Multiregional sequencing and circulating tumour DNA analysis provide complementary approaches for comprehensive disease profiling of small lymphocytic lymphoma.

We aimed at molecularly dissecting the anatomical heterogeneity of small lymphocytic lymphoma (SLL), by analysing a cohort of 12 patients for whom paired DNA from a lymph node biopsy and circulating cells, as well as plasma-circulating tumour DNA (ctDNA) was available. Notably, the analyses of the lymph node biopsy and of circulating cells complement each other since a fraction of mutations (20·4% and 36·4%, respectively) are unique to each compartment. Plasma ctDNA identified two additional unique mutations. Consistently, the different synchronous sources of tumour DNA complement each other in informing on driver gene mutations in SLL harbouring potential prognostic and/or predictive value.

Koolen-de Vries syndrome in the first adulthood patient of Southern India ancestry.

Koolen-de Vries syndrome (KdVS, MIM#610443) is a rare malformation condition mainly characterized by cognitive impairment in association with craniofacial and visceral anomalies. The core phenotype is caused by mutations in the chromatin remodeler KANSL1 (MSL1V1, KIAA1267, KAT8 Regulatory NSL Complex Subunit 1, MIM#612452), which maps to 17q21.31 critical genomic region (Koolen et al., Nature Genetics 2012;44:639-641). Considering its molecular basis, KdVS is included in the group of Developmental Disorders of Chromatin Remodeling (DDCRs), also termed chromatinopathies. We describe the first KdVS patient of Southern India ethnicity, harboring the typical de novo 17q21.31 microdeletion, including KANSL1. Observed facial features and congenital anomalies are in line with the already reported KdVS phenotype, suggesting that phenotypic features are consistent across different ethnicities.

Smith-Magenis syndrome: Clinical and behavioral characteristics in a large retrospective cohort.

Smith-Magenis syndrome (SMS), characterized by dysmorphic features, neurodevelopmental disorder, and sleep disturbance, is due to an interstitial deletion of chromosome 17p11.2 (90%) or to point mutations in the RAI1 gene. In this retrospective cohort, we studied the clinical, cognitive, and behavioral profile of 47 European patients with SMS caused by a 17p11.2 deletion. We update the clinical and neurobehavioral profile of SMS. Intrauterine growth was normal in most patients. Prenatal anomalies were reported in 15%. 60% of our patients older than 10 years were overweight. Prevalence of heart defects (6.5% tetralogy of Fallot, 6.5% pulmonary stenosis), ophthalmological problems (89%), scoliosis (43%), or deafness (32%) were consistent with previous reports. Epilepsy was uncommon (2%). We identified a high prevalence of obstipation (45%). All patients had learning difficulties and developmental delay, but ID range was wide and 10% of patients had IQ in the normal range. Behavioral problems included temper tantrums and other difficult behaviors (84%) and night-time awakenings (86%). Optimal care of SMS children is multidisciplinary and requires important parental involvement. In our series, half of patients were able to follow adapted schooling, but 70% of parents had to adapt their working time, illustrating the medical, social, educative, and familial impact of having a child with SMS.

A novel flow cytometric method for enhancing acute promyelocytic leukemia screening by multidimensional dot-plots.

Acute promyelocytic leukemia (APL) is generally characterized by t(15;17)(q24;q21). In some cases, the classic translocation cannot be identified by conventional methods, since the PML-RARA fusion protein results from complex, variant, or cryptic translocation. The diagnostic algorithm of APL starts with screening methods, such as flow cytometry (FC), followed by fluorescence in situ hybridization or polymerase chain reaction to confirm the diagnosis. Our aim was to develop a novel protocol for analyzing APL samples based on multidimensional dot-plots that can provide comprehensive information about several markers at the same time. The protocol included four optimized multidimensional dot-plots, which were tested by retrospective reanalysis of FC results in APL (n = 8) and non-APL (n = 12) acute myeloid leukemia (AML) cases. After predicting the potential position of hypergranular- and microgranular-type aberrant promyelocytes, the percentages of blast populations were examined within the gates in all AML cases. The percentage of blasts in each predefined gate was well above the cut-off value (95%) in APL cases in all tubes. In non-APL AML cases, the percentage of blasts in the same gates never reached the cut-off value in all investigated tubes, and even when it did in a single tube, the pattern was markedly different from that observed in APL cases. In conclusion, multidimensional dot-plots can be used for screening APL even in cryptic APL cases, although reproducibility across several laboratories would require standardization of antibodies and fluorochromes. This easy-to-use and quick method can support the diagnosis of APL and the prompt initiation of the appropriate treatment.

