadherin, p120, EGFR, CK5/6 and p63, positive means any intensity of either nucleic, cytoplasmic or membranous expression whereas negative means no expression.

According to the expression status of ER, PR, HER2 and Ki-67, the cases were classified into 5 different molecular subtypes: luminal A (ER-positive and/or PR-positive, HER2-negative, Ki-67 < 20%), luminal B/HER2-negative (ER-positive and/or PR-positive, HER2-negative, Ki-67 ≥ 20%), luminal B/HER2-positve (ER-positive and/or PR-positive, HER2-positive, any Ki-67), HER2-overexpression (ER-negative, PR-negative, HER2-positive) and triple-negative (ER-negative, PR-negative, HER2-negative). Of note, we hypothesize that the molecular subtype of IDC-DCIS group is commonly reported as a result of the invasive component by IHC.

Statistical analysis

Data were analyzed using SPSS 26.0 software. Normally distributed data are described as the mean (standard deviation), and non-normally distributed data are described as the median (interquartile range, IQR). In order to compare the distribution of clinical and pathological features between the three different groups, the Pearson’s Chi-squared test or Fisher exact test were used to compare categorical variables, and Kruskal-Wallis test was used to compare continuous variables. The Bonferroni adjusted P value was used for comparisons between any two groups with the chi-square test. Furthermore, the correlations between different biomarkers and clinical outcomes among the three different groups were assessed separately using the Pearson’s correlation coefficient (R). For all tests, P < 0.05 was considered statistically significant.

Clinicopathological Features

A total of 501 breast cancer patients including 107 cases of pure DCIS, 253 cases of pure IDC, and 141 cases of IDC-DCIS were involved in this study. The mean age at diagnosis of pure IDC group was 55.22 years (SD 12.04; range 24–87), which was significantly older than DCIS and IDC-DCIS patients (P < 0.001), but no significant difference was found between DCIS and IDC-DCIS under the Bonferroni adjusted p value (P = 0.213) (Table 1). Compared with DCIS and IDC-DCIS, IDC was significantly associated with a larger tumor size (P < 0.001) and no significant difference was noted between DCIS and IDC-DCIS (P = 0.096). The rates of lymph node involvement were 0% in DCIS, 26.2% in IDC-DCIS and 52.2% in IDC, with significant differences between any two groups (P < 0.001). Besides, the proportions of high histological grading were 41.1% in DCIS, 20.6% in invasive component of IDC-DCIS and 54.2% in IDC. IDC was significantly associated with a high nuclear grade (P < 0.001) compared to other groups. Interestingly, IDC-DCIS was significantly associated with a moderate grade compared to DCIS (P < 0.001).

Table 1 summarizes the immunohistochemistry results of DCIS, invasive component of IDC-DCIS and IDC. IDC was significantly associated with a more negative expression of hormone receptor compared to DCIS and IDC-DCIS (P < 0.001), while DCIS and IDC-DCIS showed no significant difference. A significant difference was observed in the expression of Ki-67 and CK5/6 in the three groups, with significant differences between any two groups (P < 0.001). EGFR expression was significantly higher in DCIS and IDC than in IDC-DCIS (P < 0.001), while DCIS and IDC showed no significant difference. p63 was significantly expressed in DCIS compared to other groups (P < 0.001). There was no significant difference in HER2, E-cadherin or p120 expression among the three groups.

Based on IHC results, the cases were classified into the 5 different subtypes and are listed in Table 1. The molecular subtypes composition was significantly different between the 3 groups (P < 0.001). Bonferroni adjusted p value was performed to make any further intergroup comparisons. Triple-negative subtype was more frequent in IDC (22.5%) than in DCIS (4.7%) and IDC-DCIS (7.1%) while luminal A subtype was more frequent in DCIS (54.2%) and IDC-DCIS (41.8%) compared to pure IDC (14.6%). Luminal B/HER2-negative subtype was dominant in IDC (accounting for 38.3%).

