Numerical and/or structural centrosome amplification (CA) is a hallmark of cancers that is often associated with the aberrant tumor karyotypes and poor clinical outcomes. Mechanistically, CA ...compromises mitotic fidelity and leads to chromosome instability (CIN), which underlies tumor initiation and progression. Recent technological advances in microscopy and image analysis platforms have enabled better-than-ever detection and quantification of centrosomal aberrancies in cancer. Numerous studies have thenceforth correlated the presence and the degree of CA with indicators of poor prognosis such as higher tumor grade and ability to recur and metastasize. We have pioneered a novel semi-automated pipeline that integrates immunofluorescence confocal microscopy with digital image analysis to yield a quantitative centrosome amplification score (CAS), which is a summation of the severity and frequency of structural and numerical centrosome aberrations in tumor samples. Recent studies in breast cancer show that CA increases across the disease progression continuum, while normal breast tissue exhibited the lowest CA, followed by cancer-adjacent apparently normal, ductal carcinoma
in situ
and invasive tumors, which showed the highest CA. This finding strengthens the notion that CA could be evolutionarily favored and can promote tumor progression and metastasis. In this review, we discuss the prevalence, extent, and severity of CA in various solid cancer types, the utility of quantifying amplified centrosomes as an independent prognostic marker. We also highlight the clinical feasibility of a CA-based risk score for predicting recurrence, metastasis, and overall prognosis in patients with solid cancers.
The response of human epidermal growth factor receptor2 (HER2)- positive breast cancer (BC) patients to anti-HER2 targeted therapy is significant. However, the response is not uniform and a ...proportion of HER2-positive patients do not respond. This study aims to identify predictors of response in the neoadjuvant treatment and to assess the discordance rate of HER2 status between pre- and post-treatment specimens in HER2-positive BC patients. The study group comprised 500 BC patients treated with neoadjuvant chemotherapy (NACT) and/or neoadjuvant anti-HER2 therapy and surgery who had tumours that were 3+ or 2+ with HER2 immunohistochemistry (IHC). HER2 IHC 2+ tumours were classified into five groups by fluorescence in situ hybridisation (FISH) according to the 2018 ASCO/CAP guidelines of which Groups 1, 2 and 3 were considered HER2 amplified. Pathological complete response (pCR) was more frequent in HER2 IHC 3+ tumours than in HER2 IHC 2+/HER2 amplified tumours, when either in receipt of NACT alone (38% versus 13%; p = 0.22) or neoadjuvant anti-HER2 therapy (52% versus 20%; p < 0.001). Multivariate logistic regression analysis showed that HER2 IHC 3+ and histological grade 3 were independent predictors of pCR following neoadjuvant anti-HER2 therapy. In the HER2 IHC 2+/HER2 amplified tumours or ASCO/CAP FISH Group 1 alone, ER-negativity was an independent predictor of pCR following NACT and/or neoadjuvant anti-HER2 therapy. In the current study, 22% of HER2-positive tumours became HER2-negative by IHC and FISH following neoadjuvant treatment, the majority (74%) HER2 IHC 2+/HER2 amplified tumours. Repeat HER2 testing after neoadjuvant treatment should therefore be considered.
E-cadherin is a tumor suppressor gene in invasive lobular breast cancer. However, a proportion of high-grade ductal carcinoma shows reduced/loss of E-cadherin. In this study, we assessed the ...underlying mechanisms and molecular implications of E-cadherin loss in invasive ductal carcinoma. This study used large, well-characterized cohorts of early-stage breast cancer-evaluated E-cadherin expression via various platforms including immunohistochemistry, microarray analysis using Illumina HT-12 v3, copy number analysis using Affymetrix SNP 6.0 arrays, and next-generation sequencing for differential gene expression. Our results showed 27% of high-grade invasive ductal carcinoma showed reduced/loss of E-cadherin membranous expression. CDH1 copy number loss was in 21% of invasive ductal carcinoma, which also showed low CDH1 mRNA expression (p = 0.003). CDH1 copy number was associated with copy number loss of TP53, ATM, BRCA1, and BRCA2 (p < 0.001). Seventy-nine percent of invasive ductal carcinoma with reduced CDH1 mRNA expression showed elevated expression of E-cadherin transcription suppressors TWIST2, ZEB2, NFKB1, LLGL2, CTNNB1 (p < 0.01). Reduced/loss E-cadherin expression was associated with differential expression of 2143 genes including those regulating Wnt (FZD2, GNG5, HLTF, WNT2, and CER1) and PIK3-AKT (FGFR2, GNF5, GNGT1, IFNA17, and IGF1) signaling pathways. Interestingly, key genes differentially expressed between invasive lobular carcinoma and invasive ductal tumors did not show association with E-cadherin loss in invasive ductal carcinoma. We conclude that E-cadherin loss in invasive ductal carcinoma is likely a consequence of genomic instability occurring during carcinogenesis. Potential novel regulators controlling E-cadherin expression in invasive ductal carcinoma warrant further investigation.
