A novel ex vivo high-throughput assay reveals antiproliferative effects of idelalisib and ibrutinib in chronic lymphocytic leukemia.

PI3Kδ (idelalisib) and BTK (ibrutinib) inhibitors have demonstrated significant clinical activity in chronic lymphocytic leukemia (CLL) interfering with the cross-talk between CLL cells and the lymph node microenviroment, yet their mechanism of action remains to be fully elucidated. Here, we developed an ex vivo model with the aim of reproducing the effects of the microenvironment that would help shed light on the in vivo mechanism of action of idelalisib and ibrutinib and predict their clinical efficacy in individual patients. First we explored the effects of various cell-extrinsic elements on CLL apoptosis and proliferation and found that the combination of CpG+IL2+HS5 stromal cell line + human serum +CLL plasma and erythrocyte fractions represented the best co-culture conditions to test the effects of the novel inhibitors. Then, using this assay, we investigated the impact of idelalisib and ibrutinib on both survival and proliferation in 30 CLL patients. While both drugs had a limited direct pro-apoptotic activity, a potent inhibition of proliferation was achieved at clinically achievable concentrations. Notably, up to 10% of CLL cells still proliferated even at the highest concentrations, likely mirroring the known difficulty to achieve complete responses in vivo. Altogether, this novel assay represents an appropriate ex vivo drug testing system to potentially predict the clinical response to novel inhibitors in particular by quantifying the antiproliferative effect.

Drug Discovery Testing Compounds in Patient Samples by Automated Flow Cytometry.

Functional ex vivo assays that predict a patient's clinical response to anticancer drugs for guiding cancer treatment have long been a goal, but few have yet proved to be reliable. To address this, we have developed an automated flow cytometry platform for drug screening that evaluates multiple endpoints with a robust data analysis system that can capture the complex mechanisms of action across different compounds. This system, called PharmaFlow, is used to test peripheral blood or bone marrow samples from patients diagnosed with hematological malignancies. Functional assays that use the whole sample, retaining all the microenvironmental components contained in the sample, offer an approach to ex vivo testing that may give results that are clinically relevant. This new approach can help to predict the patients' response to existing treatments or to drugs under development, for hematological malignancies or other tumors. In addition, relevant biomarkers can be identified that determine the patient's sensitivity, resistance, or toxicity to a given treatment. We propose that this approach, which better recapitulates the human microenvironment, constitutes a more predictive assay for personalized medicine and preclinical drug discovery.

Array-based comparative genomic hybridization of mapped BAC DNA clones to screen for chromosome 14 copy number abnormalities in meningiomas.

Chromosome 14 loss in meningiomas are associated with more aggressive tumour behaviour. To date, no studies have been reported in which the entire chromosome 14q of meningioma tumour cells has been studied by high-resolution array comparative genomic hybridization (a-CGH). Here, we used a high-resolution a-CGH to define the exact localization and extent of numerical changes of chromosome 14 in meningioma patients. An array containing 807 bacterial artificial chromosome clones specific for chromosome 14q (average resolution of approximately 130 Kb) was constructed and applied to the study of 25 meningiomas in parallel to the confirmatory interphase fluorescence in situ hybridization (iFISH) analyses. Overall, abnormalities of chromosome 14q were detected in 10/25 cases (40%). Interestingly, in seven of these cases, loss of chromosome 14q32.3 was detected by iFISH and confirmed to correspond to monosomy 14 by a-CGH. In contrast, discrepant results were found between iFISH and a-CGH in the other three altered cases. In one patient, a diploid background was observed by iFISH, while monosomy 14 was identified by a-CGH. In the remaining two cases, which showed gains of the IGH gene by iFISH, a-CGH did not detected copy number changes in one case showing a tetraploid karyotype, while in the other tumour, varying genetic imbalances along the long arm of chromosome 14 were detected. In summary, here, we report for the first time, the high-resolution a-CGH profiles of chromosome 14q in meningiomas, confirming that monosomy 14 is the most frequent alteration associated with this chromosome; other numerical abnormalities being only sporadically detected.

