Genomic description of acquired fluconazole- and echinocandin-resistance in patients with serial Candida glabrata isolates.

Candida glabrata is one of the most common causes of systemic candidiasis, often resistant to antifungal medications. To describe the genomic context of emerging resistance, we conducted a retrospective analysis of 82 serially collected isolates from 33 patients from population-based candidemia surveillance in the United States. We used whole-genome sequencing to determine the genetic relationships between isolates obtained from the same patient. Phylogenetic analysis demonstrated that isolates from 29 patients were clustered by patient. The median SNPs between isolates from the same patient was 30 (range: 7-96 SNPs), while unrelated strains infected four patients. Twenty-one isolates were resistant to echinocandins, and 24 were resistant to fluconazole. All echinocandin-resistant isolates carried a mutation either in the FKS1 or FKS2 HS1 region. Of the 24 fluconazole-resistant isolates, 17 (71%) had non-synonymous polymorphisms in the PDR1 gene, which were absent in susceptible isolates. In 11 patients, a genetically related resistant isolate was collected after recovering susceptible isolates, indicating in vivo acquisition of resistance. These findings allowed us to estimate the intra-host diversity of C. glabrata and propose an upper boundary of 96 SNPs for defining genetically related isolates, which can be used to assess donor-to-host transmission, nosocomial transmission, or acquired resistance. IMPORTANCE In our study, mutations associated to azole resistance and echinocandin resistance were detected in Candida glabrata isolates using a whole-genome sequence. C. glabrata is the second most common cause of candidemia in the United States, which rapidly acquires resistance to antifungals, in vitro and in vivo.

[Atypical purpuric oedema of the nose during granulomatosis with polyangiitis]. / Un œdème purpurique du nez atypique au cours d'une granulomatose avec polyangéite.

INTRODUCTION: Granulomatosis with polyangeitis or Wegener's disease is a necrotizing vasculitis of small and medium vessels associated with antineutrophil cytoplasmic autoantibodies (ANCA). The most frequent sites are lung, ear, nose and throat and kidney. PATIENTS AND METHODS: We report the case of a 47-year-old woman presenting purpuric oedematous plaque with bullous detachment of the nose and hospitalised for the assessment of two suspicious neoplastic lung lesions discovered as a result of a recent stroke and repeated seromucosal otitis. Granulomatosis with polyangeitis was suspected because of multiple systemic lesions. The histopathology of skin lesions and laboratory investigation results were consistent with this diagnosis. A favourable outcome was achieved with corticosteroids and rituximab. DISCUSSION: The diagnosis of GPA is based on criteria established by the American College of Rheumatology. The cutaneous clinical aspect described in our case confirms the polymorphism of the cutaneous lesions possibly associated with this disease. They are rarely isolated but, in some cases, allow early diagnosis with improved prognosis, which remains severe in the absence of treatment.

Randomized phase-III study of low-dose cytarabine and etoposide + /- all-trans retinoic acid in older unfit patients with NPM1-mutated acute myeloid leukemia.

The aim of this randomized clinical trial was to evaluate the impact of all-trans retinoic acid (ATRA) in combination with non-intensive chemotherapy in older unfit patients (> 60 years) with newly diagnosed NPM1-mutated acute myeloid leukemia. Patients were randomized (1:1) to low-dose chemotherapy with or without open-label ATRA 45 mg/m2, days 8-28; the dose of ATRA was reduced to 45 mg/m2, days 8-10 and 15 mg/m2, days 11-28 after 75 patients due to toxicity. Up to 6 cycles of cytarabine 20 mg/day s.c., bid, days 1-7 and etoposide 100 mg/day, p.o. or i.v., days 1-3 with (ATRA) or without ATRA (CONTROL) were intended. The primary endpoint was overall survival (OS). Between May 2011 and September 2016, 144 patients (median age, 77 years; range, 64-92 years) were randomized (72, CONTROL; 72, ATRA). Baseline characteristics were balanced between the two study arms. The median number of treatment cycles was 2 in ATRA and 2.5 in CONTROL. OS was significantly shorter in the ATRA compared to the CONTROL arm (p = 0.023; median OS: 5 months versus 9.2 months, 2-years OS rate: 7% versus 10%, respectively). Rates of CR/CRi were not different between treatment arms; infections were more common in ATRA beyond treatment cycle one. The addition of ATRA to low-dose cytarabine plus etoposide in an older, unfit patient population was not beneficial, but rather led to an inferior outcome.The clinical trial is registered at clinicaltrialsregister.eu (EudraCT Number: 2010-023409-37, first posted 14/12/2010).