Previous studies have reported up to 50% of ductal carcinoma in situ (DCIS), is HER2 positive, but the frequency of HER2-positive invasive breast cancer (IBC) is lower. The aim of this study is to ...characterise HER2 status in DCIS and assess its prognostic value.
HER2 status was evaluated in a large series of DCIS (n = 868), including pure DCIS and DCIS associated with IBC, prepared as tissue microarrays (TMAs). HER2 status was assessed using immunohistochemistry (IHC) and chromogenic in situ hybridisation (CISH).
In pure DCIS, HER2 protein was over-expressed in 9% of DCIS (3+), whereas 15% were HER2 equivocal (2+). Using CISH, the final HER2 status was positive in 20%. In mixed DCIS, HER2 amplification of the DCIS component was detected in 15% with amplification in the invasive component of only 12%. HER2-positive DCIS was associated with features of aggressiveness (p < 0.0001) and more frequent local recurrence (p = 0.03). On multivariate analysis, combined HER2+/Ki67+ profile was an independent predictor of local recurrence (p = 0.006).
The frequency of HER2 positivity in DCIS is comparable to IBC- and HER2-positive DCIS is associated with features of poor prognosis. The majority of HER2 over-expression in DCIS is driven by gene amplification.
Using digitalized whole slide images (WSI) in routine histopathology practice is a revolutionary technology. This study aims to assess the clinical impacts of WSI quality and representation of the ...corresponding glass slides. 40,160 breast WSIs were examined and compared with their corresponding glass slides. The presence, frequency, location, tissue type, and the clinical impacts of missing tissue were assessed. Scanning time, type of the specimens, time to WSIs implementation, and quality control (QC) measures were also considered. The frequency of missing tissue ranged from 2% to 19%. The area size of the missed tissue ranged from 1–70%. In most cases (>75%), the missing tissue area size was <10% and peripherally located. In all cases the missed tissue was fat with or without small entrapped normal breast parenchyma. No missing tissue was identified in WSIs of the core biopsy specimens. QC measures improved images quality and reduced WSI failure rates by seven-fold. A negative linear correlation between the frequency of missing tissue and both the scanning time and the image file size was observed (p < 0.05). None of the WSI with missing tissues resulted in a change in the final diagnosis. Missing tissue on breast WSI is observed but with variable frequency and little diagnostic consequence. Balancing between WSI quality and scanning time/image file size should be considered and pathology laboratories should undertake their own assessments of risk and provide the relevant mitigations with the appropriate level of caution.
The role of different subtypes of tumour infiltrating lymphocytes (TILs) in breast ductal carcinoma in situ (DCIS) is still poorly defined. This study aimed to assess the prognostic significance of B ...and T lymphocytes and immune checkpoint proteins expression in DCIS.
A well characterised DCIS cohort (n = 700) with long-term follow-up comprising pure DCIS (n = 508) and DCIS mixed with invasive carcinoma (IBC; n = 192) were stained immunohistochemically for CD20, CD3, CD4, CD8, FOXP3, PD1 and PDL1. Copy number variation and TP53 mutation status were assessed in a subset of cases (n = 58).
CD3+ lymphocytes were the predominant cell subtype in the pure DCIS cohort, while FOXP3 showed the lowest levels. PDL1 expression was mainly seen in the stromal TILs. Higher abundance of TILs subtypes was associated with higher tumour grade, hormone receptor negativity and HER2 positivity. Mutant TP53 variants were associated with higher levels of stromal CD3+, CD4+ and FOXP3+ cells. DCIS coexisting with invasive carcinoma harboured denser stromal infiltrates of all immune cells and checkpoint proteins apart from CD4+ cells. Stromal PD1 was the most differentially expressed protein between DCIS and invasive carcinoma (Z = 5.8, p < 0.0001). Dense TILs, stromal FOXP3 and PDL1 were poor prognostic factors for DCIS recurrence, while dense TILs were independently associated with poor outcome for all recurrences (HR = 7.0; p = 0.024), and invasive recurrence (HR = 2.1; p = 0.029).
Immunosuppressive proteins are potential markers for high risk DCIS and disease progression. Different stromal and intratumoural lymphocyte composition between pure DCIS, DCIS associated with IBC and invasive carcinoma play a potential role in their prognostic significance and related to the underlying genomic instability. Assessment of overall TILs provides a promising tool for evaluation of the DCIS immune microenvironment.
