Circulating tumor DNA (ctDNA) is a component of cell-free DNA (cfDNA) that is shed by malignant tumors into the bloodstream and other bodily fluids. ctDNA can comprise up to 10% of a patient's cfDNA ...depending on their tumor type and burden. The short half-life of ctDNA ensures that its detection captures tumor burden in real-time and offers a non-invasive method of repeatedly evaluating the genomic profile of a patient's tumor. A challenge in ctDNA detection includes clonal hematopoiesis of indeterminate potential (CHIP), which can be distinguished from tumor variants using a paired whole-blood control. Most assays for ctDNA quantification rely on measurements of somatic variant allele frequency (VAF), which is a mutation-dependent method. Patients with certain types of solid tumors, including colorectal cancer (CRC), can have levels of cfDNA 50 times higher than healthy patients. ctDNA undergoes a precipitous drop shortly after tumor resection and therapy, and rising levels can foreshadow radiologic recurrence on the order of months. The amount of tumor bulk required for ctDNA detection is lower than that for computed tomography (CT) scan detection, with ctDNA detection preceding radiologic recurrence in many cases. cfDNA/ctDNA can be used for tumor molecular profiling to identify resistance mutations when tumor biopsy is not available, to detect minimal residual disease (MRD), to monitor therapy response, and for the detection of tumor relapse. Although ctDNA is not yet implemented in clinical practice, studies are ongoing to define the appropriate way to use it as a tool in the clinic. In this review article, we examine the general aspects of ctDNA, its status as a biomarker, and its role in the management of early (II-III) and late (IV; mCRC) stage colorectal cancer (CRC).
Circulating tumor cells (CTCs) are tumor cells shed from either primary tumors or its metastases that circulate in the peripheral blood of patients with metastatic cancers. The molecular ...characterization of the CTCs is critical to identifying the key drivers of cancer metastasis and devising therapeutic approaches. However, the molecular characterization of CTCs is difficult to achieve because their isolation is a major technological challenge.
CTCs from two triple negative breast cancer patients were enriched using CellSearch and single cells selected by DEPArray™. A TP53 R110 fs*13 mutation identified by next generation sequencing in the breast and chest skin biopsies of both patients was studied in single CTCs.
From 6 single CTC isolated from one patient, 1 CTC had TP53 R110 delC, 1 CTC showed the TP53 R110 delG mutation, and the remaining 4 single CTCs showed the wild type p53 sequence; a pool of 14 CTCs isolated from the same patient also showed TP53 R110 delC mutation. In the tumor breast tissue of this patient, only the TP53 R110 delG mutation was detected. In the second patient a TP53 R110 delC mutation was detected in the chest wall skin biopsy; from the peripheral blood of this patient, 5 single CTC and 6 clusters of 2 to 6 CTCs were isolated; 3 of the 5 single CTCs showed the TP53 R110 delC mutation and 2 CTCs showed the wild type TP53 allele; from the clusters, 5 showed the TP53 R110 delC mutation, and 1 cluster the wild type TP53 allele. Single white blood cells isolated as controls from both patients only showed the wild type TP53 allele.
We are able to isolate uncontaminated CTCs and achieve single cell molecular analysis. Our studies showed the presence of different CTC sub-clones in patients with metastatic breast cancer. Some CTCs had the same TP53 mutation as their matching tumor samples although others showed either a different TP53 mutation or the wild type allele. Our results indicate that CTCs could represent a non-invasive source of cancer cells from which to determine genetic markers of the disease progression and potential therapeutic targets.
Inflammatory breast cancer (IBC) is a rare but aggressive carcinoma characterized by severe erythema and edema of the breast, with many patients presenting in advanced metastatic disease. The ..."inflammatory" nature is not due to classic immune-mediated inflammation, but instead results from tumor-mediated blockage of dermal lymphatic ducts. Previous work has shown that expression of PD-L1 on tumor cells can suppress T cell activation in triple-negative (TN) non-IBC breast cancer. In the present work, we investigated immune parameters in peripheral blood of metastatic IBC patients to determine whether cellular components of the immune system are altered, thereby contributing to pathogenesis of the disease. These immune parameters were also compared to PD-1 and PD-L1 expression in IBC tumor biopsies.
Flow cytometry-based immune phenotyping was performed using fresh peripheral blood from 14 stage IV IBC patients and compared to 11 healthy age-similar control women. Immunohistochemistry for CD20, CD3, PD-1, and PD-L1 was performed on tumor biopsies of these metastatic IBC patients.