Dose-escalated CHOEP for the treatment of young patients with aggressive non-Hodgkin's lymphoma: I. A randomized dose escalation and feasibility study with bi- and tri-weekly regimens.

BACKGROUND: To determine the maximum tolerated dose of a bi- and tri-weekly combination chemotherapy with cyclophosphamide, doxorubicin, vincristine and prednisone plus etoposide (CHOEP) regimen without stem-cell support. PATIENTS AND METHODS: Randomized phase I/II multicenter four-level (cyclophosphamide: 1000-1200-1400-1600 mg/m2; doxorubicin: 55-60-65-70 mg/m2; etoposide: 375-450-525-600 mg/m2) dose escalation study with CHOEP-14 and CHOEP-21 in young patients (18-60 years) with newly diagnosed aggressive non-Hodgkin's lymphoma. Dose-limiting toxicity was defined as thrombocytopenia <80,000/mm3 and leukocytopenia <2500/mm3 on days 16 (CHOEP-14) and 23 (CHOEP-21) or prolonged (>4 days) leukocytopenia (<1000/mm3) or thrombocytopenia (<20,000/mm3). RESULTS: One hundred and thirty-nine patients (high-CHOEP-14: 47, high-CHOEP-21: 92) were randomly allocated to the study. Maximal tolerated dose was level 2 for CHOEP-14 and level 4 for CHOEP-21. With a less favorable profile of patients in CHOEP-14, 4-year event-free survival was 47.9% after high-CHOEP-14 and 66.2% after high-CHOEP-21, 4-year overall survival 62.1% after high-CHOEP-14 and 73.4% after high-CHOEP-21, respectively. CONCLUSION: Significant dose escalations of CHOEP are possible with granulocyte colony-stimulating factor support, with different chemotherapy models favoring the maximally escalated bi- or tri-weekly regimen, respectively. Because a higher total dose can be achieved with six cycles of the tri-weekly compared with the biweekly regimen, CHOEP-21 at dose escalation level 3 was chosen for a nationwide randomized comparison with baseline CHOEP-21 in a subsequent phase III trial.

Dose-escalated CHOEP for the treatment of young patients with aggressive non-Hodgkin's lymphoma: II. Results of the randomized high-CHOEP trial of the German High-Grade Non-Hodgkin's Lymphoma Study Group (DSHNHL).

BACKGROUND: The addition of etoposide to combination chemotherapy with cyclophosphamide, doxorubicin, vincristine and prednisone [etoposide to combination chemotherapy with cyclophosphamide, vincristine and prednisone (CHOEP)] improved outcome of young patients with good-prognosis aggressive lymphoma. To improve results further, the maximal dose-escalated version of CHOEP-21 tolerable without stem-cell support (high CHOEP: cyclophosphamide 1400 mg/m2, doxorubicin 65 mg/m2, vincristine 2 mg, etoposide 175 mg/m2 x3, prednisone 100 mg x5) was compared with CHOEP-21. PATIENTS AND METHODS: Intention-to-treat analysis of 389 young (18-60 years) patients with good-prognosis (age-adjusted International Prognostic Index = 0, 1) aggressive lymphoma randomized to CHOEP-21 (n = 194) or high CHOEP (n = 195). RESULTS: There was no difference in 3-year event-free (64% versus 67%; P = 0.734) or overall survival (83% versus 87%; P = 0.849). Neither low-risk nor low-intermediate risk patients benefited from high CHOEP. High CHOEP was more toxic than CHOEP-21 (grades 3 and 4 leukocytopenia 100% versus 87.2%, P < 0.001; thrombocytopenia 80.8% versus 9.6%, P < 0.001; infections 35% versus 11%, P < 0.001; therapy-associated deaths 3.1% versus 0%, P = 0.03). CONCLUSION: Dose-escalated CHOEP-21 does not provide clinical benefit for young patients with good-prognosis aggressive lymphomas. Since differences between chemotherapy regimens are compressed by the addition of rituximab, the results of this trial have bearing on strategies aiming to improve outcome of good-prognosis aggressive lymphomas in the rituximab era.

