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Background: To compare the Nordic and the Memorial Sloan-Kettering Cancer Center (MSKCC) chemotherapy protocols for Overall Survival (OS) and Progression-Free Survival (PFS) for intracranial primary CNS lymphoma (PCNSL). Methods: A prospective database at Oslo University Hospital of PCNSL was reviewed over a 12-year period (2003–2014). Results: Overall, 79 patients with PCNSL were identified, of whom 57 received chemotherapy. MSKCC with Rituximab (RTX) was used in 18 patients (32%) who had median OS of 46.3 months [9.8–131.9] and median PFS of 34.6 months [6.4–131.9]. The Nordic protocol was used in 14 patients (25%) who had median OS of 30.9 months [2.7–106.3] and PFS of 14.3 months [0.0–106.3]. The MSKCC was used without RTX in 25 patients (44%) who had OS of 15.2 months [0.7–136.5] and PFS of 12.0 months [0.0–117.0]. MSKCC with RTX had a significantly longer median OS (p

Survival of patients with high-risk diffuse large B-cell lymphoma (DLBCL) is suboptimal, and the risk of central nervous system (CNS) progression is relatively high. We conducted a phase 2 trial in 139 patients aged 18 to 64 years who had primary DLBCL with an age-adjusted International Prognostic Index (aaIPI) score of 2 to 3 or site-specific risk factors for CNS recurrence. The goal was to assess whether a dose-dense immunochemotherapy with early systemic CNS prophylaxis improves the outcome and reduces the incidence of CNS events. Treatment consisted of 2 courses of high-dose methotrexate in combination with biweekly rituximab (R), cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP-14), followed by 4 courses of R-CHOP-14 with etoposide (R-CHOEP) and 1 course of high-dose cytarabine with R. In addition, liposomal cytarabine was administered intrathecally at courses 1, 3, and 5. Coprimary endpoints were failure-free survival and CNS progression rates. Thirty-six (26%) patients experienced treatment failure. Progression occurred in 23 (16%) patients, including three (2.2%) CNS events. At 5 years of median follow-up, failure-free survival, overall survival, and CNS progression rates were 74%, 83%, and 2.3%, respectively. Treatment reduced the risk of progression compared with our previous trial, in which systemic CNS prophylaxis was given after 6 courses of biweekly R-CHOEP (hazard ratio, 0.49; 95% CI, 0.31-0.77; P = .002) and overcame the adverse impact of an aaIPI score of 3 on survival. In addition, outcome of the patients with BCL2/MYC double-hit lymphomas was comparable to the patients without the rearrangements. The results are encouraging, with a low toxic death rate, low number of CNS events, and favorable survival rates. This trial was registered at www.clinicaltrials.gov as #NCT01325194.

Background: In precision cancer medicine, the challenge is to prioritize DNA driver events, account for resistance markers, and procure sufficient information for treatment that maintains patient safety. The MetAction project, exploring how tumor molecular vulnerabilities predict therapy response, first established the required workflow for DNA sequencing and data interpretation (2014–2015). Here, we employed it to identify molecularly matched therapy and recorded outcome in end-stage cancer (2016–2019). Material and methods: Metastatic tissue from 26 patients (16 colorectal cancer cases) was sequenced by the Oncomine assay. The study tumor boards interpreted called variants with respect to sensitivity or resistance to matched therapy and recommended single-agent or combination treatment if considered tolerable. The primary endpoint was the rate of progression-free survival 1.3-fold longer than for the most recent systemic therapy. The objective response rate and overall survival were secondary endpoints. Results: Both common and rare actionable alterations were identified. Thirteen patients were found eligible for therapy following review of tumor sensitivity and resistance variants and patient tolerability. The interventions were inhibitors of ALK/ROS1-, BRAF-, EGFR-, FGFR-, mTOR-, PARP-, or PD-1-mediated signaling for 2–3 cases each. Among 10 patients who received treatment until radiologic evaluation, 6 (46% of the eligible cases) met the primary endpoint. Four colorectal cancer patients (15% of the total study cohort) had objective response. The only serious adverse event was a transient colitis, which appeared in 1 of the 2 patients given PD-1 inhibitor with complete response. Apart from those two, overall survival was similar for patients who did and did not receive study treatment. Conclusions: The systematic MetAction approach may point forward to a refined framework for how to interpret the complexity of sensitivity versus resistance and patient safety that resides in tumor sequence data, for the possibly improved outcome of precision cancer medicine in future studies.

Patients with mantle cell lymphoma (MCL) generally have a dismal prognosis. Intensified induction treatment with rituximab and high dose cytarabine (R_HDAC), and consolidation with high-dose therapy with autologous stem cell support has resulted in 10-year overall survival (OS) higher than 60%. However, the clinical course varies. Diagnostic tools capable of stratifying patients include the MCL International Prognostic Index (MIPI), gene expression-based proliferation signature, Ki-67 proliferation index or tumour cell morphology. Here, we tested the performance of a newly developed Nanostring-based RNA expression-based proliferation assay (MCL35) on formalin-fixed paraffin-embedded tumour tissue from younger patients recruited in or treated according to Nordic MCL protocols compared to the prognosticators listed above. Seventy-four patients were included and the assay performed well in all cases except four, which had inadequate RNA quality. The patients were evenly distributed in the MCL35 low-, intermediate- and high-risk categories. MCL35 low- and intermediate- risk groups had overlapping progression-free survival (PFS), while patients in the high-risk category had significantly inferior PFS. Combining MCL35 with MIPI or the MIPI-C (MIPI with the addition of binary Ki67 score +/-30%) showed a better discrimination than either assessment alone. In conclusion, the MCL35 assay alone or combined with MIPI or MIPI-C scores can identify patients who still have a dismal outcome despite intensified treatment.

The purposes of this study are to study the impact of deep brain involvement on overall survival (OS) and progression-free survival (PFS) in intracranial primary CNS lymphoma (PCNSL), and to explore possible mechanisms for this impact using advanced MRI techniques. Seventy-nine patients with histologically verified PCNSL were identified from a prospective clinical database of patients treated at Oslo University Hospital between 2003 and 2014. Patients were treated per standard chemotherapeutic regimens (N = 57) or no chemotherapy (N = 22). Anatomical MRIs were available in all patients to assess tumor load and location based on contrast agent enhancement visible on T1-weighted images. Diffusion MRIs were available in 33 (42%) patients and perfusion MRI in 13 (16%) patients that received active treatment. Across all patients, OS and PFS were 16.4 and 9.8 months, respectively. In multivariate analysis, MRI-based deep brain involvement (80%) was the only negative significant factor of OS (OR = 14.2; P

BACKGROUND: The current study was conducted to expedite international standardized reporting of bone marrow disease in children with neuroblastoma and to improve equivalence of care. METHODS: A multidisciplinary International Neuroblastoma Response Criteria Bone Marrow Working Group was convened by the US National Cancer Institute in January 2012 with representation from Europe, North America, and Australia. Practical transferable recommendations to standardize the reporting of bone marrow disease were developed. RESULTS: To the authors' knowledge, the current study is the first to comprehensively present consensus criteria for the collection, analysis, and reporting of the percentage area of bone marrow parenchyma occupied by tumor cells in trephine-biopsies. The quantitative analysis of neuroblastoma content in bone marrow aspirates by immunocytology and reverse transcriptase-quantitative polymerase chain reaction are revised. The inclusion of paired-like homeobox 2b (PHOX2B) for immunohistochemistry and reverse transcriptase-quantitative polymerase chain reaction is recommended. Recommendations for recording bone marrow response are provided. The authors endorse the quantitative assessment of neuroblastoma cell content in bilateral core needle biopsies-trephines and aspirates in all children with neuroblastoma, with the exception of infants, in whom the evaluation of aspirates alone is advised. It is interesting to note that 5% disease is accepted as an internationally achievable level for disease assessment. CONCLUSIONS: The quantitative assessment of neuroblastoma cells is recommended to provide data from which evidence-based numerical criteria for the reporting of bone marrow response can be realized. This is particularly important in the minimal disease setting and when neuroblastoma detection in bone marrow is intermittent, where clinical impact has yet to be validated. The wide adoption of these harmonized criteria will enhance the ability to compare outcomes from different trials and facilitate collaborative trial design.