Table 1

Clinicopathologic characteristics of different BC groups (DCIS, IDC-DCIS and IDC)
Characteristic	DCIS (n = 107)	IDC-DCIS (n = 141)	IDC (n = 253)	P value^a
Age: Mean (SD)	49.99 ± 9.25 (Range: 30–73)	51.94 ± 11.67 (Range: 25–88)	55.22 ± 12.04 (Range: 24–87)	< 0.001^*
Tumor size: Median (IQR)	1.6 (1.5)	1.5 (1.05)	2.2 (1.4)	< 0.001^*
Lymph node involvement Yes No	0 107	37 104	132 121	< 0.001^*
Histological grading Low (I) Moderate (II) High (III)	32 31 44	23 89 29	4 112 137	< 0.001^*
ER Positive Negative	89 18	122 19	167 86	< 0.001^*
PR Positive Negative	78 29	111 30	131 122	< 0.001^*
HER2 Positive Negative	36 71	30 111	62 191	0.075
Molecular Subtypes Luminal A Luminal B/HER2- Luminal B/HER2+ HR-/HER2+ Triple-negative	58 (54.2%) 8 (7.5%) 24 (22.4%) 12 (11.2%) 5 (4.7%)	59 (41.8%) 42 (29.8%) 21 (14.9%) 9 (6.4%) 10 (7.1%)	37 (14.6%) 97 (38.3%) 33 (13.0%) 29 (11.5%) 57 (22.5%)	< 0.001^*
E-cadherin Positive Negative	107 0	141 0	253 0	1.000
P120 Positive Negative	107 0	141 0	253 0	1.000
EGFR Positive Negative	47 60	20 121	97 156	< 0.001^*
CK5/6 Positive Negative	60 47	9 132	45 208	< 0.001^*
p63 Positive Negative	102 5	7 134	5 248	< 0.001^*

ER, estrogen receptor; PR, progesterone receptor; HER2, human epidermal growth factor receptor 2; HR, hormone receptor; EGFR, epidermal growth factor receptor; SD, standard deviation; IQR, interquartile range.

^a Comparisons between DCIS, IDC-DCIS and IDC.

*Statistically significant.

Analysis of the in-situ and invasive component of IDC-DCIS

We analyzed 141 cases of IDC-DCIS with the IHC results of the in-situ component and synchronous invasive component in the archives (Table 2). There was no significant difference in the expression of ER (P = 1.000), PR (P = 0.768), HER2 (P = 0.668), E-cadherin (P = 1.000), p120 (P = 1.000) and EGFR (P = 0.416). However, the invasive component was associated with a higher Ki-67 index (P = 0.006) compared to the in-situ component when we defined Ki-67 ≥ 20% as high expression. Besides, there was a significant difference in the expression of CK5/6 (P < 0.001), p63 (P < 0.001) which were found to be positively expressed in the in-situ component compared to the invasive component.

Table 2

Analysis of in-situ and invasive component of IDC-DCIS
	IDC component (n = 141)	DCIS component (n = 141)	P value^a
ER Positive Negative	122 19	122 19	1.000
PR Positive Negative	111 30	113 28	0.768
HER2 Positive Negative	30 111	33 108	0.668
Ki-67 Low (< 20%) High (≥ 20%)	65 76	88 53	0.006^*
E-cadherin Positive Negative	141 0	141 0	1.000
P120 Positive Negative	141 0	141 0	1.000
EGFR Positive Negative	20 121	25 116	0.416
CK5/6 Positive Negative	9 132	114 27	< 0.001^*
P63 Positive Negative	7 134	136 5	< 0.001^*

ER, estrogen receptor; PR, progesterone receptor; HER2, human epidermal growth factor receptor 2; HR, hormone receptor; EGFR, epidermal growth factor receptor.

^a Comparisons between IDC component and DCIS component in IDC-DCIS.

*Statistically significant.

We also focused on different histology grading of the in-situ component to find out whether there existing any IHC clue that could explain the aggressive tendency within the group (Table 3). There were 75 cases fully contains information on both components. We discovered that high grade in-situ component of IDC-DCIS was significantly associated with positive HER2 expression (P < 0.001) and a high Ki-67 index. As for other biomarkers, no distinction was found between the two groups.

Table 3

IHC results of high grade and non-high grade in-situ component of IDC-DCIS
	In-situ component of IDC-DCIS (n = 75)
	High grade	Non-high grade	P value
ER Positive Negative	39 8	26 2	0.223
PR Positive Negative	35 12	24 4	0.250
HER2 Positive Negative	19 28	1 27	< 0.001^*
Ki-67 Low High	17 30	23 5	< 0.001^*
EGFR Positive Negative	14 33	3 25	0.056
CK5/6 Positive Negative	41 6	25 3	0.791

ER, estrogen receptor; PR, progesterone receptor; HER2, human epidermal growth factor receptor 2; HR, hormone receptor; EGFR, epidermal growth factor receptor.

*Statistically significant.

