A Practical Approach for Targeting Structural Variants Genome-wide in Plasma Cell-free DNA.

Interrogating plasma cell-free DNA (cfDNA) to detect cancer offers promise; however, no current tests scan structural variants (SVs) throughout the genome. Here, we report a simple molecular workflow to enrich a tumorigenic SV (DNA palindromes/fold-back inversions) that often demarcates genomic amplification and its feasibility for cancer detection by combining low-throughput next-generation sequencing with automated machine learning (Genome-wide Analysis of Palindrome Formation, GAPF-seq). Tumor DNA signal manifested as skewed chromosomal distributions of high-coverage 1-kb bins (HCBs), differentiating 39 matched breast tumor DNA from normal DNA with an average AUC of 0.9819. In a proof-of-concept liquid biopsy study, cfDNA from 0.5 mL plasma from prostate cancer patients was sufficient for binary classification against matched buffy coat DNA with an average AUC of 0.965. HCBs on the X chromosome emerged as a determinant feature and were associated with AR amplification. GAPF-seq could generate unique cancer-specific SV profiles in an agnostic liquid biopsy setting.

A Practical Approach for Targeting Structural Variants Genome-wide in Plasma Cell-free DNA.

Plasma cell-free DNA (cfDNA) is a promising source of gene mutations for cancer detection by liquid biopsy. However, no current tests interrogate chromosomal structural variants (SVs) genome-wide. Here, we report a simple molecular and sequencing workflow called Genome-wide Analysis of Palindrome Formation (GAPF-seq) to probe DNA palindromes, a type of SV that often demarcates gene amplification. With low-throughput next-generation sequencing and automated machine learning, tumor DNA showed skewed chromosomal distributions of high-coverage 1-kb bins (HCBs), which differentiated 39 breast tumors from matched normal DNA with an average Area Under the Curve (AUC) of 0.9819. A proof-of-concept liquid biopsy study using cfDNA from prostate cancer patients and healthy individuals yielded an average AUC of 0.965. HCBs on the X chromosome emerged as a determinant feature and were associated with androgen receptor gene amplification. As a novel agnostic liquid biopsy approach, GAPF-seq could fill the technological gap offering unique cancer-specific SV profiles.

The applications of plasma cell-free DNA in cancer detection: Implications in the management of breast cancer patients.

Liquid biopsy probes DNA, RNA, and proteins in body fluids for cancer detection and is one of the most rapidly developing areas in oncology. Tumor-derived DNA (circulating tumor DNA, ctDNA) in the context of cell-free DNA (cfDNA) in blood has been the main target for its potential utilities in cancer detection. Liquid biopsy can report tumor burden in real-time without invasive interventions, and would be feasible for screening tumor types that lack standard-of-care screening approaches. Two major approaches to interrogating ctDNA are genetic mutation and DNA methylation profiling. Mutation profiling can identify tumor driver mutations and guide precision therapy. Targeted genomic profiling of DNA methylation has become the main approach for cancer screening in the general population. Here we review the recent technological development and ongoing efforts in clinical applications. For clinical applications, we focus on breast cancer, in which subtype-specific biology demarcates the applications of ctDNA.

The fragility of a structurally diverse duplication block triggers recurrent genomic amplification.

The human genome contains hundreds of large, structurally diverse blocks that are insufficiently represented in the reference genome and are thus not amenable to genomic analyses. Structural diversity in the human population suggests that these blocks are unstable in the germline; however, whether or not these blocks are also unstable in the cancer genome remains elusive. Here we report that the 500 kb block called KRTAP_region_1 (KRTAP-1) on 17q12-21 recurrently demarcates the amplicon of the ERBB2 (HER2) oncogene in breast tumors. KRTAP-1 carries numerous tandemly-duplicated segments that exhibit diversity within the human population. We evaluated the fragility of the block by cytogenetically measuring the distances between the flanking regions and found that spontaneous distance outliers (i.e DNA breaks) appear more frequently at KRTAP-1 than at the representative common fragile site (CFS) FRA16D. Unlike CFSs, KRTAP-1 is not sensitive to aphidicolin. The exonuclease activity of DNA repair protein Mre11 protects KRTAP-1 from breaks, whereas CtIP does not. Breaks at KRTAP-1 lead to the palindromic duplication of the ERBB2 locus and trigger Breakage-Fusion-Bridge cycles. Our results indicate that an insufficiently investigated area of the human genome is fragile and could play a crucial role in cancer genome evolution.

Plasminogen activator inhibitor-2 (PAI-2) overexpression supports bladder cancer development in PAI-1 knockout mice in N-butyl-N- (4-hydroxybutyl)-nitrosamine- induced bladder cancer mouse model.

BACKGROUND: Accumulating evidence suggests that plasminogen activator inhibitor-1 (PAI-1) plays an important role in bladder tumorigenesis by regulating cell cycle. However, it remains unclear whether and how inhibition of PAI-1 suppresses bladder tumorigenesis. METHODS: To elucidate the therapeutic effect of PAI-1 inhibition, we tested its tumorigenicity in PAI-1 knockout (KO) mice exposed to a known bladder carcinogen. RESULTS: PAI-1 deficiency did not inhibit carcinogen-induced bladder cancer in mice although carcinogen-exposed wild type mice significantly increased PAI-1 levels in bladder tissue, plasma and urine. We found that PAI-1 KO mice exposed to carcinogen tended to upregulate protein C inhibitor (PAI-3), urokinase-type plasminogen activator (uPA) and tissue-type PA (tPA), and significantly increased PAI-2, suggesting a potential compensatory function of these molecules when PAI-1 is abrogated. Subsequent studies employing gene expression microarray using mouse bladder tissues followed by post hoc bioinformatics analysis and validation experiments by qPCR and IHC demonstrated that SERPING1 is further downregulated in PAI-1 KO mice exposed to BBN, suggesting that SERPING1 as a potential missing factor that regulate PAI-2 overexpression (compensation pathway). CONCLUSIONS: These results indicate that serpin compensation pathway, specifically PAI-2 overexpression in this model, supports bladder cancer development when oncoprotein PAI-1 is deleted. Further investigations into PAI-1 are necessary in order to identify true potential targets for bladder cancer therapy.

