The current study assessed the performance of the fully automated RT-PCR-based Idylla™ GeneFusion Assay, which simultaneously covers the advanced non-small cell lung carcinoma (aNSCLC) actionable ALK , ROS1 , RET , and MET exon 14 rearrangements, in a routine clinical setting involving 12 European clinical centers. The Idylla™ GeneFusion Assay detects fusions using fusion-specific as well as expression imbalance detection, the latter enabling detection of uncommon fusions not covered by fusion-specific assays. In total, 326 archival aNSCLC formalin-fixed paraffin-embedded (FFPE) samples were included of which 44% were resected specimen, 46% tissue biopsies, and 9% cytological specimen. With a total of 179 biomarker-positive cases (i.e., 85 ALK , 33 ROS1 , 20 RET fusions and 41 MET exon 14 skipping), this is one of the largest fusion-positive datasets ever tested. The results of the Idylla™ GeneFusion Assay were compared with earlier results of routine reference technologies including fluorescence in situ hybridization, immunohistochemistry, reverse-transcription polymerase chain reaction, and next-generation sequencing, establishing a high sensitivity/specificity of 96.1%/99.6% for ALK , 96.7%/99.0% for ROS1 , 100%/99.3% for RET fusion, and 92.5%/99.6% for MET exon 14 skipping, and a low failure rate (0.9%). The Idylla™ GeneFusion Assay was found to be a reliable, sensitive, and specific tool for routine detection of ALK , ROS1 , RET fusions and MET exon 14 skipping. Given its short turnaround time of about 3 h, it is a time-efficient upfront screening tool in FFPE samples, supporting rapid clinical decision making. Moreover, expression-imbalance-based detection of potentially novel fusions may be easily verified with other routine technologies without delaying treatment initiation.

Background Lynch syndrome (LS) is caused by germline mutations in DNA mismatch repair (MMR) genes being MLH1 and MSH2 the most commonly mutated. By contrast MLH1 inactivation as the result of promoter methylation is strongly indicative of a sporadic cancer, providing LS exclusion criteria for 85% of high microsatellite instability (MSI-H) tumours. Here we present a cost effective strategy enabling to test methylation of MLH1 promoter without bisulfite conversion and with minimal DNA quantity requirement. Methods After macrodissection from HE stained slides, DNA was extracted from FFPE tissue sections of colorectal carcinomas. A droplet digital PCR (ddPCR) was performed using two reactions mix. A 270-bp region of the MLH1 promoter (chr3:36993151-36993420) is amplified in the presence/absence of HinP1I, a methylation sensitive restriction enzyme, targeting 3 CpG islands. Analysis was made by the QuantaSoft™ (Bio-Rad) software which calculates amplicon concentrations between the 2 reactions to obtain a percentage of MLH1 methylation. Sensitivity of the technique was assigned to 5% of methylation with a minimal DNA concentration of 5 ng. Results Methylation analysis by ddPCR was compared to pyrosequencing combined with bisulfite conversion for 65 samples and a 100% concordance was obtained. Moreover 10 samples were analysed by ddPCR and MethylLight RealTime-PCR with the same concordance. After validation, the technique was implemented in the clinical diagnosis and, in one year, out the 79 MMR-deficient colorectal carcinomas analysed, 60 (76 %) were MLH1-methylated tumours. Conclusions This ddPCR sequencing combining methylation sensitive restriction enzyme is a cost-effective strategy, requiring less technical turn around time and minimal DNA quantity as compared to standard analysis. Moreover, this technique could be further used for other promoter methylation analysis (such as MGMT) and on circulating tumoral DNA. Legal entity responsible for the study The authors. Funding Has not received any funding. Disclosure All authors have declared no conflicts of interest.

Abstract Background Tumour mutational burden (TMB) is a measurement of DNA variants in a tumour and is a potential biomarker of response to Immune Checkpoint Inhibitor (ICI) therapy. Patients with non-small cell lung cancer (NSCLC) whose tumours have a high TMB (≥10 mutations/Mb) might benefit from upfront ICI combination therapy. Clinical trials have measured TMB using Whole Exome Sequencing (WES) and/or the FoundationOne CDx (F1CDx) assay. In parallel, several commercial next-generation sequencing (NGS) assays have become available. Before implementation of TMB testing in clinical practice, technical and clinical validation are needed. Hence, a multi-center study was organised to establish concordance between several TMB assays. Methods Fifteen resection NSCLC formalin-fixed paraffin-embedded (FFPE) samples with broad TMB range were selected. Each participant received extracted DNA and was asked to report TMB values using their own protocol. In parallel, FFPE slides were analysed with F1CDx. Eight labs participated using five different methods: Oncomine TML assay (Thermofisher; n = 4), TSO500 assay (Illumina; n = 1), NEOplus assay (NEO New Oncology; n = 1), a 0,4 Mb targeted resequencing lab-developed assay (LDT; n = 1) and WES using three different TMB calculation methods (n = 1). Correlations between each platform and F1CDx were calculated. Also, the reported TMB category (high vs low) of the different platforms in comparison to F1CDx was evaluated for the fifteen samples. Results Assessment of TMB values obtained by the platforms and F1CDx demonstrated a high correlation (R2 between 0.81 and 0.94), except for the smaller LDT (R2 = 0.53). The TMB category (high vs low) reported by each platform showed concordance with the F1CDx category for eleven (73%) to thirteen (93%) of the fifteen samples. From the fifteen samples, the same category was reported by all different platforms for seven (47%) samples. Conclusion Our data show that assays from different providers can be used to predict TMB. However, samples with a TMB value around the cut-off of 10 mut/Mb are challenging and interpretation should occur with caution. Further studies are required before implementing these assays in routine clinical diagnosis. Legal entity responsible for the study The authors. Funding Bristol-Myers Squibb. Disclosure S. Lambin: Research grant / Funding (institution): Bristol-Myers Squibb. All other authors have declared no conflicts of interest.