Follicular lymphoma (FL) constitutes a significant proportion of lymphomas and shows frequent relapses. Beyond conventional chemotherapy, new therapeutic approaches have emerged, focussing on the interplay between lymphoma cells and the microenvironment. Here we report the immunophenotypic investigation of the microenvironment of a clinically well-characterized prospective cohort (study SAKK35/10, NCT01307605) of 154 treatment-naive FL patients in need of therapy, who have been treated with rituximab only or a combination of rituximab and the immunomodulatory drug lenalidomide/Revlimid® A high ratio of CD4- to CD8-positive T cells (P = 0·009) and increased amounts of PD1+ tumour-infiltrating T cells (P = 0·007) were associated with inferior progression-free survival in the whole cohort. Interestingly, the prognostic impact of PD1+ T cells and the CD4/CD8 ratio lost its significance in the subgroup treated with R2 . In the latter group, high amounts of GATA3+ T helper (Th2) equivalents were associated with better progression-free survival (P < 0·001). We identified tumour microenvironmental features that allow prognostic stratification with respect to immuno- and combined immuno- and immunomodulatory therapy. Our analysis indicates that lenalidomide may compensate the adverse prognostic implication of higher amounts of CD4+ and, particularly, PD1+ T cells and that it has favourable effects mainly in cases with higher amounts of Th2 equivalents. [Correction added on 11 February 2020, after online publication: The NCT-trial number was previously incorrect and has been updated in this version].

Introduction Relapses of diffuse large B-cell lymphoma typically occur within 2-3 years and only 10% of these patients reach a 3-year progression-free survival compared to 65% at diagnosis. Our ability to distinguish patients at risk for relapse remains based on clinical staging. We hypothesized that identifying genetic alterations in serial tumour biopsies at diagnosis and relapse would improve our ability to identify high-risk patients, make therapeutic selections and reveal molecular markers for chemo-immunotherapy resistant tumours. However, relatively few relapsed/refractory biopsies have been sequenced. A unique, clinically annotated, Nordic DLBCL cohort was used to identify significantly mutated genes, assess potential driver genes, comprehensively examine clonal evolution, and gauge the importance of clinical relapsed sampling. Methods To address the lack of information on the molecular foundations of relapsed/refractory DLBCL, we performed whole exome sequencing (WES) on 42 DLBCL cases, with 34% representing relapsed/refractory biopsies and 13 serially sample cases. Enriched with relapsed/refractory diffuse large B-cell lymphoma cases, we performed multiple computational analyses to identify significantly mutated genes (MutSig2CV), mutational signatures (NMF and DeConstructsSig), driver genes (IntOgen and CADD), clonal evolution architecture (SciClone and ClonEvol), druggable gene analysis (DGIdb), and HLA-inference and mutation calling (Polysolver). Results Clonal evolution analysis of 13 paired diagnostic and relapsed biopsies revealed that relapsed/refractory biopsies have remarkable similarities to diagnostic biopsies and often present with late divergent clonal evolution of the tumor. Mutational analysis of 18 serially sampled tumors determined that in the majority of cases druggable oncogenic variants do arise at relapse. In addition, time to relapse correlated with divergence of mutations from the diagnostic biopsy. In addition to being identified as a significantly mutated gene, mutations in HLA-A had an increased incidence in cases that ultimately relapsed. This result led to an in-depth investigation into the mutational prevalence, timing, impact on prognosis, and loss of heterozygosity in the human leukocyte antigen (HLA) haplotypes of relapsed/refractory DLBCL. HLA-A mutagenesis and loss of heterozygosity was discovered as mechanisms of immune evasion in cases that go on to relapse from R-CHOP like therapies (Figure 1). Conclusions Our results yield insight into the development of chemo-immunotherapy resistant diffuse large B-cell lymphoma, and highlight the clinical importance of sampling relapsed biopsies. Analysis of immune evasion through MHC Class I/II, specifically HLA-A, may provide better characterization of patients for relapse prediction. In the age of personalized medicine it will be instrumental to determine if relapsed biopsies offer additional insight for salvage therapy treatment. Divergence of biopsies, as characterized by shared genomic mutations, increase with time and the majority of cases present with new alterations in druggable genes post-therapy. Disclosures Leppa: Roche: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Celgene: Consultancy; Bayer: Research Funding. Holte:Novartis: Honoraria, Other: Advisory board.

Background: High-grade B-cell lymphoma with MYC and BCL2 and/or BCL6 rearrangements (HGBL-DH/TH) occurs in approximately 8% of de novo diffuse large B-cell lymphoma (DLBCL) cases, with BCL2 translocations (HGBL-DH/TH-BCL2) being a germinal center B-cell like (GCB) phenomenon (Scott et al. Blood 2018). HGBL-DH/TH is associated with poor outcome when treated with standard R-CHOP-therapy, but retrospective studies suggest that more aggressive treatment may improve outcome (Petrich et al. Blood 2014; Oki et al. BJH 2014). To overcome limitations with identification of HGBL-DH/TH by fluorescence in situ hybridization (FISH), Ennishi et al. developed a gene expression signature (DHITsig) that identified tumors with a HGBL-DH/TH-BCL2 gene expression phenotype. This DHITsig was translated into a new assay (DLBCL90) for application on formalin-fixed paraffin-embedded (FFPE) biopsies (Ennishi et al. J Clin Oncol 2019), and included identification of cell-of-origin (COO) and primary mediastinal B-cell lymphoma (Scott et al. Blood 2014; Mottok et al. Blood 2018). The DHITsig roughly doubled the number of cases in the HGBL-DH/TH-group compared with FISH, and was associated with a poor prognosis in patients treated with R-CHOP. In our study we aimed to validate the DLBCL90 assay in an independent cohort of young high-risk patients treated with dose-intensive immunochemotherapy within two Nordic trials. Patients and methods: RNA was extracted from pretreatment FFPE biopsies from 88 high-risk de novo DLBCL patients treated with dose-dense immunochemotherapy with systemic CNS prophylaxis in two Nordic trials (Holte et al. Ann. Oncol. 2013; Leppä et al. 15-ICML 2019). In the first trial patients received 6 courses of R-CHOEP-14 followed by 1 course of HD-Mtx and HD-Ara-C. In the second trial 2 courses of HD-Mtx were given in combination with R-CHOP-14 at the start of treatment, followed by 4 courses of R-CHOEP-14 and 1 course of R-HD-Ara-C at the end. Liposomal Ara-C was also administered intrathecally at courses 1, 3 and 5. Digital gene expression was performed, applying the DLBCL90 assay on the NanoString platform (NanoString Technologies, Seattle, WA) and FISH break-apart probes for MYC, BCL2 and BCL6 were used for identification of HGBL-DH/TH. Results: The COO assay assigned 54%, 31% and 15% of the tumors to the GCB, activated B-cell like (ABC) and unclassified group, respectively. The ABC- and unclassified DLBCLs showed a trend towards inferior outcome when compared to the GCB-group (OS: 59%, 66%, 91%, p=0.076, PFS: 74%, 59%, 85%, p=0.122, FFS: 63%, 51%, 83%, p=0.052, respectively). The DHITsig was only seen in the GCB subtype. Of the patients with the GCB subtype, 5 (10%) and 11 patients (23%) were assigned to the DHITsig-positive and DHITsig-indeterminate group, respectively. FISH results were available for 71 samples, and 6 were identified as HGBL-DH/TH-BCL2. Four of them were assigned to the DHITsig-positive group, while 1 case was assigned to the DHITsig-indeterminate group with a 79% probability of belonging to the DHITsig-positive group (cut-off 80%). The last case was a triple-hit tumor that was assigned to the DHITsig-negative group. This patient had a favorable outcome with a complete remission after first line therapy, and was still in remission at the last follow-up 45 months after diagnosis. Overall, after a median follow-up of 64 months, 17 patients (19%) experienced relapse and 13 patients (15%) had died. Within the GCB subgroup, there were no significant differences in clinical outcome (OS, PFS, FFS) between the DHITsig positive, negative and indeterminate groups when analyzed as separate groups, or when the positive/indeterminate groups were merged (figure). Conclusion: We confirm that the DLBCL90 gene expression assay identifies HGBL-DH/TH-BCL2 in FFPE biopsies. This, together with a rapid turnaround time, makes it an attractive alternative to FISH for double-hit assignment in the clinical setting. In our cohort of young-high risk patients treated with dose-dense immunochemotherapy, the DHITsig was not associated with inferior survival. Of note, neither was the HGBL-DH/TH defined by FISH. This may be due to the intensified treatment, overcoming the poor prognostic impact of the double-hit biology. Disclosures Jørgensen: Gilead: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees. Jerkeman:Roche: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Gilead: Honoraria, Research Funding; Acerta: Honoraria, Research Funding; Celgene: Honoraria, Research Funding. Scott:Roche/Genentech: Research Funding; Celgene: Consultancy; Janssen: Consultancy, Research Funding; NanoString: Patents & Royalties: Named inventor on a patent licensed to NanoSting [Institution], Research Funding. Holte:Novartis: Honoraria, Other: Advisory board.