Diagnostic significance of intratumoral CD8+ tumor-infiltrating lymphocytes in medullary carcinoma.

Invasive ductal carcinomas of breast with marked stromal lymphocytic infiltration have come to be classified as lymphocyte-predominant breast cancer (LPBC) because it obtains high pathological complete response rates with neoadjuvant chemotherapy. Medullary carcinoma (MC), which is independent from LPBC, is a rare histological subtype of invasive breast carcinoma accompanied by abundant lymphoplasmacytic infiltration as LPBC. Although MC shows marked cellular and structural atypia, it usually has a favorable outcome. It is occasionally difficult to distinguish MC from LPBC because both subtypes have nonspecific morphological features according to the present diagnostic criteria. Herein, we adopted multiplexed fluorescent immunohistochemistry to perform quantitative and simultaneous analyses of tumor-infiltrating lymphocytes (TILs) considering their spatial distribution and examined focal immune reaction differences between MC and LPBC. We found that CD8+ TILs are predominant in the intratumoral region, whereas CD4+ TILs are less common in MC. In non-luminal-type cancers, the numbers of stromal and intratumoral CD8+ TILs were significantly higher in MC than in LPBC. Stratified analyses by CD4+ TIL subsets showed robust infiltration of intratumoral CD8+ TILs in non-luminal-type MC even in suppressive environments, such a low T helper 1-to-regulatory T cell ratio. Our results suggest that extensive intratumoral CD8+ TIL infiltration might well be a promising biomarker for distinguishing MC from LPBC, especially in non-luminal-type cancers. Intratumoral CD8+ TILs and nonluminal intrinsic subtypes may serve as diagnostic characteristics allowing reliable histological criteria to be established for reproducibly diagnosing MC.

Menstrual cycle could affect Ki67 expression in estrogen receptor-positive breast cancer patients.

BACKGROUND: Ki67 is a potent prognostic marker for determining systemic treatment of patients with hormone receptor-positive breast cancer. However, evaluation of Ki67 expression can be difficult, due mostly to its heterogeneity. The Ki67 expression level, which indicates that a cell is undergoing division (cell cycle), rises when proliferation activity increases. Thus, Ki67 expression might be affected by hormonal stimuli. We hypothesised that Ki67 expression level might change during the menstrual cycle. We examined pairs of biopsy and surgical specimens from individual patients to evaluate this hypothesis. METHODS: First, the effects of estradiol on Ki67 expression in breast cancer cell lines were examined employing immunocytochemistry and Western blotting. Next, differences in Ki67 expression between biopsy and surgical specimens from 131 patients with estrogen receptor-positive tumours were retrospectively examined. RESULTS: In vitro experiments showed Ki67 expression in estrogen receptor-positive cancer cells to be dependent on estradiol stimulation. Ki67 expression was higher in biopsy samples collected in the luteal phase than in those from other phases. When biopsy and surgical samples were obtained at different times during the menstrual cycle in the same individual, there were differences in Ki67 expression between these samples. Those collected in the luteal phase showed higher Ki67 expression than samples obtained during other phases (p<0.01). CONCLUSIONS: Ki67 expression varied in the same patients according to menstrual cycle phase. Our results suggest that Ki67 expression in estrogen receptor-positive breast cancer should be carefully assessed bearing in mind the patient's menstrual cycle, since the interpretation of expression could affect treatment decisions.

Ki67 expression and the effect of neo-adjuvant chemotherapy on luminal HER2-negative breast cancer.

BACKGROUND: Patients with luminal HER2-negative tumours have a favourable prognosis. However, there is a subpopulation in which poorer outcomes are obtained with endocrine therapy alone. This subpopulation is considered to benefit from chemotherapy. However, the significance of chemotherapy for those with luminal tumours has decreased due to recent changes in treatment strategies. Thus, it is often difficult to determine whether we should recommend chemotherapy to such patients in clinical practice. We investigated Ki67 expression, as a means of predicting the responses of luminal HER2-negative breast cancer patients to neo-adjuvant chemotherapy (NAC), in order to identify a subpopulation that would benefit from these treatments. METHODS: We enrolled 114 luminal HER2-negative breast cancer patients undergoing surgery after NAC. Biomarkers were examined using biopsy specimens obtained prior to treatment, to avoid any chemotherapy-related effects. Chemotherapy effects were determined employing operative specimens and we defined pathological complete response (pCR) as invasive nest disappearance, based only on the primary breast tumour. We applied receiver operating characteristic curve analysis to data from our 114 patients, to investigate Ki67 expression as a predictor of pCR. RESULTS: The pCR rate was significantly higher for tumours with high Ki67 expression (p < 0.01) and all patients who obtained pCR remained recurrence-free during the median 58-month observation period. We identified 35% as the Ki67 cut-off value which distinguishes those with a pCR from other cases. Another dataset, comprised of 196 patients with a median 29-month observation period, was recruited for validation. Disease-free survival was found to be significantly (p < 0.01) lower in the patients with tumours in which Ki67 expression was higher than 35%. CONCLUSION: Our results raise the possibility of the luminal HER2-negative subpopulation with Ki67 expression higher than 35% benefiting from chemotherapy, as evidenced by improved survival.