Chronic myeloid leukemia (CML) and acute lymphoblastic leukemia (ALL) are, respectively, a myeloproliferative and a lymphoproliferative neoplasm that can be characterized by the chimeric fusion oncogene BCR-ABL1. Tyrosine Kinase Inhibitors (TKI) are the standard therapy for patients with CML/ALL. However, mutations of the BCR-ABL1 kinase domain constitute a major cause of treatment failure in CML and ALL receiving TKI therapy. While 2nd and 3rd generation TKI have proven their efficacy against mutated BCR-ABL1-mediated clonal expansion, the presence of compound mutations can produce high level of resistance to these TKIs. Even the last addition to the TKI armamentarium, ponatinib, remains ineffective against some BCR-ABL1 compound mutations (Zabriskie, M.S., et al., BCR-ABL1 Compound Mutations Combining Key Kinase Domain Positions Confer Clinical Resistance to Ponatinib in Ph Chromosome-Positive Leukemia. Cancer Cell, 2014. 26(3):p.428-442). Therefore, the distinction between compound (different mutations present on 1 unique malignant clone) and polyclonal mutations (different mutations present on 2 or more different clones) is of great clinical importance in order to select the most suitable treatment and to estimate outcomes. The objective of this study is to determine in a straightforward way whether BCR-ABL1 mutations discovered by Next Generation Sequencing are compound mutations or polyclonal mutations. A simple proof-of-concept experiment was first performed by using 3 synthetic oligonucleotides (gBlocks, IDT) mimicking the presence of compound mutations versus polyclonal mutations in resistant leukemia cells. The first oligo harbored the M237I mutation, the second oligo mutations E255K, E279K, V299L, T315I, F359V, A380S, H396R, S417Y, F459K and F486S and the third one contained all the mutations. Dual-color probes assays have been set up to target specifically 2 different mutations. Mixtures of 2 oligonucleotides harboring 1 mutation each versus 1 oligonucleotide harboring 2 mutations have been compared by performing duplex droplet digital PCR (ddPCR) reactions on the Bio-Rad ddPCR QX200 System. Linkage detection is based on the observation that the presence of 2 targets on the same DNA molecule increases the number of double-positive droplets relative to the number expected due to chance. Automatic linkage evaluation was made by the QuantaSoft Software and mathematical calculations refer to (Regan, J.F., et al., A rapid molecular approach for chromosomal phasing. PLoS One, 2015. 10(3): p. e0118270). The first experiment successfully validated the detection of mutations residing on two different oligonucleotides (polyclonal mutations) versus mutations on the same molecule (compound mutations). When performing serial dilutions of 2 oligonucleotides containing different mutations, a sensitivity of 10%:90% was achieved with a good linearity (r2=0.97). Mixing experiment also showed that ddPCR phasing could distinguish between a mixture of compound and polyclonal mutations versus and the sole presence of polyclonal mutations at the same sensitivity and linearity levels. Moreover, no influence of the genomic distance between mutations (from position 255 to position 562) was observed. The strategy was further applied to 20 clinical samples from CML/ALL patients characterized by multiple resistance mutations. Drop-phase is a rapid (< 4 hours), scalable (100 samples), technically easy to perform and cost-effective method. This strategy will help to identify compound mutations in patients with TKI-resistant CML/ALL and allow to modulate the patient's drug strategy and to prevent progression and therapeutic failure. Disclosures Vannuffel: Incyte: Consultancy. Soverini:Incyte: Consultancy.

Background: About 80% to 85% of lung cancers are non-small cell lung cancer (NSCLC). EGFR tyrosine kinase inhibitors as well as several other targeting molecules have been demonstrated to be effective in treating patients with activating mutations. We investigated the use of circulating tumor DNA (ctDNA) and high sensitive detection techniques for mutational profiling to improve the diagnosis and monitoring of NSCLC patients. Methods: ctDNA was extracted from plasma using the QIAamp Circulating Nucleic Acid kit (Qiagen). A custom panel was designed to cover EGFR, KRAS, NRAS, BRAF, PIK3CA, DDR2, AKT1, PTEN, MEK1 and ERBB2 hotspot mutations. Libraries, constructed according to the AmpliSeq protocol, were sequenced on the semiconductor Ion Torrent S5XL platform. The presence of the EGFR T790M mutation was also assessed by a digital PCR assay. Results: A total of 120 patients from 30 Belgian institutions were enrolled in this prospective study. The majority of patients presented with stage IV adenocarcinoma and progression. Forty-six (46) patients had a mutation detected on a former biopsy: EGFR exon 19 (26), EGFR exon 21 (8), KRAS (10), PIK3CA (1) and ERBB2 (1). Among those patients, 28 (61%) harbored the same mutation when their ctDNA was sequenced with our NGS panel: EGFR exon 19 (15), EGFR exon 21 (6), KRAS (5), PIK3CA (1) and ERBB2 (1). For 7 patients, for which no mutation had not been previously detected, 4 EGFR, 2 KRAS and 1 NRAS mutations were found after ctDNA analysis. As far as the ddPCR detection of EGFR T790M was concerned, the mutation was detected on 7 (21%) of the 34 patients presenting EGFR mutations in their prior biopsy (5 in exon 19 and 2 in exon 21). Patients with acquired T790M mutation were previously treated by Afatinib (3), Erlotinib (2) or Gefitinib (1). Conclusions: Our results indicate that ctDNA can be an alternative and noninvasive source of tumor DNA, a surrogate to classical biopsies, particularly when access to tumor tissue is limited. NGS and ddPCR assays are sensitive enough to promote a clinical translation of ctDNA analysis into disease management and therapeutic decision. Supported by a grant from Boehringer Ingelheim. Legal entity responsible for the study: Institut de Pathologie et de Génétique Funding: Boehringer Ingelheim Institut de Pathologie et de Génétique Disclosure: All authors have declared no conflicts of interest.