Background In neuroblastoma (NB), the most powerful prognostic marker, the MYCN amplification (MNA), occasionally shows intratumoural heterogeneity (ITH), i.e. coexistence of MYCN-amplified and non-MYCN-amplified tumour cell clones, called heterogeneous MNA (hetMNA). Prognostication and therapy allocation are still unsolved issues. Methods The SIOPEN Biology group analysed 99 hetMNA NBs focussing on the prognostic significance of MYCN ITH. Results Patients 18 m: 0.67 ± 0.14, p = 0.011; metastatic: 18 m: 0.28 ± 0.09, p = 0.084). The genomic 'background’, but not MNA clone sizes, correlated significantly with relapse frequency and OS. No relapses occurred in cases of only numerical chromosomal aberrations. Infiltrated bone marrows and relapse tumour cells mostly displayed no MNA. However, one stage 4s tumour with segmental chromosomal aberrations showed a homogeneous MNA in the relapse. Conclusions This study provides a rationale for the necessary distinction between heterogeneous and homogeneous MNA. HetMNA tumours have to be evaluated individually, taking age, stage and, most importantly, genomic background into account to avoid unnecessary upgrading of risk/overtreatment, especially in infants, as well as in order to identify tumours prone to developing homogeneous MNA.

Purpose To report a bilateral uveal melanoma detected in a micrometastasis study. Method Case report. Results At enucleation of a circumpapillary amelanotic mixed melanoma in a patient with ocular melanocytosis, a pigmented lesion in the other eye was detected, thought to be a naevus. BAP1 was positive showing nuclear staining of the tumour cells. Seven years later the naevus showed growth and development of a retinal detachment. FNAB disclosed monosomy 3 in the spindle tumour cells. Conclusion A case of bilateral melanoma with long-term survival without metastatic diseases is reported. Different gene expressions in the two eyes were revealed. The case is a reminder that follow-up over years is essential in patients with a uveal melanoma, especially with ocular melanocytosis.

Background: The prognostic impact of segmental chromosome alterations (SCAs) in children older than 1 year, diagnosed with localised unresectable neuroblastoma (NB) without MYCN amplification enrolled in the European Unresectable Neuroblastoma (EUNB) protocol is still to be clarified, while, for other group of patients, the presence of SCAs is associated with poor prognosis. Methods: To understand the role of SCAs we performed multilocus/pangenomic analysis of 98 tumour samples from patients enrolled in the EUNB protocol. Results: Age at diagnosis was categorised into two groups using 18 months as the age cutoff. Significant difference in the presence of SCAs was seen in tumours of patients between 12 and 18 months and over 18 months of age at diagnosis, respectively (P = 0.04). A significant correlation (P = 0.03) was observed between number of SCAs per tumour and age. Event-free (EFS) and overall survival (OS) were calculated in both age groups, according to both the presence and number of SCAs. In older patients, a poorer survival was associated with the presence of SCAs (EFS = 46% vs 75%, P = 0.023; OS = 66.8% vs 100%, P = 0.003). Moreover, OS of older patients inversely correlated with number of SCAs (P = 0.002). Finally, SCAs provided additional prognostic information beyond histoprognosis, as their presence was associated with poorer OS in patients over 18 months with unfavourable International Neuroblastoma Pathology Classification (INPC) histopathology (P = 0.018). Conclusions: The presence of SCAs is a negative prognostic marker that impairs outcome of patients over the age of 18 months with localised unresectable NB without MYCN amplification, especially when more than one SCA is present. Moreover, in older patients with unfavourable INPC tumour histoprognosis, the presence of SCAs significantly affects OS.

A 15-month-old boy was diagnosed with malignant hypertension caused by a catecholamine excreting retroperitoneal paraganglioma consisting of a composite phaeochromocytoma/differentiating neuroblastoma. After alpha-blockade the tumor was excised. No adjuvant treatment was given, and he is doing well eight years after the diagnosis. The patient is the first child known to have an extra-adrenal retroperitoneal composite tumor, and also the youngest child with a composite phaeochromocytoma/neuroblastoma reported in the English literature.

The tumour microenvironment influences outcome in patients with follicular lymphoma (FL), but its impact on transformation is less studied. We investigated the prognostic significance of the tumour microenvironment on transformation and survival in FL patients treated in the rituximab era. We examined diagnostic and transformed biopsies from 52 FL patients using antibodies against CD3, CD4, CD8, CD21 (CR2), CD57 (B3GAT1), CD68, FOXP3, TIA1, PD-1 (PDCD1), PD-L1 (CD274) and PAX5. Results were compared with a second cohort of 40 FL patients without signs of transformation during a minimum of five years observation time. Cell numbers and localization were semi-quantitatively assessed. Better developed CD21+ follicular dendritic cell (FDC) meshworks at diagnosis was a negative prognostic factor for overall survival (OS), progression-free survival (PFS) and time to transformation (TTT) in patients with subsequently transformed FL. Remnants of FDC meshworks at transformation were associated with shorter OS and PFS from transformation. High degrees of intrafollicular CD68+ and PD-L1+ macrophage infiltration, high total area scores and an extrafollicular/diffuse pattern of FOXP3+ T cells and high intrafollicular scores of CD4+ T cells at diagnosis were associated with shorter TTT. Scores of several T-cell subset markers from the combined patient cohorts were predictive for transformation, especially CD4 and CD57.

Background Of 4,706 peripheral neuroblastic tumors (pNTs) registered on the Children’s Cancer Group and Children’s Oncology Group Neuroblastoma Study between 1989 and 2010, 51 cases (1.1%) had genotype-phenotype discordance characterized by MYCN amplification (indicating poor prognosis) and Favorable Histology (indicating better prognosis).

BACKGROUND The clinical courses of chronic lymphocytic leukaemia (CLL) are very heterogeneous. Biological markers that provide good prognostic information at the time of diagnosis are available. The aim of the study was to determine the prevalence of these markers in a population-based material. MATERIAL AND METHOD Biological markers were examined using standard laboratory methods after obtaining an informed consent statement from patients diagnosed with chronic lymphocytic leukaemia in the period 1.10.2007-31.12.2009. RESULTS There were 388 new cases of chronic lymphocytic leukaemia during the study period, and 236 patients (61%) were included in the study. Of 222 patients, 178 (80%) were in Binet's stage A, 26 (12%) in stage B and 18 (8%) in stage C. The V(H) gene was mutated in 69% and unmutated in 31% of cases. Cytogenetic aberrations were found in 68%: del(13q14) in 48%, trisomy 12 in 13%, del(11q22) in 10% and del(17p13) in 7%. CD38-positive disease was found in 28% of the patients. The V(H) gene was mutated in 67% of the patients in Binet's stage A, and in the majority of these a mutated V(H) gene was associated with non-expression of CD38 and del(13q14). INTERPRETATION At the time of diagnosis, most patients are asymptomatic and do not need treatment. The biological markers that indicate a favourable prognosis occur most frequently in this group. Markers that indicate a poor prognosis occur more frequently in the group that has symptoms at the time of diagnosis.

BACKGROUND: In neuroblastoma (NB), the presence of segmental chromosome alterations (SCAs) is associated with a higher risk of relapse. METHODS: In order to analyse the role of SCAs in infants with localised unresectable/disseminated NB without MYCN amplification, we have performed an array CGH analysis of tumours from infants enrolled in the prospective European INES trials. RESULTS: Tumour samples from 218 out of 300 enroled patients could be analysed. Segmental chromosome alterations were observed in 11%, 20% and 59% of infants enroled in trials INES99.1 (localised unresectable NB), INES99.2 (stage 4s) and INES99.3 (stage 4) (P

We present seven cases of mantle cell lymphoma with morphological features of marginal-zone lymphoma. Of particular interest, four of the patients had predominant involvement of the gastrointestinal tract. All cases displayed the translocation t(11;14)(q13;q32) and expressed cyclin D1. Cytogenetic analysis revealed trisomy 3 in one case and somatic hypermutation of immunoglobulin heavy genes could be demonstrated in two out of four cases. The latter features are reminiscent of marginal-zone lymphoma. The localization of these lymphomas mainly in the gastrointestinal tract and the higher exposure to antigens in this area may explain why this variant of mantle cell lymphoma harbours features of marginal-zone lymphoma.

