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Purpose:EGFR exon 20 insertions account for up to 10% of all EGFR mutations in lung adenocarcinomas, representing the third most common cluster of mutations. The management of advanced cancers with these mutations remains elusive, without an approved inhibitor.Experimental Design:Preclinical models of a representative set of EGFR exon 20 insertion mutations to evaluate the efficacy of different inhibitors and description of the clinical outcome of an advanced lung cancer.Results:We show that select first-, second-, and third-generation EGFR inhibitors are unable to deter common EGFR exon 20 insertion mutants in concentrations that spare the wild-type kinase. Nonetheless, EGFR exon 20 insertion mutants associate with the Hsp90 chaperone system. We exploit this vulnerability to show that the nongeldanamycin Hsp90 inhibitor luminespib (formerly AUY922) degrades EGFR exon 20 mutations, downstream targets, and induces apoptosis. In addition, a patient whose EGFR inhibitor–insensitive lung adenocarcinoma harbored an EGFR exon 20 insertion mutation had a confirmed radiographic response to luminespib.Conclusions:The report confirms that EGFR exon 20 mutations are dependent on Hsp90 and are readily inhibited by the Hsp90 inhibitor luminespib; a treatment strategy that has been pursued in a confirmatory clinical trial (NCT01854034) for this group of lung adenocarcinomas that currently represent an unmet clinical need in precision oncology.

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Background: Brazil became the South American epicenter for coronavirus disease (COVID-19) soon after the first case was diagnosed in February 2020 with the highest infection rate occurring in the state of Sao Paulo. COVID-19 is characterized by marked thrombo-inflammation mechanisms, and neutrophil-lymphocyte ratio (NLR) among many clinical and laboratory data, is becoming an inflammatory marker of severity and mortality of COVID-19. We evaluated the serial weekly lymphocyte ratios, which are easily derivable from the routine blood counts, in the survivors and non-survivors of COVID-19 at the Clinical Hospital of University of Campinas (UNICAMP), Campinas, Sao Paulo, Brazil, from time of diagnosis to the 3 rd week of care. This hospital is one of the referral centers for COVID-19 patients in this state. Methods: In this retrospective study, we reviewed the medical notes of 320 adults hospitalized patients with PCR-confirmed COVID-19 at the Clinical Hospital of UNICAMP, from March 2020 to March 2021. The serial weekly hematological parameters (analyzed using automated counter - XN 9000™, Sysmex, Japan) from the time of diagnosis were analyzed and lymphocytes ratios (neutrophil-lymphocyte, NLR, platelet-lymphocyte PLR, and monocyte-lymphocyte MLR) were calculated. The survivors (n=257) were those who recovered from the disease and were discharged from the hospital, while the non-survivors (n=63) were those who died in the course of treatment. Statistical analyses were performed using SPSS (version 22). Unpaired data of Survivors and Non-survivors with COVID-19 were compared using Mann-Whitney tests. Repeated measures were compared within and between groups using univariate and multivariate tests in general linear models. All results were considered significant if p Results: Of the 320 patients, 257 (80.3%) were survivors and had lower mean age than the non-survivors (57.73 vs 64.65 years, p We used general linear models for repeated measures and corrected for the patients who did not stay long enough to have a complete series of blood counts, Figure 1 A-G. Multivariate tests between the survivor and non-survivor groups showed significant variations with serial weekly lymphocyte count (p Conclusions: The non-survivors were older and their NLR and MLR tend to increase from the time of diagnosis while their PLR tend to decrease after the 2 nd week post-COVID-19 diagnosis and treatment. On the other hand, all three ratios significantly decrease in the survivors. While neutrophilia and lymphopenia improved in the survivor, they worsen in non-survivors. These cells may have contributed towards the recovery by ameliorating the inflammatory response in survivors, and death by worsening the response in non-survivors of COVID-19. This study shows that serial lymphocyte count, neutrophil count, NLR, PLR, and MLR could serve as good and easily accessible markers of outcomes in patients with COVID-19 and could be used for monitoring of response to treatment. Figure 1 Figure 1. Disclosures Costa: Novartis: Consultancy.