Risk factors for clinical outcomes

Pearson’s correlation coefficient (R) was used to assess the correlations between biomarkers and clinical outcomes (Table 4). In DCIS, all biomarkers were not significantly associated with tumor size except for PR, which was inversely correlated with both tumor size and high histology grading (P < 0.001, R= -0.337; P = 0.025, R= -0.217). Besides, high histology grading was associated with HER2 expression (P < 0.001, R = 0.370) and a high Ki-67 index (P < 0.001, R = 0.453). In IDC-DCIS, both tumor size and grade were positively correlated with HER2 expression (R = 0.284 and R = 0.250, respectively) and a high Ki-67 index (R = 0.268 and R = 0.259, respectively). In IDC, lymph node involvement was positively correlated with ER (P < 0.001, R = 0.232) and PR (P = 0.015, R = 0.153), and inversely correlated with EGFR (P = 0.003, R= -0.189) and CK5/6 (P = 0.002, R= -0.196).

Table 4

Correlation between biomarkers and clinical outcomes
		ER	PR	HER2	Ki-67	EGFR	CK 5/6	p63
DCIS	Tumor size P value^a R^b	0.068 -0.177	< 0.001^* -0.337	0.079 0.170	0.519 -0.063	0.274 0.107	0.401 0.082	0.202 -0.124
	Lymph node involvement P value R	0.438 -0.075	0.805 -0.024	0.991 -0.001	0.775 0.028	0.421 0.078	0.421 -0.078	0.697 0.038
	Grade P value R	0.401 -0.081	0.025 -0.217	< 0.001^* 0.370	< 0.001^* 0.453	0.599 -0.051	0.897 0.013	0.326 0.095
IDC-DCIS	Tumor size P value R	0.654 0.038	0.707 0.032	0.001^* 0.284	0.001^* 0.268	0.132 0.127	0.191 -0.111	0.512 -0.056
	Lymph node involvement P value R	0.581 0.047	0.683 0.034	0.144 0.123	0.052 0.164	0.217 -0.104	0.777 -0.024	0.461 -0.062
	Grade P value R	0.505 -0.056	0.150 -0.121	0.003^* 0.250	0.002^* 0.259	0.260 0.095	0.899 0.011	0.673 -0.036
IDC	Tumor size P value R	0.015 0.152	0.263 0.071	0.322 -0.062	0.220 0.077	0.033 -0.134	0.047 -0.125	0.058 -0.119
	Lymph node involvement P value R	< 0.001^* 0.232	0.015 0.153	0.327 -0.062	0.545 -0.038	0.003^* -0.189	0.002^* -0.196	0.723 0.022
	Grade P value R	0.002^* -0.191	0.003^* -0.189	0.676 0.026	< 0.001^* 0.360	< 0.001^* 0.252	< 0.001^* 0.221	0.241 0.074

ER, estrogen receptor; PR, progesterone receptor; HER2, human epidermal growth factor receptor 2; HR, hormone receptor; EGFR, epidermal growth factor receptor.

^aP value of the Pearson correlation coefficient.

^b Pearson correlation (R): R > 0: positive correlation; R < 0: negative correlation; 0.1–0.3: weak correlation, 0.3–0.5: medium correlation, 0.5-1: strong correlation.

*Statistically significant.

Despite a string of controversies, DCIS is recognized as a precursor lesion to IDC, but the exact mechanism of progression or transition is poorly understood and debatable. Some researchers speculated a sequential progression from in-situ to invasive disease, whereas others have proposed that DCIS already acquired invasive characteristics from inception (Champion et al. 2019), such that high-grade DCIS are predisposed to progress to invasive disease, in contrast with low-grade DCIS (Lesurf et al. 2016; Hughes et al. 2019). The positive correlation and high concordance rate among biological markers between the invasive and in-situ component in IDC-DCIS acknowledge a clonal relationship (Leong et al. 2001). In IDC-DCIS, evolving from a gradual accumulation of mild tumor suppressor gene mutations, pre-existing DCIS lesions have been speculated to give rise to IDC, whereas pure IDC is postulated to arise de novo from a more drastic tumor suppressor gene defect (Wong et al. 2010). Such a model is evocative of colorectal carcinoma that may arise through an adenomatous polyp pathway or de-novo pathway (Hisabe et al. 2014). Conversely, some studies advocated that DCIS may not be a precursor for IDC as a result of major changes in gene expression during transition from normal breast tissue to DCIS rather than in the transition from DCIS to IDC (Schnitt et al. 2009). In addition, not all DCIS lesions are able to progress to invasive disease as some DCIS lesions are genetically programmed to remain DCIS only, resulting in overtreatment in many cases (Steinman et al. 2007; Lips et al. 2022).