Condensed versus standard schedule of high-dose cytarabine consolidation therapy with pegfilgrastim growth factor support in acute myeloid leukemia.

The aim of this cohort study was to compare a condensed schedule of consolidation therapy with high-dose cytarabine on days 1, 2 and 3 (HDAC-123) with the HDAC schedule given on days 1, 3 and 5 (HDAC-135) as well as to evaluate the prophylactic use of pegfilgrastim after chemotherapy in younger patients with acute myeloid leukemia in first complete remission. One hundred and seventy-six patients were treated with HDAC-135 and 392 patients with HDAC-123 with prophylactic pegfilgrastim at days 10 and 8, respectively, in the AMLSG 07-04 and the German AML Intergroup protocol. Time from start to chemotherapy until hematologic recovery with white blood cells >1.0 G/l and neutrophils >0.5 G/l was in median 4 days shorter in patients receiving HDAC-123 compared with HDAC-135 (P<0.0001, each), and further reduced by 2 days (P<0.0001) by pegfilgrastim. Rates of infections were reduced by HDAC-123 (P<0.0001) and pegfilgrastim (P=0.002). Days in hospital and platelet transfusions were significantly reduced by HDAC-123 compared with HDAC-135. Survival was neither affected by HDAC-123 versus HDAC-135 nor by pegfilgrastim. In conclusion, consolidation therapy with HDAC-123 leads to faster hematologic recovery and less infections, platelet transfusions as well as days in hospital without affecting survival.

Assessment of imatinib as first-line treatment of chronic myeloid leukemia: 10-year survival results of the randomized CML study IV and impact of non-CML determinants.

Chronic myeloid leukemia (CML)-study IV was designed to explore whether treatment with imatinib (IM) at 400 mg/day (n=400) could be optimized by doubling the dose (n=420), adding interferon (IFN) (n=430) or cytarabine (n=158) or using IM after IFN-failure (n=128). From July 2002 to March 2012, 1551 newly diagnosed patients in chronic phase were randomized into a 5-arm study. The study was powered to detect a survival difference of 5% at 5 years. After a median observation time of 9.5 years, 10-year overall survival was 82%, 10-year progression-free survival was 80% and 10-year relative survival was 92%. Survival between IM400 mg and any experimental arm was not different. In a multivariate analysis, risk group, major-route chromosomal aberrations, comorbidities, smoking and treatment center (academic vs other) influenced survival significantly, but not any form of treatment optimization. Patients reaching the molecular response milestones at 3, 6 and 12 months had a significant survival advantage. For responders, monotherapy with IM400 mg provides a close to normal life expectancy independent of the time to response. Survival is more determined by patients' and disease factors than by initial treatment selection. Although improvements are also needed for refractory disease, more life-time can currently be gained by carefully addressing non-CML determinants of survival.

[Fatal giant cell arteritis with severe bilateral involvement of the vertebral arteries]. / Maladie de Horton d'évolution fatale avec atteinte sévère bilatérale des artères vertébrales.

With steroid therapy, it is commonly considered that prognosis is good in giant cell arteritis. However serious or even fatal complications may occur. Here we report the case of a patient who developed fatal giant cell arteritis with severe stenosis of both vertebral arteries and right carotid siphon. Several similar cases have been reported in the literature. Initially diagnosis may be difficult because neurological manifestations are intermittent and classical signs of giant cell arteritis may be lacking. In such condition the reason of poor outcome is unknown and therapy remains empiric.