Human hemoglobin (Hb) Coimbra (βAsp99Glu) is one of the seven βAsp99 Hb variants described to date. All βAsp99 substitutions result in increased affinity for O2 and decreased heme-heme cooperativity and their carriers are clinically characterized by erythrocytocis, caused by tissue hypoxia. Since βAsp99 plays an important role in the allosteric α1β2 interface and the mutation in Hb Coimbra only represents the insertion of a CH2 group in this interface, the present study of Hb Coimbra is important for a better understanding of the global impact of small modifications in this allosteric interface. We carried out functional, kinetic and dynamic characterization of this hemoglobin, focusing on the interpretation of these results in the context of a growth of the position 99 side chain length in the α1β2 interface. Oxygen affinity was evaluated by measuring p50 values in distinct pHs (Bohr effect), and the heme-heme cooperativity was analyzed by determining the Hill coefficient (n), in addition to the effect of the allosteric effectors inositol hexaphosphate (IHP) and 2,3-bisphosphoglyceric acid (2,3-BPG). Computer simulations revealed a stabilization of the R state in the Coimbra variant with respect to the wild type, and consistently, the T-to-R quaternary transition was observed on the nanosecond time scale of classical molecular dynamics simulations. Fil: Jorge, S. E.. Universidade Estadual de Campinas; Brasil Fil: Bringas, Mauro. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentina Fil: Petruk, Ariel Alcides. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentina Fil: Arrar, Mehrnoosh. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentina Fil: Marti, Marcelo Adrian. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina Fil: Skaf, M. S.. Universidade Estadual de Campinas; Brasil Fil: Costa, F. F.. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentina Fil: Capece, Luciana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentina Fil: Sonati, M. F.. Universidade Estadual de Campinas; Brasil Fil: Estrin, Dario Ariel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentina

Hb Bristol-Alesha [HBB: c.202G>A; β 67 Val>Met] is a rare structural variant of hemoglobin (Hb) resulting from a GTG>ATG substitution at codon 67 of the β-globin gene that leads to the replacement of valine by methionine in the corresponding position of the β-globin chain. The methionine residue is subsequently modified to aspartic acid [β67(E11)Val-Met→Asp], possibly by autoxidation mechanisms. This substitution prevents normal non-polar binding of Val67 to the heme group, resulting in molecular instability and severe hemolysis. We identified Hb Bristol-Alesha (in the heterozygous state), as the cause of severe congenital hemolytic anemia in an 11-month-old girl of mixed (native Indian and European) ethnic origin from the Midwestern region of Brazil, whose parents were clinically and hematologically normal. The mutation on the β-globin gene was found to have been coinherited with the α212 patchwork allele.

A hemoglobina (Hb) e a hemeproteina das hemacias caracterizada pela ligacao reversivel ao O2 e, consequentemente, o transporte deste gas aos tecidos perifericos. Complementar aos estudos de funcao da Hb extraida de bolsas de sangue, este trabalho tem por objetivo a analise de parâmetros bioquimicos das bolsas de sangue (concentrado de hemacias, CH) apos processos de retirada de leucocitos (por filtracao, exposicao de irradiacao ? e processo misto de filtracao e irradiacao), com os intervalos de armazenamento de 48 horas, 21 dias e 42 dias. Resultados aqui apresentados parecem apontar para alteracoes bioquimicas da bolsa de sangue e modificacoes funcionais da Hb tanto em relacao ao tempo de armazenamento quanto pelos processos de leucorreducao.

