Real-world performance of integrative clinical genomics in pediatric precision oncology.

Despite significant improvement in the survival of pediatric cancer patients, treatment outcomes for high-risk, relapsed, and refractory cancers remain unsatisfactory. Moreover, prolonged survival is frequently associated with long-term adverse effects due to intensive multimodal treatments. Accelerating the progress of pediatric oncology requires both therapeutic advances and strategies to mitigate the long-term cytotoxic side effects, potentially through targeting specific molecular drivers of pediatric malignancies. In this report, we present the results of integrative genomic and transcriptomic profiling of 230 patients with malignant solid tumors (the "primary cohort") and 18 patients with recurrent or otherwise difficult-to-treat nonmalignant conditions (the "secondary cohort"). The integrative workflow for the primary cohort enabled the identification of clinically significant single-nucleotide variants, small insertions/deletions, and fusion genes, which were found in 55% and 28% of patients, respectively. For 38% of patients, molecularly informed treatment recommendations were made. In the secondary cohort, known or potentially driving alteration was detected in 89% of cases, including a suspected novel causal gene for patients with inclusion body infantile digital fibromatosis. Furthermore, 47% of findings also brought therapeutic implications for subsequent management. Across both cohorts, changes or refinements to the original histopathological diagnoses were achieved in 4% of cases. Our study demonstrates the efficacy of integrating advanced genomic and transcriptomic analyses to identify therapeutic targets, refine diagnoses, and optimize treatment strategies for challenging pediatric and young adult malignancies and underscores the need for broad implementation of precision oncology in clinical settings.

Efficacy of ixazomib, lenalidomide, dexamethasone regimen in daratumumab-exposed relapsed/refractory multiple myeloma patients: A retrospective analysis.

We performed retrospective analysis of relapsed/refractory multiple myeloma (RRMM) patients previously exposed to daratumumab treated with ixazomib, lenalidomide, dexamethasone (IRd) regimen in real clinical practice. Our aim was to evaluate efficacy of IRd in these patients and select a subset of patients that would benefit from this treatment the most. In total, we analyzed 43 daratumumab-exposed RRMM patients treated in our center. Minimal response or better was achieved by 53.5% of patients from the cohort. Median progression free survival (PFS) was 4.56 months (95% CI: 2.56, 8.03) and median overall survival (OS) was 28.92 months (95% CI: 5.4, NR). Duration of response (DOR) was evaluable in 28 patients and reached a median of 21.3 months (95% CI: 6.85, NR). Next, we evaluated hazard ratios (HR) for OS and PFS. There was improved OS in patients that were not-triple refractory or worse (HR = 0.39, 95%Cl (0.14; 1.10), p = .07) and in patients, that had less than three previous lines of treatment (LOT) (HR = 0.13, 95%Cl (0.03; 0.6) p = .003). Similar to OS, there was improved PFS in patients, that were not triple-refractory or worse (HR = 0.52, 95%Cl (0.25; 1.10), p = .08). We concluded, that the best survival benefit for RRMM patients pretreated with daratumumab to IRd regimen was observed in patients that were not triple-refractory and had less than three previous lines of treatment (LOT). The DOR in these patients was 21.3 months (95% CI: 6.85, NR).

De novo accelerated phase of chronic myeloid leukemia should be recognized even in the era of tyrosine kinase inhibitors.

Overall survival of patients classified according to the European LeukemiaNet 2020 classification. Chronic phase (CP), accelerated phase (AP), blast crisis (BC), low risk (LR), intermediate risk (IR), high risk (HR).

Analysis of rare driving events in pediatric acute myeloid leukemia.

Elucidating genetic aberrations in pediatric acute myeloid leukemia (AML) provides insight in biology and may impact on risk-group stratification and clinical outcome. This study aimed to detect such aberrations in a selected series of samples without known (cyto)genetic aberration using molecular profiling. A cohort of 161 patients was selected from various study groups: DCOG, BFM, SJCRH, NOPHO and AEIOP. Samples were analyzed using RNA sequencing (n=152), whole exome (n=135) and/or whole genome sequencing (n=100). In 70 of 156 patients (45%), of whom RNA sequencing or whole genome sequencing was available, rearrangements were detected, 22 of which were novel; five involving ERG rearrangements and four NPM1 rearrangements. ERG rearrangements showed self-renewal capacity in vitro, and a distinct gene expression pattern. Gene set enrichment analysis of this cluster showed upregulation of gene sets derived from Ewing sarcoma, which was confirmed comparing gene expression profiles of AML and Ewing sarcoma. Furthermore, NPM1-rearranged cases showed cytoplasmic NPM1 localization and revealed HOXA/B gene overexpression, as described for NPM1 mutated cases. Single-gene mutations as identified in adult AML were rare. Patients had a median of 24 coding mutations (range, 7-159). Novel recurrent mutations were detected in UBTF (n=10), a regulator of RNA transcription. In 75% of patients an aberration with a prognostic impact could be detected. Therefore, we suggest these techniques need to become standard of care in diagnostics.