SummaryIntroductionStandardization of BCR‐ABL1 messenger RNA quantification by real‐time PCR on the International Scale (IS) is critical for monitoring therapy response in chronic myelogenous leukaemia. Since 2006, BCR‐ABL1 IS standardization is propagated along reference laboratories by calculating a laboratory‐specific conversion factor (CF), co‐ordinated in Europe through the European Treatment and Outcome Study project. Although this process has proven successful to some extent, it has not been achievable for all laboratories due to the complexity of the process and the stringent requirements in terms of numbers of samples to be exchanged. In addition, several BCR‐ABL1 IS quantification methods and secondary reference materials became commercially available. However, it was observed that different IS methods generate consistently different results.MethodsTo overcome these difficulties, we have developed an alternative and simple approach of CF calculation, based on the retrospective analysis of existing external quality assessment (EQA) data. Our approach does not depend on the exchange of samples and is solely based on the mathematical CF calculation using EQA results.Results and conclusionWe have demonstrated by thorough statistical validation that this approach performs well in converting BCR‐ABL1 measurements to improve IS estimation. In expectation of a true golden standard method for BCR‐ABL1 IS quantification, the proposed method is a valuable alternative.

Abstract BACKGROUD: The immunogenicity of some human tumors towards T lymphocytes is well established. Recently, encouraging results have been obtained with immunotherapies inhibiting immune checkpoints in cancers such as melanoma, NSCLC and bladder cancer. Fewer studies explored these treatments in breast cancer (BC) as these tumors are often considered to be poorly immunogenic. METHODS: We analysed the T cell receptor β-chains variable genes (TCRBV) repertoires of tumor-infiltrating T cells in 17 early BC. We looked for clonally amplified T cells as their presence is an expected consequence of tumor immunogenicity. RNA was extracted and reverse-transcribed from formalin-fixed, paraffin-embedded tumor tissues. A short random sequence was added to the cDNA and used as a unique molecular identifier (UMI) for each cDNA molecule. cDNA encoding TCRBV genes was then amplified and sequenced using high throughput sequencing. Usage of UMIs during this procedure strongly improved the accuracy of the analysis by avoiding amplification biases inherent to the construction of the TCRBV library and by allowing an absolute quantification of TCRBV mRNA molecules normalized with the RPP30 housekeeping gene. TCRBV sequences were aligned using IMGT/HighV-QUEST. The Simpson's index was used to evaluate TCRBV repertoires diversity (ranging from 0 = infinite diversity to 1 = no diversity). For 3 patients, the same procedure was applied on blood T cells collected a few days before tumor resection and the analysis was also carried out on 3 normal tissues obtained from breast reduction surgery. RESULTS: T cell infiltration varied strongly from one tumor to another ranging from 5 to 2498 TCRBV/103 RPP30 mRNA molecules. TCRBV repertoires analysis indicated that infiltrated T cells corresponded to oligoclonal populations. We observed 3 clonotypes in the smaller repertoire and 74 in the largest one and the Simpson's index ranged from 0.01 to 0.65. Most tumors (16/17) contained at least one clonotype that made up ≥10% of the infiltrating T cells, with the highest observed proportions reaching 80%. Normal breast samples were infiltrated by a more diverse repertoire: 130 to 368 clonotypes were identified in those tissues and Simpson's index ranged from 0.002 to 0.008. Highest observed frequency among those clonotypes was 2%. For 3 BC patients, the frequencies of the most prevalent clonotypes in the tumor were compared to those of the same clonotypes in blood prior to surgery. These T cell clones were 250 to >34000 times more frequent in the tumor than in the blood. CONCLUSIONS: Some early BC are infiltrated by oligoclonal T cell populations that are highly enriched relative to the blood. Quantitative T cell repertoire analysis allows to distinguish 3 types of BC: (1) tumors without T cell infiltration, (2) tumors with a high T cell infiltration and a small T cell repertoire, and (3) tumors with a high T cell infiltration and a large repertoire. Our observations suggest that anti-tumor T cell responses are ongoing in some early BC and this warrants boosting such responses with immune checkpoint inhibitors in selected patients. T cell repertoire evaluation could be used as a predictive biomarker to identify patients who will benefit from this treatment. Citation Format: Carrasco J, Schröder D, Coulie PG, Godelaine D, Berlière M, Theate I, Delrée P, Vannuffel P, Galant C, Duhoux FP, Machiels J-P, Canon J-L. Early-stage breast carcinomas are infiltrated by oligoclonal T cell populations highly enriched relative to the blood. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P4-04-10.

