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Background Fluoropyrimidines are used in chemotherapy combinations for multiple cancers. Deficient dihydropyrimidine dehydrogenase activity can lead to severe life-threatening toxicities. DPYD*2A polymorphism is one of the most studied variants. The study objective was to document the impact of implementing this test in routine clinical practice. Methods We retrospectively performed chart reviews of all patients who tested positive for a heterozygous or homozygous DPYD*2A mutation in samples obtained from patients throughout the province of Quebec, Canada. Results During a period of 17 months, 2,617 patients were tested: 25 patients tested positive. All were White. Twenty-four of the 25 patients were heterozygous (0.92%), and one was homozygous (0.038%). Data were available for 20 patients: 15 were tested upfront, whereas five were identified after severe toxicities. Of the five patients confirmed after toxicities, all had grade 4 cytopenias, 80% grade ≥3 mucositis, 20% grade 3 rash, and 20% grade 3 diarrhea. Eight patients identified with DPYD*2A mutation prior to treatment received fluoropyrimidine-based chemotherapy at reduced initial doses. The average fluoropyrimidine dose intensity during chemotherapy was 50%. No grade ≥3 toxicities were observed. DPYD*2A test results were available in an average of 6 days, causing no significant delays in treatment initiation. Conclusion Upfront genotyping before fluoropyrimidine-based treatment is feasible in clinical practice and can prevent severe toxicities and hospitalizations without delaying treatment initiation. The administration of chemotherapy at reduced doses appears to be safe in patients heterozygous for DPYD*2A. Implications for Practice Fluoropyrimidines are part of chemotherapy combinations for multiple cancers. Deficient dihydropyrimidine dehydrogenase activity can lead to severe life-threatening toxicities. This retrospective analysis demonstrates that upfront genotyping of DPYD before fluoropyrimidine-based treatment is feasible in clinical practice and can prevent severe toxicities and hospitalizations without delaying treatment initiation. This approach was reported previously, but insufficient data concerning its application in real practice are available. This is likely the first reported experience of systematic DPYD genotyping all over Canada and North America as well.

Introduction Genotyping circulating tumor DNA (ctDNA) is a promising noninvasive clinical tool to identify the EGFR T790M resistance mutation in patients with advanced NSCLC with resistance to EGFR inhibitors. To facilitate standardization and clinical adoption of ctDNA testing across Canada, we developed a 2-phase multicenter study to standardize T790M mutation detection using plasma ctDNA testing. Methods In phase 1, commercial reference standards were distributed to participating clinical laboratories, to use their existing platforms for mutation detection. Baseline performance characteristics were established using known and blinded engineered plasma samples spiked with predetermined concentrations of T790M, L858R, and exon 19 deletion variants. In phase II, peripheral blood collected from local patients with known EGFR activating mutations and progressing on treatment were assayed for the presence of EGFR variants and concordance with a clinically validated test at the reference laboratory. Results All laboratories in phase 1 detected the variants at 0.5 % and 5.0 % allele frequencies, with no false positives. In phase 2, the concordance with the reference laboratory for detection of both the primary and resistance mutation was high, with next-generation sequencing and droplet digital polymerase chain reaction exhibiting the best overall concordance. Data also suggested that the ability to detect mutations at clinically relevant limits of detection is generally not platform-specific, but rather impacted by laboratory-specific practices. Conclusions Discrepancies among sending laboratories using the same assay suggest that laboratory-specific practices may impact performance. In addition, a negative or inconclusive ctDNA test should be followed by tumor testing when possible.