Evolution of TP53 abnormalities during CLL disease course is associated with telomere length changes.

BACKGROUND: Telomeres are protective structures at chromosome ends which shorten gradually with increasing age. In chronic lymphocytic leukemia (CLL), short telomeres have been associated with unfavorable disease outcome, but the link between clonal evolution and telomere shortening remains unresolved. METHODS: We investigated relative telomere length (RTL) in a well-characterized cohort of 198 CLL patients by qPCR and focused in detail on a subgroup 26 patients who underwent clonal evolution of TP53 mutations (evolTP53). In the evolTP53 subgroup we explored factors influencing clonal evolution and corresponding changes in telomere length through measurements of telomerase expression, lymphocyte doubling time, and BCR signaling activity. RESULTS: At baseline, RTL of the evolTP53 patients was scattered across the entire RTL spectrum observed in our CLL cohort. RTL changed in the follow-up samples of 16/26 (62%) evolTP53 cases, inclining to reach intermediate RTL values, i.e., longer telomeres shortened compared to baseline while shorter ones prolonged. For the first time we show that TP53 clonal shifts are linked to RTL change, including unexpected RTL prolongation. We further investigated parameters associated with RTL changes. Unstable telomeres were significantly more frequent among younger patients (P = 0.032). Shorter telomeres were associated with decreased activity of the B-cell receptor signaling components p-ERK1/2, p-ZAP-70/SYK, and p-NFκB (P = 0.04, P = 0.01, and P = 0.02, respectively). CONCLUSIONS: Our study revealed that changes of telomere length reflect evolution in leukemic subclone proportion, and are associated with specific clinico-biological features of the explored cohort.

Cytogenetic complexity in chronic lymphocytic leukemia: definitions, associations, and clinical impact.

Recent evidence suggests that complex karyotype (CK) defined by the presence of ≥3 chromosomal aberrations (structural and/or numerical) identified by using chromosome-banding analysis (CBA) may be relevant for treatment decision-making in chronic lymphocytic leukemia (CLL). However, many challenges toward the routine clinical application of CBA remain. In a retrospective study of 5290 patients with available CBA data, we explored both clinicobiological associations and the clinical impact of CK in CLL. We found that patients with ≥5 abnormalities, defined as high-CK, exhibit uniformly dismal clinical outcomes, independently of clinical stage, TP53 aberrations (deletion of chromosome 17p and/or TP53 mutations [TP53abs]), and the expression of somatically hypermutated (M-CLL) or unmutated immunoglobulin heavy variable genes. Thus, they contrasted with CK cases with 3 or 4 aberrations (low-CK and intermediate-CK, respectively) who followed aggressive disease courses only in the presence of TP53abs. At the other end of the spectrum, patients with CK and +12,+19 displayed an exceptionally indolent profile. Building upon CK, TP53abs, and immunoglobulin heavy variable gene somatic hypermutation status, we propose a novel hierarchical model in which patients with high-CK exhibit the worst prognosis, whereas those with mutated CLL lacking CK or TP53abs, as well as CK with +12,+19, show the longest overall survival. Thus, CK should not be axiomatically considered unfavorable in CLL, representing a heterogeneous group with variable clinical behavior. High-CK with ≥5 chromosomal aberrations emerges as prognostically adverse, independent of other biomarkers. Prospective clinical validation is warranted before ultimately incorporating high-CK in risk stratification of CLL.

Genomic arrays identify high-risk chronic lymphocytic leukemia with genomic complexity: a multi-center study.

