Manuela Zappa

Erythropoietin production and erythropoiesis in compensated and anaemic states of hereditary spherocytosis

A compensated haemolytic state is defined by decreased red cell life‐span without anaemia, i.e. by increased erythropoiesis in the absence of the physiological stimulus for erythropoietin (Epo) production. We evaluated s‐Epo levels and the expansion of erythropoiesis (as measured by circulating transferrin receptor, s‐TfR) in 32 patients with hereditary spherocytosis (HS) with the aim of verifying whether the enhanced erythropoiesis of compensated haemolysis was Epo‐dependent. 20 of the patients (62.5%) had normal Hb values (> 12 g/dl in females and > 13 g/dl in males). Their compensated haemolytic state was the result of up to 8.2 times normal s‐Epo and up to 3.9 times normal s‐TfR levels, which were maintained by physiological regulation of erythropoiesis, as documented by the inverse dependence of Hb on s‐Epo levels. Considering that patients with iron‐deficiency anaemia represented the predicted physiological Epo response to anaemia, the observed/predicted ln s‐Epo ratio (O/P ratio) was calculated in HS patients with anaemia and was used as an index of the adequateness of Epo production. All the anaemic HS patients had an O/P ratio > 1, documenting inappropriately high s‐Epo levels. This work demonstrates that the compensated haemolytic state of HS patients is produced by an inappropriately high s‐Epo level, and that the pattern of Epo overproduction is a biological characteristic of the disease.

A case of complete adenylate kinase deficiency due to a nonsense mutation in AK-1 gene (Arg 107 --> Stop, CGA --> TGA) associated with chronic haemolytic anaemia.

Two siblings of Italian origin with mild chronic haemolytic anaemia, psychomotor impairment and undetectable adenylate kinase (AK) activity are reported. The other red cell enzyme activities were normal except for a slight decrease of PFK. 2,3‐DPG levels were increased in both siblings, and AMP decreased in one only. The parents were not consanguineous and displayed intermediate AK activity. The sequence of complete erythrocyte AK‐1 cDNA showed the presence of a nonsense homozygous mutation at codon 107 (CGA → TGA, Arg → Stop) in the siblings. The mutation results in a truncated protein of 107 amino acids in comparison with the 194 of the normal one. Moreover a 37 bp deletion in the first part of exon 6 (from nt 326 to nt 362 of the cDNA sequence) was detected in one allele; this deletion is not likely to further affect the enzyme structure, being localized after the stop codon. The new variant was named AK Fidenza, from the origin of the patients.

A variant of the EPB3 gene of the anti‐Lepore type in hereditary spherocytosis

The EPB3 gene encodes band 3 (anion exchanger 1) of the red cell membrane. A subset of hereditary spherocytosis (HS) is associated with EPB3 gene mutations and band 3 deficiency. We report a large Italian family in which 10 of the 27 members investigated displayed an autosomal dominant HS. SDS‐PAGE revealed a reduction in band 3 in the patients. Screening of the Pst I polymorphic site confirmed the linkage of HS with the EPB3 gene. Analysis of complementary and genomic DNA showed a large additional segment. Nucleotide sequencing disclosed an in‐frame duplication of 69 nucleotides (nt) including a triplet of intronic origin and a genuine exonic duplication of 66 nt. Two CCTGC sequences occurred close to one another, one near the intron 12 acceptor splice site (nt −7 to −3), and the other within exon 13 (nt 1494–1498). We assumed that the abnormal allele arose from an unequal recombination event of the anti‐Lepore type between the two CCTGC sequences.