6580 Background: The combination of carboplatin and 5-fluorouracil (5-FU) is effective when used concurrently with radiotherapy for locoregionally advanced oropharyngeal carcinomas (Calais et al. 1999). DPYD polymorphisms can be associated with an increased risk of severe toxicity to fluoropyrimidines (Deenen et al. 2016). Upfront screening for the DPYD*2A allele is available in the province of Québec, Canada since March 2017. This study aimed to determine the effect of upfront genotyping on grade ≥3 toxicities. Methods: The studied population included all consecutive cases of oropharyngeal carcinomas treated with 5-FU based chemoradiotherapy one year before and after the implementation of upfront DPYD*2A genotyping. All patients were treated at the Centre Hospitalier de l’Université de Montréal (CHUM) between March 2016 and April 2018. Clinical data were extracted from chart review. Extended screening for 3 supplemental at-risk DPYD variants was also retrospectively performed in August 2019. Results: 181 patients were included in the analysis (87 patients before and 94 patients after DPYD*2A screening implementation). 91% of patients (n = 86) were prospectively genotyped for the DPYD*2A allele. Of those screened, 2% (n = 2/87) demonstrated a heterozygous DPYD*2A mutation. Those two patients received cisplatin-based treatment and thus avoided 5-FU toxicities. Extended genotyping of DPYD*2A-negative patients later allowed for the retrospective identification of 6 additional patients with alternative DPYD variants (two c.2846A > T and four c.1236G > A allele mutations). Conclusions: The DPYD*2A, c.2846A > T and c.1236G > A polymorphisms are associated with an increased risk of G3-4 toxicity to 5-FU, as well as higher hospitalization rates. Upfront DPYD genotyping can identify patients in whom fluoropyrimidine-related toxicity should be avoided. This represents an interesting addition in terms of pharmacovigilance. [Table: see text]

(1) To assess the ability of flow cytometric immunophenotyping to detect and quantitate eosinophils in patients with eosinophilic otitis media (EOM). (2) to evaluate the association of EOM to bronchial asthma.Twenty-one patients with chronic otorrhea or middle ear effusion (MEE) were included in this prospective cohort study. Group I composed of 10 patients (14 ears) and associated to bronchial asthma. Group II included 11 patients (11 ears) without bronchial asthma. Samples of MEE were sent for flow cytometric analysis at initial presentation. Patients with positive eosinophils on flow cytometric immunophenotyping were analyzed after one-month course of dexamethasone eardrops.EOM was diagnosed in all patients of group I and in three patients of group II. The mean eosinophils percentage was 43.5% and 14.2% for group I and group II, respectively (p = .006). Those patients showed a significant response to dexamethasone eardrops, both on clinical examination and on flow cytometric analysis with a decrease in eosinophil levels post-treatment. However, this improvement was temporary and symptoms recurred after treatment cessation. Bronchial asthma was not associated to all patients with EOM.Diagnosis of EOM remained mostly clinical; flow cytometry immunophenotyping of MEE may be helpful as an additional tool in diagnosis and monitoring the response to treatment, particularly in non-asthmatic patients.

Introduction The association of venous thromboembolic events (VTE) and lung cancer is highly prevalent. Additionally, the occurrence of a VTE with cancer has been associated with a worse prognosis and a poor quality of life. Underlying cancer biological features such as tumour mutations may contribute to VTE risk and cancer prognosis. Since preclinical data suggest a link between thrombosis and KRAS mutations in tumours, we aimed to validate this association in a patient registry cohort. Methods: A retrospective case control study was performed using the CHUM NSCLC registry. Cases had VTE occurring 6 months previous to or after a diagnosis of NSCLC. Diagnosis of VTE (venous thrombosis, pulmonary embolism, and migratory superficial thrombophlebitis) was confirmed by a review of the imaging reports. Controls were patients with NSCLC without thrombosis matched for age and stage (I-IIIA/IIIB-IV). Exclusion criteria included insufficient tissue for KRAS/EGFR mutation analysis or insufficient clinical information. Results Between Jan 2000 and Dec 2009 a total of 57 cases with VTE and 102 controls without VTE were included. The OR for thrombosis in KRAS and EGFR mutated NSCLC patients are respectively 2.67 (1.12-6.42; p = 0.014) and 0.99 (0.27-3.48; p = 0.99). Conclusions KRAS mutation is associated with an increased risk of VTE in this NSCLC cohort. These findings are consistent with preclinical studies. Prospective data on VTE rates from clinical trials with molecularly defined NSCLC are needed to confirm these findings.