Complex karyotype (CK) identified by chromosome-banding analysis (CBA) has shown prognostic value in chronic lymphocytic leukemia (CLL). Genomic arrays offer high-resolution genome-wide detection of copy-number alterations (CNAs) and could therefore be well equipped to detect the presence of a CK. Current knowledge on genomic arrays in CLL is based on outcomes of single center studies, in which different cutoffs for CNA calling were used. To further determine the clinical utility of genomic arrays for CNA assessment in CLL diagnostics, we retrospectively analyzed 2293 arrays from 13 diagnostic laboratories according to established standards. CNAs were found outside regions captured by CLL FISH probes in 34% of patients, and several of them including gains of 8q, deletions of 9p and 18p (p<0.01) were linked to poor outcome after correction for multiple testing. Patients (n=972) could be divided in three distinct prognostic subgroups based on the number of CNAs. Only high genomic complexity (high-GC), defined as ≥5 CNAs emerged as an independent adverse prognosticator on multivariable analysis for time to first treatment (Hazard ratio: 2.15, 95% CI: 1.36-3.41; p=0.001) and overall survival (Hazard ratio: 2.54, 95% CI: 1.54-4.17; p<0.001; n=528). Lowering the size cutoff to 1 Mb in 647 patients did not significantly improve risk assessment. Genomic arrays detected more chromosomal abnormalities and performed at least as well in terms of risk stratification compared to simultaneous chromosome banding analysis as determined in 122 patients. Our findings highlight genomic array as an accurate tool for CLL risk stratification.

Genomic landscape of B-other acute lymphoblastic leukemia in an adult retrospective cohort with a focus on BCR-ABL1-like subtype.

INTRODUCTION: BCR-ABL1-like acute lymphoblastic leukemia (ALL) is a high-risk disease with a complex genomic background. Though extensively studied, data on the frequency and mutual associations of present mutations are still incomplete in adult patients. This retrospective study aims to map the genomic landscape of B-other ALL in a cohort of adult patients with a focus on the BCR-ABL1-like ALL subtype. METHODS: We analyzed bone marrow and peripheral blood samples of adult B-other ALL patients treated consecutively at three major Czech teaching hospitals. Samples were analyzed by cytogenetic methods, gene expression profiling, multiplex ligation-dependent probe amplification (MLPA), and next-generation sequencing (NGS). RESULTS: Fifty-eight B-other ALL patients (not BCR-ABL1, KMT2A-rearranged, ETV6-RUNX1, TCF3-PBX1, or iAMP21) were included in the study. Median follow-up was 23.8 months. Samples from 33 patients were available for a gene expression analysis, 48.9% identified as BCR-ABL1-like ALL. Of the BCR-ABL1-like ALL cases, 18.8% harbored IGH-CRLF2 and 12.5% P2RY8-CRLF2 fusion gene. We observed a higher MRD failure rate in BCR-ABL1-like than in non-BCR-ABL1-like ALL patients after the induction treatment (50.0 vs. 13.3%, p=.05). There was a trend to worse progression-free and overall survival in the BCR-ABL1-like group, though not statistically significant. Deletions in IKZF1 gene were found in 31.3% of BCR-ABL1-like cases. Patients with concurrent IKZF1 and CDKN2A/B, PAX5 or PAR1 region deletions (IKZF1plus profile) had significantly worse progression-free survival than those with sole IKZF1 deletion or IKZF1 wild-type (p=.02). NGS analysis was performed in 54 patients and identified 99 short variants in TP53, JAK2, NRAS, PAX5, CREBBP, NF1, FLT3, ATM, KRAS, RUNX1, and other genes. Seventy-five of these gene variants have not yet been described in B-cell precursor ALL to date. CONCLUSION: This study widens existing knowledge of the BCR-ABL1-like and B-other ALL genomic landscape in the adult population, supports previous findings, and identifies a number of novel gene variants.

miR-150 downregulation contributes to the high-grade transformation of follicular lymphoma by upregulating FOXP1 levels.