Background: The first phase of the MetAction trial established the required diagnostic infrastructure, implemented security-approved systems for handling of sensitive information, educated the Trial Team within the context of tumor boards, and estimated costs of the initiative within public health services. The endeavor enabled expedited and safe mutation profiling of metastatic tumors in order to offer molecularly matched medication for end-stage cancer (Ree et al., ESMO Open 2017;2:e000158). The aim of the second trial phase was to investigate the utility of the MetAction pipeline in clinical practice. Procedures: An eligible patient with end-stage metastatic disease from any origin had been on the previous line of systemic therapy for 6 or more weeks with radiologic evaluation intervals of 6-12 weeks and disease progression according to the Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1. Biopsy-sampled metastatic tissue was analyzed by DNA sequencing (Ion Oncomine™ Comprehensive Panel), where called variants were filtered prior to assessment and prioritization, supplemented with fluorescence in situ hybridization to cover relevant biomarkers. The Molecular Tumor Board interpreted the findings within the likelihood of signaling pathway activity, for the sequential Clinical Tumor Board to conclude on potential systemic tumor-directed medication. On study therapy, radiologic work-up was performed every 8 weeks. The primary objective was to compare progression-free survival (PFS) on study treatment, termed Period-B, with PFS for the most recent therapy, termed Period-A. If Period-B/Period-A was ≥1.3, the study therapy was deemed to be of benefit. The incidence of diagnostic adverse events and treatment-related grade 3-5 Common Terminology Criteria for Adverse Events (CTCAE) toxicities was secondary end points. Results: 26 patients were enrolled. Biopsy procedures were undertaken at lung or pleural sites (6 cases), liver or peritoneal sites (19 cases), and an inguinal lymph node (1 case), and did not cause adverse events. Histologic entities were 18 adenocarcinomas (AC), 2 undifferentiated carcinomas, 1 case each of cholangiocarcinoma and squamous cell carcinoma, and 4 different sarcoma entities. 13 patients were found eligible for off-label use of molecularly matched therapy (inhibitor of ALK-, BRAF-, EGFR-, FGFR-, mTOR-, PARP-, ROS1-, or PD-1-mediated signaling). Among the 10 individuals who received study treatment until radiologic evaluation, 5 met the primary end point. The patient with cholangiocarcinoma and a patient with rectal AC primaries, both given crizotinib, obtained Period-B/Period-A outcome slightly better than 1.3. Notably, 3 patients with colon AC primaries, receiving either a combination of panitumumab with vemurafenib or chemotherapy or single-agent pembrolizumab, obtained long-lasting responses. In addition, 1 colon AC patient receiving pembrolizumab with RECIST progression (i.e., primary end point failure) before a long-lasting response to off-protocol continuation, reported CTCAE grade 3 toxicity (a colitis event that immediately resolved on high-dose prednisolone). Conclusion: MetAction procedures and treatments were safe. 15% (4/26) of patients with progressing end-stage cancer had the disease course substantially reversed by this biomarker-directed therapy approach. Citation Format: Anne Hansen Ree, Kjersti Flatmark, Vigdis Nygaard, Daniel Heinrich, Kjetil Boye, Svein Dueland, Vegard Nygaard, Eivind Hovig, Klaus Beiske, Marius Lund-Iversen, Vivi A. Flørenes, Christin Johansen, Inger Riise Bergheim, Menaka Sathermugathevan, Sigve Nakken, Gry A. Geitvik, Ole C. Lingjærde, Anne-Lise Børresen-Dale, Hege G. Russnes, Gunhild M. Mælandsmo. The MetAction trial: long-lasting responses to molecularly matched therapy in end-stage cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A101.

Summary Background More accurate prognostic assessment of patients with neuroblastoma is required to better inform the choice of risk-related therapy. The aim of this study is to develop and validate a gene-expression signature to improve outcome prediction. Methods 59 genes were selected using an innovative data-mining strategy, and were profiled in the largest neuroblastoma patient series (n=579) to date using real-time quantitative PCR starting from only 20 ng of RNA. A multigene-expression signature was built using 30 training samples, tested on 313 test samples, and subsequently validated in a blind study on an independent set of 236 tumours. Findings The signature has a performance, sensitivity, and specificity of 85·4% (95% CI 77·7–93·2), 84·4% (66·5–94·1), and 86·5% (81·1–90·6), respectively, to predict patient outcome. Multivariate analysis indicates that the signature is a significant independent predictor of overall survival and progression-free survival after controlling for currently used risk factors: patients with high molecular risk have a higher risk of death from disease and higher risk of relapse or progression than patients with low molecular risk (odds ratio 19·32 [95% CI 6·50–57·43] and 3·96 [1·97–7·97] for overall survival and progression-free survival, respectively, both p MYCN status, age, International Neuroblastoma Staging System stage, ploidy, International Neuroblastoma Pathology Classification grade of differentiation, and mitosis karyorrhexis index (odds ratios between 4·81 and 10·53 depending on the model for overall survival and 3·68 [95% CI 2·01–6·71] for progression-free survival). Interpretation The 59-gene expression signature is an accurate predictor of outcome in patients with neuroblastoma. The signature is an independent risk predictor, identifying patients with an increased risk of poor outcome in the current clinical-risk groups. The method and signature is suitable for routine laboratory testing, and should be evaluated in prospective studies. Funding The Belgian Foundation Against Cancer, the Children Cancer Fund Ghent, the Belgian Society of Paediatric Haematology and Oncology, the Belgian Kid's Fund and the Fondation Nuovo-Soldati (JV), the Fund for Scientific Research Flanders (KDP, JH), the Fund for Scientific Research Flanders, the Institute for the Promotion of Innovation by Science and Technology in Flanders, Strategisch basisonderzoek, the Fondation Fournier Majoie pour l'Innovation, the Instituto Carlos III, the Italian Neuroblastoma Foundation, the European Community under the FP6, and the Belgian programme of Interuniversity Poles of Attraction.

BACKGROUND: Large granular lymphocytic leukaemia (LGL-leukaemia) is considered a rare disease. LGL-leukaemia is usually of the T-cell type, but a minority displays an NK-cell phenotype. Incidence and prevalence are unknown. MATERIAL AND METHODS: We identified patients with LGL-leukaemia (with well-defined diagnostic criteria) diagnosed at Rikshospitalet University Hospital between 01.10.2001 and 31.12.2007. Their medical records were assessed retrospectively. RESULTS: LGL-leukaemia was diagnosed in 52 patients, 26 women and 26 men, median age of 59 (26 - 86) years, during the study period. The leukaemia displayed NK-cell phenotype in one patient and T-cell phenotype in the remaining 51 patients. Slightly more than one third of the patients were asymptomatic. Cytopenia, mostly neutropenia, was usually the cause of the clinical phenotype in symptomatic patients. Co-morbidity with autoimmune disease was common, and we also found a high prevalence of clonal B-cell disease (17 %). INTERPRETATION: Our data support the notion that LGL-leukaemia is under-diagnosed. Unexplained cytopenias should suggest the possibility of LGL-leukaemia, and appropriate diagnostic measures should be undertaken. An early diagnosis may save patients an extensive and unnecessary diagnostic work-up and ensure that a simple and effective treatment is offered.

Aims: To investigate the relationship between phenotype and genotype in oligodendroglial tumours and evaluate whether 1p/19q status can be reliably predicted from histological findings. Methods and results: Three neuropathologists reviewed the association between 10 histological variables, location and genetic losses at 1p, 19q and 17p13 in 63 oligodendroglial tumours (cohort 1). Based on these findings, a multiple logistic regression model for prediction of 1p/19q status was constructed. The ability of this model to predict 1p/19q status was tested on cohort 2, comprising 20 oligodendroglial tumours. Loss of heterozygosity at 1p, 19q and 17p13 was analysed using polymerase chain reaction. Combined 1p/19q loss and losses at 17p13 were mutually exclusive (P

Chronic cold agglutinin disease (CAD) is a subgroup of autoimmune hemolytic anemia. Primary CAD has traditionally been defined by the absence of any underlying or associated disease. The results of therapy with corticosteroids, alkylating agents and interferon-alpha have been poor. Cold reactive immunoglobulins against erythrocyte surface antigens are essential to pathogenesis of CAD. These cold agglutinins are monoclonal, usually IgMkappa autoantibodies with heavy chain variable regions encoded by the V(H)4-34 gene segment. By flowcytometric and immunohistochemical assessments, a monoclonal CD20+kappa+B-lymphocyte population has been demonstrated in the bone marrow of 90% of the patients, and lymphoplasmacytic lymphoma is a frequent finding. Novel attempts at treatment for primary CAD have mostly been directed against the clonal B-lymphocytes. Phase 2 studies have shown that therapy with the chimeric anti-CD20 antibody rituximab produced partial response rates of more than 50% and occasional complete responses. Median response duration, however, was only 11 months. In this review, we discuss the clinical and pathogenetic features of primary CAD, emphasizing the more recent data on its close association with clonal lymphoproliferative bone marrow disorders and implications for therapy. We also review the management and outline some perspectives on new therapy modalities.