Abstract Background. Among myeloproliferative diseases, development of chronic myeloid leukaemia (CML) is associated with the emergence of the fusion oncogene BCR-ABL1 resulting from a t(9,22) chromosomal translocation (Philadelphia chromosome). Mutations of the BCR-ABL1 kinase domain constitute a major cause of treatment failure in CML patients receiving tyrosine kinase inhibitor (TKI) treatment. Moreover, the occurrence of cells with multiple mutations is frequently associated with a higher resistance rate to the different TKI (Imatinib, Dasatinib or Nilotinib). So far, the gold standard procedure to detect BCR-ABL1 mutations remains the conventional Sanger Sequencing (SS), endowed with an analytical sensitivity of 10 to 20 %. The recent implementation of Next Generation Sequencing (NGS) allows lowering the sensitivity level and quantitative follow-up of the mutated subclone(s), which probably improves CML patient's treatments management. Aims. In this retrospective study, we evaluate the advantage of NGS approach to i) identify patients harbouring (low level) mutations that have not been not assessed by conventional methods, ii) detect the emergence of mutated clones earlier than SS and iii) monitor evolution of mutations. Methods. Total BCR-ABL1 RNA was transcribed into a long range cDNA covering the kinase, the regulatory, and the SH2/SH3 domains of either p190 or p210 BCR-ABL1 transcripts (exons 4 to 10). From primers designed with the AmpliseqTM Designer Software, a set of 10 amplicons was generated according to the AmpliSeqTM protocol. Bar-coded libraries were sequenced on the Ion Torrent PGM platform and data were analysed with Torrent Suite and NextGene softwares. Serial dilutions of samples harbouring mutations at different levels were used to determine a variant frequency cut-off. Our methodology was applied to a group of 36 patients presenting with poor response to TKI and with no mutation detected by SS and to a set of 100 samples, corresponding to 20 mutated patients, at different time points before the time of mutation identification by SS. Results. From the serial dilutions experiment, the detection limit of the assay was set up to 2 % (R² > 0.997). An overall coverage ranging from 20 000 to 50 000 reads can be achieved for the hotspot mutations when up to 12 samples were tested together on a 316 Ion chip. On the 36 patients tested by NGS versus SS, no one was found to harbour TKI-resistance mutation. NGS successfully detected all mutations identified by SS; mutations were typically detected within 4 months (18/20 patients) and were also detected up to 9 months prior to detection by SS, even in patients with a low abundance of BCR-ABL1 transcripts and in sequencing failure by SS. In 2 patients presenting with up to 3 mutations, evolution of mutations (emergence, expansion or depletion) correlates with clinical data of treatment decisions, i.e. E255K (patient-1) and L248V (patient-2) depletions when switching from Imatinib to Dasatinib, F317L (patient-1), G250E (patient-1) and T315I (patient-2) expansions under Dasatinib and a complete but transient depletion of T315I (patient-2) with the protein synthesis inhibitor homoharringtonine (Omacetaxine). Finally, assessment of the mutation status of one patient with compound mutations following an Illumina protocol on a MiSeq platform had allowed comparison of technologies performances. Conclusions. NGS did not detect mutations in 36 patients poorly responding to TKI with no detectable BCR-ABL1 TK mutation by SS. For 20 patients showing BCR-ABL1 TK mutation by SS, NGS was able to detect the mutation in samples taken up to 9 months prior to the moment when the mutation was observed by SS. Advances in sequencing technologies and further lowering sensitivity levels can contribute even more to earlier detection of mutations and guide an earlier switch of TKI. Quantitative and sensitive monitoring of mutation evolution can also inform the most appropriate and optimized treatment algorithms. A prospective evaluation of the clinical impact of NGS-based BCR-ABL1 mutation detection is ongoing. Disclosures Vannuffel: ARIAD Pharmaceuticals: Research Funding.

Introduction: Colorectal cancer is the third most common cause of cancer and cancer death, and approximately 20% of all patients will present with metastatic disease. Treatment has become increasingly complex with the emergence of new chemotherapy drugs and targeted agents. To date, in metastatic colorectal cancer (mCRC), KRAS testing has been restricted to codons 12 and 13. However, phase II and III trials now suggest including both KRAS and NRAS codons 12, 13, 61, 117, and 146. Searching for these additional codons could help screen 20% more patients with mCRC for treatment with EGFR inhibitors. Other finding suggests that testing for the BRAF V600 mutation compliments KRAS and NRAS mutations analysis and may be as important for treatment decisions. Here we present a multiplexed and cost effective strategy enabling to test simultaneously these 11 hotspot mutations. Methods: A custom panel (CP-KRAS), covering exons 2, 3 and 4 of KRAS and NRAS and exon 15 of BRAF, was designed using the Ion AmpliSeq Designer module. FFPE tissue sections were macro-dissected and DNA was extracted using the Maxwell FFPE Tissue LEV purification kit. Sequencing was performed according to the AmpliSeq protocol on the Ion Torrent PGM, starting from as less as 6 ng of DNA. Data were analysed with both Torrent Suite v4 and NextGENe software v2.3 with a hotspot positions-targeting bed file. Sensitivity of the technique was assigned from 2.5% at 500 x coverage to 10% at 100 x coverage, with a forward to reverse reads ratio >0.25. Results: After validation of the CP-KRAS panel on well-characterized EQC samples, more than 250 FFPE specimens (corresponding to a 8 months of CP-KRAS testing) were sequenced. 29% of the samples were mutated for KRAS codon 12 and 6% for codon 13. Of the wild-type samples tested for additional mutations, 3% of samples were mutated for KRAS codon Q61, 4% for KRAS codon A146, 4% for NRAS G12 and 4% for NRAS Q61. 9% additional samples were mutated for BRAF V600. Each additional mutation was confirmed by pyrosequencing, mini-sequencing (SNaPshot) or Sanger sequencing. In total, 24% tumours wild type for KRAS exon 2 harboured a mutation in another of the RAS pathway genes. Conclusion: This next generation and high throughput sequencing workflow on a single-tube multiplexed amplification of those KRAS, NRAS and BRAF hotspot mutations could help to identify additional patients who possibly fail to respond to anti-EGFR treatment. Moreover, this strategy is cost-effective, less fastidious and requires minimal DNA quantity from FFPE tissue as compared to standardanalysis.