Follicular lymphoma (FL) is a common indolent B-cell malignancy with a variable clinical course. An unfavorable event in its course is histological transformation to a high-grade lymphoma, typically diffuse large B-cell lymphoma. Recent studies show that genetic aberrations of MYC or its overexpression are associated with FL transformation (tFL). However, the precise molecular mechanisms underlying tFL are unclear. Here we performed the first profiling of expression of microRNAs (miRNAs) in paired samples of FL and tFL and identified 5 miRNAs as being differentially expressed. We focused on one of these miRNAs, namely miR-150, which was uniformly downmodulated in all examined tFLs (∼3.5-fold), and observed that high levels of MYC are responsible for repressing miR-150 in tFL by binding in its upstream region. This MYC-mediated repression of miR-150 in B cells is not dependent on LIN28A/B proteins, which influence the maturation of miR-150 precursor (pri-miR-150) in myeloid cells. We also demonstrated that low miR-150 levels in tFL lead to upregulation of its target, namely FOXP1 protein, which is a known positive regulator of cell survival, as well as B-cell receptor and NF-κB signaling in malignant B cells. We revealed that low levels of miR-150 and high levels of its target, FOXP1, are associated with shorter overall survival in FL and suggest that miR-150 could serve as a good biomarker measurable in formalin-fixed paraffin-embedded tissue. Overall, our study demonstrates the role of the MYC/miR-150/FOXP1 axis in malignant B cells as a determinant of FL aggressiveness and its high-grade transformation.

Clinicopathological correlations of the immunoprofile in diffuse large B-cell lymphoma NOS - a single institutions experience.

The experimental platform in hematooncology is still searching for more valid prognostic and predictive factors on clinical, morphological and molecular levels. The bridge closer to daily practice is so-called translation medicine and from this point of view we have tried to sort diffuse large B-cell lymphoma not otherwise specified. The applied methodological approaches are morphology, indirect immunohistochemistry on formaline-fixed, parrafin-embeded tissue, Hans classifier sorting, expression of Bcl-2, CD5, CD20, CD30 and NfκB proteins in comparison with the clinical (Ann Arbor stage, IPI, aa-IPI, PFS, OS), laboratory and cytogenetic results (complex and simplex karyotypes). Statistical analysis included Cox regressive analysis, Mann-Whitney and Kruska-Wallis test. The interval of PFS and OS has been assessed according to Kaplan-Meier analysis. According to Hans classifier 11 cases (18.7 %) could not be sorted exactly into GCB/nonGCB- like subgroups. All relapsing cases bear negative expression of CD10 and 28 cases of non-relapsing cases showed positive expression of CD10. The "third" - GCB-like/nonGCB-like unsortable subgroups shared a very similar course of PFS with the nonGCB-like subgroup and a worse clinical course of OS. Statistically nonsignificantly better response to chemotherapy was shown by cases with positive Bcl-2 expression of more than 30 %. Statistically nonsignificantly better OS and PFS was shown by cases with a proliferation index Ki67 more than 70 %. The study detected 17 cases (28.8 %) with a nuclear expression of p50 and one case with nuclear expression of p65 (1.7 %) which may imply the possibility of NfκB signaling pathway activation. A statistically nonsignificant realationship of p50 expression and OS/PFS was indicated.

[Analysis of serum free light chains κ/λ ratio and heavy/light chain pairs of immunoglobulin to the stratification of multiple myeloma according to Mayo Stratification of Myeloma and Revised International Staging System]. / Analýza vztahu volných lehkých retezcu κ/λ a páru tezkých/lehkých retezcu imunoglobulinu ke stratifikaci mnohocetného myelomu podle Mayo Stratification of Myeloma and Revised International Staging System.

