[The early expressive vocabulary size in simultaneous bilingual growing-up infants - a diagnostic relevant criterion?]. / Der frühe expressive Wortschatzumfang simultan mehrsprachig aufwachsender Kinder - ein diagnostisch relevantes Kriterium?

AIM: Bilingual young children's early expressive vocabulary size and its composition (as one domain of the language development) should be examined to find out whether children with a risk for delayed language development may be identified in this way. METHOD: 30 bilingual kindergarten infants from Berlin (with simultaneous language acquisition; second language German) and 30 monolingual German infants from the greater areas of Stuttgart and Heidelberg were pair matched (mean chronological age 22.5 [SD 3.1] months; min 16; max 26). The German expressive vocabulary checklist Elternfragebogen zur Wort-schatzentwicklung im frühen Kindesalter (ELAN; Bockmann & Kiese-Himmel, 2006) was filled out by all parents. In addition, parents of bilingual infants completed the adaption of the German vocabulary checklist Sprachbeurteilung durch Eltern (SBE-2-KT; v. Suchodoletz & Sachse, 2008) for the second mother tongue. RESULTS: The monolinguals' word sum in the ELAN (145.7; SD 75.8) differed significantly (p=0.001) from the bilinguals' word sum (78.3; SD 78.9 words). In contrast, bilinguals did not significantly differ in their overall expressive vocabulary size (ELAN+SBE-2-KT: 101.2; SD 77.0 words) from their monolingual counterparts (ELAN). CONCLUSION: Because bilinguals had a similar sized overall early vocabulary (both languages) like monolingual German-learning infants, the diagnostic criterion to identify late talkers with 24 months of age (less than 50 German words and no word combinations) should not be applied to bilingually infants with simultaneously double language acquisition.

EBMT prospective observational study on allogeneic hematopoietic stem cell transplantation in T-prolymphocytic leukemia (T-PLL).

Preliminary data suggest that allogeneic stem cell transplantation (allo-SCT) may be effective in T-prolymphocytic leukemia (T-PLL). The purpose of the present observational study was to assess the outcome of allo-SCT in patients aged 65 years or younger with a centrally confirmed diagnosis of T-PLL. Patients were consecutively registered with the EBMT at the time of transplantation and followed by routine EBMT monitoring but with an extended dataset. Between 2007 and 2012, 37 evaluable patients (median age 56 years) were accrued. Pre-treatment contained alemtuzumab in 95% of patients. Sixty-two percent were in complete remission (CR) at the time of allo-SCT. Conditioning contained total body irradiation with 6 Gy or more (TBI6) in 30% of patients. With a median follow-up of 50 months, the 4-year non-relapse mortality, relapse incidence, progression-free (PFS) and overall survival were 32, 38, 30 and 42%, respectively. By univariate analysis, TBI6 in the conditioning was the only significant predictor for a low relapse risk, and an interval between diagnosis and allo-SCT of more than 12 months was associated with a lower NRM. This study confirms for the first time prospectively that allo-SCT can provide long-term disease control in a sizable albeit limited proportion of patients with T-PLL.

Drug-based perturbation screen uncovers synergistic drug combinations in Burkitt lymphoma.

Burkitt lymphoma (BL) is a highly aggressive B-cell lymphoma associated with MYC translocation. Here, we describe drug response profiling of 42 blood cancer cell lines including 17 BL to 32 drugs targeting key cancer pathways and provide a systematic study of drug combinations in BL cell lines. Based on drug response, we identified cell line specific sensitivities, i.e. to venetoclax driven by BCL2 overexpression and partitioned subsets of BL driven by response to kinase inhibitors. In the combination screen, including BET, BTK and PI3K inhibitors, we identified synergistic combinations of PI3K and BTK inhibition with drugs targeting Akt, mTOR, BET and doxorubicin. A detailed comparison of PI3K and BTKi combinations identified subtle differences, in line with convergent pathway activity. Most synergistic combinations were identified for the BET inhibitor OTX015, which showed synergistic effects for 41% of combinations including inhibitors of PI3K/AKT/mTOR signalling. The strongest synergy was observed for the combination of the CDK 2/7/9 inhibitor SNS032 and OTX015. Our data provide a landscape of drug combination effects in BL and suggest that targeting CDK and BET could provide a novel vulnerability of BL.

Discovery of novel drug sensitivities in T-PLL by high-throughput ex vivo drug testing and mutation profiling.

