Summary
Chronic/recurrent behaviour may be encountered in some distinct atypical or malignant lymphoproliferations, while recurrences are not generally observed in reactive/benign lymphadenopathies. ...We retrospectively analysed a consecutive series of 486 human immunodeficiency virus‐negative adults, who underwent lymphadenectomy. Neoplastic and benign/reactive histopathological pictures were documented in 299 (61·5%) and 187 (38·5%) cases, respectively. Of note, seven of the 111 (6·3%) patients with benign lymphadenopathy without well‐defined aetiology, showed chronic/recurrent behaviour, without constitutional symptoms. Enlarged lymph nodes were round in shape and hypoechoic, mimicking lymphoma. Reactive follicular hyperplasia and paracortical expansion were observed. Human herpesvirus (HHV)‐6B positive staining in follicular dendritic cells (FDCs) was documented in all seven patients. Serological, molecular and immunological examinations suggested HHV‐6B reactivation. Among the remaining 104 cases with reactive lymphoid hyperplasia in the absence of well‐known aetiology and without recurrences, positivity for HHV‐6B on FDCs was found in three cases, whereas in seven further patients, a scanty positivity was documented in rare, scattered cells in inter‐follicular regions. Immunohistochemistry for HHV‐6A and HHV‐6B was invariably negative on 134 lymph nodes, with either benign pictures with known aetiology or malignant lymphoproliferative disorders, tested as further controls. Future studies are warranted to investigate a potential association between HHV‐6B reactivation and chronic/recurrent benign lymphadenopathy.
Highlights • Invasive fusariosis (IF) is an emerging mould infection in hematologic patients. • Fusarium -specific T cells are significantly higher in IF patients. • In disseminated IF, specific T ...lymphocytes secretes pro-inflammatory cytokines. • In pulmonary IF, we documented only the presence of IL10-secriting T cells. • Fusarium -specific T cells could discriminate between different fungal pneumonias.
Bone marrow has already been described as an enrichment site for several antigen-specific T lymphocytes, but the presence of mould-specific T cells has never been investigated in the bone marrow. We ...have previously demonstrated that mould-specific T cells emerge in the peripheral blood of patients with invasive fungal infections (IFI) but tend to become undetectable after disease resolution. In seven patients with a history of IFI, we investigated the presence of mould-specific T cells secreting different cytokines in bone marrow and peripheral blood paired samples. The results showed that the frequencies of mould-specific T cells secreting the protective cytokine IFNγ are significantly higher in bone marrow (BM) and are mainly represented by CD8+ T lymphocytes with effector phenotype. A putative disappearance of such protective BM responses after myeloablative therapy could contribute to the increased risk of IFI in hematologic patients.
Abstract 284▪▪This icon denotes a clinically relevant abstract
Selection of drug-resistant mutations in the Bcr-Abl kinase domain (KD) is a critical problem undermining the long-term efficacy of ...tyrosine kinase inhibitor (TKI)-based therapies in Philadelphia-positive (Ph+) acute lymphoblastic leukemia (ALL) patients.
Bcr-Abl KD mutation screening is routinely performed by Sanger sequencing (SS). Before the advent of ultra-deep sequencing (UDS) technologies, no method was available that could conjugate the possibility to scan the KD for the so many mutations known to be associated with TKI resistance with a sensitivity higher than that of SS. UDS technologies also allow high throughputness and accurate quantitation of mutated clones and their application in a diagnostic setting is not far to come.
We used an UDS strategy for Bcr-Abl KD mutation screening in order to study the dynamics of expansion of mutated clones in Ph+ ALL patients receiving TKI-based therapies and to test the ability of UDS to highlight emerging clones harboring critical mutations.
72 samples from 25 Ph+ ALL patients who had developed resistance to one or multiple lines of TKI (imatinib, dasatinib, nilotinib, bosutinib, ponatinib) therapy were selected for this retrospective analysis. All the patients had previously been analyzed by Sanger sequencing (SS) and were known to have developed one or more TKI-resistant Bcr-Abl KD mutations on treatment. In order to reconstruct the dynamics of mutation emergence, longitudinal re-analysis of monthly collected samples was perfomed with UDS on a Roche GS Junior. UDS allowed to achieve a lower detection limit of at least 0.1% (by generating a minimum of 5,000 sequence reads/patient), as compared to 20% of SS.
39 samples were known to harbor one (n=27 samples) or more (n=12 samples) TKI-resistant mutations with >20% abundance, as assessed by SS. UDS could successfully detect all the 54 mutations previously identified by SS. In addition, UDS detected one or multiple lower-level (<20%) mutations in 42/72 (58%) samples, demonstrating that in more than half of the cases SS may misclassify Bcr-Abl KD mutation status or underestimate its complexity. Lower-level mutations were indeed found both in samples that had been scored as wild-type by SS and in samples already harboring mutations with >20% abundance. The type of lower-level mutations detected by UDS could easily be accounted for by TKI exposure history, since the majority were known to be poorly sensitive either to the TKI being administered or to the previous TKI received. Overall, 44 samples turned out to carry multiple (two to five) mutations at any level, distributed in the same and/or in different subpopulations with a complex clonal architecture that UDS allowed to reconstruct.