Introduction: Gastrointestinal stromal tumours (GIST) occur in the gastrointestinal track, most commonly in the stomach or small intestine. These sporadic tumours are associated with genetic changes. In GIST subsets, c-KIT or PDGFRa mutations correlate with distinct anatomical site, clinical phenotype and sensitivity to tyrosine kinase inhibitors. Here we depict a multiplexed evaluation strategy based on Next Generation Sequencing (NGS) to investigate for the most relevant regions of c-KIT and PDGFRa in a GIST context. Methods: A custom panel, covering exons 9, 11, 13 and 17 of c-KIT and exons 12, 14 and 18 of PDGFRa, was designed using the Ion AmpliSeq Designer module. FFPE tissue sections were macro-dissected and DNA was extracted using the Maxwell FFPE Tissue LEV purification kit. Sequencing was performed according to the AmpliSeq protocol on the Ion Torrent PGM, from as less as 6 ng of input DNA. Data were analysed with both Torrent Suite v4 and NextGENe software v2.3 with a whole exons-targeting bed file. Sensitivity of the technique was assigned from 2.5% at 500 x coverage to 10% at 100 x coverage, assuming a forward to reverse reads ratio >0.25. Results: DNA from samples for which c-KIT and PDGFRa mutations have been previously identified by Sanger sequencing (10 providing from EQC schemes and 30 from our regular practice), as well as from 10 wild-type samples, were tested with the designed panel. For all samples, there is a perfect correlation between our multiplexed sequencing strategy and the « gold standard » Sanger sequencing. Mutations - point mutations, duplications, deletions, insertions or both, mainly located in exons 9 and 11 of c-KIT and in exon 18 of PDGFRa – were found in all 40 mutated samples by both techniques, while no critical variant was detected in the 10 normal samples. Conclusion: This multiplexed amplification of 7 exons in GIST samples demonstrates that custom NGS panels can be designed to identify mutations in genes or regions more relevant to diagnosis, prognosis or therapeutic choices. For any tumour setting or pathway, NGS panels allow more comprehensive sequence coverage than standard techniques and are scalable in term of regions of interest and number of samples.

To the Editor: Myelodysplastic ⁄myeloproliferative neoplasm (MDS ⁄MPN) resulting from an acquired t(8;9)(p22;p24), that fuses the human autoantigen pericentriolar material (PCM1) gene to the janus-activated kinase 2 (JAK2) gene, is a very rare disease (1–4). Only few cases with poorly characterised pathology have been described so far (1–4). We report here the pathological features observed in a patient with MDS ⁄MPN presenting this peculiar translocation. A 57-yr-old man was admitted to the Department of Haematology because of fatigue, weight loss and dyspnoea for 3 months. Clinical examination only revealed a splenomegaly. Peripheral blood (PB) analysis demonstrated macrocytic anaemia (haemoglobin: 10.5 g ⁄dL), leucocytosis (31.2 · 10 ⁄L) and normal platelets. The leucocyte count showed 66% neutrophils, 8% eosinophils, 1% basophils, 13% immature granulocytes and 14% erythroblasts. Haematocrit level was 32.9%. The bone marrow (BM) aspirate exhibited a hypercellular marrow with an increase in both granulocytes and their precursors, and eosinophils. Erythropoiesis and megakaryocytes were reduced. Macrophages as well as some sea-blue histiocytes were found. Very few ring sideroblasts could be identified and Perls’ stain for iron was reported as grade 1. No dysplastic features were recorded. Overall, these morphological findings were suggestive of chronic myelogenous leukaemia (CML). Microscopic examination of a BM trephine biopsy led to the same diagnosis. Cytogenetic analysis was initially performed on cell suspensions obtained from the BM aspirate. This analysis did not disclose any obvious chromosomal abnormality, and the karyotype was reported as normal. In particular, no t(9;22)(q34;q11.2) could be seen, a finding confirmed by fluorescence in situ hybridisation (FISH) analysis. Likewise, no BCR-ABL1 fusion gene, JAK2 V617F (exon 14) mutation or JAK2 exon 12 anomaly (mutation or deletion) could be demonstrated by molecular studies. A splenectomy was performed for diagnostic purposes. Microscopic examination revealed features indicative of extramedullary haemopoiesis, which mostly involved precursors of erythropoiesis and granulopoiesis. Interestingly, only a few megakaryocytes could be seen within splenic tissue. Also, a subcapsular nodule exhibited morphological and immunohistochemical characteristics highly reminiscent of littoral cell angioma. However, this surgical procedure did not result in a dramatic improvement of PB abnormalities. On a subsequent cytogenetic analysis performed on unstimulated PB cultures, an abnormality at chromosome 9p was suspected. Using a probe specific for the telomere of chromosome 9, FISH analysis detected a translocation between 9p and the p arm of another C group chromosome, further identified as a chromosome 8 (Fig. 1A). To confirm that this translocation involved the JAK2 gene, additional FISH analysis was carried out, using two bacterial artificial chromosome probes encompassing this gene (RP11125K10 and RP11-39K24, Resources for Molecular Cytogenetics, University of Bari, Italy). The breakpoint was localised within probe 39K24, thereby confirming the involvement of JAK2 in this translocation (Fig. 1B– D). Review of the BM trephine biopsy sections revealed a hypercellular marrow with loss of fat cells. Megakaryopoiesis was significantly decreased but did not display dysplastic features. There was a marked increase in neutrophils and eosinophils and their precursors, with relatively preserved maturation (Fig. 2A, B). However, in contrast to CML, maturation of granulopoiesis from paratrabecular or perivascular regions towards the central areas of intertrabecular marrow was lost. No accumulation of immature granulocytic precursors, monocytes, mast cells, or basophils could be demonstrated. Erythropoiesis exhibited dysplastic features. Notably, large nodules composed almost exclusively of proerythroblasts or early erythroblasts were found in the paratrabecular areas (Fig. 2C, D). These cells exhibited variable atypias and significant mitotic activity. In these nodules, microvessel density was particularly prominent. Gomori stain revealed a diffuse argyrophilic fibrosis with a slight increase in reticulin (Fig. 2E), graded as MF-1, according to the European consensus for grading of BM fibrosis and assessment of cellularity (5). Immunohistochemical studies using monoclonal antibodies directed against CD61, myeloperoxydase and glycophorin A illustrated further the abnormalities noted in megakaryopoiesis, granulopoiesis and erythropoiesis, respectively (Fig. 2F). Stainings for CD68, CD117 and doi:10.1111/j.1600-0609.2010.01525.x

Sanfilippo syndrome type B (mucopolysaccharidosis IIIB) is an autosomal recessive disease that is caused by a deficiency of the lysosomal enzyme alpha-N-acetylglucosaminidase (NAGLU). Over 100 different mutations in the NAGLU gene have been identified in Sanfilippo syndrome type B patients; however, no large deletions have been reported. Here we present the first case of a large homozygous intragenic NAGLU gene deletion identified in an affected child of consanguineous parents. Long range and multiplex PCR methods were used to characterize this deletion which encompasses exons 3 and 4 and is 1146 base pairs long. We propose that Alu element-mediated unequal homologous recombination between an Alu-Y in intron 2 and an Alu-Sx in intron 4 is the likely mechanism for this deletion, thereby contributing further insight into the molecular etiology of this disorder and providing additional evidence of its allelic heterogeneity.