T-cell prolymphocytic leukemia (T-PLL) is a rare and aggressive neoplasm of mature T-cells with an urgent need for rationally designed therapies to address its notoriously chemo-refractory behavior. The median survival of T-PLL patients is <2 years and clinical trials are difficult to execute. Here we systematically explored the diversity of drug responses in T-PLL patient samples using an ex vivo drug sensitivity and resistance testing platform and correlated the findings with somatic mutations and gene expression profiles. Intriguingly, all T-PLL samples were sensitive to the cyclin-dependent kinase inhibitor SNS-032, which overcame stromal-cell-mediated protection and elicited robust p53-activation and apoptosis. Across all patients, the most effective classes of compounds were histone deacetylase, phosphoinositide-3 kinase/AKT/mammalian target of rapamycin, heat-shock protein 90 and BH3-family protein inhibitors as well as p53 activators, indicating previously unexplored, novel targeted approaches for treating T-PLL. Although Janus-activated kinase-signal transducer and activator of transcription factor (JAK-STAT) pathway mutations were common in T-PLL (71% of patients), JAK-STAT inhibitor responses were not directly linked to those or other T-PLL-specific lesions. Overall, we found that genetic markers do not readily translate into novel effective therapeutic vulnerabilities. In conclusion, novel classes of compounds with high efficacy in T-PLL were discovered with the comprehensive ex vivo drug screening platform warranting further studies of synergisms and clinical testing.

GvL effects in T-prolymphocytic leukemia: evidence from MRD kinetics and TCR repertoire analyses.

Allogeneic stem cell transplantation (alloSCT) is used for treating patients with T-prolymphocytic leukemia (T-PLL). However, direct evidence of GvL activity in T-PLL is lacking. We correlated minimal residual disease (MRD) kinetics with immune interventions and T-cell receptor (TCR) repertoire diversity alterations in patients after alloSCT for T-PLL. Longitudinal quantitative MRD monitoring was performed by clone-specific real-time PCR of TCR rearrangements (n=7), and TCR repertoire diversity assessment by next-generation sequencing (NGS; n=3) Although post-transplant immunomodulation (immunosuppression tapering or donor lymphocyte infusions) resulted in significant reduction (>1 log) of MRD levels in 7 of 10 occasions, durable MRD clearance was observed in only two patients. In all three patients analyzed by TCR-NGS, MRD responses were reproducibly associated with a shift from a clonal, T-PLL-driven profile to a polyclonal signature. Novel clonotypes that could explain a clonal GvL effect did not emerge. In conclusion, TCR-based MRD quantification appears to be a suitable tool for monitoring and guiding treatment interventions in T-PLL. The MRD responses to immune modulation observed here provide first molecular evidence for GvL activity in T-PLL which, however, may be often only transient and reliant on a poly-/oligoclonal rather than a monoclonal T-cell response.

Thiotepa-based high-dose therapy for autologous stem cell transplantation in lymphoma: a retrospective study from the EBMT.

Clinical information about thiotepa-based autologous stem cell transplantation (auto-SCT) outside the primary central nervous system lymphoma (PCNSL) field is sparse. In this registry-based retrospective study, we evaluated potential risks and benefits of thiotepa-based preparative regimens compared with BEAM (carmustine, etoposide, cytarabine, melphalan) in auto-SCT for diffuse large B-cell lymphoma (DLBCL, excluding PCNSL), follicular lymphoma (FL) or Hodgkin lymphoma (HL). A total of 14 544 patients (589 thiotepa and 13 955 BEAM) met the eligibility criteria, and 535 thiotepa- and 1031 BEAM-treated patients were matched in a 1:2 ratio for final comparison. No significant differences between thiotepa and BEAM groups for any survival end point were identified in the whole sample or disease entity subsets. For a more detailed analysis, 47 TEAM (thiotepa, etoposide, cytarabine, melphalan)-treated patients were compared with 75 matched BEAM patients with additional collection of toxicity data. Again, there were no significant differences between the two groups for any survival end point. In addition, the frequency of common infectious and non-infectious complications including secondary malignancies was comparable between TEAM and BEAM. These results indicate that thiotepa-based high-dose therapy might be a valuable alternative to BEAM in DLBCL, HL and FL. Further evaluation by prospective clinical trials is warranted.

p53-dependent non-coding RNA networks in chronic lymphocytic leukemia.