Purpose: EGFR exon 20 insertions account for up to 10% of all EGFR mutations in lung adenocarcinomas, representing the third most common cluster of mutations. The management of advanced cancers with these mutations remains elusive, without an approved inhibitor. Experimental Design: Preclinical models of a representative set of EGFR exon 20 insertion mutations to evaluate the efficacy of different inhibitors and description of the clinical outcome of an advanced lung cancer. Results: We show that select first-, second-, and third-generation EGFR inhibitors are unable to deter common EGFR exon 20 insertion mutants in concentrations that spare the wild-type kinase. Nonetheless, EGFR exon 20 insertion mutants associate with the Hsp90 chaperone system. We exploit this vulnerability to show that the nongeldanamycin Hsp90 inhibitor luminespib (formerly AUY922) degrades EGFR exon 20 mutations, downstream targets, and induces apoptosis. In addition, a patient whose EGFR inhibitor–insensitive lung adenocarcinoma harbored an EGFR exon 20 insertion mutation had a confirmed radiographic response to luminespib. Conclusions: The report confirms that EGFR exon 20 mutations are dependent on Hsp90 and are readily inhibited by the Hsp90 inhibitor luminespib; a treatment strategy that has been pursued in a confirmatory clinical trial (NCT01854034) for this group of lung adenocarcinomas that currently represent an unmet clinical need in precision oncology.

Congenital erythropoietic porphyria is a rare autosomal recessive disease caused by a deficiency of uroporphyrinogen III synthase, owing to mutations in UROS in chromosome 10. Occasionally, patients show a mild, late-onset disease, without germline UROS mutations, associated with haematological malignancies. We report a 65-year-old patient with photosensitivity, overexcretion of porphyrins and thrombocytopenia. Bone marrow analysis gave a diagnosis of myelodysplastic syndrome (MDS) with the presence of a derivative chromosome 3, possibly due to an inversion including 3q21 and 3q26 break points. After allogeneic stem-cell transplantation, complete remission of MDS and uroporphyria was achieved. To our knowledge, this is the first reported case of acquired erythropoietic uroporphyria associated with MDS, with chromosome 3 alterations.

Hb S-São Paulo (SP) [HBB:c.20A > T p.Glu6Val; c.196A > G p.Lys65Glu] is a new double-mutant hemoglobin that was found in heterozygosis in an 18-month-old Brazilian male with moderate anemia. It behaves like Hb S in acid electrophoresis, isoelectric focusing and solubility testing but shows different behavior in alkaline electrophoresis, cation-exchange HPLC and RP-HPLC. The variant is slightly unstable, showed reduced oxygen affinity and also appeared to form polymers more stable than the Hb S. Molecular dynamics simulation suggests that the polymerization is favored by interfacial electrostatic interactions. This provides a plausible explanation for some of the reported experimental observations Fil: Jorge, Susan E. D. C.. Universidade Estadual de Campinas; Brasil Fil: Petruk, Ariel Alcides. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina Fil: Kimura, Elza M.. Universidade Estadual de Campinas; Brasil Fil: Oliveira, Denise M.. Universidade Estadual de Campinas; Brasil Fil: Caire, Lucas. Universidade Estadual de Campinas; Brasil Fil: Suemasu, Cintia N.. Universidade Estadual de Campinas; Brasil Fil: Silveira, Paulo A. A.. Albert Einstein Institute; Brasil Fil: Albuquerque, Dulcineia M.. Universidade Estadual de Campinas; Brasil Fil: Costa, Fernando F.. Universidade Estadual de Campinas; Brasil Fil: Skaf, Munir S.. Universidade Estadual de Campinas; Brasil Fil: Martínez, Leandro. Universidade de Sao Paulo; Brasil Fil: De Fatima Sonati, Maria. Universidade Estadual de Campinas; Brasil