Of note, in 14/25 (56%) patients with molecularly detectable disease but not yet evidence of cytogenetic or hematologic relapse, UDS could identify emerging TKI-resistant mutations 1 to 2 months before they became detectable by SS. These outgrowing mutations were detected at 1–19% abundance in 12 patients and at 0.1–1% abundance in 2 patients. In the remaining 11 patients, dynamics of outgrow of the TKI-resistant mutations (five T315I, two Y253H, two E255K, one E255V and one F317L) was so rapid that not even strict monthly monitoring could allow to pick them up before they became dominant.
Now that multiple options are available, Bcr-Abl KD mutation monitoring has become a precious tool for rational decision-making in order to maximize the efficacy of TKI-based regimens as induction or salvage therapy for Ph+ ALL patients. UDS proved as reliable as SS for the detection of mutations with >20% abundance and to have comparable costs. As a key advantage, UDS added precious quantitative and qualitative information on the full repertoire of mutated populations, that SS failed to appreciate in more than half of the samples analyzed. TKI-resistant mutations leading to patient relapse were not necessarily preexisting at low levels at diagnosis or at the time of switchover to another TKI, underlining the importance of regular monitoring of patients. Although TKI-resistant populations may arise and take over very rapidly, in approximately half of the patients monthly monitoring with UDS would have allowed to identify them earlier than SS and well in advance of clinical relapse, thus allowing a more timely therapeutic intervention.
Soverini:Novartis: Consultancy; Bristol-Myers Squibb: Consultancy; ARIAD: Consultancy. Luppi:CELGENE CORPORATION: Research Funding. Baccarani:ARIAD, Novartis, Bristol Myers-Squibb, and Pfizer: Consultancy, Honoraria, Speakers Bureau. Martinelli:NOVARTIS: Consultancy, Honoraria, Speakers Bureau; BMS: Consultancy, Honoraria, Speakers Bureau; PFIZER: Consultancy; ARIAD: Consultancy.
Abstract 4222
Paroxysmal nocturnal hemoglobinuria (PNH) is an uncommon acquired disorder, consequence of a clonal expansion of haematopoietic stem cells that have acquired a somatic mutation of PIGA ...gene and is characterized by intravascular haemolysis, susceptibility to infections and arterial and venous thromobophilia. PNH frequently arises in association with disorders of bone marrow failure, particularly aplastic anaemia. Venous thrombo-embolism (VTE) is a major cause of morbidity and mortality in accounting for about 1/3 of all death. Pathogenesis of VTE is multifactorial but may be related to the ongoing haemolysis and the increased platelets activation by release of ADP and consumption of nitric oxide. Thromboembolic risk is known to be related to the clone size being higher in patients with PNH granulocytes > 50%, and probably to the ongoing haemolysis. PNH often affects the middle aged adults, of reproductive age, but women with PNH have to be generally discouraged from becoming pregnant because of the increased risk of complication both for the mother and the foetus. Twenty-six published clinical reports describe pregnancy outcome in 43 women with PNH: in the majority of cases no prophylactic anticoagulant therapy was administrated. Major maternal complication (mainly thromboembolic events) are reported in 16,3% of cases during pregnancy and in 30.2% of cases during puerperium. Five patients died as a consequence of thromboembolic events (n=3) and infections (n=2). The pregnancy terms with a pre-term delivery in 38% of cases, usually as a consequence of maternal complication: no foetal abnormalities were reported. Anticoagulation with low molecular weight heparin (LMWH) is generally recommended during pregnancy and puerperium, however, it provide only partial protection from thrombotic complications. Eculizumab is a humanized monoclonal antibody directed against the C5 complement protein, with inhibition of complement-mediated cell lysis. This drug is effective in controlling intravascular haemolysis, stabilizes haemoglobin levels, reduces transfusion requirements and thrombotic events and improves quality of life of PNH patients. It is composed by a hybrid IgG2-IgG4 constant fraction portion without antibody effector mechanism, moreover IgG2 isotypes do not cross placenta but few and partial experiences have been reported about its effects during pregnancy. We report the case of a 35-year old woman with a diagnosis of PNH establish in 2003 evolved from aplastic anaemia (1995). She needed about one unit of packed red blood cells monthly. She started eculizumab treatment at standard dosage (900 mg every other week) in April 2005 obtaining a rapid normalization of haemoglobin and LDH levels. She hadn't further haemolytic events and she never presented vascular complications. The patient became pregnant in October 2008: flow-cytometry analysis demonstrated that 35% of neutrophils and monocytes had loss of CD55 and CD59. Although the patient had a relatively small PNH clone and she hadn't previous thrombotic event, we decided to continue eculizumab during pregnancy in order to avoid haemolysis and the consequent VTE risk. Moreover, we started a prophylactic anticoagulant therapy with LMWH and anti-platelet therapy with acetylsalicylic acid at 16 weeks of gestation. During the entire pregnancy the patient never required transfusional support and the minimal haemoglobin dosage was 9 g/dl. Anti-platelet therapy was stopped at 36 weeks of gestation. At 38th weeks of gestation the patient delivered vaginally a child male, 3430 g, with Apgar score of 9 and 10. Both the mother and the newborn didn't experience any complication in particular the mother didn't need transfusional support and haemoglobin level maintained stable in spite of a mild LDH increase. Eculizumab treatment was continued also in the puerperium. In absence of data about the possible presence of eculizumab in breast milk and its possible effect on the newborn, we decided to avoid breast-feeding. At this time, after 2 months from the childbirth, both the mother and the newborn haven't medical complication. This is the first report of a pregnancy carried out during eculizumab treatment without significant toxicity for both the mother and the baby. The role of eculizumab in pregnancy and puerperium has to be further investigate in order to reduce the disease related maternal and foetal risk.