Background. HPV is a positive prognostic factor in HNSCC. We studied the prevalence and prognostic impact of HPV on survival parameters and treatment toxicity in patients with locally advanced HNSCC treated with concomitant chemoradiation therapy.Methods. Data on efficacy and toxicity were available for 560 patients. HPV was detected by PCR. Analysis was performed using Kaplan-Meier survival curves, Fisher’s test for categorical data, and log-rank statistics for failure times.Results. Median follow-up was 4.7 years. DNA extraction was successful in 255 cases. HPV prevalence was 68.6%, and 53.3% for HPV 16. For HPV+ and HPV−, median LRC was 8.9 and 2.2 years (P=0.0002), median DFS was 8.9 and 2.1 years (P=0.0014), and median OS was 8.9 and 3.1 years (P=0.0002). Survival was different based on HPV genotype, stage, treatment period, and chemotherapy regimen. COX adjusted analysis for T, N, age, and treatment remained significant (P=0.004).Conclusions. Oropharyngeal cancer is increasingly linked to HPV. This study confirms that HPV status is associated with improved prognosis among H&N cancer patients receiving CRT and should be a stratification factor for clinical trials including H&N cases. Toxicity of CRT is not modified for the HPV population.

Immune checkpoint inhibitors are novel agents in the process of revolutionising cancer care. These agents have become a very appealing therapeutic alternative since they are much better tolerated than cytotoxic therapy, due to their relatively favorable toxicity profile. However, adverse events associated with these agents usually involve the immune system and can have serious implications jeopardising survival. Herein we report the first case of immune-mediated agranulocytosis due to Nivolumab therapy. The patient suffered from Staphylococcus infection during her agranulocytosis which only responded to high dose corticosteroid therapy.

650 Background: Fluoropyrimidines (FU) are part of chemotherapy combinations for multiple gastrointestinal cancers. Deficient dihydropyrimidine deshydrogenase (DPD) activity can lead to severe life-threatening toxicities in 3-5% of populations tested. The DPYD*2A polymorphism leading to deficient DPD activity is one of the most studied variants. Methods: We retrospectively performed chart reviews of all patients that tested positive for a heterozygous or homozygous DPYD*2A mutation in samples obtained from patients throughout the province of Quebec (Canada). The study objective was to document the impact of implementing this test in routine clinical practice, including the effects on treatment types, delays and toxicities. Results: 2,617 patients were tested by PCR for the presence of DPYD*2A in a period of 17 months: 25 patients tested positive. All were Caucasian. 24 of the 25 patients were heterozygous (0.92%) and one was homozygous (0.038%). A chart review was available for 20 patients: 15 were tested upfront while five were performed following severe toxicities on the first cycle of FU-based chemotherapy. Of the five patients identified following toxicities, all had grade 4 cytopenias, 80% grade ≥ 3 mucositis, 20% grade 3 rash and 20% grade 3 diarrhea, with an average of 15 days of hospitalisations due to febrile neutropenia. Seven of the 13 patients identified upfront received FU-based chemotherapy at reduced initial doses ranging from 25 to 50%. The average dose during the course of chemotherapy was 50% and two patients reached 100% dosage. No grade ≥ 3 toxicities were observed. DPYD*2A test results were available in an average of 6 days, causing no significant delays in treatment initiation according to 99% of queried physicians. Conclusions: Upfront genotyping before FU-based treatment is feasible in clinical practice. It may prevent severe toxicities and hospitalisations without delaying treatment initiation. The administration of chemotherapy at reduced doses appears to be safe in patients heterozygous for DPYD*2A.

e21064Background: First/second generation EGFR-TKI are eventually met with therapeutic failure in EGFRm aNSCLC, but treatment continuation beyond initial progression can delay the need for second l...

Increasing multi-ethnicity is likely to make alpha-thalassemia (alpha-thal) more prevalent in Western metropolitan areas. Multiplex polymerase chain reaction (m-PCR) allows rapid and precise identification of most of alpha-thal carriers. With this method, we sought to determine the prevalence of alpha-thal and the corresponding genotype, among all non repetitive consecutive blood samples that had an unexplained microcytosis. These specimens had been sent to the hematology laboratory for a blood count analysis, found to be microcytic, and secondarily tested for ferritin level and hemoglobin (Hb) high performance liquid chromatography (HPLC) profile. Five hundred and sixteen microcytic blood samples were evaluated and 197 samples with normal ferritin and Hb HPLC were studied by m-PCR. Among 196 interpretable PCRs, 48 alpha-thal cases (24.5%) were identified: 28 with a single alpha-globin gene deletion and 20 with two alpha-globin gene deletions. Of these 20 cases, six showed two deletions in cis. None of the erythrocytic parameters studied predicted the presence of alpha-thal deletions. We conclude that a significant proportion (24.5%) of blood counts with microcytosis not explained by an iron deficiency, an inflammatory state or an abnormal Hb on HPLC, are caused by an alpha-globin gene deletion. The pertinence of genetic counseling for alpha-thal based on molecular diagnosis should be evaluated more formally in urban centers where this genetic condition is likely to have an increasing prevalence and clinical relevance.