Background: Reliable detection of neuroblastoma cells in bone marrow (BM) is critical because BM involvement influences staging, risk assessment, and evaluation of therapeutic response in neuroblastoma patients. Standard cytomorphologic examination of BM aspirates is sensitive enough to detect single tumor cells. Consequently, more sensitive and specific detection methods are indispensable. Methods: We used real-time quantitative reverse transcription-PCR (QPCR) of the tyrosine hydroxylase (TH), GD2 synthetase (GALGT), and embryonic lethal, abnormal vision, Drosophila-like 4 (ELAVL4) genes to detect disseminated neuroblastoma cells. We assessed assay sensitivity by addition experiments and then analyzed 97 neuroblastic tumor, BM, peripheral blood (PB), or peripheral blood stem cell (PBSC) samples from 30 patients. The QPCR results were compared with those of a standardized immunocytochemical assay. Results: The molecular markers were highly expressed in all evaluated tumor samples. In addition, 32%, 11%, and 38% of all BM, PB, and PBSC samples scored positive for TH, GALGT, or ELAVL4, respectively. The TH and ELAVL4 assays could detect 1 neuroblastoma cell in 106 mononuclear cells. By contrast, the GALGT QPCR assay could detect 1 neuroblastoma cell in 104 mononuclear cells. We assessed the potential prognostic value of TH, GALGT, and ELAVL4 QPCR by analyzing subsequent samples from 3 patients with stage 4 disease. Preliminary results indicated that persistence of high ELAVL4 expression has prognostic value. Conclusions: ELAVL4 QPCR can be used to detect residual neuroblastoma cells in clinical samples. However, combination of several molecular markers and screening techniques should be considered to ensure reliable detection of rare neuroblastoma cells.

e14033 Background: The MetAction project consists of two clinical trial phases. The completed first phase established the required diagnostic infrastructure, implemented security-approved systems for handling of sensitive information, educated the entire project staff within the context of tumor boards, and estimated costs of the initiative within public health services. The endeavor enabled expedite and safe mutation profiling of metastatic tumors in order to offer biomarker-based treatment with molecularly matched medication to patients with end-stage cancer, as reported in Ree et al., ESMO Open 2017. The ongoing second trial phase investigates the feasibility of the established MetAction pipeline in clinical practice. Methods: An eligible patient has end-stage metastatic disease from a solid cancer. Metastatic tissue is analyzed by DNA sequencing (Ion Oncomine™ Comprehensive Panel), where called variants are filtered prior to assessment and prioritization, supplemented with fluorescence in situ hybridization to cover relevant biomarkers. The Molecular Tumor Board interprets the findings within the likelihood of signaling pathway activity, and the sequential Clinical Tumor Board (CTB) may conclude on treatment with any systemic tumor-directed medication. Results: At the time of writing, 19 patients enrolled onto the second trial phase have accomplished the diagnostic procedures from sampling of metastatic tissue to CTB conclusion. Biopsy procedures were undertaken at lung or pleural sites (five cases), liver or superficial or deep peritoneal sites (13 cases), and an inguinal lymph node (one case) and did not cause adverse events. Histologic entities were 12 adenocarcinomas and one case each of squamous cell and undifferentiated carcinoma, cholangiocarcinoma, and four different sarcoma entities. Twelve patients have been found eligible for off-label use of molecularly matched therapy (inhibitor of ALK-, BRAF-, EGFR-, FGFR-, mTOR-, PARP-, or PD-1-mediated signaling). Conclusions: We will report on patient outcome (progression-free survival, overall response rate, and tolerance) to this biomarker-directed treatment in end-stage cancer. Clinical trial information: NCT02142036.

Objective Through the conduct of an individual-based intervention study, the main purpose of this project was to build and evaluate the required infrastructure that may enable routine practice of precision cancer medicine in the public health services of Norway, including modelling of costs. Methods An eligible patient had end-stage metastatic disease from a solid tumour. Metastatic tissue was analysed by DNA sequencing, using a 50-gene panel and a study-generated pipeline for analysis of sequence data, supplemented with fluorescence in situ hybridisation to cover relevant biomarkers. Cost estimations compared best supportive care, biomarker-agnostic treatment with a molecularly targeted agent and biomarker-based treatment with such a drug. These included costs for medication, outpatient clinic visits, admission from adverse events and the biomarker-based procedures. Results The diagnostic procedures, which comprised sampling of metastatic tissue, mutation analysis and data interpretation at the Molecular Tumor Board before integration with clinical data at the Clinical Tumor Board, were completed in median 18 (8-39) days for the 22 study patients. The 23 invasive procedures (12 from liver, 6 from lung, 5 from other sites) caused a single adverse event (pneumothorax). Per patient, 0–5 mutations were detected in metastatic tumours; however, no actionable target case was identified for the current single-agent therapy approach. Based on the cost modelling, the biomarker-based approach was 2.5-fold more costly than best supportive care and 2.5-fold less costly than the biomarker-agnostic option. Conclusions The first project phase established a comprehensive diagnostic infrastructure for precision cancer medicine, which enabled expedite and safe mutation profiling of metastatic tumours and data interpretation at multidisciplinary tumour boards for patients with end-stage cancer. Furthermore, it prepared for protocol amendments, recently approved by the designated authorities for the second study phase, allowing more comprehensive mutation analysis and opportunities to define therapy targets.

Primary chronic cold agglutinin disease is a rare hemolytic disease mediated by monoclonal IGHV4-34-encoded cold agglutinins with a predominant specificity for the blood group antigen I. Bone marrow from 54 patients was studied to type the underlying lymphoproliferative disorder better. Bone marrow biopsies showed circumscribed intra-parenchymatous nodules with small monotonous monoclonal B cells in 40/54 patients (median infiltration: 10% of marrow cells) with a CD20(+), IgMs(+), IgDs(+), CD27(+), CD5(-/+), CD11c(-), CD23(-), CD38(-) immunophenotype. Neither plasmacytoid cytological features nor expression of plasma cell differentiation-associated transcription factors MUM1, XBP1 and BLIMP1 were noted in these B cells. However, a limited number of mature monoclonal IgM(+), IgD(-) plasma cells were present outside the lymphoid nodules and were diffusely scattered throughout the marrow. Of interest, the MYD88 L265P mutation, typical of lymphoplasmacytic lymphoma, was not detected (17/17 cases). Somatically mutated monoclonal IGHV4-34 gene rearrangement was demonstrated in eight patients with frozen samples (mean sequence homology 95.4%). However, mutations of BCL6 intron 1 were not demonstrated, except in one patient, suggesting that the lymphoma cells had not matured in the germinal center. In conclusion, cold agglutinin-associated lymphoproliferative disease displays homogeneous histological and immunophenotypic features. The absence of plasmacytoid cells, the presence of plasma cells predominantly outside the nodular lymphoid infiltrates, IGHV4-34 restriction and absence of MYD88 L265P mutation strongly suggest that cold agglutinin-associated lymphoproliferative disease is a distinct entity that is different from lymphoplasmacytic lymphoma.

Immunocytochemical detection (ICC) of isolated tumor cells in bone marrow (BM) is currently the most established method for monitoring early dissemination in epithelial cancer. However, the low sample size that can practically be analyzed restricts the sensitivity and reliability of the ICC method. To be able to analyze larger samples, a negative immunomagnetic separation (IMS) technique, utilising anti-CD45-conjugated Dynabeads, has been developed. Tumor-cell enrichment by depletion of CD45-expressing mononuclear cells (MNC) is followed by ICC for detection of the cytokeratin (CK)-positive (+) epithelial cells. In this study, bone-marrow samples (n = 165) and peripheral-blood-progenitor-cell (PBPC) apheresis products (n = 22) from breast-cancer patients were analyzed. The negative IMS analysis of 1 to 2 x 10(7) MNC was compared with ICC analysis of 2 x 10(6) unseparated MNC. Negative IMS resulted in 85% mean depletion of MNC. The results showed that 11.7% of the samples were positive by ICC analysis of unseparated MNC, as compared with 23.5% after negative IMS. In samples presenting10 CK+ cells, a 4-fold higher number of positive cells was detected by the negative IMS technique. Moreover, there was no evidence for general enrichment of false-positive cells. Altogether our results show that negative IMS is an efficient enrichment method for sensitive detection of CK+ cells in BM/PBPC products from breast-cancer patients. This opens the possibility for further characterization of micrometastatic tumor cells.

Detection of isolated tumour cells (TCs) in bone marrow (BM) from epithelial cancer patients by immunocytochemical (ICC) analysis seems to predict future relapse, but the reported percentages of positive BMs among patients with localized cancer show large variations and the number of detected TCs is low. This emphasizes the importance of thoroughly testing the methods in use. This study was performed to clarify to what extent positive staining of haematopoietic cells (HCs) interferes with the ICC detection of epithelial cells in BM. BM mononuclear cells (MNCs) from normal donors and stage I-II breast cancer patients were stained with anti-cytokeratin (CK) and isotype control monoclonal antibodies (MAbs) followed by alkaline phosphatase (AP)-based visualization of immunolabelled cells. In the ICC staining of normal donors by the anti-CK MAbs AE1/AE3 or A45-B/B3, rare immunoreactive cells were detected in 7/20 and 8/19 BMs, respectively. Morphological examination recognized all these cells as typical HCs. In the breast cancer patients (n = 257), anti-CK-positive cells were detected in 26.6 per cent, excluding cells with HC morphology. Using the same morphological criteria, isotype control-positive cells were detected in 5.4 per cent of patients. Some of the false-positive events were further analysed and cells with strong reactivity against the AP enzyme alone were detected. Double ICC staining recognized the majority of these AP directly-reactive cells as CD45-negative and human Ig kappa/lambda-positive, in accordance with the phenotype of mature plasma cells. Morphological evaluation and adequate controls are important to ensure the diagnostic specificity of micrometastases in BM. It is recommended that the number of BM MNCs included in negative controls should equal the number of cells in the diagnostic specimens.