Most of the 1,300 human hemoglobin (Hb) variants are characterized by normal O2 binding, including here the highly prevalent Hbs S [HBB:c.20A>T; p.Glu6Val] and C [HBB:c.19G>A; p.Glu6Lys]. Hb S and Hb C carriers are generally asymptomatic and might be admitted as blood donors. However, the functional efficiency of these variants in standard blood bags - red blood cells (RBC) concentrates - under the interference of anticoagulant and storage is not well established. In this prospective study, three samples of RBC concentrates (phenotypes AA, AS and AC), with citrate phosphate dextrose adenine (CPDA-1) as anticoagulant, were analyzed for equilibrium tests and compared to fresh Hb A without anticoagulant. The Hb samples were extracted after 48 hours and 35 days of storage and purified by exclusion and ion exchange chromatography (Sephadex G-25 and Amberlite MB-3 columns in Hepes buffer without NaCl). O2 affinity was evaluated by spectrophotometry-tonometry method, by determining the p50 in stripped lysates ([Hb]=70 μM/Heme), in pHs of 6.5, 7.0, 7.5, 8.0 and 8.5, for Bohr effect calculations. Heme-heme cooperativity was determined by Hill coefficient (n) in all studied pHs. The activity of allosteric effector in the Hb-O2 ligation was also tested, using inositol hexaphosphate ([IHP]=1mM), a mimetic compound of the intraerythrocytic 2,3-BPG. The p50 of Hb A after 35 days in stored blood bag was decreased in all pHs, in both presence and absence of IHP, when compared to fresh Hb A, suggesting that the anticoagulant and the storage might affect HbA-O2 binding, increasing its affinity for the ligand. No other differences were observed: heme-heme cooperativity and Bohr effect were similar to fresh Hb A without anticoagulant. The same was found when stored Hb AS was compared to Hb A from blood bag (35 days storage, in the stripped state and under the interference of IHP). However, the stripped lysate containing Hb AC revealed an important reduction of the O2 affinity after both 48 hours and 35 days of storage, mainly at pHs ³ 7, thus interfering in the Bohr effect. The heme-heme cooperativity resulted in normal n coefficients, indicating that the Hb-O2 ligation remained cooperative, in spite of the significant reduction of O2 affinity. Similar results were observed in the presence of IHP (48 hours and 35 days), although Bohr effect was reestablished, indicating that the supplementation with a 2,3-BPG-like allosteric effector might restore some Hb-ion interactions, and therefore, Hb C function. As the 2,3-BPG is consumed with storage of RBC concentrates, Hb AC showed an even lower affinity for O2 after 35 days of storage. Although still preliminary, our results indicate a significant functional reduction of Hb C in RBC concentrates in standard blood bag (CPDA-1 anticoagulant), proportional to the time of storage. Considering that the screening for hemoglobinopathies in the blood banks routine is mainly focused in the detection of Hb S, a greater understanding about the properties of native Hb and its most frequent variants in stored RBC concentrates could also contribute with the determination of safe parameters in hemotherapy based on the functional efficiency of each hemoglobin. Financial Support: Fapesp, CNPq, Capes, Faepex-Unicamp. Disclosures No relevant conflicts of interest to declare.

Phosphatidylinositol-phosphate kinases (PIPKins) belong to a family of lipid kinase enzymes that generate lipid messengers derivate from inositol, including the second messenger phosphatidylinositol-4,5-biphosphate [PI(4,5)P2], which participates in several cell regulation processes including gene expression. Previous studies developed in our laboratory suggested that the enzyme phosphatidylinositol-4-phosphate kinase-II-alpha (PIP4KIIα) may be related with the expression of the globin genes. Thus, the aim of this work was to evaluate the expression levels of the α, β and γ globin genes after disruption and overexpression of the PIP4KIIα gene (PIP4KIIA) in KU812 erythroleukemic cell line. The PIP4KIIα gene disruption was performed by using CRISPR/Cas9 and CRISPR/dCas9 techniques: KU812 cells were electro-transfected (Lonza 4D Nucleofector) with CRISPR/Cas9 lentiviral plasmid construct containing 3 gRNA targets to exon 8 of PIP4KIIA to knockout the gene; RNA samples from the pool of cells were collected and quantified by qPCR after 9 (t1) and 14 days (t2) of transfection. To overexpress PIP4K2A, CRISPR/dCas9 system (five lentiviral plasmids - 3 gRNAs targeting 214 nt upstream the transcriptional start site of PIP4K2A and 2 SAM accessorial factors) was transfected (Lipofectamine 3000) and RNA quantified by qPCR 24 (h1), 48 (h2) and 72 (h3) hours after the procedure. β-ACTIN and GAPDH genes were used as endogenous controls for qPCR. Our results with transfected cells without selection revealed, as expected, a reduction of PIP4KIIA compared to the control (no transfected cells - t1 and t2). The α globin genes presented a variation of 42% (increase in t1) and a 60% reduction in t2 (RQ = 1.42; RQ = 0.40, respectively), while β globin genes presented a 123% increase in t1 and remained similar to the control at t2 (RQ = 2.23; RQ = 1.01). The γ globin genes presented variation of 18% and 59% reduction in t1 and t2, respectively (RQ = 0.82; RQ = 0.41). The overexpression results suggested that PIP4KIIA leads to a higher γ globin expression in h2 and h3 (RQ = 3.26; RQ = 2.01) as well as a lower β globin expression in h1, h2 and h3 (RQ = 0.54; RQ = 0.002; RQ = 0.18), while a globin remained similar to the control at all analysed times (RQ variation of 1.26; 0.82 and 1.25, respectively). These results, although preliminary, suggest a possible relationship between PIP4KIIα and the regulation of expression of the globin genes. Financial Support: Fapesp, CNPq, CAPES, Faepex-Unicamp. Disclosures No relevant conflicts of interest to declare.