INTRODUCTION: Assessment of serum levels of free light chains (FLC-κ and FLC-λ) and recently heavy/light chain pairs of immunoglobulin (HLC-κ and HLC-λ) and their ratio (FLC-r and HLC-r) has significantly enriched traditional algorithm of multiple myeloma (MM) evaluation. The aim of the presented study was to assess the relationship of classical prognostic parameters of MM, standard FLC-κ/λ and HLC-κ/λ ratio ((s)FLC-r and (s)HLC-r), modified ratio of "involved/uninvolved" FLC and HLC ((m)FLC-r and (m)HLC-r ), the difference between "involved - uninvolved" FLC and HLC (FLC-dif. and HLC-dif.) to current stratification models of MM based on the result of cytogenetic analysis. PATIENTS AND METHODS: In a group of 97 patients with MM we assessed serum levels of FLC by FreeliteTM method, and we calculated (s)FLC-r, (m)FLC-r and FLC-dif. indices by HevyliteTM method. For cytogenetic analysis we used FICTION (fluorescence immunophenotyping and interphase cytogenetics as a tool for the investigation of neoplasms). For MM stratification we used standard staging systems according to Durie-Salmon (D-S) and International Staging System (ISS) as well as novel stratification systems based on the results of cytogenetic analysis, ie. "Mayo Stratification of Myeloma and Risk-Adapted Therapy" (mSMART) and "Revised International Staging System" (R-ISS). RESULTS: Stratification mSMART and R-ISS has significantly different representation of "standard" or "low-risk" (71, 15.5, 11.3 a 29.9 %), "intermediate risk" (15.5, 53.6, 34 a 33 %) and "high risk" patients (13.4, 30.9, 54.7 a 37.1 %) compared to standard staging systems. mSMART stratification was compared to prognostic factors of MM (Hb, albumin, ß(2)-M, creatinine and LDH), and the only significant relationship was found in the case of ß(2)-M, R-ISS had relationship only to Hb and creatinine. In the case of D-S staging we found significant relationship of stages 1-3 and substages A and B to the levels of (m)FLC-r, FLC-dif. and (m)HLC-r, ISS had moreover relationship to k HLC-dif. and MIg concentration. Analysis of mSMART stratification showed primarily significant relationship of risk categories 1-3 to (m)FLC-r and (s)HLC-r indices, and R-ISS to (m)HLC-r index and MIg concentration. In both cytogenetics-based stratifications there was a lack of relationship to (s)FLC-r, FLC-dif. and HLC-dif. indices. CONCLUSION: Comparison of the results of standard staging systems according to D-S and ISS with cytogenetics based models mSMART and R-ISS showed different representation of risk groups, and significantly different relationship to classical prognostic factors together with original relationship of sMART stratification to (m)FLC-r and (s)HLC-r, and R-ISS to (m)HLC-r and MIg concentration.

[The prospects for children with acute lymphoblastic leukemia of being cured has increased in the Czech Republic in the 21st century to 90% - outcome of the ALL-IC BFM 2002 trial]. / Sance na vylécení detí s akutní lymfoblastickou leukémií stoupla v Ceské republicev 21. století na 90 % - výsledky studie ALL IC-BFM 2002.

BACKGROUND: Acute lymphoblastic leukemia (ALL) is the most frequent childhood malignancy. Treatment has been unified in the middle of 1980 in the Czech Republic. In 2002-2007 children and adolescents with acute lymphoblastic leukemia were treated in an international randomized trial ALL-IC BFM 2002 in the Czech Republic. 291 patients aged 1-18 years were enrolled; infants below 1 year entered a separate trial. METHODS AND RESULTS: Patients were stratified into three risk groups according to their age, initial leukocyte count, prednisone response, presence of fusion genes BCR/ABL or MLL/AF4, bone marrow D+15 and remission status D+33. The whole therapy took 24 months. Randomized late intensification compared standard BFM therapy with extended, usually more intensive experimental treatment. The median follow-up was 8.7 years. Complete remission was achieved in 97.9% patients, 1% died in remission. 11% of children relapsed, 1.7% with CNS involvement. Six children (2.1%) developed secondary malignancy. Event free survival (EFS) 8 years from diagnosis was 83.5%, overall survival (OS) 91.4%. EFS and OS of the risk groups were: standard risk: 89.4%; 98.1%; intermediate risk: 82.6%; 89.6%; high risk: 68.8%; 78.1%. Male sex and age above 10 years were adverse prognostic factors. CONCLUSIONS: In comparison with the previous trial ALL-BFM 95, significant improvement was achieved.

[Use of archival formalin-fixed, paraffin-embedded (FFPE) tissue samples for molecular genetic analysis in diffuse large B-cell lymphoma (DLBCL)]. / Vyuzití archivovaných parafínových blocku s formalinem fixovanou tkání (FFPE) pro molekulárne genetickou analýzu u difuzního velkobunecného lymfomu (DLBCL).

The currently valid molecular genetic subclassification of patients with diffuse large B-cell lymphoma (DLBCL) into three prognostic subgroups based on expression profiling has been the objective of numerous genetic studies. In routine clinical practice, however, expression profiling technology remains unavailable for the most of centers. Apart from the technology, in some cases molecular genetic laboratories have problems obtaining high-quality material, i.e. fresh tissues, for RNA isolation to determine gene expression. One possibility is to determine the gene expression from RNA obtained by isolation from formalin-fixed, paraffin-embedded (FFPE) tissue. This pilot study aimed at isolating RNA from FFPE in patients diagnosed with DLBCL and verifying the potential use of such RNA for the expression analysis of 7 selected genes. Although the study showed that it is possible to isolate RNA and determine the expression of the selected genes from archival material, the values of relative expression of some genes in the set were too variable to be used for unambiguous prognostic classification. It was confirmed that retrospective analyses of selected genes may be performed with sufficient material obtained, and that properly archived blocks may be used for molecular biology analyses even after 8 years.

