Rose Marie Amini

Molecular diversity in ductal carcinoma in situ (DCIS) and early invasive breast cancer

Ductal carcinoma in situ (DCIS) is a non‐invasive form of breast cancer where cells restricted to the ducts exhibit an atypical phenotype. Some DCIS lesions are believed to rapidly transit to invasive ductal carcinomas (IDCs), while others remain unchanged. Existing classification systems for DCIS fail to identify those lesions that transit to IDC. We studied gene expression patterns of 31 pure DCIS, 36 pure invasive cancers and 42 cases of mixed diagnosis (invasive cancer with an in situ component) using Agilent Whole Human Genome Oligo Microarrays 44k. Six normal breast tissue samples were also included as controls. qRT‐PCR was used for validation. All DCIS and invasive samples could be classified into the “intrinsic” molecular subtypes defined for invasive breast cancer. Hierarchical clustering establishes that samples group by intrinsic subtype, and not by diagnosis. We observed heterogeneity in the transcriptomes among DCIS of high histological grade and identified a distinct subgroup containing seven of the 31 DCIS samples with gene expression characteristics more similar to advanced tumours. A set of genes independent of grade, ER‐status and HER2‐status was identified by logistic regression that univariately classified a sample as belonging to this distinct DCIS subgroup. qRT‐PCR of single markers clearly separated this DCIS subgroup from the other DCIS, and contains samples from several histopathological and intrinsic molecular subtypes. The genes that differentiate between these two types of DCIS suggest several processes related to the re‐organisation of the microenvironment. This raises interesting possibilities for identification of DCIS lesions both with and without invasive characteristics, which potentially could be used in clinical assessment of a womans risk of progression, and lead to improved management that would avoid the current over‐ and under‐treatment of patients.

Molecular subtypes in ductal carcinoma in situ of the breast and their relation to prognosis: a population-based cohort study

BackgroundDifferent molecular subtypes of breast cancer have been identified based on gene expression profiling. Treatment suggestions based on an approximation of these subtypes by immunohistochemical criteria have been published by the St Gallen international expert consensus panel. Ductal carcinoma in situ (DCIS) can be classified into the same molecular subtypes. Our aim was to study the relation between these newly defined subtypes and prognosis in DCIS.MethodsTMA including 458 women from a population-based cohort with DCIS diagnosed 1986–2004 was used. Stainings for ER, PR, HER2 and Ki67 were used to classify the surrogate molecular subtypes according to the St Gallen criteria from 2011. The associations with prognosis were examined using Kaplan-Meier analyses and Cox proportional hazards regression models.ResultsSurrogate molecular subtyping could be done in 381 cases. Mean follow up was 164xa0months. Of the classified DCIS 186 were Luminal A (48.8%), 33 Luminal B/HER2- (8.7%), 74 Luminal B/HER2+ (17.4%), 61 HER2+/ER- (16.0%) and 27 Triple Negative (7.1%). One hundred and two women had a local recurrence of which 58 were invasive. Twenty-two women had generalised disease, 8 without a prior local recurrence. We could not find a prognostic significance of the molecular subtypes other than a higher risk of developing breast cancer after more than 10xa0years of follow-up among women with a Triple Negative DCIS (OR 3.2; 95% CI 1.1-9.8).ConclusionsThe results from this large population-based cohort, with long-term follow up failed to demonstrate a prognostic value for the surrogate molecular subtyping of DCIS using the St Gallen criteria up to ten years after diagnosis. More than ten years after diagnosis Triple Negative DCIS had an elevated risk of recurrence.

Role of microRNAs and microRNA machinery in the pathogenesis of diffuse large B-cell lymphoma

Deregulation of microRNA (miRNA) expression has been documented in diffuse large B-cell lymphoma (DLBCL). However, the impact of miRNAs and their machinery in DLBCL is not fully determined. Here, we assessed the role of miRNA expression and their processing genes in DLBCL development. Using microarray and RT-qPCR approaches, we quantified global miRNAs and core components of miRNA-processing genes expression in 75 DLBCLs (56 de novo and 19 transformed) and 10 lymph nodes (LN). Differential miRNA signatures were identified between DLBCLs and LNs, or between the de novo and transformed DLBCLs. We also identified subsets of miRNAs associated with germinal center B-cell phenotype, BCL6 and IRF4 expression, and clinical staging. In addition, we showed a significant over-expression of TARBP2 in de novo DLBCLs as compared with LNs, and decreased expression of DROSHA, DICER, TARBP2 and PACT in transformed as compared with de novo cases. Interestingly, cases with high TARBP2 and DROSHA expression had a poorer chemotherapy response. We further showed that TARBP2 can regulate miRNA-processing efficiency in DLBCLs, and its expression inhibition decreases cell growth and increases apoptosis in DLBCL cell lines. Our findings provide new insights for the understanding of miRNAs and its machinery in DLBCL.