Hemoglobin (Hb) Zurich-Albisrieden (ZA) [α2 59(E8) Gly > Arg; HBA2:c.178G > C] is a rare and highly unstable α-chain variant. A few simple and compound heterozygotes (αZA α/αα and -/αZA α, respectively) have been described so far in Switzerland and China. We describe here a case of homozygosity for the Hb ZA mutation (αZA α/αZA α) in a Brazilian child with severe congenital hemolytic anemia and ineffective erythropoiesis.

Human hemoglobin (Hb) is the erythrocyte hemeprotein resulting from the combination of one pair of α-like (α or ζ) chains and another pair of β-like (β, δ, γ or e) chains. Each of these chains is associated with a heme prosthetic group, a tetrapyrrole ring (protoporphyrin IX) containing a central ferrous atom (Fe2+), which can reversibly bind to a molecule of O2, being, therefore, responsible for its transport from the lungs to the tissues. This introductory chapter summarizes these important aspects, including findings of protein structure, synthesis and function, as well as its gene organization and regulation. We also describe the developmental switches in globin chain production (from the embryonic period until hematological adult life), heme synthesis and globin gene expression/regulation, besides functional aspects of the hemoglobin molecule. The chapter also includes models that predict the mechanisms of Hb-O2 ligation, mediated by the presence of allosteric effectors, such as H+/CO2, Cl− and organic phosphates, such as 2,3-biphosphoglycerate (2,3-BPG, from erythrocyte metabolism).

Three novel α-globin variants were found during a screening program for hemoglobinopathies in blood donors at the UNICAMP Hematology and Hemotherapy Center, Campinas, State of Sao Paulo, Southeastern Brazil. They were named for the town of origin of the carrier as Hb Itapira [α30(B11)Glu→Val], Hb Bom Jesus da Lapa [α30(B11)Glu→Ala] and Hb Boa Esperanca [α16(A14)Lys→Thr]. Hb Itapira, like Hb Bom Jesus da Lapa, shows an electrophoretic mobility similar to that of Hb S [β6(A3)Glu→Val, GAG→GTG] at alkaline pH; it is associated with a triplicate α-globin allele (αααanti 3.7) and corresponds to only 5.5% of the total hemoglobin (Hb). Hb Boa Esperanca, found in two different individuals, moves faster than Hb A and exhibits an abnormal functional performance.