[Ortner's syndrome and giant-cell vasculitis]. / Syndrome d'Ortner et maladie de Horton.

INTRODUCTION: Ortner's syndrome was first described as a left laryngeal nerve palsy caused by a dilated left atrium in mitral stenosis. Aortic aneurysm is another well-documented etiology. CASE RECORD: We report the case of a 90 year-old woman with temporal arteritis with recent onset hoarseness, and simultaneous discovery of aortic arch aneurysm and left vocal cord palsy. DISCUSSION: The occurrence of hoarseness and aortic aneurysm in Giant-cell vasculitis is discussed. We suggest to consider Horton's disease (GCA) as a possible etiology of Ortner's cardio-vocal syndrome.

Mapping of chromosomal imbalances in pancreatic carcinoma by comparative genomic hybridization.

To identify recurrent chromosomal imbalances in pancreatic adenocarcinoma, 27 tumors were analyzed by using comparative genomic hybridization. In 23 cases chromosomal imbalances were found. Gains of chromosomal material were much more frequent than losses. The most common overrepresentations were observed on chromosomes 16p (eight cases), 20q (seven cases), 22q (six cases), and 17q (five cases) and under-representations on a subregion of chromosome 9p (eight cases). Distinct high-level amplifications were found on 1p32-p34, 6q24, 7q22, 12p13, and 22q. These data provide evidence for a number of new cytogenetically defined recurrent aberrations which are characteristic of pancreatic carcinoma. The overrepresented or underrepresented chromosomal regions represent candidate regions for potential oncogenes and tumor suppressor genes, respectively, possibly involved in pancreatic tumorigenesis.

Alterations of the cyclin D1/p16-pRB pathway in mantle cell lymphoma.

Mantle cell lymphoma (MCL) has recently become generally accepted as a subentity of malignant lymphomas that is characterized by the chromosomal translocation t(11;14)(q13;q32), resulting in the overexpression of cyclin D1. Cyclin D1 forms a complex with cell cycle-dependent kinase (cdk) 4, which inactivates the retinoblastoma protein (pRB) via phosphorylation. However, in transgenic mice, the overexpression of cyclin D1 alone is not sufficient for the development of malignant lymphoma. To determine whether other members of the pRB pathway contribute to the malignant transformation of MCL, we analyzed 37 cases of MCL that were well characterized by morphology, immunophenotype, and/or interphase cytogenetics [detection of t(11;14)(q13;q32)]. Interphase fluorescence in situ hybridization was performed using a cosmid contig (250 kb) of the CDKN2/p16 region (encoding an inhibitor of the cyclin D1/cdk4 complex) and a phage contig (200 kb) of the Rb region. CDKN2/p16 deletion was detected in 15 cases (41%), including 6 homozygous deletions; Rb was deleted in 15 cases (41%), all of which were hemizygous deletions. Nine cases (24%) had deletions of both CDKN2/p16 and Rb. Further analysis of a subset of 17 MCLs revealed a highly significant correlation between CDKN2/p16 deletion and proliferation index, determined by the rate of Ki67 expression (P = 0.014; t test). No significant correlation was found between CDKN2/p16 deletion and the blastoid variant of MCL (P = 0.23; Fisher's test) or between proliferation index and blastoid morphology (P = 0.51; t test). Deletion of Rb did not have any impact on cell proliferation in addition to CDKN2/p16 deletion (P = 0.76; t test). Additional analysis of 13q14 deletions suggests that these deletions may target another gene telomeric to Rb. We conclude that deletion of CDKN2/p16 occurs in approximately one-half of MCLs and is a more relevant indicator of the proliferative features as compared to morphological criteria. In contrast, although deletions of chromosomal band 13q14 are frequent in MCL, inactivation of Rb seems not to be involved in the pathogenesis of MCL.

Genomic imbalances including amplification of the tyrosine kinase gene JAK2 in CD30+ Hodgkin cells.