In our study, a good concordance and high similarity in receptor expression between the invasive and co-existing in-situ component in IDC-DCIS postulate a clonal relationship that DCIS is the likely progenitor. However, we found that the invasive component was significantly associated with a higher Ki-67 index. Noteworthy, Kim et Al. revealed that in IDC-DCIS, the grade of the accompanying DCIS component is a significant prognostic factor and also proposed a parallel progression such that IDC with accompanying high grade DCIS inclines to be of high histologic grade (Kim et al. 2013). We observed that high grade DCIS was present along both non-high grade and high grade IDC, suggesting a higher propensity of high-grade DCIS to transit to invasive disease. Additionally, HER2 and a high Ki-67 index were significantly associated with the high-grade in-situ component of IDC-DCIS, highlighting their impact in promoting invasion, and thus HER2 targeting therapies could be implemented in further DCIS clinical trials to assess their potential relevance in preventing DCIS progression (Roses et al. 2009; Lopez et al. 2019).

We found that DCIS and IDC-DCIS patients were significantly younger and presented with more favorable biological tumor characteristics such as smaller size, lower nuclear grade and less lymph node metastasis compared to pure IDC. Besides, the concomitant presence of DCIS with IDC (IDC-DCIS) was a marker of reduced aggressiveness and is associated with lower local recurrence (Dieterich et al. 2014). The differences in expression of hormone receptors (ER and PR), HER2 and Ki-67 among DCIS, IDC-DCIS and IDC are still controversial. A study by Schorr et al. (2010) revealed a lower steroid receptor status in IDC-DCIS compared to DCIS, while Liu et Al. found a higher expression in IDC-DCIS compared to DCIS (2020). Our study revealed higher positive expression of hormone receptor (ER and PR) in DCIS and IDC-DCIS compared to IDC. The increase in ER and PR expression in IDC-DCIS in turn insinuate that pure IDC carcinogenesis pathway is related with the basaloid phenotype, which is associated with multiple defects in tumor suppressor genes such as TP53 and the BRCA DNA repair genes (Wong et al. 2010). Ki-67 index varied significantly among the three groups: IDC possessed the highest Ki-67 index while DCIS had the lowest expression. Different distribution of biomarkers and receptor expression led to the classification of 5 molecular subtypes (Muggerud et al. 2010). Different roles and speeds of progression according to different subtypes have been speculated, with HER2-overexpression subtype having the slowest progression and Triple-negative subtype being the fastest (Kurbel et al. 2013). Based on our findings, Luminal A was the dominant subtype in DCIS and triple-negative subtype was more frequent in IDC. Due to the elevated high Ki-67 index in IDC-DCIS, Luminal B/HER2- accounted for a significant number of cases in IDC-DCIS. A previous study by Wong et Al. postulated that co-existing DCIS in IDC-DCIS had no effect on Ki-67, while the predominant high-risk factors are mutated BRCA1/TP53 and HER2 (2012).

Pure DCIS, IDC-DCIS and pure IDC have different gene expression pattern and these genes may pinpoint driver pathways involved in DCIS transition. The progression from in-situ disease to invasive disease is a multi-step process. Genes associated with epithelial-to-mesenchymal transition (EMT) and myoepithelial cell-specific genes were enriched in invasive cancer, speculating that disruption of myoepithelial cell-layer leads to DCIS transition (Knudsen et al. 2012). EGFR and its downstream pathway regulate EMT, migration and tumor invasion, and EGFR overexpression in breast cancer is associated with a larger tumor size, poor differentiation and clinical outcomes (Masuda et al. 2012). Meijnen et Al. showed no evidence of expression of EGFR and only 2% of CK5/6 in their DCIS series (2008). We found a higher expression of EGFR among pure DCIS and IDC cases, triple-negative subtype (IDC-DCIS and IDC) and high-grade IDC. Our study revealed that positive CK5/6 expression was significantly associated with pure DCIS cases compared to IDC-DCIS and IDC. In IDC-DCIS, we also observed a significant loss of CK5/6 expression in invasive component, highlighting that malignant transformation might be accompanied by loss of CK5/6 expression (Otterbach et al. 2000). CK5/6 expression is a recognized characteristic of poorly differentiated basal-like invasive breast carcinoma and is associated with a poor clinical outcome (Nielsen et al. 2004). In our study, CK5/6 expression was associated with a high grade in IDC, consistent with previous studies (Volkel et al. 2022; Abdelrahman et al. 2017). Moreover, in both IDC-DCIS and IDC, a positive correlation was observed between CK5/6 and EGFR. A study by Sutton et Al. revealed that CK5/6 and EGFR expression was associated with lymph node metastasis in triple-negative breast carcinoma (2010). However, our study revealed an inverse correlation between expressions of EGFR and CK5/6 and lymph node involvement as well as tumor size in IDC and no significant association was found between lymph node metastasis and EGFR and CK5/6 expressions in triple-negative IDC. A recent study revealed a worse prognosis of EGFR and CK5/6 positive patients after neoadjuvant chemotherapy (Wang et al. 2020). In our study, although there was good concordance of EGFR expression between co-existing in-situ component and invasive component in IDC-DCIS, a positive EGFR expression was frequent among high grade in-situ component of IDC-DCIS. In addition, there were significant differences in molecular subtypes, pathological grading and expressions of Ki-67, EGFR and CK5/6 between pure DCIS and in-situ component of IDC-DCIS, implying a different biological behavior and molecular mechanisms between pure DCIS and DCIS associated with IDC-DCIS (Park et al. 2006). Based on our findings, we can speculate that high histology grade is crucial in the transition of DCIS to invasive disease. We also hypothesize that some pure DCIS in our study were either genetically constituted to be only DCIS or did not acquire the genetic changes required to progress to IDC by the time of diagnosis.