Introduction Genomic aberrations involving ALK , ROS1 and MET can be driver oncogenes in lung adenocarcinomas. Identification of tyrosine kinase inhibitors (TKIs) with activity against these tumors and of preclinical systems to model response are warranted. Methods We analyzed cases with lung adenocarcinomas for representative genomic aberrations, evaluated the response to the multitargeted MET/ALK/ROS1 crizotinib TKI in cases with MET aberrations and profiled lung cancer cell lines with the aforementioned genomic changes. Results Lung cancer cell lines with ALK rearrangement, ROS1 rearrangement or MET amplification had expected in vitro responses to crizotinib and the ALK/ROS1 TKI ceritinib. However, a commercially-available cell line with MET exon 14 skipping mutation and co-occurring PIK3CA -p.Glu545Lys mutation did not respond to crizotinib; suggesting the latter abrogated response. 10% of MET exon 14 skipping mutation co-occurred with PIK3CA mutation in the TCGA cohort. Putative crizotinib-responsive somatic mutations ( ALK rearrangements, ROS1 rearrangements, high level MET amplification or MET exon 14 skipping mutations) were present in 10% of lung adenocarcinomas analyzed at our service and in 9.5% of the TCGA lung adenocarcinoma database. One patient each whose advanced tumors harbored high level MET amplification with wild-type PIK3CA or MET exon 14 skipping mutation with PIK3CA -p.Glu542Lys had significant responses to crizotinib; suggesting that PIK3CA co-mutation did not affect clinical response. Conclusions Approximately 10% of lung adenocarcinomas harbor aberrations that are targetable using the approved multitargeted TKI crizotinib. MET exon 14 skipping mutation predicts for response to MET TKIs in human lung adenocarcinomas but co-occurrence of PIK3CA mutation needs to be better evaluated as a modifier of response to TKI therapy. MET TKIs should not be omitted from MET exon 14 skipping mutated tumors until further preclinical and clinical data can confirm or refute mechanisms of primary or acquired resistance to crizotinib and other MET TKIs in these recalcitrant cancers.

Introduction Crizotinib is an oral multitargeted tyrosine kinase inhibitor (TKI) with activity against lung cancers driven by ALK-rearrangements, ROS1-rearrangements and MET-amplification. Comprehensive genomic profiling (CGP) based on clinical next generation sequencing (NGS) can detect crizotinib-sensitive genomic changes. We describe use of CGP to identify tumors responsive to crizotinib. Methods Retrospective review of representative lung adenocarcinomas treated with crizotinib and assayed with a clinical NGS assay. Results We report 3 cases of lung adenocarcinoma; one each identified to harbor an ALK-rearrangement (EML4-ALK), ROS1-rearrangement (SDC4-ROS1) and MET-amplification by genomic profiling. Notably, the MET-amplification was only detected by CGP as subsequent FISH testing did not show amplification. CGP also revealed other common genomic changes (somatic mutations [TP53 in 2 cases], deletions [CDKN2A in 1 case], amplifications [MCL1 in 1 case] and variants of unknown significance) in these cases. All patients received crizotinib 250 mg twice daily and achieved radiographic tumor reduction for months. The case harboring MET amplification of 10 copies achieved partial response and is one of the first MET-amplified lung cancer responsive to crizotinib in which the sole detection method was CGP. Conclusions CGP holds the promise of detecting predictive genomic alterations (somatic mutations, copy number changes and rearrangements) that may underlie tumor dependency in an oncogene and govern response to clinically-available TKIs for lung adenocarcinomas.

Recent progress of genetic studies has dramatically unveiled pathogenesis of acute myeloid leukemia (AML). However, overall survival of AML still remains unsatisfactory, and development of novel therapeutics is required. CCAAT/enhancer binding protein α (C/EBPα) is one of the crucial transcription factors that induce granulocytic differentiation, and its activity is perturbed in human myeloid leukemias. As its reexpression can induce differentiation and subsequent apoptosis of leukemic cells in vitro, we hypothesized that chemical compounds that restore C/EBPα expression and/or activity would lead to myeloid differentiation of leukemic cells. Using a cell-based high-throughput screening, we identified 2-[(E)-2-(3,4-dihydroxyphenyl)vinyl]-3-(2-methoxyphenyl)-4(3H)-quinazolinone as a potent inducer of C/EBPα and myeloid differentiation. Leukemia cell lines and primary blast cells isolated from human patients with AML treated with ICCB280 demonstrated evidence of morphological and functional differentiation, as well as massive apoptosis. We performed conformational analyses of the high-throughput screening hit compounds to postulate the spatial requirements for high potency. Our results warrant a development of novel differentiation therapies and significantly affect care of patients with AML with unfavorable prognosis in the near future.