Molecular characterization of the PK-LR gene in sixteen pyruvate kinase-deficient patients

We studied the PK‐LR gene in 16 unrelated patients with congenital haemolytic anaemia associated with erythrocyte pyruvate kinase deficiency. Fifteen different mutations were detected among the 28 mutated alleles identified: two deletions (del 1010G, del 1042–1044); one four nucleotide duplication (nt 1515–1518, GGTC); one splice site [IVS6(−2)t]; nine missense (991A, 1003A, 1151T, 1160G, 1181T, 1181A, 1456T, 1483A, 1529A); and two nonsense (721T, 1675T) mutations. Eight of them [del 1010G, del 1042–1044, dupl 1515–1518, IVS6(−2)t, 1003A, 1160G, 1181T, 1181A] were novel. The deletion 1042–1044 causes the loss of Lys 348. Deletion 1010G and duplication 1515–1518 determine a frameshift and the creation of a stop codon at nucleotides 1019 and 1554 respectively. Mutation IVS6(−2)t leads to an alteration of the 5′ and 3′ splice site consensus sequence; the cDNA analysis shows a 67‐bp deletion in the first part of exon 11 (del 1437–1503). All the four new missense mutations involve highly conserved amino acids. The most frequent mutation in Italy would appear to be 1456T. Correlation was made between mutations, biochemical characteristics of the enzyme and clinical course of the disease.

Reactive follicular hyperplasia on dasatinib treatment for chronic myeloid leukemia

Dear Editor, Dasatinib (DAS) is an oral dual Abl and Src tyrosine kinase inhibitor licensed in the treatment of chronic myeloid leukemia (CML) [1, 2] and is well known to exert an immunomodulatory effect both in vivo and in vitro [3]. DAS shows a distinct toxicity profile among which a previously unrecognized adverse event (AE) is represented by persistent lymphadenopathy with reactive follicular hyperplasia (FLH). Recently, lymphadenopathy with morphologic features of reactive FLH has been described in two small series of longtermDAS-treated CML patients [4, 5], which globally encompass twelve patients, even though only three in the first-line setting. In addition, only in a few patients complete FLHmorphologic and immunophenotypic features were reported. Herein, we describe two cases of patients with chronic phase (CP)-CML, intermediate risk according to Sokal score, and e14a2 transcript type, who presented with unexplained lymphadenopathy during front-line DAS therapy. Case 1: in October 2012, a 30-year-old man was diagnosed with CP-CML and he was front-line treated with DAS 100 mg QD, rapidly obtaining a major and subsequently a deep molecular response. DAS was well tolerated but approximately after 48months, bilateral cervical lymphadenopathy appeared. At a subsequent ultrasound examination, multiple enlarged lymph nodes were detected both in the cervical and submandibular area. Case 2: in April 2016, a 49-year-old man was diagnosed with CP-CML and he was front-line treated with DAS 100 mg QD. The drug was well tolerated and after 12 months of therapy he obtained a major molecular response. Nevertheless, in April 2017 a swelling at the angle of the left mandible and concomitant bilateral cervical, preauricular, and sovraclavear lymphadenopathy appeared. At a subsequent ultrasound examination, multiple enlarged lymph nodes were detected both in the cervical and left submandibular area. For both patients, no generalized lymphadenopathy was noted and no constitutional symptoms were reported. As screening for active viral infection was negative and no signs of local or systemic infectious disease were detected, an excisional biopsy was performed, showing in both cases enlarged lymph nodes with overall preserved architecture, marked follicular hyperplasia with evident reactive germinal centers (CD10+, BCL6+, BCL2and very high Ki-67 immunoreactivity), and moderate expansion of the paracortical T-zone in which a predominance of small CD3+, CD5+ T lymphocytes was identifiable together with scattered enlarged CD45+, CD30−/+, CD15−, and CD20+/− immunoblasts (Fig. 1). In situ hybridization for EBV-encoded RNA was negative. A diagnosis of FLH was made, ruling out an extramedullary blastic transformation of CML. Then, both patients definitely discontinued DAS and started ponatinib at a daily dose of 15 mg, achieving complete clinical resolution of lymphadenopathy. For what concerns FLH pathogenesis, it should be underlined that Lyn, a Src family protein, is expressed in B lymphocytes and regulates their activity via Akt/PKB signaling pathway. Thus, DAS inhibitory impact on Src kinases and the consequent upregulation of Akt/PKB pathway could contribute to the B lymphocytes proliferation and FLH development. * Alessandra Iurlo aiurlo@policlinico.mi.it