p63 is recognized as a marker of non-invasive epithelial tumors such as DCIS (Melino et al. 2011). Theoretically and histologically, invasive carcinoma is defined by absence of the myoepithelial cell layer. We found that the majority of IDC-DCIS and IDC presented with a loss of p63, and when comparing to the in-situ component of IDC-DCIS, the invasive component was significantly associated with a loss of p63, supporting that loss of p63 is associated with invasion (Bonacho et al. 2020). However, we found a small percentage of DCIS lacking the myoepithelial marker p63, yet, the underlying mechanism in such cases is still unknown (Mohan et al. 2016). One potential explanation could be the heterogeneity in expression of markers in myoepithelial cells (Mardekian et al. 2016), thus prompting the use of more than one single marker for its identification and differentiation between DCIS and IDC. It has been reported that double positive status of p63 and CK5/6 could predict a lower subsequent breast carcinoma incidence in the occurrence of intraductal papilloma (Yang et al. 2015).

This study may have several limitations should be noted. Our study was a single institutional study, and grade of some in-situ components in IDC-DCIS were unavailable, possibly because the in-situ component was too small to adequately grade. Besides, due to the short follow-up time and the better prognosis of breast cancer compared with other cancers, relevant information such as patient overall-survival will continue to be updated for the following research. Overall, this study provides valuable clinicopathological clues to the progression of DCIS to IDC.

To our knowledge, this is the first retrospective studies comparing different clinicopathological features and distinctive clinical outcomes simultaneously in DCIS, IDC-DCIS and IDC. Presence of in-situ component in IDC-DCIS is a marker of reduced aggressiveness compared to pure IDC. In IDC-DCIS, concordance of biomarker expression between in-situ and invasive component supports that DCIS is the precursor lesion. The synchronous association of high grade DCIS with both non-high and high grade invasive components in IDC-DCIS suggests a higher propensity of invasive potential. This study indicates that HER2 and Ki-67 are associated with high grade DCIS and propound that HER2 targeted therapies could be of potential use in DCIS clinical trials.

BC Breast cancer

CK5/6 Cytokeratin 5/6

DCIS Ductal carcinoma in situ

EGFR Epidermal growth factor receptor

ER Estrogen receptor

HER2 Human epidermal growth factor receptor 2

HR Hormone receptor

IDC Invasive ductal carcinoma

IQR Interquartile range

PR Progesterone receptor

SD Standard deviation

Acknowledgements The authors thank Department of Oncology Surgery, and the Sir Run Run Shaw Hospital.

Author contributions All authors contributed to the data collection. The first draft of the manuscript was written by ZJH, and data analysis were performed by Nadire and Saad. WLB, ZJC, JFY and CYX reviewed and edited the draft. All authors read and approved the final manuscript.

Funding The research was supported by the National Natural Science Foundation of China (No.81972597, No.82203610).

Data availability The data in this study are available from the corresponding authors upon reasonable request.

Conflict of interest The authors declared that they have no conflicts of interest to this work.

Ethics approval The study was approved by the Ethics Committee of Sir Run Run Shaw Hospital (Hangzhou, China; approval no. 2023-832-01).

Consent to participate The Ethics Committee of Sir Run Run Shaw Hospital waived the requirement for written informed consent because the study was an observational, retrospective study, and the patients’ identification information was removed.

Consent to publish Not applicable

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No competing interests reported.

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Clinicopathological features of breast cancer progression: From DCIS to invasive ductal carcinoma

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Clinicopathological Features

Analysis of the in-situ and invasive component of IDC-DCIS

Risk factors for clinical outcomes

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