The cellular and molecular mechanisms of dysfunction and depletion of CD4+T lymphocytes over the course of human immunodeficiency virus type 1 (HIV-1) infection are still incompletely understood, but chronic immune activation is thought to play an important role in disease progression. We studied CD4+T-cell biology in CD4C/HIV transgenic (Tg) mice, in which Nef expression is sufficient to induce a severe AIDS-like disease including a preferential decrease of CD4+T cells. We show here that Nef-expressing Tg CD4+T cells exhibit an activated/memory-like phenotype which appears to be independent of antigenic stimulation, as documented in experiments involving breeding with AD10 TcR Tg mice. In addition, in vivo bromodeoxyuridine incorporation showed that a larger proportion of Tg than non-Tg CD4+T cells entered the S phase. However, in vitro, Tg CD4+T cells were found to have a very limited capacity to divide in response to stimulation with anti-CD3 and anti-CD28 or in allogeneic mixed leukocyte reactions. Interestingly, despite these observations, the deletion of Tg CD4+T cells had little impact on the development of other AIDS-like organ phenotypes. Thus, the Nef-induced chronic activation of CD4+T cells may exhaust the T-cell pool and may contribute to the thymic atrophy and the low number of CD4+T cells observed in these Tg mice.

CD4C/human immunodeficiency virus (HIV) transgenic mice develop an AIDS-like disease. We used this model to study the effects of HIV-1 on dendritic cells (DC). We found a progressive decrease in total DC numbers in the lymph nodes, with a significant accumulation of CD11bHiDC. In the thymus, the recovery of transgenic CD8α+DC had a tendency to be lower. Spleen DC were augmented in the marginal zone. Transgenic DC showed a decreased capacity to present antigen in vitro, consistent with their reduced major histocompatibility complex class II expression and impaired maturation profile. The accumulation of immature DC may contribute to disease and may reflect an adaptive advantage for the virus by favoring its replication and preventing the generation of fully functional antiviral responses.

Chronic myeloid leukemia (CML) is characterised by the presence of the Philadelphia chromosome (Ph), which results in the production of the constitutively active tyrosine kinase BCR-ABL1, with tyrosine kinase inhibitors (TKI)

The mechanisms responsible for degeneration of germinal centers (GC) and follicular dendritic cell (FDC) networks during progression to AIDS remain elusive. Here, we show that CD4+ T cells from CD4C/HIV-1 Tg mice, which develop a severe AIDS-like disease, express low levels of CD40 ligand. Accordingly, GC formation, FDC networks, and immunoglobulin isotype switching are impaired in these animals. However, Tg B cells respond to in vitro CD40 stimulation. Total serum IgG levels are reduced in Tg mice, whereas total IgM levels are increased with a significant amount showing DNA specificity. IFN-γ- and IL-6-deficient CD4C/HIV Tg mice also develop the AIDS-like disease and produce auto-Ab. Thus, CD4C/HIV Tg mice have immune dysfunction accompanied by autoimmune responses.

Compound heterozygotes for β-thalassemia and high oxygen affinity hemoglobin (Hb) have been documented, but experience in the management of such rare cases is minimal. Although hydroxyurea (HU) has never been used in a heterozygote with high oxygen affinity Hb and β-thalassemia, we hypothesized that it would decrease erythrocytosis through a lowered production of abnormal cells and increase of P 50 by induction of fetal hemoglobin (HbF). We present the case of a patient with compound high oxygen affinity Hb mutation with β-thalassemia. PCR analysis revealed combined Hb Regina and IVS1-110 G/A mutations. Treatment with HU caused a decrease in Ht (61.1% to 38.6%) and erythrocyte volume (74.87 mUkg to 40.65 mUkg), as well as an increase in P 50 (6 mmHg to 10 mmHg ) and HbF level (3.6% to 29.8%) at 12-month follow-up. Effects of HU on 2,3 DPG and HbNO levels did not appear to be predictable. Corroboration with other cases is needed to establish solid evidence on the clinical efficacy of HU in this population.