There is a well-known association in male patients between mediastinal germ cell tumors (GCT) and hematologic malignancies, with a propensity towards acute megakaryoblastic leukemia. These rare malignancies have been shown to share a common clonal origin, often deduced from the finding of isochromosome 12p, i(12p), in cells from both the solid tumor and the leukemia, and thus are now known to represent different manifestations of the same clonal process. We treated a young female patient with a malignant phyllodes tumor followed by an acute megakaryoblastic leukemia and found several of the same marker chromosomes by karyotype analysis of cells from both the tumor and the leukemia implying a common clonal origin of the two. To the best of our knowledge, this has not been demonstrated in phyllodes tumors before, but indicates that the same type of leukemization may occur of this tumor as has been described in mediastinal GCT.

Background In children older than 1 year with localised unresectable neuroblastoma (NB), treatment strategies are heterogeneous according to the national groups. The objective of this phase III non-randomised study was to evaluate the efficacy of conventional chemotherapy followed by surgery. Patients and methods In the presence of surgical risk factors (SRF), six courses of chemotherapy alternating Carboplatin–Etoposide and Vincristin–Cyclophosphamide–Doxorubicin were given, and surgical resection was attempted after four. Survival analyses were performed using an intention-to-treat approach. The main objective was to achieve a 5-year survival over 80%. Results Out of 191 registered children, 160 were evaluable. There were 62.5% older than 18 months and 52.5% had unfavourable histology according to International Neuroblastoma Pathology Classification (INPC). Chemotherapy reduced the number of SRFs by one third. Delayed surgery was attempted in 86.3% of patients and was complete or nearly complete in 74%. The 5-year EFS and OS were 76.4% and 87.6% respectively, with significant better results for patients younger than 18 months or with favourable histology. Conclusion This strategy provides encouraging results in children older than 1 year or 12 months with localised unresectable NB without MYCN amplification. However, in children older than 18 months and with unfavourable histology, additional treatment is recommended.

Background Metastatic progression due to development or enrichment of therapy-resistant tumor cells is eventually lethal. Molecular characterization of such chemotherapy resistant tumor cell clones may identify markers responsible for malignant progression and potential targets for new treatment. Here, in a case of stage IV adenocarcinoma of the gastroesophageal junction, we report the successful genome wide analysis using array comparative genomic hybridization (CGH) of DNA from only fourteen tumor cells using a bead-based single cell selection method from a bone metastasis progressing during chemotherapy. Case presentation In a case of metastatic adenocarcinoma of the gastroesophageal junction, the progression of bone metastasis was observed during a chemotherapy regimen of epirubicin, oxaliplatin and capecitabine, whereas lung-, liver and lymph node metastases as well as the primary tumor were regressing. A bone marrow aspirate sampled at the site of progressing metastasis in the right iliac bone was performed, and single cell molecular analysis using array-CGH of Epithelial Specific Antigen (ESA)-positive metastatic cells, and revealed two distinct regions of amplification, 12p12.1 and 17q12-q21.2 amplicons, containing the KRAS (12p) and ERBB2 (HER2/NEU) (17q) oncogenes. Further intrapatient tumor heterogeneity of these highlighted gene copy number changes was analyzed by fluorescence in situ hybridization (FISH) in all available primary and metastatic tumor biopsies, and ErbB2 protein expression was investigated by immunohistochemistry. ERBB2 was heterogeneously amplified by FISH analysis in the primary tumor, as well as liver and bone metastasis, but homogenously amplified in biopsy specimens from a progressing bone metastasis after three initial cycles of chemotherapy, indicating a possible enrichment of erbB2 positive tumor cells in the progressing bone marrow metastasis during chemotherapy. A similar amplification profile was detected for wild-type KRAS, although more heterogeneously expressed in the bone metastasis progressing on chemotherapy. Correspondingly, the erbB2 protein was found heterogeneously expressed by immunohistochemical staining of the primary tumor of the gastroesophageal junction, while negative in liver and bone metastases, but after three initial cycles of palliative chemotherapy with epirubicin, oxaliplatin and capecetabine, the representative bone metastasis stained strongly positive for erbB2. Conclusion Global analysis of genetic aberrations, as illustrated by performing array-CGH analysis on genomic DNA from only a few selected tumor cells of interest sampled from a progressing bone metastasis, can identify relevant therapeutic targets and genetic aberrations involved in malignant progression, thus emphasizing the importance and feasibility of this powerful tool on the road to more personalized cancer therapies in the future.

We analyzed the relationships among clinical variables, histology, 1p/19q status, and outcome in 95 patients with oligodendroglial tumors. The studyenrolled adult patients who underwent firsttime surgery for a supratentorial oligodendroglial tumor at Oslo University Hospital, Rikshospitalet. Tumors were: 27 oligodendrogliomas, WHO grade II; 32 oligoastrocytomas, WHO grade II; 16 anaplastic oligodendrogliomas, WHO grade III; 14 anaplastic oligoastrocytomas, WHO grade III; and 6 glioblastomas with a major oligodendroglial component, WHO grade IV. The clinical files were reviewed. Three neuropathologists evaluated the histological slides independently. Loss-of-heterozygosity analysis for 1p and 19q was performed by PCR. Favorable prognostic factors from univariate analyses included seizures as presenting symptom, female sex, location in the frontal lobe, low WHO grade, classic histology, absence of gemistocytic cells, and combined 1p/19q loss. Solitary 19q loss was a negative prognostic marker. 1p/19q status was of prognostic significance in both tumors with classic and nonclassic oligodendroglial histology. In the multivariate analysis, WHO grade II (P < .001), frontal tumor location (P 5 .002), and combined 1p/19q loss (P < .001) remained favorable prognostic variables. Our results suggest that tumor location, WHO grade, and 1p/19q status are important independent variables associated with survival in oligodendroglial tumors. The study suggests that solitary 19q loss is a negative prognostic variable and that 1p/19q loss is associated with prolonged survival also in oligodendroglial tumors without classic histology.

Purpose: Precise and comprehensive analysis of neuroblastoma genetics is essential for accurate risk evaluation and only pangenomic/multilocus approaches fulfill the present-day requirements. We present the establishment and validation of the PCR-based multiplex ligation-dependent probe amplification (MLPA) technique for neuroblastoma. Experimental Design: A neuroblastoma-specific MLPA kit was designed by the SIOP Europe Neuroblastoma Biology Committee in cooperation with MRC-Holland. The contained target sequences cover 19 chromosomal arms and reference loci. Validation was performed by single locus and pangenomic techniques (n = 174). Dilution experiments for determination of minimal tumor cell percentage were performed and testing of reproducibility was checked by interlaboratory testing (n = 15). Further 156 neuroblastomas were used for establishing the amplification cutoff level. Results: The MLPA technique was tested in 310 neuroblastomas and 8 neuroblastoma cell lines (including validation and amplification cutoff level testing). Intertechnique validation showed a high concordance rate (99.5%). Interlaboratory MLPA testing (κ = 0.95, P < 0.01) revealed 7 discrepant of 1,490 results (0.5%). Validation by pangenomic techniques showed a single discordance of 190 consensus results (0.5%). The test results led to formulation of interpretation standards and to a kit revision. The minimal tumor cell percentage was fixed at 60%. Conclusions: The recently designed neuroblastoma-specific MLPA kit covers all chromosomal regions demanded by the International Neuroblastoma Risk Group for therapy stratification and includes all hitherto described genetic loci of prognostic interest for future studies and can be modified or extended at any time. Moreover, the technique is cost effective, reliable, and robust with a high interlaboratory and intertechnique concordance. Clin Cancer Res; 17(4); 792–804. ©2011 AACR.

Most patients diagnosed with primary chronic cold agglutinin disease (CAD) have a clonal lymphoproliferative bone marrow disorder. Treatment with rituximab is the only well-documented effective therapy, leading to 45%-60% partial responses (PR). Complete responses (CR) are rare, and median response duration is only 11 months. In a prospective multicenter trial, 29 patients received rituximab 375 mg/m2 on days 1, 29, 57 and 85; and fludarabine orally, 40 mg/m2 on days 1-5, 29-34, 57-61 and 85-89. Twenty-two patients (76%) responded, 6 (21%) achieving CR and 16 (55%) PR. Among 10 patients nonresponsive to rituximab monotherapy, 1 achieved CR and 6 PR. Median increase in hemoglobin level was 3.1 g/dL among the responders and 4.0 g/dL in those who achieved CR. Lower quartile of response duration was not reached after 33 months. Estimated median response duration was more than 66 months. Grade 3-4 hematologic toxicity occurred in 12 patients (41%). In conclusion, fludarabine and rituximab combination therapy is very efficient in patients with CAD. Toxicity may be a concern, and benefits should be carefully weighed against risks in very old and comorbid patients. It remains to be established whether the combination should be first-line or an efficient second-line therapy in CAD patients requiring treatment. This study is registered at http://www.clinicaltrials.gov as NCT00373594.