The impact of cytogenetics on duration of response and overall survival in patients with relapsed multiple myeloma (long-term follow-up results from BSBMT/UKMF Myeloma X Relapse [Intensive]): a randomised, open-label, phase 3 trial.

The Myeloma X trial (ISCRTN60123120) registered patients with relapsed multiple myeloma. Participants were randomised between salvage autologous stem cell transplantation (ASCT) or weekly cyclophosphamide following re-induction therapy. Cytogenetic analysis performed at trial registration defined t(4;14), t(14;16) and del(17p) as high-risk. The effect of cytogenetics on time to progression (TTP) and overall survival was investigated. At 76 months median follow-up, ASCT improved TTP compared to cyclophosphamide (19 months (95% confidence interval [95% CI] 16-26) vs. 11 months (9-12), hazard ratio [HR]: 0·40, 95% CI: 0·29-0·56, P < 0·001), on which the presence of any single high-risk lesion had a detrimental impact [likelihood ratio test (LRT): P = 0·011]. ASCT also improved OS [67 months (95% CI 59-not reached) vs. 55 months (44-67), HR: 0·64, 95% CI: 0·42-0·99, P = 0·0435], with evidence of a detrimental impact with MYC rearrangement (LRT: P = 0·021). Twenty-one (24·7%) cyclophosphamide patients received an ASCT post-trial, median OS was not reached (95% CI: 39-not reached) for these participants compared to 31 months (22-39), in those who did not receive a post-trial ASCT. The analysis further supports the benefit of salvage ASCT, which may still be beneficial after second relapse in surviving patients. There is evidence that this benefit reduces in cytogenetic high-risk patients, highlighting the need for targeted study in this patient group.

Clinical and genetic investigation in Chinese patients with demyelinating Charcot-Marie-Tooth disease.

Demyelinating Charcot-Marie-Tooth disease (CMT) is the most common subtype of CMT. It is caused mainly by 17p11.2 heterozygous duplication, but also by mutations in more than 20 genes which affect development and function of Schwann cells. To investigate the profile of genes mutated and clinical features in demyelinating CMT of Chinese descent, we collected a cohort of 44 demyelinating CMT patients and screened them using multiplex ligation-dependent probe amplification (MLPA) and targeted next-generation sequencing (NGS) technology. The MLPA technology revealed that 77.3% demyelinating CMT patients harbored 17p11.2 heterozygous duplication and 6.8% patients harbored heterozygous deletion of exon 6 of MPZ gene, that was further confirmed a novel c.674_675insA mutation in MPZ gene. In the patients with 17p12 heterozygous duplication, 3 sets of independent families were discordant for the CMT phenotype within the same family. The targeted NGS technology revealed that 6 candidate mutations including 1 previously reported mutation (GDAP1: c.571C>T) and 5 novel mutations (SBF2: c.415T>C, c.619G>T, c.1258A>G; GDAP1: c.589delC; PMP22: c.318delT) were found. In conclusion, combined MLPA technique with targeted NGS, the demyelinating CMT genetic diagnostic success rate was increased.

An uncommon case of chronic myeloid leukemia with variant cytogenetic.

Chronic Myeloid Leukemia (CML) is myeloproliferative neoplasm characterized by Philadelphia chromosome which is a balanced translocation between chromosome 9 and 22 in 90% of cases. However, variant cytogenetic still happens in 5-10 % of cases, the importance of which is controversial as well as its response to therapy, prognosis and progression to acute leukemias. Here we report a male patient with CML and variant cytogenetic who responded to low dose of Dasatinib (50 mg daily).

"Double-hit" chronic lymphocytic leukemia: An aggressive subgroup with 17p deletion and 8q24 gain.