Mutations of the tumor suppressor p53 lead to chemotherapy resistance and a dismal prognosis in chronic lymphocytic leukemia (CLL). Whereas p53 targets are used to identify patient subgroups with impaired p53 function, a comprehensive assessment of non-coding RNA targets of p53 in CLL is missing. We exploited the impaired transcriptional activity of mutant p53 to map out p53 targets in CLL by small RNA sequencing. We describe the landscape of p53-dependent microRNA/non-coding RNA induced in response to DNA damage in CLL. Besides the key p53 target miR-34a, we identify a set of p53-dependent microRNAs (miRNAs; miR-182-5p, miR-7-5p and miR-320c/d). In addition to miRNAs, the long non-coding RNAs (lncRNAs) nuclear enriched abundant transcript 1 (NEAT1) and long intergenic non-coding RNA p21 (lincRNA-p21) are induced in response to DNA damage in the presence of functional p53 but not in CLL with p53 mutation. Induction of NEAT1 and lincRNA-p21 are closely correlated to the induction of cell death after DNA damage. We used isogenic lymphoma cell line models to prove p53 dependence of NEAT1 and lincRNA-p21. The current work describes the p53-dependent miRNome and identifies lncRNAs NEAT1 and lincRNA-p21 as novel elements of the p53-dependent DNA damage response machinery in CLL and lymphoma.

Comparison of three RT-PCR methods.

Various approaches can now be taken for amplification of RNA transcripts using the polymerase chain reaction (PCR). Here, we compare three such methods: (i) uncoupled reverse transcription (RT)-PCR (using separate reactions for cDNA synthesis and PCR), (ii) continuous RT-PCR (in which RT and DNA amplification occur in an uninterrupted reaction) using either a single enzyme for both RT and DNA amplification or (iii) using two enzymes, one for each task. We have found that the continuous two-enzyme RT-PCR method is the most sensitive, followed by the uncoupled RT-PCR and then the continuous single-enzyme method. The continuous methods require less sample handling than the uncoupled method, and hence are less labor-intensive and less prone to contamination. The continuous single-enzyme method is the most expensive to perform in terms of reagents due to the quantity of DNA polymerase required; however, it does have advantages over the two enzyme methods in that the use of a thermostable enzyme for RT can alleviate certain problems by allowing RT to occur at higher temperatures than those tolerable by viral reverse transcriptases.

Single-tube protocol for the extraction of DNA or RNA from paraffin-embedded tissues using a starch-based adhesive.

We describe a method using an inexpensive craft glue to routinely isolate specific areas of tissue as small as 1 mm2 from paraffin sections. The tissue may be digested to release nucleic acid suitable for PCR or reverse transcription PCR. The use of this procedure obviates the requirement for manual microdissection or ultraviolet light irradiation. Tissue remaining on the slide can be stained and analyzed, allowing the precision of the extraction to be determined. The slide can be stored as a permanent record of the material taken for analysis.

The use of optimal cutting temperature compound can inhibit amplification by polymerase chain reaction.

Optimal cutting temperature (OCT) is a widely used embedding medium for tissues for histopathologic analysis. This investigation examined the effects that OCT storage can have upon the ability to perform subsequent molecular biological analyses. Tumor material was dissected into small pieces and stored at approximately 20 degrees C both with and without OCT. DNA and RNA were then extracted from the tissue fragments and analyzed by the polymerase chain reaction (PCR), using primer sets designed to amplify a range of product sizes, and also by reverse transcriptase-PCR (RT-PCR). The storage of pathological specimens in OCT compound was found to affect significantly and irreversibly the ability to amplify DNA in the PCR, particularly as the size of the amplified fragment increased. This effect appeared to occur as a result of greater degradation of DNA extracted from tissue embedded in OCT compared to DNA extracted from tissue stored without OCT. RNA quality appeared unaffected, which may be because of the extraction protocol employed. Our results suggest that OCT-embedded frozen-tissue samples may be used for RNA isolation for subsequent RT-PCR and for the in vitro amplification of DNA targets of approximately < 300 base pairs only. We strongly advise against the routine storage of any tissue biopsy material in OCT if molecular analyses may be required.

Sensitive detection of Ross River virus--a one-tube nested RT-PCR.

A sensitive nested RT-PCR that can be carried out in a single tube is described. The sensitivity of this system was determined, and compared to that of a single round of PCR, and a single round of PCR followed by hybridisation with a radiolabelled oligonucleotide probe. We found that with the one-tube nested RT-PCR we were able to detect 0.1 pfu/ml of Ross River virus. The nested RT-PCR was 100-times more sensitive than a single round of RT-PCR followed by hybridisation, and 10,000-times more sensitive than a single round of RT-PCR alone. This system provides a sensitive detection of Ross River virus, and can be adapted for detection of RNA from any source. The test material is added to a single tube at the outset, and by subsequent addition of two sets of reagents, the entire nested RT-PCR can be carried out in the same tube. This system has maximum sensitivity, minimises risk of contamination, and is amenable to automation.

A one-tube, one manipulation RT-PCR reaction for detection of Ross River virus.