Quality aspects of the tissue microarray technique in a population-based cohort with ductal carcinoma in situ of the breast

Aims:u2002 Tissue microarray (TMA) is an efficient technique for analysis of molecular markers. Prospectively collected samples have been reported to give excellent concordance between TMA data and corresponding whole‐sections. The aim was to evaluate the usefulness of TMA in a population‐based cohort of 213 women with ductal carcinoma in situ of the breast (DCIS).

The prognostic role of HER2 expression in ductal breast carcinoma in situ (DCIS); a population-based cohort study

BackgroundHER2 is a well-established prognostic and predictive factor in invasive breast cancer. The role of HER2 in ductal breast carcinoma in situ (DCIS) is debated and recent data have suggested that HER2 is mainly related to in situ recurrences. Our aim was to study HER2 as a prognostic factor in a large population based cohort of DCIS with long-term follow-up.MethodsAll 458 patients diagnosed with a primary DCIS 1986–2004 in two Swedish counties were included. Silver-enhanced in situ hybridisation (SISH) was used for detection of HER2 gene amplification and protein expression was assessed by immunohistochemistry (IHC) in tissue microarrays. HER2 positivity was defined as amplified HER2 gene and/or HER2 3+ by IHC. HER2 status in relation to new ipsilateral events (IBE) and Invasive Breast Cancer Recurrences, local or distant (IBCR) was assessed by Kaplan-Meier survival analyses and Cox proportional hazards regression models.ResultsPrimary DCIS was screening-detected in 75.5xa0% of cases. Breast conserving surgery (BCS) was performed in 78.6xa0% of whom 44.0xa0% received postoperative radiotherapy. No patients received adjuvant endocrine- or chemotherapy. The majority of DCIS could be HER2 classified (Nu2009=u2009420 (91.7xa0%)); 132 HER2 positive (31xa0%) and 288 HER2 negative (69xa0%)). HER2 positivity was related to large tumor size (Pu2009=u20090.002), high grade (Pu2009<u20090.001) and ER- and PR negativity (Pu2009<u20090.001 for both). During follow-up (mean 184xa0months), 106 IBCRs and 105 IBEs were identified among all 458 cases corresponding to 54 in situ and 51 invasive recurrences. Eighteen women died from breast cancer and another 114 had died from other causes. The risk of IBCR was statistically significantly lower subsequent to a HER2 positive DCIS compared to a HER2 negative DCIS, (Log-Rank Pu2009=u20090.03, (HR) 0.60 (95xa0% CI 0.38–0.94)). Remarkably, the curves did not separate until after 10 years. In ER-stratified analyses, HER2 positive DCIS was associated with lower risk of IBCR among women with ER negative DCIS (Log-Rank Pu2009=u20090.003), but not for women with ER positive DCIS.ConclusionsImproved prognostic tools for DCIS patients are warranted to tailor adjuvant therapy. Here, we demonstrate that HER2 positive disease in the primary DCIS is associated with lower risk of recurrent invasive breast cancer.

U-2932 : two clones in one cell line, a tool for the study of clonal evolution

Genetic heterogeneity is common in tumors, explicable by the development of subclones with distinct genetic and epigenetic alterations. We describe an in vitro model for cancer heterogeneity, comprising the diffuse large B-cell lymphoma cell line U-2932 which expresses two sets of cell surface markers representing twin populations flow-sorted by CD20 vs CD38 expression. U-2932 populations were traced to subclones of the original tumor with clone-specific immunoglobulin IgVH4–39 hypermutation patterns. BCL6 was overexpressed in one subpopulation (R1), MYC in the other (R2), both clones overexpressed BCL2. According to the combined results of immunoglobulin hypermutation and cytogenetic analysis, R1 and R2 derive from a mother clone with genomic BCL2 amplification, which acquired secondary rearrangements leading to the overexpression of BCL6 (R1) or MYC (R2). Some 200 genes were differentially expressed in R1/R2 microarrays including transcriptional targets of the aberrantly expressed oncogenes. Other genes were regulated by epigenetic means as shown by DNA methylation analysis. Ectopic expression of BCL6 in R2 variously modulated new candidate target genes, confirming dual silencing and activating functions. In summary, stable retention of genetically distinct subclones in U-2932 models tumor heterogeneity in vitro permitting functional analysis of oncogenes against a syngenic background.