Comparative genomic hybridization was applied for a comprehensive screening of frequently occurring net gains and losses of chromosomal subregions in small populations of CD30+ Hodgkin cells and their morphological variants. In 12 Hodgkin's lymphomas, recurrent gains were detected on chromosomal arms 2p, 9p, and 12q (in six, four, and five tumors, respectively) and distinct high-level amplifications were identified on chromosomal bands 4p16, 4q23-q24, and 9p23-p24. In Hodgkin cells with 9p23-p24 amplification, fluorescence in situ hybridization revealed an increased copy number of chromosomal sequences spanning the tyrosine kinase gene JAK2. Several of the imbalances described, in particular a gain in chromosomal arm 9p that includes JAK2 amplification, are similar to the genomic changes detected in primary mediastinal B-cell lymphoma.

Heterogeneity of deletions involving RB-1 and the D13S25 locus in B-cell chronic lymphocytic leukemia revealed by fluorescence in situ hybridization.

Recently, the D13S25 locus, which is in close proximity to the retinoblastoma gene (RB-1) on chromosome band 13q14, was discussed to play a role in the pathogenesis of B-CLL. In the present study, we isolated two overlapping genomic DNA clones (termed c13S25) containing the D13S25 DNA segment and used them as probes to analyze 85 B-CLL cases by fluorescence in situ hybridization; of the 55 cases with two RB-1 copies, 13 exhibited hemizygous (n = 7) or homozygous (n = 6) deletion of D13S25. Of 29 cases with hemizygous deletion of RB-1, all but two also showed loss of D13S25 (hemizygous, n = 25; homozygous, n = 2). One case had a homozygous deletion of both loci. We conclude that deletion of D13S25 occurs in a substantial number of B-CLL without deletion of RB-1. However, in some cases there is deletion of RB-1 without loss of D13S25, suggesting that D13S25 is not the locus of the putative tumor suppressor gene. According to our data, such a gene is most likely located within the genomic region between D13S25 and RB-1.

Fluorescence in situ hybridization in leukemias: 'the FISH are spawning!'.

Fluorescence in situ hybridization (FISH) is a powerful tool for the analysis of chromosomal abnormalities in metaphase and interphase cells. Interphase cytogenetics has become an important technique for the analysis of leukemias, since in many cases it may be difficult to obtain metaphase spreads representative for the malignant clone(s). Using suitable DNA probes, leukemia samples can be screened for the most relevant chromosomal abnormalities. Alternatively, chromosomal imbalances can be identified by applying the new approach of comparative genomic hybridization. In this review, recent advances in the analysis of leukemia made possible by FISH are presented and future prospects of molecular cytogenetics are discussed.

Cytogenetic and molecular cytogenetic analysis of B cell chronic lymphocytic leukemia: specific chromosome aberrations identify prognostic subgroups of patients and point to loci of candidate genes.

The most frequent chromosome aberrations in B cell chronic lymphocytic leukemia (B-CLL) detected by conventional chromosome banding analysis are trisomy 12 followed by structural abnormalities of the long arms of chromosomes 13, 14, and 11. Complex karyotypes, trisomy 12, and a '14q+' abnormality have been associated with inferior prognosis, whereas aberrations of 13q have been found in patients with a favorable outcome. However, the cytogenetic analysis of B-CLL by conventional banding techniques has remained a difficult task mainly due to the low in vitro mitotic activity of the tumor cells. Although B cell mitogens are used for cell culture, clonal chromosome aberrations are detected in only half of the B-CLL tumors. 'Interphase cytogenetics' by means of fluorescence in situ hybridization (FISH) circumvents this problem, because there is no need to induce the malignant cells to proliferate in vitro. Numerical and structural chromosome aberrations can be detected in non-dividing interphase cells as well as in metaphase spreads. By FISH, the most common chromosome abnormalities are deletions of 13q followed by deletions of 11q, trisomy 12, and deletions of 17p. Except for the TP53 gene at 17p13, no candidate gene affected by these aberrations has so far been identified. FISH will be instrumental for the identification of such genes because the recurrent aberrations, especially deletions, can be systematically delineated to the resolution level of several kb. Furthermore, based on the sensitive detection of chromosome abnormalities by FISH, more accurate correlations between chromosome abnormalities and prognosis can be performed. Deletion of the TP53 gene at 17p13 have already been shown to be one of the most important independent prognostic factors for survival. Other specific aberrations of clinical significance will likely be identified by the systematic application of interphase cytogenetics on a large series of patients.