Detection of isolated tumor cells (TC) in BM from carcinoma patients can predict future relapse. Various molecular and immunocytochemical (ICC) methods have been used to detect these cells, which are present at extremely low frequencies of 10(-5) - 10(-6). The specificity and sensitivity of these techniques may vary widely. In 1996, a European ISHAGE Working Group was founded to standardize and optimize procedures used for the detection of minimal residual disease. We have attempted to develop objective criteria for the evaluation of immunocytochemically identifiable cancer cells.An interlaboratory ring experiment was performed, to compare the screening and detection of micrometastasis-positive events between different laboratories. The discrepant results induced us to establish a common consensus on morphological criteria applicable to the identification of immunostained micrometastatic TC.Bared on this consensus evaluation, we propose a classification of stained elements into three groups: (1) 'TC's show pathognomonic signs of epithelial TC-nature, as defined by a clearly enlarged nucleus or clusters ofor = 2 immunopositive cells. (2) 'Probable TC's represent morphological overlap between hematopoietic cells (HC) and TC which lack pathognomonic signs of TC-nature, but do not exhibit clear morphological features of HC. These cells are considered as TC if control staining with an isotype-specific, unrelated Ab is negative. (3) 'TC-negative' cells are defined as 'false positive' HC, skin squamous epithelial cells and artefacts.The proposed classification of immunostained events is a first step towards the development of standardized immunocytochemical assays for the detection of occult micrometastatic TC in BM or blood.

Disseminating disease is a predictive and prognostic indicator of poor outcome in children with neuroblastoma. Its accurate and sensitive assessment can facilitate optimal treatment decisions. The International Neuroblastoma Risk Group (INRG) Task Force has defined standardised methods for the determination of minimal disease (MD) by immunocytology (IC) and quantitative reverse transcriptase-polymerase chain reaction (QRT-PCR) using disialoganglioside G(D2) and tyrosine hydroxylase mRNA respectively. The INRG standard operating procedures (SOPs) define methods for collecting, processing and evaluating bone marrow (BM), peripheral blood (PB) and peripheral blood stem cell harvest by IC and QRT-PCR. Sampling PB and BM is recommended at diagnosis, before and after myeloablative therapy and at the end of treatment. Peripheral blood stem cell products should be analysed at the time of harvest. Performing MD detection according to INRG SOPs will enable laboratories throughout the world to compare their results and thus facilitate quality-controlled multi-centre prospective trials to assess the clinical significance of MD and minimal residual disease in heterogeneous patient groups.

Immunological phenotyping of acute leukaemias is important for a more precise diagnosis with respect to both cell lineage and maturation level. We have developed a rapid and reliable method for immunophenotyping, based on the use of magnetic monodisperse beads coated with monoclonal antibodies. After only a 10-min incubation of immunomagnetic beads (IMB) with mononuclear cells isolated from bone marrow or peripheral blood, the percentage of rosetting cells can be counted in the microscope. A panel of 16 monoclonal antibodies against haematopoietic cell-surface antigens was applied on 29 cases of acute myelogenic (AML) or lymphocytic (ALL) leukaemias, in order to compare immunological typing by immunomagnetic beads with immunofluorescence staining (IF). In all the cases tested, the two methods showed a virtually identical antigen distribution. The procedure described offers the advantages of being fast and simple to perform. Moreover, it has a high specificity and is easy to interpret in cases with low antigen expression.

Translocations involving an immunoglobulin (IG) locus are a recurring theme in B cell neoplasia. The rearrangements lead to the joining of an IG gene with a (proto)oncogene, whereby the latter comes under the influence of transcription-stimulating sequences in the constitutively active IG locus resulting in deregulation of the oncogene and neoplastic growth. We present here three cases of B cell neoplasia that showed a t(14;19)(q32;p13) by karyotypic analysis. Detailed molecular cytogenetic characterization of the breakpoints on chromosomes 14 and 19 in the two cases from which extra material was available, demonstrated the involvement of the immunoglobulin heavy-chain (IGH@)-variable region on chromosome 14 in both and, in one case, that the breakpoint was within the BRD4 gene on chromosome 19. Against the background of what one knows about IGH@ involvement in lymphatic malignancies, it is difficult to envisage a fusion gene with qualitatively altered protein product as the crucial pathogenetic outcome of the translocation. In spite of the fact that we found BRD4 split by the t(14;19)(q32;p13) in one of the two informative cases, we cannot be sure that this was the pathogenetically relevant target gene. Other pathogenetic possibilities could be deregulation of the neighboring NOTCH3 and/or ABHD9 genes, located distal to BRD4 in 19p13.

Combined loss of heterozygosity (LOH) on 1p and 19q is reported in 50% to 90% of oligodendroglial tumors and has emerged as a strong and favorable prognostic factor. Fluorescence in situ hybridization (FISH) and polymerase chain reaction (PCR) are the most widely used techniques. The aim of this study was to evaluate the reliability of FISH to predict LOH at 1p and 19q when performed on touch preparations from 40 oligodendroglial tumors, even if the majority of the nuclei showed chromosomal imbalance. PCR was used as the gold standard. The presence of none or one target signal was reported as FISH-LOH, whereas all other losses were defined as FISH-imbalance. The sum of nuclei with FISH-LOH and imbalance was calculated in each case (FISH-sum) and cut-off values were defined as the mean FISH-sum value in controls plus 3 standard deviations; 27.7% for 1p and 33.2% for 19q. These corresponded well with the optimal cut-off values for our data, calculated using the minimum error rate classification procedure (35.6% for 1p and 33.1% for 19q). Concurrent FISH and PCR results were encountered in 95% for 1p and 87.5% for 19q. FISH-sum was the best and simplest discriminating variable for correct classification of LOH status. Under these conditions, even a dominant population of nuclei showing FISH-imbalance represented an LOH status in the tumor cells. FISH on touch preparations is a quick and reliable method for 1p/19q testing, does not require normal DNA and can be easily performed in an immunohistochemistry unit.

SummaryCommon variable immunodeficiency (CVID) is the most frequent symptomatic primary immunodeficiency disease, characterized by low levels of circulating immunoglobulins and recurrent bacterial infections, particularly of the respiratory tract. T cell dysfunction is often present, and lymphoproliferative and autoimmune disorders as well as haematological cytopenias are frequently observed. In this study, we report a polyclonal expansion of large granular lymphocytes (LGL) in a substantial proportion of CVID patients, associated with splenomegaly, increased numbers of CD8+ T cells, inverted CD4 : CD8 T cell ratios and neutropenia. CVID patients who had both increased numbers of LGL and granulocytopenia had elevated levels of soluble Fas ligand (sFasL). Our observations indicate that CVID may be added to the list of inflammatory diseases associated with increased numbers of LGL. Furthermore, our findings suggest common pathogenic mechanisms of granulocytopenia in CVID and lymphoproliferative disease of granular lymphocytes.

Purpose To assess the prognostic value of clinical, biologic, and morphologic data in peripheral neuroblastic tumors, International Neuroblastoma Staging System (INSS) stages 2A and 2B MYCN nonamplified, a multinational protocol entitled Localized Neuroblastoma European Study Group trial 94.01, with a trial of surgery as the only treatment, was initiated in 1995. We present the prognostic value of the revised International Neuroblastoma Pathology Classification (INPC) applied to the patients included in this protocol until its closure in 1999. Materials and Methods A total of 120 neuroblastic tumors from trial patients were reviewed by the European International Society of Pediatric Oncology neuroblastoma pathology panel and assigned to a favorable or unfavorable prognostic category according to the INPC guidelines. Overall survival and relapse-free survival (RFS) were estimated by the Kaplan-Meier method and compared by the log-rank test. Results A total of 115 of 120 patients were assessable and were assigned to the favorable (90 patients; 78.3%) or unfavorable (25 patients; 21.7%) category. The 60-month survival rate was 97.7% in favorable patients compared with 73.8% in unfavorable patients (P = .0002). RFS analysis showed a 60-month relapse rate of 13.4% and 32% in favorable and unfavorable patients (P < .025), respectively. Statistical analysis demonstrated a significant association of unfavorable INPC category and high lactate dehydrogenase level (P < .045). Conclusion This European study shows for the first time that the INPC prognostic categorization has a significant impact on outcome prediction in INSS stage 2 localized peripheral neuroblastic tumors.