No relevant conflicts of interest to declare.
Abstract
Background & Aims - In chronic myeloid leukemia (CML) and Philadelphia-positive (Ph+) acute lymphoblastic leukemia (ALL), tyrosine kinase inhibitor (TKI) therapy may select for ...drug-resistant BCR-ABL mutants, routinely assessed by Sanger sequencing (SS). We took advantage of ultra-deep sequencing (UDS) in order to: 1) resolve qualitatively and quantitatively the complexity of mutated populations surviving TKIs; 2) investigate their clonal structure and evolution in relation to time and treatment.
Methods - We retrospectively performed a longitudinal analysis of 111 samples from 32 CML or Ph+ ALL patients who had received sequential treatment with multiple TKIs and had experienced sequential relapses accompanied by selection of TKI-resistant mutations. All samples had already been scored by SS. UDS of the BCR-ABL KD was done using Roche 454 technology (lower detection limit, 0.1%). Seqnext software was used for alignment and variant identification; Jalview and Figtree softwares were used for haplotype and phylogenetic reconstruction.
Results - UDS showed that SS often misclassifies or underestimates BCR-ABL KD mutation status. In more than half of the cases, UDS revealed that up to five ‘minor’ mutations with 1-20% abundance were present, either in samples scored as wild-type by SS or in samples already bearing one or more dominant mutations. The high degree of complexity was even more evident when the clonal relationships of multiple mutations were reconstructed and the relative abundance of all mutant subclones coexisting at each timepoint was assessed. This revealed that identical mutations may be acquired in parallel by independent subclones (e.g., one wild-type and one already harboring a mutation), via the same or different nucleotide changes leading to the same amino acid substitution (convergent evolution). Longitudinal quantitative follow-up showed that the landscape of all competing mutant subclones is highly dynamic, and that dominant subclones may be replaced as quickly as within one month in case of selective pressure change. Earlier identification of emerging TKI-resistant mutants was made possible by UDS.
Conclusions - 1) sequential changes in the selective pressure exerted by TKIs may result in a heterogeneous mosaic of subclones harbouring different mutations or mutation combinations
2) The ‘ecosystem’ of mutant subclones is highly dynamic: acquisition of additional mutations dictates quick shrinkage or expansion of subclones over time
3) Inherent sensitivity to a specific TKI is necessary but not sufficient to determine the ‘fitness’ of a mutant subclone: competition with coexisting subclones also concurs to shape its fate
4) Reasoning on the basis of mutations detectable by SS may not always be sufficient to predict responsiveness to a TKI.
Supported by Fondazione CARISBO, PRIN, IGA MZCR NT11555.
Citation Format: Simona Soverini, Caterina De Benedittis, Katerina Machova Polakova, Adela Brouckova, Fausto Castagnetti, Cristina Papayannidis, Gabriele Gugliotta, Francesca Palandri, Hana Klamova, Ilaria Iacobucci, Claudia Venturi, Federica Cattina, Paola Bresciani, Valeria Coluccio, Marzia Salvucci, Mario Tiribelli, Gianni Binotto, Tamara Intermesoli, Mario Luppi, Maria Teresa Bochicchio, Emanuela Ottaviani, Domenico Russo, Rosti Gianantonio, Michele Baccarani, Giovanni Martinelli. Unraveling the complexity of tyrosine kinase inhibitor-resistant populations by ultra-deep sequencing of the BCR-ABL kinase domain in Philadelphia-positive leukemias. abstract. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4632. doi:10.1158/1538-7445.AM2013-4632
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