Estrogen receptor β1 in diffuse large B-cell lymphoma growth and as a prognostic biomarker

Abstract Diffuse large B-cell lymphoma (DLBCL) shows a higher incidence in males versus females. Epidemiological studies have shown that female gender is a favorable prognostic factor, which may be explained by estrogens. Here we show that when grafting human DLBCL cells to immunocompromised mice, tumor growth in males is faster. When treating mice grafted with either germinal center or activated B-cell like DLBCL cells with the selective estrogen receptor β (ERβ) agonist diarylpropionitrile, tumor growth was significantly inhibited. Furthermore, nuclear ERβ1 expression analysis in primary DLBCL’s by immunohistochemistry revealed expression in 89% of the cases. Nuclear ERβ1 expression was in a univariate and multivariate analysis, an independent prognostic factor for adverse progression-free survival in Rituximab-chemotherapy treated DLBCL (pu2009=u20090.02 and pu2009=u20090.04, respectively). These results suggest that estrogen signaling through ERβ1 is an interesting future therapeutic target for treatment of DLBCL, and that ERβ1 expression can be used as a prognostic marker.

Chromosome 11q23 aberrations activating FOXR1 in B-cell lymphoma

Recurrent chromosome 11q23 abnormalities, including focal gains and losses have been described in mantle cell lymphoma, diffuse large B-cell lymphoma (DLBCL) and in a subset of high-grade B-cell lymphomas lacking MYC rearrangements. We describe a novel fusion of FOXR1 forkhead box gene, located at 11q23, with a neighboring gene in B-cell lymphoma. RNAseq and sequencing of cloned PCR products revealed fusion transcripts of 5′ Ribosomal Protein S25 (RPS25) with FOXR1 in the DLBCL cell line U-2932, both genes located at the amplified chromosomal region 11q23 (Figure 1a, Supplementary Figure S1A). Genomic cloning localized the breakpoint to intron 2/3 of RPS25 and to the promoter region of FOXR1 (bp − 3532). Cell line U-2932 comprises two distinct clones traceable to subclones present in the patient ́s tumor. These differences also affected 11q32, FOXR1 and RPS25 being tetraploid in subclone R1, and triploid in subclone R2 (3n) (Supplementary Figure S1A). In accordance with the genomic data, the RPS25/FOXR1 fusion was detected in subclone R1 but not in R2 (Figure 1b). RPS25/FOXR1 was also verified in the patient ́s DNA, which collectively suggested that the fusion had occurred at some later stages of tumor development (Figure 1b). Physiological FOXR1 (formerly FOXN5) expression is restricted to the early stages of embryogenesis. Ectopic expression as result of 11q23 intrachromosomal deletion-fusion has hitherto been described in neuroblastoma only. In-frame fusions with the 5′ MLL or PAFAH1B2 genes led to overexpression of FOXR1. In accordance with the notion that a constitutively expressed 5′ gene (RPS25) might be responsible for the ectopic expression of FOXR1 in B-cell lymphoma also, FOXR1 levels were 1000 × higher in the fusion-positive than in the fusion-negative U-2932 subclone. Expression array analyses showed that the RPS25/FOXR1-positive U-2932 subclone had the highest FOXR1 expression level of 55 B lymphoma cell lines tested, three log-scales higher than average (Figure 2a). Quantitative PCR analysis conducted to verify the expression arrays included 17 additional B lymphoma cell lines, revealing that the primary effusion lymphoma cell line CRO-AP3

A Phase I/IIa Trial Using CD19-Targeted Third-Generation CAR T Cells for Lymphoma and Leukemia

Purpose: The chimeric antigen receptor (CAR) T-cell therapy has been effective for patients with CD19+ B-cell malignancies. Most studies have investigated the second-generation CARs with either CD28 or 4-1BB costimulatory domains in the CAR receptor. Here, we describe the first clinical phase I/IIa trial using third-generation CAR T cells targeting CD19 to evaluate safety and efficacy. Patients and Methods: Fifteen patients with B-cell lymphoma or leukemia were treated with CAR T cells. The patients with lymphoma received chemotherapy during CAR manufacture and 11 of 15 were given low-dose cyclophosphamide and fludarabine conditioning prior to CAR infusion. Peripheral blood was sampled before and at multiple time points after CAR infusion to evaluate the persistence of CAR T cells and for immune profiling, using quantitative PCR, flow cytometry, and a proteomic array. Results: Treatment with third-generation CAR T cells was generally safe with 4 patients requiring hospitalization due to adverse reactions. Six of the 15 patients had initial complete responses [4/11 lymphoma and 2/4 acute lymphoblastic leukemia (ALL)], and 3 of the patients with lymphoma were in remission at 3 months. Two patients are still alive. Best predictor of response was a good immune status prior to CAR infusion with high IL12, DC-Lamp, Fas ligand, and TRAIL. Responding patients had low monocytic myeloid-derived suppressor cells (MDSCs; CD14+CD33+HLA−DR−) and low levels of IL6, IL8, NAP3, sPDL1, and sPDL2. Conclusions: Third-generation CARs may be efficient in patients with advanced B-cell lymphoproliferative malignancy with only modest toxicity. Immune profiling pre- and posttreatment can be used to find response biomarkers.