Ponatinib as a Valid Alternative Strategy in Patients with Blast Crisis-Chronic Myeloid Leukemia Not Eligible for Allogeneic Stem Cells Transplantation and/or Conventional Chemotherapy: Report of a Case

Currently, imatinib and dasatinib are the only tyrosine-kinase inhibitors approved in the US and Europe for the treatment of blast crisis of chronic myeloid leukemia (BC-CML) at diagnosis, while ponatinib is the only inhibitor used in patients bearing T315I mutation. Here we report the case of a 61-year-old man diagnosed with B-cell lymphoid BC-CML, initially treated with imatinib 800 mg day and then with dasatinib 140 mg day because of intolerance. A complete cytogenetic response (CCyR) was achieved at three months; however, three months later a relapse was observed, and the T315I mutation was detected. Ponatinib 45 mg once daily was then started together with a short course of chemotherapy. Bone marrow evaluation after six months of therapy showed the regaining of CCyR, together with the achievement of a deep molecular response. However, one year from ponatinib start the patient experienced a new disease relapse; he was effectively treated with ponatinib and chemotherapy once again, but in the meanwhile an ischemic stroke was detected. This case report confirms the high efficacy of ponatinib monotherapy in BC-CML patients, representing a valid option for non-allogeneic stem cells transplantation eligible cases and the only one available for those carrying the T315I mutation.

Comprehensive Molecular Analyses in a Case of Masked Philadelphia Chronic Myeloid Leukemia.

Here, we report the case of an 80-year-old woman with masked Philadelphia chronic myeloid leukemia (Ph CML). At diagnosis, qualitative PCR demonstrated the presence of a typical e14a2 configuration, and chromosome analysis showed an apparently normal female karyotype. However, FISH with BCR-ABL1 dual fusion probes gave a positive signal in 152/200 analyzed nuclei, with the fusion signal detected on the long arm of a cytogenetically normal chromosome 9. Using locus-specific probes for chromosome 9 and 22 telomeres, a third chromosome involvement was excluded. Furthermore, microarray analysis from the same specimens showed a normal result. Due to a high Charlson Comorbidity Index, the patient was treated with a reduced dose of imatinib, achieving a rapid hematological response after 1 month. However, after 6 months of imatinib therapy, she had to be considered as warning (Ph+ 26.5%, BCR-ABL1 >1%) according to the European LeukemiaNet 2013 recommendations. In conclusion, we confirmed the importance of a combination of cytogenetic and molecular techniques for the diagnosis and therapy monitoring of masked Ph CML, but, different from what has been reported in the literature so far, we cannot completely exclude the fact that the unusual cytogenetic pattern of this patient may have negatively influenced her response to tyrosine kinase inhibitor therapy.

Analysis of Pig-a Gene mutations in paroxysmal nocturnal hemoglobinuria

Abstract Paroxysmal nocturnal hemoglobinuria (PNH) is a clonal stem cell disorder characterized by an acquired PIG-A gene mutation. The clinical symptoms are intravascular hemolysis and hemoglobinuria. A variety of mutations in PIG-A gene have been so far reported. The aim of the study was to investigate the molecular defect in 8 patients with hemolytic PNH and in a 51 years old female who spontaneously recovered from severe hemolytic PNH (SL). The study of the entire codifying region and flanking intronic sequences of PIG-A gene was done by SSCP analysis and sequencing. 8 new and one known mutations were detected. Results are reported in the table. Mutation C55T has been already described in 5 PNH patients, but its nature was controversial. C55T was the only mutation found in patient SL. It was also detected in patient DTI, in association with T728C. To exclude the somatic origin of this variant, the mucous membrane cells were analyzed and found to be positive. C55T was also present in 1/100 normal alleles investigated, thus confirming its polymorphic nature. Mutations so far identified could be useful as molecular markers for monitoring the course of the disease during therapy.