Patient gender is associated with distinct patterns of chromosomal abnormalities and sex chromosome linked gene-expression profiles in meningiomas.

The female predominance of meningiomas has been established, but how this is affected by hormones is still under discussion. We analyzed the characteristics of meningiomas from male (n = 53) and female (n = 111) patients by interphase fluorescence in situ hybridization (iFISH). In addition, in a subgroup of 45 (12 male and 33 female) patients, tumors were hybridized with the Affymetrix U133A chip. We show a higher frequency of larger tumors (p = .01) and intracranial meningiomas (p = .04) together with a higher relapse rate (p = .03) in male than in female patients. Male patients had a higher percentage of del(1p36) (p < .001), while loss of an X chromosome was restricted to tumors from female patients (p = .008). In turn, iFISH studies showed a higher frequency of chromosome losses, other than monosomy 22 alone, in meningiomas from male patients (p = .002), while female patients displayed a higher frequency of chromosome gains (p = .04) or monosomy 22 alone (p = .03) in the ancestral tumor clone. Interestingly, individual chromosomal abnormalities had a distinct impact on the recurrence-free survival rate of male versus female patients. In turn, gene expression showed that eight genes (RPS4Y1, DDX3Y, JARID1D, DDX3X, EIF1AY, XIST, USP9Y, and CYorf15B) had significantly different expression patterns (R(2) > 0.80; p < .05) in tumors from male and female patients. In summary, we show the existence of different patterns of chromosome abnormalities and gene-expression profiles associated with patient gender, which could help to explain the slightly different clinical behavior of these two patient groups.

Microarray-based analysis of spinal versus intracranial meningiomas: different clinical, biological, and genetic characteristics associated with distinct patterns of gene expression.

It has long been recognized that spinal meningiomas show particular clinical and histological features. Here, we compare the clinico-biological characteristics as well as the genetic abnormalities and patterns of gene expression of spinal and intracranial meningiomas. Fourteen spinal and 141 intracranial meningioma patients were analyzed at diagnosis. In all tumors, interphase fluorescence in situ hybridization (iFISH) studies were performed for the detection of quantitative abnormalities for 11 different chromosomes. Additionally, microarray analyses were performed on a subgroup of 18 histologically benign meningiomas (7 spinal and 11 intracranial). Upon comparison with intracranial tumors, spinal meningiomas showed a marked predominance of psammomatous and transitional tumors (p = 0.001), together with a higher proportion of cases displaying a single tumor cell clone by iFISH (p = 0.004). In 86% of the spinal versus 56% of the intracranial tumors (p = 0.01), the ancestral tumor cell clone detected showed either absence of any chromosomal abnormality or monosomy 22/22q- alone. Analysis of gene expression profiles showed differential expression between spinal and intracranial meningiomas for a total of 1555 genes, 35 of which allowed a clear distinction between both tumor types. Most of these 35 genes (n = 30) showed significantly higher expression among spinal tumors and corresponded to genes involved in signal transduction pathways, which did not show a significantly different expression according to tumor histopathology. In summary, we show the occurrence of unique patterns of genetic abnormalities and gene expression profiles in spinal as compared to intracranial meningiomas that provide new insights into the molecular pathways involved in the tumorigenesis and progression of spinal meningiomas, and could help explain their particular clinical and histological features.

Lineage involvement in chronic myeloid leukaemia: comparison between MBCR/ABL and mBCR/ABL cases.