Precision immuno-oncology approach for four malignant tumors in siblings with constitutional mismatch repair deficiency syndrome.

Constitutional mismatch repair deficiency (CMMRD) is a rare syndrome characterized by an increased incidence of cancer. It is caused by biallelic germline mutations in one of the four mismatch repair genes (MMR) genes: MLH1, MSH2, MSH6, or PMS2. Accurate diagnosis accompanied by a proper molecular genetic examination plays a crucial role in cancer management and also has implications for other family members. In this report, we share the impact of the diagnosis and challenges during the clinical management of two brothers with CMMRD from a non-consanguineous family harbouring compound heterozygous variants in the PMS2 gene. Both brothers presented with different phenotypic manifestations and cancer spectrum. Treatment involving immune checkpoint inhibitors significantly contributed to prolonged survival in both patients affected by lethal gliomas. The uniform hypermutation also allowed immune-directed treatment using nivolumab for the B-cell lymphoma, thereby limiting the intensive chemotherapy exposure in this young patient who remains at risk for subsequent malignancies.

Stromal cells engineered to express T cell factors induce robust CLL cell proliferation in vitro and in PDX co-transplantations allowing the identification of RAF inhibitors as anti-proliferative drugs.

Several in vitro models have been developed to mimic chronic lymphocytic leukemia (CLL) proliferation in immune niches; however, they typically do not induce robust proliferation. We prepared a novel model based on mimicking T-cell signals in vitro and in patient-derived xenografts (PDXs). Six supportive cell lines were prepared by engineering HS5 stromal cells with stable expression of human CD40L, IL4, IL21, and their combinations. Co-culture with HS5 expressing CD40L and IL4 in combination led to mild CLL cell proliferation (median 7% at day 7), while the HS5 expressing CD40L, IL4, and IL21 led to unprecedented proliferation rate (median 44%). The co-cultures mimicked the gene expression fingerprint of lymph node CLL cells (MYC, NFκB, and E2F signatures) and revealed novel vulnerabilities in CLL-T-cell-induced proliferation. Drug testing in co-cultures revealed for the first time that pan-RAF inhibitors fully block CLL proliferation. The co-culture model can be downscaled to five microliter volume for large drug screening purposes or upscaled to CLL PDXs by HS5-CD40L-IL4 ± IL21 co-transplantation. Co-transplanting NSG mice with purified CLL cells and HS5-CD40L-IL4 or HS5-CD40L-IL4-IL21 cells on collagen-based scaffold led to 47% or 82% engraftment efficacy, respectively, with ~20% of PDXs being clonally related to CLL, potentially overcoming the need to co-transplant autologous T-cells in PDXs.

Current survival measures reliably reflect modern sequential treatment in CML: correlation with prognostic stratifications.

Using the data of 723 chronic myeloid leukemia (CML) patients in the chronic phase, we analyzed the prognostic value of the Sokal, Euro, and EUTOS scores as well as the level of BCR-ABL1 and the achievement of complete cytogenetic response (CCgR) at 3 months of imatinib therapy in relation to the so-called current survival measures: the current cumulative incidence (CCI) reflecting the probability of being alive and in CCgR after starting imatinib therapy; the current leukemia-free survival (CLFS) reflecting the probability of being alive and in CCgR after achieving the first CCgR; and the overall survival. The greatest difference between the CCI curves at 5 years after initiating imatinib therapy was observed for the BCR-ABL1 transcripts at 3 months. The 5-year CCI was 94.3% in patients with BCR-ABL1 transcripts ≤ 10% and 57.1% in patients with BCR-ABL1 transcripts > 10% (P = 0.005). Therefore, the examination of BCR-ABL1 transcripts at 3 months may help in early identification of patients who are likely to perform poorly with imatinib. On the other hand, CLFS was not significantly affected by the considered stratifications. In conclusion, our results indicate that once the CCgR is achieved, the prognosis is good irrespective of the starting prognostic risks.