Chronic lymphocytic leukemia (CLL) with 17p deletion (17p-) is associated with a lack of response to standard treatment and thus the worst possible clinical outcome. Various chromosomal abnormalities (including unbalanced translocations, deletions, ring chromosomes and isochromosomes) result in the loss of 17p and one copy of the TP53 gene. The objective of the present study was to determine whether the type of chromosomal abnormality leading to 17p- and the additional aberrations influenced the prognosis in a series of 195 patients with 17p-CLL. Loss of 17p resulted primarily from an unbalanced translocation (70%) with several chromosome partners (the most frequent being chromosome 18q), followed by deletion 17p (23%), monosomy 17 (8%), isochromosome 17q [i(17q)] (5%) and a ring chromosome 17 (2%). In a univariate analysis, monosomy 17, a highly complex karyotype (≥5 abnormalities), and 8q24 gain were associated with poor treatment-free survival, and i(17q) (P = .04), unbalanced translocations (P = .03) and 8q24 gain (P = .001) were significantly associated with poor overall survival. In a multivariate analysis, 8q24 gain remained a significant predictor of poor overall survival. We conclude that 17p deletion and 8q24 gain have a synergistic impact on outcome, and so patients with this "double-hit" CLL have a particularly poor prognosis. Systematic, targeting screening for 8q24 gain should therefore be considered in cases of 17p- CLL.

A Paediatric Acute Promyelocytic Leukaemia Patient Harbouring a Cryptic PML-RARA Insertion due to a Complex Structural Chromosome 17 Rearrangement.

Acute promyelocytic leukaemia with PML-RARA fusion is usually associated with the t(15;17)(q24.1;q21.1) translocation but may also arise from complex or cryptic rearrangements. The fusion usually resides on chromosome 15 but occasionally on others. We describe a cryptic PML-RARA fusion within a novel chromosome 17 rearrangement. We performed interphase fluorescence in situ hybridisation (FISH) using a dual-fusion PML-RARA probe, followed by reverse transcriptase-polymerase chain reaction (RT-PCR) for PML-RARA, karyotyping, and metaphase FISH using RARA break-apart, locus-specific, and subtelomere probes for chromosome 17. An 850K SNP microarray was also employed. Interphase and metaphase FISH showed atypical results involving a single PML-RARA fusion, no second fusion, but instead separate diminished PML and RARA signals. RT-PCR confirmed PML-RARA fusion; however, karyotyping detected only an altered chromosome 17. Metaphase FISH showed the single fusion and diminished 5' RARA signals located unexpectedly in the subtelomeric short-arm and long-arm regions of the rearranged chromosome 17, respectively. SNP microarray revealed no copy number abnormality. This paediatric patient with PML-RARA fusion reflects a cryptic insertion that resides within a complex and novel chromosome 17 rearrangement. This rearrangement likely arose via 7 chromosome breaks with the insertion occurring first followed by sequential paracentric and then pericentric inversions.

Applications of imaging flow cytometry in the diagnostic assessment of acute leukaemia.

Automated imaging flow cytometry integrates flow cytometry with digital microscopy to produce high-resolution digital imaging with quantitative analysis. This enables cell identification based on morphology (cell size, shape), antigen expression, quantification of fluorescence signal intensity and localisation of detected signals (i.e. surface, cytoplasm, nuclear). We describe applications of imaging flow cytometry for the diagnostic assessment of acute leukaemia. These bone marrow malignancies are traditionally diagnosed and classified by cell morphology, phenotype and cytogenetic abnormalities. Traditionally morphology is assessed by light microscopy, phenotyping by conventional flow cytometry and genetics by karyotype and fluorescence in situ hybridisation (FISH) on interphase nuclei/metaphase spreads of cells on slides. Imaging flow cytometry adds a new dimension to the diagnostic assessment of these neoplasms. We describe three specific applications: From this we conclude that imaging flow cytometry offers benefits over conventional diagnostic methods. Specifically the ability to visualise the cells of interest, the pattern and localisation of expressed antigens and assess cytogenetic abnormalities in one integrated automated high-throughput test. Imaging flow cytometry presents a new paradigm for the diagnostic assessment of leukaemia.

Survival of Del17p CLL Depends on Genomic Complexity and Somatic Mutation.

PURPOSE: Chronic lymphocytic leukemia (CLL) with 17p deletion typically progresses quickly and is refractory to most conventional therapies. However, some del(17p) patients do not progress for years, suggesting that del(17p) is not the only driving event in CLL progression. We hypothesize that other concomitant genetic abnormalities underlie the clinical heterogeneity of del(17p) CLL. EXPERIMENTAL DESIGN: We profiled the somatic mutations and copy number alterations (CNA) in a large group of del(17p) CLLs as well as wild-type CLL and analyzed the genetic basis of their clinical heterogeneity. RESULTS: We found that increased somatic mutation number associates with poor overall survival independent of 17p deletion (P = 0.003). TP53 mutation was present in 81% of del(17p) CLL, mostly clonal (82%), and clonal mutations with del(17p) exhibit shorter overall survival than subclonal mutations with del(17p) (P = 0.019). Del(17p) CLL has a unique driver mutation profile, including NOTCH1 (15%), RPS15 (12%), DDX3X (8%), and GPS2 (6%). We found that about half of del(17p) CLL cases have recurrent deletions at 3p, 4p, or 9p and that any of these deletions significantly predicts shorter overall survival. In addition, the number of CNAs, but not somatic mutations, predicts shorter time to treatment among patients untreated at sampling. Indolent del(17p) CLLs were characterized by absent or subclonal TP53 mutation and few CNAs, with no difference in somatic mutation number. CONCLUSIONS: We conclude that del(17p) has a unique genomic profile and that clonal TP53 mutations, 3p, 4p, or 9p deletions, and genomic complexity are associated with shorter overall survival. Clin Cancer Res; 23(3); 735-45. ©2016 AACR.