The relationship between different Abelson/breakpoint cluster region (BCR/ABL+) gene rearrangements and the involvement of different haematopoietic cell lineages were investigated in 15 chronic myeloid leukaemia patients. Analysis of purified cell populations confirmed the involvement of the neutrophil (89%), monocytic (89%), eosinophil (88%), erythroid (100%), and CD34(+) cells (100%) in virtually all patients, without differences between minor BCR/ABL+ and major BCR/ABL+ cases; BCR/ABL+ B- and natural killer (NK)-cells were detected in 43% and 31% of cases, respectively, whereas BCR/ABL+ T-cells were rare (7%). All three minor BCR/ABL+ patients showed involvement of both B- and NK-cells, which was infrequent (27%, P = 0.06 and 10%, P = 0.01) among major BCR/ABL+ cases.

The cytogenetic relationship between primary and recurrent meningiomas points to the need for new treatment strategies in cases at high risk of relapse.

PURPOSE: Recurrence is the major factor influencing the clinical outcome of meningioma patients although the exact relationship between primary and recurrent tumors still needs to be clarified. The aim of the present study is to analyze the cytogenetic relationship between primary and subsequent recurrent meningiomas developed within the same individual. EXPERIMENTAL DESIGN: Multicolor interphase fluorescence in situ hybridization was done for the identification of numerical abnormalities of 12 chromosomes in single-cell suspensions from 59 tumor samples corresponding to 25 recurrent meningioma patients. In 47 of these tumors, the distribution of different tumor cell clones was also analyzed in paraffin-embedded tissue sections. In parallel, 132 nonrecurrent cases were also studied. RESULTS: Most recurrent meningiomas showed complex cytogenetic aberrations associated with two or more tumor cell clones in the first tumor analyzed. Interestingly, in most individuals (74%), exactly the same tumor cell clones identified in the initial lesion were also detected in the subsequent recurrent tumor samples. In the recurrent tumor samples of the remaining cases (26%), we observed tumor cell clones related to those detected in the initial lesion but which had acquired one or more additional chromosome aberrations associated with either the emergence of new clones with more complex karyotypes or the disappearance of the most representative clones from the primary lesions. Multivariate analysis of prognostic factors showed that the Maillo et al. prognostic score, based on age of patient, tumor grade, and monosomy 14, together with tumor size was the best combination of independent variables for predicting tumor recurrence at diagnosis. CONCLUSION: Overall, our results indicate that the development of recurrent meningiomas after complete tumor resection is usually due to regrowth of the primary tumor and rarely to the emergence of an unrelated meningioma, underlining the need for alternative treatment strategies in cases at high risk of relapse, particularly those with a high Maillo et al. prognostic score and larger tumors.

Characterization of chromosome 14 abnormalities by interphase in situ hybridization and comparative genomic hybridization in 124 meningiomas: correlation with clinical, histopathologic, and prognostic features.

We analyzed quantitative chromosome 14 abnormalities in 124 meningiomas by interphase fluorescence in situ hybridization (iFISH) and confirmed the nature of abnormalities by comparative genomic hybridization (CGH). We correlated the abnormalities with clinical, histopathologic, and prognostic factors. Of 124 cases, 50 (40.3%) showed loss (14.5%) or gain (25.8%) of the 14q32 chromosome region by iFISH. Most corresponded to numeric abnormalities: monosomy (12.9%), trisomy (1.6%), or tetrasomy (24.2%); in only 2 cases (1.6%), chromosome 14 loss did not involve the whole chromosome and was restricted to the 14q31-q32 region (confirmed by CGH). Cases with gain or monosomy corresponded more frequently to histologically malignant tumors (P = .009). Patients with monosomy 14/14q-, but not those with gain, more often were male (P = .04) and had a greater incidence of recurrence (P = .003) and shorter relapse-free survival (P = .03). The 2 patients with loss limited to 14q31-q32 had histologically benign tumors and no relapse after more than 5 years' follow-up. Most meningiomas with chromosome 14 abnormalities have numeric changes, with interstitial deletions of 14q31-q32 present in few cases. Of the abnormalities detected, only monosomy 14 showed an adverse prognostic impact.