Report from the European Myeloma Network on interphase FISH in multiple myeloma and related disorders.

The European Myeloma Network has organized two workshops on fluorescence in situ hybridization in multiple myeloma. The first aimed to identify specific indications and consensus technical approaches of current practice. A second workshop followed a quality control exercise in which 21 laboratories analyzed diagnostic cases of purified plasma cells for recurrent abnormalities. The summary report was discussed at the EHA Myeloma Scientific Working Group Meeting 2010. During the quality control exercise, there was acceptable agreement on more than 1,000 tests. The conclusions from the exercise were that the primary clinical applications for FISH analysis were for newly diagnosed cases of MM or frank relapse cases. A range of technical recommendations included: 1) material should be part of the first draw of the aspirate; 2) samples should be sent at suitable times to allow for the lengthy processing procedure; 3) most importantly, PCs must be purified or specifically identified; 4) positive cut-off levels should be relatively conservative: 10% for fusion or break-apart probes, 20% for numerical abnormalities; 5) informative probes should be combined to best effect; 6) in specialist laboratories, a single experienced analyst is considered adequate; 7) at least 100 PC should be scored; 8) essential abnormalities to test for are t(4;14), t(14;16) and 17p13 deletions; 9) suitable commercial probes should be available for clinically relevant abnormalities; 10) the clinical report should be expressed clearly and must state the percentage of PC involved and the method used for identification; 11) a retrospective European based FISH data bank linked to clinical data should be generated; and 12) prospective analysis should be centralized for upcoming trials based on the recommendations made. The European Myeloma Network aims to build on these recommendations to establish standards for a common European data base to define subgroups with prognostic significance.

Imatinib trough plasma levels do not correlate with the response to therapy in patients with chronic myeloid leukemia in routine clinical setting.

Association of trough imatinib plasma levels (IPL) with cytogenetic or molecular response to treatment in patients with chronic myeloid leukemia (CML) was repeatedly reported. We analyzed their value in the routine clinical setting in 131 patients with chronic phase CML in whom imatinib was applied as first- or second-line treatment. A total of 1,118 measurements were obtained by ultra-performance liquid chromatography-tandem mass spectrometry assay in patients treated with daily dose of imatinib ranging from 100 to 800 mg. Samples were obtained from 1 to 96 h after drug ingestion. High inter (36%) and intraindividual variability (9-33%) of IPL was observed. For analysis of correlation of IPL with treatment response, two sets of samples were selected according to the European LeukemiaNet (ELN) criteria. The first set consisted of 241 samples taken 24 ± 2 h after dosing in 54 patients, and the second one consisted of 329 samples taken 24 ± 4 h after imatinib ingestion in 84 patients. In both sets, only patients treated with 400 mg imatinib once daily for at least 18 months were included. From multiple measurements in individual patients, mean IPL were used. In both sets, we were not able to demonstrate a statistically significant correlation between IPL and response to treatment according to the ELN. We believe that this was due to the differences in patients' compliance, leukemia biology, and other variables that are difficult to eliminate in the routine clinical practice. The use of IPL for prognostic estimation in CML treatment outside the clinical trials is probably limited.

Cytogenetics in Chronic Lymphocytic Leukemia: ERIC Perspectives and Recommendations.

Mounting evidence underscores the clinical value of cytogenetic analysis in chronic lymphocytic leukemia (CLL), particularly as it allows the identification of complex karyotype, that has recently emerged as a prognostic and potentially predictive biomarker. That said, explicit recommendations regarding the methodology and clinical interpretation of either chromosome banding analysis (CBA) or chromosome microarray analysis (CMA) are still lacking. We herein present the consensus of the Cytogenetic Steering Scientific Committee of ERIC, the European Research Initiative on CLL, regarding methodological issues as well as clinical interpretation of CBA/CMA and discuss their relevance in CLL. ERIC considers CBA standardized and feasible for CLL on the condition that standards are met, extending from the use of novel mitogens to the accurate interpretation of the findings. On the other hand, CMA, is also standardized, however, robust data on its clinical utility are still scarce. In conclusion, cytogenetic analysis is not yet mature enough to guide treatment choices in CLL. That notwithstanding, ERIC encourages the wide application of CBA, and potentially also CMA, in clinical trials in order to obtain robust evidence regarding the predictive value of specific cytogenetic profiles towards refining risk stratification and improving the management of patients with CLL.