Detection of secondary genetic aberrations in follicle center cell derived lymphomas: assessment of the reliability of comparative genomic hybridization and standard chromosome analysis.

Secondary chromosomal aberrations in follicle center cell derived lymphomas (FCDL) usually involve gains and losses of genetic material and may be an important prognostic value. In the present study, we aimed to determine the power of comparative genomic hybridization (CGH) as compared to standard chromosome analysis (CA) to detect such secondary aberrations. The same lymph node cell suspensions prepared from 30 patients with FCDL were analyzed in parallel by CGH and CA based on R banding. In all, 73 discrepancies were found. Sixty-two imbalances were detected only by CA and 11 only by CGH. In cases with completely resolved karyotypes (n= 17), the median number of discrepancies between CGH and CA was one. However, when the karyotype was partially resolved (n = 12), the median was four (P < 0.01). Discrepant results were further studied by fluorescence in situ hybridization using locus-specific probes. These data confirm, that not only for the detection of balanced aberrations, but also for the detection of unbalanced aberrations in FCDL, standard chromosome analysis is still the 'gold standard'. In contrast, CGH is useful to detect chromosomal imbalances when no metaphases are found or no fresh material is available.

BCL10 is not the gene inactivated by mutation in the 1p22 deletion region in mantle cell lymphoma.

The BCL10 gene has recently been cloned from the 1p22 breakpoint of the translocation t(1 ;14)(p22;q32) observed in mucosa-associated lymphoid tissue (MALT) lymphoma. BCL10 was shown to be a proapoptotic-signaling gene encoding a protein that contains an amino-terminal caspase recruitment domain (CARD). Mutations within the BCL10 coding region resulting in truncated BCL10 proteins with loss of their proapoptotic function and preservation of their NF-kappaB activating function were detected in MALT lymphoma. Based on these findings it was proposed that BCL10 might have tumor suppressor function. Deletions involving 1p22 are commonly observed in mantle cell lymphoma (MCL). To investigate its role in MCL we have analyzed a series of 15 MCL for deletion and mutation of BCL10. Monoallelic 1p22 deletions were detected by fluorescence in situ hybridization in five of the 15 cases and were shown to affect BCL10 in all cases. BCL10 was screened for mutations by DNA sequencing of RT-PCR amplified transcripts. In none of the 15 MCL cases studied were mutations found in the BCL10 coding region. A previously reported polymorphism exhibiting a silent 24C > G substitution was found in eight MCL cases and in four healthy probands. A missense mutation 13G >T resulting in a substitution of a serine by an alanine was seen in one of the controls. Our results strongly suggest that BCL10 is not the candidate tumor suppressor gene inactivated by deletion or mutation in band 1p22 in MCL.

Detection of trisomy 8 on blood smears using fluorescence in situ hybridization.

In this study, fluorescence in situ hybridization (ISH) with an alphoid probe was used for the detection of trisomy 8 on archival blood smears (BS). The results were compared with hybridization experiments performed on methanol/acetic acid fixed cells of cytogenetic preparations (CP) which are widely used for ISH. Five controls and 20 patients with myeloid leukemias were examined. In the controls, CP and BS had the same percentages of cells with two or three fluorescence signals. In 5/20 patients, trisomy 8 was detected both on CP and BS. Two of the patients had 7 to 10% interphase cells with three hybridization signals, indicating the presence of small subclones with trisomy 8; one of the subclones was not detected by G-banding analysis. The remaining 15 patients were disomic for chromosome 8; hybridization results were within the range of the controls both on CP and BS. We conclude that using a chromosome 8 specific alphoid probe, fluorescence ISH to interphase cells can be performed on BS with the same efficiency that is reached on methanol/acetic acid fixed cells of CP.