IBC patients with Stage IV disease had lymphopenia with significant reductions in circulating T, B, and NK cells. Reductions were observed in all subsets of CD4
T cells, whereas reductions in CD8
T cells were more concentrated in memory subsets. Immature cytokine-producing CD56
NK cells expressed higher levels of FcγRIIIa and cytolytic granule components, suggesting accelerated maturation to cytolytic CD56
cells. Immunohistochemical analysis of tumor biopsies demonstrated moderate to high expression of PD-1 in 18.2% of patients and of PD-L1 in 36.4% of patients. Interestingly, a positive correlation was observed between co-expression levels of PD-L1 and PD-1 in tumor biopsies, and higher expression of PD-L1 in tumor biopsies correlated with higher expression of cytolytic granule components in blood CD4
T cells and CD56
NK cells, and higher numbers of CD8
effector memory T cells in peripheral blood. PD-1 expression in tumor also correlated with increased infiltration of CD20
B cells in the tumor.
Our results suggest that while lymphocyte populations are severely compromised in stage IV IBC patients, an immune response toward the tumor had occurred in some patients, providing biological rationale to evaluate PD-1/PD-L1 immunotherapies for IBC.
A modified version of the PGDx elioTM Plasma Resolve assay was validated as a laboratory-developed test (LDT) for clinical use in the Molecular Diagnostics Laboratory at Fox Chase Cancer Center. The ...test detects single nucleotide variants (SNVs) and small insertions and deletions (indels) in 33 target genes using fragmented genomic DNA extracted from plasma. The analytical performance of this assay was assessed with reference standard DNA and 29 samples from cancer patients and detected 66 SNVs and 23 indels. Using 50 ng of input DNA, the sensitivity was 95.5% to detect SNVs at 0.5% allele frequency, and the specificity was 92.3%. The sensitivity to detect indels at 1% allele frequency was 70.4%. A cutoff of 0.25% variant allele frequency (VAF) was set up for diagnostic reporting. An inter-laboratory study of concordance with an orthologous test resulted in a positive percent agreement (PPA) of 91.7%.
Purpose
The molecular characterization of circulating tumor cells (CTCs) is critical to identify the key drivers of cancer metastasis and devising therapeutic approaches, particularly for ...inflammatory breast cancer (IBC) which is usually diagnosed at advance stages and progresses rapidly.
Methods
Genomic alterations in tumor tissue samples were studied using Foundation One™. Single CTCs were isolated using CellSearch followed by single-cell isolation by DEPArray™. Samples with 20 or more CTCs were chosen to isolate single CTCs using the DEPArray™.
Results
Genomic alterations were studied in primary tumor or metastatic sites from 32 IBC patients. Genes with high-frequency mutations were as follows: TP53 (69%), RB1 (16%), PIK3CA (13%), and also ErbB2 (3%). At least once during treatment, CTCs were detected in 26 patients with metastatic IBC, in two patients with locally advanced IBC, and four patients had no detectable CTCs. Per 7.5 mL of blood, fifteen patients (47%) had ≥20 CTCs and six of them were chosen at random to isolate single CTCs. These cells were tested for the presence of TP53, RB1, PIK3CA, and/or ErbB2 mutations previously found in matching tissue biopsies. The isolated CTCs showed the same mutations as primary or metastatic tumor samples. Intra-patient CTC heterogeneity was found by the presence of different CTC subclones, with some CTCs harboring different combinations of mutated and wild-type genes.
Conclusions
Our results indicate that CTCs could represent a non-invasive source of cancer cells from which to determine genetic markers as the disease progresses and identify potential therapeutic targets in IBC patients.
We studied genomic alterations in 19 inflammatory breast cancer (IBC) patients with advanced disease using samples of tissue and paired blood serum or plasma (cell-free DNA, cfDNA) by targeted next ...generation sequencing (NGS). At diagnosis, the disease was triple negative (TN) in eleven patients (57.8%), ER+ Her2- IBC in six patients (31.6%), ER+ Her2+ IBC in one patient (5.3%), and ER- Her2+ IBC in one other patient (5.3%). Pathogenic or likely pathogenic variants were frequently detected in
(47.3%),
(26.3%),
(26.3%),
(10.5%),
(10.5%),
(10.5%) and
(10.5%); other affected genes included
,
,
,
,
,
,
,
,
,
,
and
In 15 of the 19 patients in which tissue and paired blood were collected at the same time point, 80% of the variants detected in tissue were also detected in the paired cfDNA. Higher concordance between tissue and cfDNA was found for variants with higher allele fraction in tissue (AF
≥ 5%). Furthermore, 86% of the variants detected in cfDNA were also detected in paired tissue. Our study suggests that the genetic profile measured in blood cfDNA is complementary to that of tumor tissue in IBC patients.