A sensitive, single tube reverse transcription-polymerase chain reaction (RT-PCR) protocol for the detection of Ross River virus (RRV) is described. All components necessary for both reverse transcription and PCR were combined in a single tube, and reverse transcription and PCR carried out sequentially in a single, non-interrupted thermal cycling program. The antisense oligonucleotide from the two primers selected for use in the PCR also served to prime specifically for the reverse transcription. The 549 bp product was detected by electrophoresis and ethidium bromide staining. The detection limit using this system was 18 fg of purified viral RNA or 1.3 pfu of whole virus. Greater sensitivity cannot reasonably be expected unless a more sensitive method than electrophoresis and ethidium bromide staining is used for PCR product detection, such as nested PCR or hybridisation with labelled probe. This PCR detection system will be adapted for detection of RRV in mosquito populations for virus surveillance programs.

Variant chromophobe renal cell carcinoma.

Separation of renal cell tumors into different prognostic groups is an imperative function of the diagnostic pathologist. Recently, chromophobe renal carcinoma has been described as a tumor that is morphologically distinct from conventional "clear cell" carcinoma and that has a low metastatic potential. Identification is based on routine light microscopic features and is confirmed by special stains, immunohistochemistry, and electron microscopy. We present a variant of chromophobe renal carcinoma that did not show the typical cytomorphologic features on light microscopy after formaldehyde fixation. After fixation in Solufix (a commercial fixative), these features were recognized and the diagnosis was confirmed. The tumor also showed an unusual form of calcification and psammoma body formation not previously recognized in chromophobe tumors. Molecular biological assessment was inconclusive, but excluded a chromosome 3p deletion usually found in conventional renal carcinoma. The use of a different primary fixative may provide a cost-effective screening tool to detect variant renal tumors and may have important prognostic implications.

Sarcomatoid renal cell carcinoma of papillary origin. A case report and cytogenic evaluation.

Sarcomatoid renal cell carcinoma (SRCC) is an aggressive tumor variant thought to arise predominantly from dedifferentiation of clear cell carcinoma. A few reports of SRCC associated with non-clear cell tumors led to the presumption that SRCC may arise from any renal cell carcinoma, although direct evidence of this is lacking. Cytogenetic studies on 3 previously documented SRCCs associated with papillary renal cancers showed either 3p deletions or absence of trisomy 7, 17 in the sarcomatoid tumors, suggesting origin from a coexistent clear cell tumor. The present case represents the first conclusive evidence of direct progression of non-clear cell carcinoma to SRCC with both tumor components containing multiple copies of chromosomes 7 and 17. Many genetic anomalies, including p53 mutations, frequently recognized in SRCC were not recognized in this case, highlighting the importance of cytogenetic evaluation of all SRCC. The patient is well and without evidence of tumor progression 1 year after surgery, and the sinister outlook of SRCC in association with clear cell carcinoma may not apply in SRCC of non-clear cell origin.

Chaetoglobosin A preferentially induces apoptosis in chronic lymphocytic leukemia cells by targeting the cytoskeleton.

Chronic lymphocytic leukemia (CLL) is an incurable malignancy of mature B cells. One of the major challenges in treatment of CLL is the achievement of a complete remission to prevent relapse of disease originating from cells within lymphoid tissues and subsequent chemoresistance. In search for novel drugs that target CLL cells in protective microenvironments, we performed a fungal extract screen using cocultures of primary CLL cells with bone marrow-derived stromal cells. A secondary metabolite produced by Penicillium aquamarinium was identified as Chaetoglobosin A (ChA), a member of the cytochalasan family that showed preferential induction of apoptosis in CLL cells, even under culture conditions that mimic lymphoid tissues. In vitro testing of 89 CLL cases revealed effective targeting of CLL cells by ChA, independent of bad prognosis characteristics, like 17p deletion or TP53 mutation. To provide insight into its mechanism of action, we showed that ChA targets filamentous actin in CLL cells and thereby induces cell-cycle arrest and inhibits membrane ruffling and cell migration. Our data further revealed that ChA prevents CLL cell activation and sensitizes them for treatment with PI3K and BTK inhibitors, suggesting this compound as a novel potential drug for CLL.

What do we do with chronic lymphocytic leukemia with 17p deletion?

Chronic lymphocytic leukemia (CLL) with 17p deletion or mutations of the TP53 gene has a very poor outcome. Optimal treatment of these patients remains a major clinical challenge, and disagreement on the optimal treatment approach exists. Conventional chemo-immunotherapy with rituximab in combination with purine analogues yields lower response-rates and less satisfactory results than for CLL patients with intact p53. Allogeneic stem cell transplantation may allow long-term remissions in this challenging group of patients. In this review, we will discuss current treatment options as well as experimental approaches in clinical trials for CLL patients with deleted or mutated TP53. Particular emphasis will be placed on novel agents with the potential to change clinical practice and future perspectives for the management of these "highest risk" patients.