Standard cytomorphological examination of bone marrow (BM) aspirates does not appear to be sensitive enough to detect single neuroblastoma cells. The SIOPEN Neuroblastoma Bone Marrow Committee developed a sensitive and reproducible anti-GD2 immunocytochemical assay and introduced morphological and immunocytological criteria for the interpretation of results. Fixed cytospins were incubated with a commercially available anti-GD2 monoclonal antibody and an APAAP kit. Cells fulfilling all morphological and immunocytological criteria were called criteria-positive cells (CPCs). Not convincingly interpretable cells fulfilled some, but not all, criteria, and negative cells displayed only exclusion criteria. The genetic profile of doubtful cells was checked by fluorescence in situ hybridization. Ideally, 3 × 106 cells were analyzed to reach a 95% probability of detecting one tumor cell in 1 × 106 mononuclear cells. Four quality control rounds were organized to validate the method. A total of 111 quality control samples were analyzed. Two main improvements were achieved: in discordant cases, the range between the lowest and highest reported result was reduced by half, and discordant results were only found in samples with less than 10 CPCs per 1 × 106. This article describes the first internationally standardized protocol to detect and quantify rare neuroblastoma cells by immunocytochemistry. This method is an indispensable tool for multicenter studies evaluating the clinical significance of minimal residual disease in neuroblastoma.

While cyto- and histological screening of bone marrow samples are still accepted as the gold standard for initial staging of neuroblastoma patients, these applications are insufficient during or after therapy because it is not always possible to detect tumour cell infiltration below the level of 1% by morphology alone. For monitoring of minimal residual disease, techniques offering a considerably higher sensitivity have been developed. Immunocytology, RT-PCR and flow cytometry are most frequently used, but differ with regard to targets (single cells, RNA transcripts), measured parameters (tumour cell number, antigen expression, cytomorphology, cytogenetic aberrations, level/number of RNA transcripts), specificity (uni-/multi-parameter analysis) and sensitivity (number of investigated cells). The pros and cons of these methods are reviewed. Precise quantification of residual tumour cells in bone marrow and blood may show a future impact on risk grouping and therapeutic strategies for patients with disseminated disease, but the potential clinical application of these techniques has to be preceded by thorough standardisation and validation in multi-centre studies.

Introduction Tyrosine kinase inhibitor (TKI) induced neutropenia and thrombocytopenia may be relieved with G-CSF and trombopoietin analogs. To characterize the signaling response in a CML patient treated with hematopoietic growth factors and TKI, we employed single cell analysis of phosphoproteins in serial samples. Methods We characterized the single cell phosphoprotein responses to treatment with dasatinib, eltrombopag, and pegfilgrastim in CML-like cell lines K562 and KCL-22, normal peripheral blood leukocytes and leukocytes from a patient starting on nilotinib. These data formed a basis for comparison with serial samples (Figure 1) from a CML patients after extensive wash-out before restarted on third-choice TKI, combined with eltrombopag and pegfilgrastim. For characterization of in vivo transformational potential CML patient bone marrow cells were engrafted in duplicate in NOG mice by femoral injection. Basal phosphorylation was determined using 17 phosphorylation specific antibodies: All staining panels contain the same 4 surface antibodies and a live-dead discriminator; CD33(P67.6) PerCP-Cy5.5, CD38(HB7) PE-Cy7, CD34(581) PE, CD45(MEM-28) PE-Dynamics590 and phospho c-PARP(Asp214) AlexaFlour-700. Two phosphospecific antibodies were added to each panel with the respective direct conjugated dye Alexa Flour 488 and Alexa Flour 647. The lowest median signal for each phosphoprotein in CML cells and lymphocytes, respectively, were used as reference value for calculation of basal phosphorylation. Results A 56 y old male was diagnosed with CML (Sokal RR 0.75, Hasford RR 929, karyotype 46,XY,t(9;22)(q34;q11)[20], FISH 9q34 ABL/22q11.2 BCR dual fusion 62% of cells, PCR positive BCR-ABL e13e2/e14a2), developed prolonged thrombocytopenia and neutropenia on various dose levels of imatinib and nilotinib (neutrophile The CML bone marrow engrafted without symptomatic disease in NOG mice over 6 months monitoring, in contrast to engraftment with disseminated disease in an accelerated phase CML patient sample and the KCL-22 cell line. Phosphorylation of patient myeloid cells indicated STAT3(pY705) and STAT5(pY694) phosphorylation 2 and 3 hours after intake of eltrombopag. Dasatinib modestly inhibited SRC(pY418) and STAT3(pY705), but only transiently blunted the effect of eltrombopag. Pegfilgrastim strongly induced STAT3(pY705), STAT3(pS727), and SRC(pY418). Further analyses of cellular subsets and phospho-Abl will be presented. Conclusion Eltrombopag kept stable levels of platelets on dasatinib treatment, and cell phosphorylation reflected persisting signaling induced by eltrombopag after seven days on combination therapy. Pegfilgrastim induced strong phosphorylation of STAT3(pY705) during dasatinib co-therapy. Phosphoprotein signal analysis of patient leukemic cells reflected the hematological response of dasatinib, eltrombopag, and pegfilgrastim and may be used to monitor therapy responses of and growth factors and targeted therapy. Phosphoprotein modulated early after start of treatment. (A) Each day 1-23 is analysed separately with its pretreatment sample as control and signal baseline. (B) Various treatment days are compared, using pretreatment control at day 1 used as baseline. (A) and (B) were analysed by flow cytometry at the same day, using bar code labelling, sample processing and analysis as previously described (Skavland et al. Blood Cancer J 2012). On day 16 dasatinib and eltrombopag is administrated concomitantly. Signalling responses in lymphocytes were distinct different from myeloid cells. Further determination of cellular subsets and their signalling responses will be presented. Disclosures: Popa: KinN Therapeutics AS: Employment.

Detection of isolated tumor cells (TC) in bone marrow (BM) from patients with breast cancer is usually accomplished by immunocytochemical (ICC) analysis of up to 2 X 10(6) mononuclear cells (MNC). However, this method is cumbersome if large numbers of BM cells (i.e. > 1 X 10(7) cells) are to be analyzed. This emphasizes the need for TC enrichment strategies. This report describes immunomagnetic separation (IMS) techniques for enrichment and detection of viable breast carcinoma cells in BM and peripheral blood (PB). The positive IMS technique was performed by incubation of MNC with 2.8 microns magnetic particles (rat antimouse IgG1 M280-Dynabeads) coated with monoclonal antibody (mAb) against epithelial surface antigens. The rosetted tumor cells were then visualized by ICC staining using alkaline phosphatase-conjugated A45-B/B3 anticytokeratin mAb (Fab). The negative IMS technique was performed by incubation of MNC with anti-CD45-coated M450-Dynabeads (4.5 microns), followed by ICC staining of the nonrosetted cells. When 1000, 100, and 10 breast carcinoma cells were mixed with 1 X 10(7) MNC, an average of 748 (n = 9), 70 (n = 10), and 7.8 TC (n = 8), respectively, were detected with the positive IMS technique. With the negative IMS technique, 648 (n = 8), 57.8 (n = 6), and 7.3 TC (n = 6), respectively, were detected. The analysis of 1 X 10(7) MNC with the IMS techniques was compared with the ICC analysis of 2 X 10(6) unseparated MNC. A mean 3.7-fold (range 1.5-6.4) to 4.2-fold (2.5-8.2) (positive IMS) and 3.1-fold (range 2.0-5.0) to 3.8-fold (2.0-6.0) (negative IMS) higher TC detection frequency was achieved after enrichment by IMS in experiments with 100 and 1000 TC/10(7) MNC. The IMS techniques were used for examination of BM samples from locally advanced breast cancer patients. A 5.3-fold mean increase (range 2.1-13.3) in the number of TC detected was obtained when the use of positive and negative IMS together was compared with the direct ICC analysis of unseparated MNC (n = 11). Enrichment of TC by IMS techniques enables us to examine large numbers of MNC from BM or PB, which can result in the detection and characterization of minimal residual disease with increased sensitivity and specificity.

In this paper we present a further case of a new clinicopathological entity combining a rare type of non-Hodgkin lymphoma with a myelomonocytic leukaemia. The characteristic feature of the lymphoma is massive infiltration of the T-zones of lymph nodes by plasmacytoid cells originally described by Lennert1 in non-specific lymphadenitis. Two lymphoma cases of this type have recently been published, by Muller-Hermelink et al.2 who named the cells ‘plasmacytoid T-cells’ (PTC), and by Prasthofer et al.3 These three cases have similar clinical and pathological features and appear to form a distinct clinicopathological entity. In contrast to the two previously published cases the present lymphoma also contained irregular lymphoid cells accompanying the PTC in the lymph node lesion and focally infiltrating the bone marrow. An accumulation of polytypic IgG positive plasma cells was observed in the remaining lymph node follicles. Immunohistological analysis with a range of monoclonal antibodies showed the PTC of our case to be CD5(T1) +, CD4(T4) +, CD3(T3)-, CD8(T8)−, CD2(T11)−, and CD25(TAC)−, but HLA-DR + and transferrin receptor positive. The nature of this peculiar lymphoid lesion and its relationship to myelomonocytic leukaemia are discussed.