The functional evaluation of ataxia telangiectasia mutated (ATM) and p53 was recently developed in B-cell chronic lymphocytic leukaemia (B-CLL), a disease in which the response to DNA damage is frequently altered. We identified a novel biomarker of chemosensitivity based on the induction of DNA damage by the purine nucleoside analogues (PNA) fludarabine and 2-chlorodeoxyadenosine (CdA). Using genome-wide expression profiling, it was observed that, in chemosensitive samples, PNA predominantly increased the expression of p53-dependent genes, among which PLK2 was the most highly activated at early time points. Conversely, in chemoresistant samples, p53-dependent and PLK2 responses were abolished. Using a quantitative real time polymerase chain reaction, we confirmed that PNA dose- and time-dependently increased PLK2 expression in chemosensitive but not chemoresistant B-CLL samples. Analysis of a larger cohort of B-CLL patients showed that cytotoxicity induced by PNA correlated well with PLK2 mRNA induction. Interestingly, we observed that failure to up-regulate PLK2 following PNA and chemoresistance were not strictly correlated with structural alterations in the TP53 gene. In conclusion, we propose that testing PLK2 activation after a 24-h incubation with PNA could be used to investigate the functional integrity of DNA damage-response pathways in B-CLL cells, and predict clinical sensitivity to these drugs.

To the Editor: Kimura disease (KD) is a chronic inflammatory condition of unknown etiology.1-5 This rare disorder, which occurs mainly in oriental Asians, generally involves the soft tissues of the head and neck area.1-5 Typically, peripheral blood eosinophilia and elevated serum levels of immunoglobulin (Ig) E are found in these patients.1-5 A nephrotic syndrome due to glomerulonephritis may also occur in a certain number of cases.1-5 To date, the pathogenesis of such disorder is still enigmatic; an autoimmune or allergic origin has been suggested by some authors, owing to the eosinophilic hyperplasia, raised IgE amounts, and T helper 2 (Th2) cytokine profile of T lymphocytes, seen in this condition.2-5 We report the case of a 47-year-old North African male who developed a KD involving the subcutis of the retroauricular area. The presence of numerous plasmacytoid dendritic cells (PDCs) in the lesion, a feature rarely described in KD, may unravel in part the pathophysiology of the disease. The patient had no medical history apart from a right chronic maxillary sinusitis treated by surgery several years ago. He spontaneously developed a 25 × 15 × 10-mm subcutaneous swelling in the right postauricular area. Physical examination and imaging studies did not disclose any other anomaly. The mass was excised for diagnostic purposes. Microscopic examination demonstrated a well-circumscribed lymph node-like lymphoid nodule located in the subcutis (Fig. 1A). Lymphoid follicles were present and featured variably developed germinal centers. These structures occasionally contained multinucleate follicular dendritic cells (DCs) or small deposits of proteinaceous material. There was a prominent eosinophilic infiltrate involving the lymphoid follicles and the interfollicular areas. Fibrotic changes were seen within the interfollicular zones and around blood vessels. In some places, there were occasional epithelioid histiocytes or epithelioid granulomas with central eosinophilic abscesses. Interestingly, numerous clusters of PDCs were found in the vicinity of high endothelial venules or at the periphery of lymphoid follicles (Fig. 1B). These clusters were usually loose in comparison with the dense clusters normally observed in reactive lymph nodes and contained lymphocytes and eosinophils (Fig. 1B). The overlying skin contained a moderate lymphoid infiltrate made of small lymphocytes, histiocytes, eosinophils, and PDCs, in varying proportions. This lymphoid infiltrate was mostly located around dermal vessels and cutaneous adnexae. Notably, the epidermis did not demonstrate any significant changes. On immunohistochemical analysis, CD20-positive B cells formed the lymphoid follicles, whereas most of interfollicular lymphocytes showed immunoreactivity with antibodies directed against CD3, CD4, or CD8. As a rule, CD4-positive cells outnumbered CD8-positive lymphocytes. Epithelioid histiocytes or epithelioid granulomas consistently expressed CD68 and at variable extent also displayed CD163 immunoreactivity. DCs expressing S100 protein or CD1a were found throughout the lymphoid tissue, and CD1a-positive DCs were focally relatively numerous. PDCs expressed CD4, CD68, granzyme B, and CD123. These cells were also positive for BCL11a, CD2-associated protein (CD2AP), and blood dendritic cell antigen 2 but were negative for CD56 (Figs. 1C, D). Polymerase chain reaction analysis of DNA extracted from paraffin-embedded sections did not demonstrate any clonal rearrangement of the genes coding for the Ig heavy (IgH) chain and for the γ subunit of the T-cell receptor.FIGURE 1: Illustrations of the lesion. A, This low-power view shows the lymphoid nodule located in the subcutis. Hematoxylin and eosin (H&E) staining, ×2. B, A cluster of PDCs mixed up with eosinophils is seen. H&E staining, ×400. C, D, On immunohistochemical analysis, PDCs strongly express the BDCA2. Note the peculiar location of these cells within the lymphoid tissue, as shown in (C). Immunoperoxidase staining using diaminobenzidine (DAB) as chromogen, ×25 and ×400, respectively. BDCA2, blood dendritic cell antigen 2.Based on these data, a diagnosis of KD featuring a significant number of PDCs was rendered. Additional investigations revealed the presence of a moderate increase in eosinophil counts in the peripheral blood (white blood cells: 9.5 × 109/L: 15% eosinophils) and raised IgE serum levels (198 IU/mL), which confirmed the diagnosis. Serum titers of IgM, IgG, and IgA, and serum levels of several proteins of the complement system (C1q, C3, and C4), were within normal limits. Renal function tests were normal. Serologic studies for rheumatoid factor or anti-DNA antibodies were negative. Last, flow cytometric investigations did not demonstrate any abnormal lymphocytic population in the peripheral blood. To the best of our knowledge, only 1 study mentions the presence of PDCs in KD.1 However, the authors of this study did not give considerable emphasis on this finding because of the presence of such cells in other reactive lesions.1 In fact, PDCs represent a distinct cellular subset of the immune system that plays a significant role in both innate and adaptive immunity.6-8 These cells are preferentially located in T-cell areas of lymphoid tissues, close to high endothelial venules.6-8 They secrete high amounts of type 1 interferon (IFN), mainly IFN-α, in response to various viral or bacterial stimulations.6,7 They can also differentiate into DCs that regulate the function of T lymphocytes by exerting a Th1 or Th2 polarization on these cells.7 As shown in this case, PDCs express CD4; CD68; granzyme B; and CD123, the receptor for interleukin-3 α chain.6-8 They also display the adapter protein CD2AP; the transcription regulator BCL11a; and blood dendritic cell antigen 2, a negative regulator of the secretion of IFN-α.6,8 PDCs are present in various inflammatory disorders of the skin including cutaneous lymphoid hyperplasia, lupus erythematosus, Jessner lymphocytic infiltrate, psoriasis, and contact dermatitis.6,8 They also accumulate in 2 rare forms of lymphadenopathy such as Kikuchi-Fujimoto disease and the hyaline-vascular variant of Castleman disease.6,8 Recently, PDCs have been demonstrated to be recruited in allergen-challenged nasal mucosa where they influence T cells to produce Th2 cytokines, which illustrates further the role of these cells in the development of allergic reactions.6 In our patient, the accumulation of PDCs in both lymphoid nodule and overlying skin may indicate a skin-based antigenic challenge. Interestingly, a close association between PDCs and eosinophils was found. Furthermore, the presence of a dermal inflammatory infiltrate rich in eosinophils and that of numerous CD1a-positive DCs within the lymphoid nodule also supports such hypothesis. In this regard, it is worth noting that our patient did not suffer from any chronic skin disease or atopy. Therefore, the nature of the triggering factor(s) remains yet to be definitely determined. In summary, we have illustrated further the presence of PDCs in lymphoid hyperplasia associated with KD. Because these cells are involved in peculiar subtypes of immune reaction, including autoimmune and allergic ones, their presence in KD may provide further insight into the pathophysiology of this uncommon disease. Jean-Louis Dargent, MD Pascal Vannuffel, PhD Institut de Pathologie et de Génétique Gosselies, Belgium Jean-Marie Saint-Remy, MD Center for Molecular and Vascular Biology University of Leuven Leuven, Belgium Simona Fisogni, MD Fabio Facchetti, MD Department of Pathology University of Brescia Brescia, Italy