Analogues structurally related to anaplastic lymphoma kinase (ALK) inhibitor 1 were optimized for metabolic stability. The results from this endeavor not only led to improved metabolic stability, ...pharmacokinetic parameters, and in vitro activity against clinically derived resistance mutations but also led to the incorporation of activity for focal adhesion kinase (FAK). FAK activation, via amplification and/or overexpression, is characteristic of multiple invasive solid tumors and metastasis. The discovery of the clinical stage, dual FAK/ALK inhibitor 27b, including details surrounding SAR, in vitro/in vivo pharmacology, and pharmacokinetics, is reported herein.
Inflammatory breast cancer (IBC) is a distinct clinicopathologic entity that carries a worse prognosis relative to non-IBC breast cancer even when matched for standard biomarkers (ER/PR/HER2). The ...objective of this study was to identify opportunities for benefit from targeted therapy, which are not currently identifiable in the standard workup for advanced breast cancer. Comprehensive genomic profiling on 53 IBC formalin-fixed paraffin-embedded specimens (mean, 800× + coverage) using the hybrid capture-based FoundationOne assay. Academic and community oncology clinics. From a series of 2208 clinical cases of advanced/refractory invasive breast cancers, 53 cases with IBC were identified. The presence of clinically relevant genomic alterations (CRGA) in IBC and responses to targeted therapies. CRGA were defined as genomic alterations (GA) associated with on label targeted therapies and targeted therapies in mechanism-driven clinical trials. For the 44 IBCs with available biomarker data, 19 (39 %) were ER−/PR−/HER2− (triple-negative breast cancer, TNBC). For patients in which the clinical HER2 status was known, 11 (25 %) were HER2+ with complete (100 %) concordance with
ERBB2
(
HER2
) amplification detected by the CGP assay. The 53 sequenced IBC cases harbored a total of 266 GA with an average of 5.0 GA/tumor (range 1–15). At least one alteration associated with an FDA approved therapy or clinical trial was identified in 51/53 (96 %) of cases with an average of 2.6 CRGA/case. The most frequently altered genes were
TP53
(62 %),
MYC
(32 %),
PIK3CA
(28 %),
ERBB2
(26 %),
FGFR1
(17 %),
BRCA2
(15 %), and
PTEN
(15 %). In the TNBC subset of IBC, 8/19 (42 %) showed
MYC
amplification (median copy number 8X, range 7–20) as compared to 9/32 (28 %) in non-TNBC IBC (median copy number 7X, range 6–21). Comprehensive genomic profiling uncovered a high frequency of GA in IBC with 96 % of cases harboring at least 1 CRGA. The clinical benefit of selected targeted therapies in individual IBC cases suggests that a further study of CGP in IBC is warranted.
It has been suggested that xenoestrogens, a group of agents termed endocrine disruptors, may contribute to the development of hormone-dependent cancers, such as breast and endometrial cancers. We ...previously demonstrated that the xenoestrogen, bisphenol A (BPA), was able to induce the transformation in vitro of human breast epithelial cells. The normal-like human breast epithelial cell line, MCF-10F, formed tubules in collagen (3-D cultures), although after treatment with BPA (10-5 M and 10-6 M BPA) the cells produced less tubules (73% and 80%, respectively) and some spherical masses (27% and 20%, respectively). In the present study, expression and DNA methylation analyses were performed in these cells after exposure to BPA. These cells showed an increased expression of BRCA1, BRCA2, BARD1, CtIP, RAD51 and BRCC3, all of which are genes involved in DNA repair, as well as the downregulation of PDCD5 and BCL2L11 (BIM), both of which are involved in apoptosis. Furthermore, DNA methylation analysis showed that the BPA exposure induced the hypermethylation of BCL2L11, PARD6G, FOXP1 and SFRS11, as well as the hypomethylation of NUP98 and CtIP (RBBP8). Our results indicate that normal human breast epithelial cells exposed to BPA have increased expressions of genes involved in DNA repair in order to overcome the DNA damage induced by this chemical. These results suggest that the breast tissue of women with BRCA1 or BRCA2 mutations could be more susceptible to the effects of BPA.
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