Aims
Oncotype DX recurrence score (RS) is a clinically validated assay, which predicts the likelihood of disease recurrence in oestrogen receptor‐positive/HER2‐negative (ER+/HER2−) breast cancer ...(BC). In this study we aimed to compare the performance of Oncotype DX against the conventional clinicopathological parameters using a large BC cohort diagnosed in a single institution.
Methods and results
A cohort (n = 430) of ER+/HER2− BC patients who were diagnosed at the Nottingham University Hospitals NHS Trust and had Oncotype DX testing was included. Correlation with the clinicopathological and other biomarkers, including the proliferation index, was analysed. The median Oncotype DX RS was 17.5 (range = 0–69). There was a significant association between high RS and grade 3 tumours. No grade 1 BC or grade 2 tumours with mitosis score 1 showed high RS. Low RS was significantly associated with special tumour types where none of the patients with classical lobular or tubular carcinomas had a high RS. There was an inverse association between RS and levels of ER and progesterone receptor (PR) expression and a positive linear correlation with Ki67 labelling index. Notably, six patients who developed recurrence had an intermediate RS; however, four of these six cases (67%) were identified as high‐risk disease when the conventional clinical and molecular parameters were considered.
Conclusion
Oncotype DX RS is correlated strongly with the conventional clinicopathological parameters in BC. Some tumour features such as tumour grade, type, PR status and Ki67 index can be used as surrogate markers in certain scenarios.
Based on our findings, there are some patients who are low risk, such as patients with low tumour grade, special tumour types (classical lobular carcinoma, tubular, or invasive cribriform), low Ki67 <10% with high progesterone receptor (PR) >10%. Those patients should be treated as low‐risk patients.
Background
Lymphovascular invasion (LVI) is a prognostic factor in early-stage invasive breast cancer (BC). Through bioinformatics, data analyses of multiple BC cohorts revealed the positive ...association between interferon-stimulated gene 15 (
ISG15
) LVI status. Thus, we explored the prognostic significance of ISG15 in BC.
Methods
The prognostic significance of
ISG15
mRNA was assessed in METABRIC (
n
= 1980), TCGA (
n
= 854) and Kaplan–Meier Plotter (
n
= 3951). ISG15 protein was evaluated using immunohistochemistry (
n
= 859) in early-stage invasive BC patients with long-term follow-up. The associations between ISG15 expression and clinicopathological features, expression of immune cell markers and patient outcome data were evaluated.
Results
High mRNA and protein ISG15 expression were associated with LVI, higher histological grade, larger tumour size, hormonal receptor negativity, HER2 positivity, p53 and Ki67. High ISG15 protein expression was associated with HER2-enriched BC subtypes and immune markers (CD8, FOXP3 and CD68). High
ISG15
mRNA and ISG15 expressions were associated with poor patient outcome. Cox proportional multivariate analysis revealed that the elevated ISG15 expression was an independent prognostic factor of shorter BC-specific survival.
Conclusion
This study provides evidence for the role of ISG15 in LVI development and BC prognosis. Further functional studies in BC are warranted to evaluate the therapeutic potential of ISG15.
Targeting PARP1 Poly(ADP-Ribose) Polymerase 1 for synthetic lethality is a new strategy for BRCA germ-line mutated or platinum sensitive ovarian cancers. However, not all patients respond due to ...intrinsic or acquired resistance to PARP1 inhibitor. Development of alternative synthetic lethality approaches is a high priority. DNA polymerase β (Polβ), a critical player in base excision repair (BER), interacts with PARP1 during DNA repair. Here we show that polβ deficiency is a predictor of platinum sensitivity in human ovarian tumours. Polβ depletion not only increased platinum sensitivity but also reduced invasion, migration and impaired EMT (epithelial to mesenchymal transition) of ovarian cancer cells. Polβ small molecular inhibitors (Pamoic acid and NSC666719) were selectively toxic to BRCA2 deficient cells and associated with double-strand breaks (DSB) accumulation, cell cycle arrest and increased apoptosis. Interestingly, PARG Poly(ADP-Ribose) Glycohydrolase inhibitor (PDD00017273) but not PARP1 inhibitor (Olaparib) was synthetically lethal in polβ deficient cells. Selective toxicity to PDD00017273 was associated with poly (ADP-ribose) accumulation, reduced nicotinamide adenine dinucleotide (NAD
) level, DSB accumulation, cell cycle arrest and increased apoptosis. In human tumours, polβ-PARG co-expression adversely impacted survival in patients. Our data provide evidence that polβ targeting is a novel strategy and warrants further pharmaceutical development in epithelial ovarian cancers.
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