This paper summarizes the minimal workout of chronic lymphoproliferative disorders in a routine laboratory of haematology as recommended by a team of experienced laboratory supervisors in Belgium, taking into account the specific organisation of healthcare in Belgium, the innovations in the field of molecular analyses and related reimbursement. The starting point was essentially based upon clinical and/or haematological indications and it is emphasized that conclusions should be drawn in close dialogue with the clinician and experts in cytogenetics and histopathology. Reports made in the laboratory should be based upon an integration of cytomorphological, immunophenotypical and molecular data.These guidelines are not intended to be used as universal ‘diagnostic pathways’, but should be useful in developing local diagnostic pathways. It is well understood that this consensus, being valid anno 2009, may rapidly change with new technologies being introduced and new targets discovered.

Numerous studies have shown that the presence, number and type of chromosomal aberrations represent an independent predictor of prognosis in B-cell chronic lymphocytic leukemia (CLL). Consequently, cytogenetic analysis is routinely performed in this disease. However, CLL lymphocytes have a poor mitotic index, generating only 40–50% of abnormal karyotypes. The rate of detection can be increased to 80% by interphase FISH analysis. Since some aberrations, i.e. those not involving the classical regions (13q, 12, 11q, 17p, and 6q), can escape FISH detection, there has been great interest in improved culturing methods with an immunostimulatory CpG oligonucleotide (CpG). We performed a multicentric cytogenetic study to assess the impact of 2 different culturing procedures on the detection of clonal abnormalities in 159 consecutive unselected cases with CLL referred to our centers for routine analysis from October 2007 to July 2008. Dual 72 hours cultures of bone marrow or peripheral blood were set up with the addition of either a conventional B cell mitogen (TPA) or CpG and interleukin-2 (IL2). Cytogenetic analysis was performed on both cultures. FISH analysis using a CLL probe panel analyzing 1–6 regions (13q, centromere 12, 11q, 17p, 6q and 14q32) was also applied on uncultured material, on CpG and/or on TPA culture in 146 cases. Quality of banding and proliferation capacity were assessed in 38 cases. The quality was good in 17 (CpG) and 8 (TPA), intermediate in 12 (CpG) and 18 (TPA), and poor in 9 (CpG) and 12 (TPA) cases. The mitotic index was slightly higher in CpG cultures: <15 mitoses/slide were seen in 12 (CpG) and 15 (TPA) cases, 15–20 mitoses/slide in 13 (CpG) and 14 (TPA) cases, and >20 mitoses/slide in 13 (CpG) and 9 (TPA) cases, respectively. Clonal abnormalities were identified in 89 cases (56%). In 56 cases, the aberrant clone was detected in both cultures. Of these, the percentage of aberrant metaphases was similar in both cultures in 13, higher in CpG culture in 32 and higher in TPA culture in 11 cases. In 29 and 4 additional cases, a clonal abnormality was detected only in CpG or TPA culture, respectively. Thus, the percentage of abnormal karyotypes with CpG and TPA was 53 and 38%, respectively (p=0,005). “Typical” aberrations, including del(13q), +12, del(11q), del(17p) and del(6q), were detected in 16, 26, 15, 9, and 7 (CpG) cases, and in 10, 20, 16, 8, and 3 (TPA) cases, respectively. Translocations (balanced and unbalanced) were observed in 46 (CpG) and 27 (TPA) cases, whereas aberrations involving 14q32 were seen in 5 (CpG) and 4 (TPA) cases, respectively. Interphase FISH detected del(13q), +12, del(11q), del(17p), del(6q) and del(14q) in 53, 1, 5, 0, 0 and 1 cases, respectively, in which cytogenetic analysis was either normal or abnormal but did not show the specific change. Conversely, FISH did not detect del(13q), +12, del(11q), del(17p), del(6q) and del(14q) in 1, 1, 4, 1, 2 and 0 cases in which the specific change was visible at karyotypic level. FISH was performed on both CpG and TPA cultures in 9 selected cases harbouring an aberration for which a specific commercial probe was available (+12, n=3; del(13q), n=4; del(11q), n=1; and del(17p), n=1) and showing different percentages of aberrant cells in both cultures. In 3 of these cases, fresh uncultured material was also analyzed. The highest percentage of abnormal nuclei was observed in TPA culture in all cases. In the 3 uncultured samples the percentage of aberrant nuclei was similar to the CpG culture. In conclusion, our results confirm an increased detection rate of abnormalities in CLL by using CpG/IL2 stimulation. Interphase FISH can further increase the detection rate of recurrent abnormalities. However, neither cytogenetics nor FISH detected all aberrations, demonstrating the complementary nature of these techniques and the necessity of performing both.