Targeted sequencing using a 47 gene multiple myeloma mutation panel (M(3) P) in -17p high risk disease.

We constructed a multiple myeloma (MM)-specific gene panel for targeted sequencing and investigated 72 untreated high-risk (del17p) MM patients. Mutations were identified in 78% of the patients. While the majority of studied genes were mutated at similar frequency to published literature, the prevalence of TP53 mutation was increased (28%) and no mutations were found in FAM46C. This study provides a comprehensive insight into the mutational landscape of del17p high-risk MM. Additionally, our work demonstrates the practical use of a customized sequencing panel, as an easy, cheap and fast approach to characterize the mutational profile of MM.

Human epidermal growth factor receptor 2 testing in primary breast cancer in the era of standardized testing: a Canadian prospective study.

PURPOSE: Therapies that target overexpression of human epidermal growth factor receptor 2 (HER2) rely on accurate and timely assessment of all patients with new diagnoses. This study examines HER2 testing of primary breast cancer tissue when performed with immunohistochemistry (IHC) and additional in situ hybridization (ISH) for negative cases (IHC 0/1+). The analysis focuses on the rate of false-negative HER2 tests, defined as IHC 0/1+ with an ISH ratio ≥ 2.0, in eight pathology centers across Canada. PATIENTS AND METHODS: Whole sections of surgical resections or tissue microarrays (TMAs) from invasive breast carcinoma tissue were tested by both IHC and ISH using standardized local methods. Samples were scored by the local breast pathologist, and consecutive HER2-negative IHC results (IHC 0/1+) were compared with the corresponding fluorescence or silver ISH result. RESULTS: Overall, 711 surgical excisions of primary breast cancer were analyzed by IHC and ISH; HER2 and chromosome 17 centromere (CEP17) counts were available in all cases. The overall rate of false-negative samples was 0.84% (six of 711 samples). Interpretable IHC and ISH scores were available in 1,212 cases from TMAs, and the overall rate of false-negative cases was 1.6% (16 of 978 cases). CONCLUSION: Our observation confirms that IHC is an adequate test to predict negative HER2 status in primary breast cancer in surgical excision specimens, even when different antibodies and IHC platforms are used. The study supports the American Society of Clinical Oncology/College of American Pathologists and Canadian testing algorithms of using IHC followed by ISH for equivocal cases.

Understanding the molecular pathogenesis of acute promyelocytic leukemia.

Acute promyelocytic leukemia (APL) is a distinct subset of acute myeloid leukemia (AML) associated with peculiar biologic and clinical features and requiring specific management. At the genetic level, APL is featured by a unique chromosome translocation t(15;17) which results in the PML-RARα gene fusion and chimeric protein. APL is the first example of differentiation therapy targeted to a defined genetic target i.e. PML-RARα. PML-RARα behaves as an altered retinoic acid receptor with an ability of transmitting oncogenic signaling leading to accumulation of undifferentiated promyelocytes. All-trans-retinoic acid (ATRA) induces disease remission in APL patients by triggering terminal differentiation of leukemic promyelocytes. More recently, arsenic trioxide (ATO) has been shown to contribute degradation of the PML-RARα oncoprotein through bonding the PML moiety and has shown excellent synergism with ATRA in clinical trials. Elucidating the oncogenic signaling of PML-RARα through various transcription factors and the study of APL mouse models have greatly helped to understand the molecular pathogenesis of APL. However, the precise molecular mechanism by which t(15;17) is formed and initiates leukemia remains unknown. While transforming oncogenic potential of PML-RARα has been described extensively, the mechanistic events important for the formation of t(15;17) have been taken from the model of Therapy-related APL (t-APL).