BackgroundBrazil has a multiethnic population with a high diversity of hemoglobinopathies. While screenings for beta-globin mutations are far more common, alterations affecting alpha-globin genes are usually more silent and less well known. The aim of this study was to describe the results of a screening program for alpha-globin gene mutations in a representative sample of the Southeastern Brazilian population.MethodsA total of 135,000 individuals, including patients with clinical suspicion of hemoglobinopathies and their family members, randomly chosen individuals submitted to blood tests and blood donors who were abnormal hemoglobin carriers were analyzed. The variants were screened by alkaline and acid electrophoreses, isoelectric focusing and cation-exchange high performance liquid chromatography (HPLC) and the abnormal chains were investigated by reverse-phase high performance liquid chromatography (RP-HPLC). Mutations were identified by molecular analyses, and the oxygen affinity, heme–heme cooperativity and Bohr effect of the variants were evaluated by functional tests.ResultsFour new and 22 rare variants were detected in 98 families. Some of these variants were found in co-inheritance with other hemoglobinopathies. Of the rare hemoglobins, Hasharon, Stanleyville II and J-Rovigo were the most common, the first two being S-like and associated with alpha-thalassemia.ConclusionThe variability of alpha-globin alterations reflects the high degree of racial miscegenation and an intense internal migratory flow between different Brazilian regions. This diversity highlights the importance of programs for diagnosing hemoglobinopathies and preventing combinations that may lead to important clinical manifestations in multiethnic populations.

Objectives: To investigate the existence of an association between haptoglobin (Hp) genotypes and the severity of heart complications in Chagas' disease. Design and methods: Hp genotyping was performed by PCR in 107 Brazilian patients sub-classified in asymptomatic, with mild heart disease and with severe heart disease. Results: Multiple logistic regression ( R 2 = 24%) indicated that patients with the Hp1-1 genotype have lower probability of developing the severe heart complications. Conclusion: The Hp polymorphism may influence the clinical evolution of Chagas' disease.

We describe here two new unstable beta-globin variants, Hb Caruaru and Hb Olinda, found in northeastern Brazil, both associated with chronic haemolytic anaemia. Haemoglobin Caruaru is caused by a single base substitution at codon 122 (TTC-->TCC), possibly originating from the germ line cells of the patient's grandmother. Haemoglobin Olinda is also a de novo mutation, caused by a 12 bp deletion leading to the removal of the 22nd to the 25th residues of the normal beta-globin chain.

Hb H Disease is caused by the loss or inactivation of three of the four functional α-globin genes. Patients present chronic hemolytic anemia and splenomegaly. In some cases, occasional blood transfusions are required. Deletions are the main cause of this type of thalassemia ( α-thalassemia). We describe here an unusual case of Hb H disease caused by the combination of a common α(0) deletion [-( α) (20.5) ] with a rare point mutation (c.427T > A), thus resulting in an elongated and unstable α-globin variant, Hb Icaria, (X142K), with 31 additional amino-acid residues. Very high levels of Hb H and Hb Bart's were detected in the patient's red blood cells (14.7 and 19.0%, respectively). This is the first description of this infrequent association in the Brazilian population.

As anormalidades estruturais da hemoglobina estão entre as doenças genéticas mais comumente encontradas nas populações humanas. O Laboratório de Hemoglobinopatias do Departamento de Patologia Clínica da Faculdade de Ciências Médicas da Universidade Estadual de Campinas - Unicamp, localizado em Campinas, no estado de São Paulo, região Sudeste do Brasil, realizou, em seus 27 anos de existência, cerca de 130.000 diagnósticos. Entre as variantes estruturais detectadas, as hemoglobinas S, C e D-Punjab foram, como esperado, as mais freqüentes, porém um número expressivo de outras hemoglobinas anômalas, novas e raras, também foi encontrado. Esses achados estão sumarizados no presente artigo. Hemoglobin structural abnormalities are among the most commonly found human genetic diseases. The Laboratory of Hemoglobinopathies in the Clinical Pathology Department of the Medical Sciences School of the State University in Campinas - Unicamp, São Paulo, Southeastern Brazil, carried out, in its 27 years of activity, about 130,000 diagnoses. As expected, hemoglobins S, C and D were the most frequently observed variants, but an expressive number of other abnormal, novel and rare hemoglobins, was also detected. These findings are summarized in the present article.