Taking advantage of the high specificity of double-dye hydrolysis-locked nucleic acid (LNA) probes (Ugozzoli et al, 2004), we developed a qualitative three-colour real-time polymerase chain reaction (PCR) assay for the genotyping of sickle cell disease (SCD). The term SCD encompasses a group of disorders associated with mutations in the Haemoglobin Beta gene (HBB). The most common forms are defined by the presence, in codon 6, of an E6V mutation (GAG > GTG; Haemoglobin S; Hb S) on both alleles (sickle cell anaemia) and co-inheritance of Hb S and Hb C (E6K mutation; GAG > AAG; Hb SC disease). The highest prevalence of SCD is found in sub-Saharan Africa (sickle-cell gene carrier prevalence varying between 5% and 40% of the population), and most affected children die in the early years of life without being diagnosed or because of non-adapted treatments (Weatherall & Clegg, 2001). The methodological approach presented here combined the use of a primer pair designed to amplify the region of interest in the HBB gene and three double-dye probes containing LNA nucleotides. Each probe was labelled with a different fluorochrome to enable the different alleles to be discriminated (Fig 1). Primers were designed with the freeware meltcalc, ver. 2.0 (http://www.meltcalc.de). The Tm of each double-dye LNA probe was calculated by using the Exiqon Tm prediction tool (http://lna-tm.com). Specificity of the probes used in this approach. 20 ng of genomic DNA from three patients homozygous for the wild-type (WT/WT), S (S/S) and C (C/C) alleles respectively was tested in this experiment. The results given by the each probe are illustrated on different panels. The Cy5 channel was applied for the analysis of the WT probe, while FAM and Yakima Yellow were used for the S and C probes, respectively. The three probes showed absolute specificity because there was no signal detected when amplified genomic DNA did not contain the nucleotide targeted by the probe. The no template control did not give any signal for the three probes either. Samples from patients that were homozygous for each of the three aforementioned alleles were used to demonstrate the specificity of the probes (Fig 1). We used unique PCR conditions on the ABI 7500 Fast machine (Applied Biosystems, Foster City, CA, USA), which generated specific results for each probe in <40 min (see Table SI, Fig S1). A cohort of 37 genomic DNA representing the different possible genotypes was used to compare our new technique with either a PCR followed by restriction endonuclease cleavage to detect the A and S alleles (Saiki et al, 1988), or an allele-specific PCR designed to detect A and C alleles (Fischel-Ghodsian et al, 1990). A 100% concordance was observed between our technology and the two published methods. Moreover, the deduced genotypes matched perfectly with the available phenotypic analysis (Gulbis et al, 2006). To investigate the sensitivity of this analysis, we tested different amounts of genomic DNA per PCR reaction. The results were perfectly interpretable from 100 to 1 ng of starting material (data not shown), and enabled the testing of prenatal diagnosis samples even when there was scarce fetal tissue (demonstrated on 18 gDNA obtained from fetal samples; data not shown). Finally, to demonstrate the robustness of our approach, we tested 10 representative samples on currently available instruments that are widely used for multicolour real-time PCR analysis. The results obtained on the LC480 (Roche, Basel, Switzerland), the iCycler (Bio-Rad, Hercules, CA, USA) and the M × 3000 p (Stratagene, La Jolla, CA, USA) were perfectly comparable from one machine to another (see Fig S1). Although a fast PCR protocol was not applicable on all these machines, a longer PCR protocol did not affect the results. In summary, we have developed a very robust and rapid method that takes advantage of the high specificity of double-dye LNA probes, which enabled the identification of the two main mutations in SCD in <40 min. The protocol developed in our laboratory is easily transferable to other real-time PCR machines. The high sensitivity of our approach represents a great advantage for the analysis of samples containing small amounts of DNA. Moreover, this single closed tube method offers another obvious advantage for prenatal diagnosis as it prevents risk of cross contamination. We gratefully acknowledge the skillful technical assistance of Sandrine Delbauve. Table SI. Oligonucleotides used for the three-colour real-time polymerase chain reaction (PCR) assay in this study. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.