The beta-globin gene of 306 newly diagnosed beta-thalassemia (thal) minor patients were sequenced. Analysis revealed that only one amongst all the identified mutations had not been previously reported. This new mutation, causing a beta(+)-thal minor phenotype, was found in a patient of Arabic origin. The insertion frameshift mutation (+A) between codons 45 and 46 [codons 45/46 (+A)] results in a premature termination signal at codon 52. No truncated beta-globin or abnormal hemoglobin (Hb) was identified.

SummaryBackground. Numerous studies have demonstrated that elevated concentrations of acute-phase proteins affect red blood cell (RBC) aggregation. Plasma lipids and lipoproteins were also shown to be correlated with RBC aggregation in hypercholesterolemia. However, whether acute-phase proteins promote RBC hyperaggregation in hyper-lipidemic patients is unknown. The main objective of the study was to identify the impact of acute-phase proteins such as fibrinogen (Fib), haptoglobin (Hp), ceruloplasmin (Cp), α1-acid glycoprotein (AGP), α1-antitrypsin (AT), immunoglobulin G (IgG), and albumin (Alb) on RBC aggregation in 35 hyperlipidemic patients. The influence of these proteins in 32 normolipidemic subjects was also determined.Methods and Results. RBC aggregation parameters reflecting the kinetics of rouleau formation and the adhesive strength between RBCs were measured by laser reflectometry. Multivariate forward stepwise linear regression analyses were performed to study the relationship between RBC aggregation and these acute-phase proteins, total cholesterol (TC), triglycerides (TG), high (HDL-C) and low (LDL-C) density lipo-protein cholesterol, age, gender, body mass index (BMI), mean blood pressure (Mpressure), and smoking habit. The kinetics of rouleau formation was positively correlated with the linear combination of IgG and Hp (r = 0.76, p Conclusion. This study suggests that acute-phase proteins such as IgG, Hp, AGP and Alb influence significantly and in an independent way the level of RBC aggregation. The close association between RBC aggregation and cardiovascular risk factors further strengthens its clinical importance.

The objectives of this study were to detect differences in the Doppler power backscattered by blood in vivo, and to identify factors affecting the backscattered power. The main hypothesis was that variations in the erythrocyte aggregation level between veins and arteries of normolipidemic and hyperlipidemic individuals can be detected with power Doppler ultrasound. Doppler measurements were performed at 5 MHz, with an Acuson 128 XP/10 system, over the carotid artery and jugular vein, external iliac artery and vein, common femoral artery and vein and popliteal artery and vein. Doppler signals were recorded at the center of each vessel to optimize the detection of erythrocyte aggregation, and processed off-line to obtain the backscattered power. The power of each recording was compensated for Doppler gain differences, tissue attenuation with depth and transmitted power variations occurring with pulse-repetition interval modifications. Results showed statistically stronger backscattered power in veins compared to arteries for the iliac, femoral and popliteal sites. In comparison with healthy subjects, stronger powers were observed in hyperlipidemic patients for the femoral and popliteal sites. Power differences were also found between peripheral measurements. On the other hand, no difference was observed between the power measured in the carotid artery and jugular vein for both groups of individuals. Multiple linear regression analyses were performed to identify factors affecting the backscattered power. Results showed a correlation (r) of 71.2% between the Doppler power in the femoral vein and the linear combination of two parameters: an erythrocyte aggregation index S10 measured with a laser scattering method, and the diameter of the vessel measured on B-mode images. Statistically significant linear correlation levels were also found between S10 and the Doppler power in various vessels. In conclusion, this study showed that power Doppler differences exist in vivo in large vessels between veins and arteries of normolipidemic and hyperlipidemic individuals. The Doppler power variations were also shown to be related to erythrocyte aggregation.

Background. RAS gene mutations have an impact on treatment response and overall prognosis for certain types of cancer.Objectives. To determine the prevalence and impact of K-RAS codons 12 and 13 mutations in patients with locally advanced HNSCC treated with primary or adjuvant chemo-radiation.Methods. 428 consecutive patients were treated with chemo-radiation therapy and followed for a median of 37 months. From these, 199 paraffin embedded biopsy or surgical specimens were retrieved. DNA was isolated and analyzed for K-RAS mutational status.Results. DNA extraction was successful in 197 samples. Of the 197 specimens, 3.5% presented K-RAS codon 12 mutations. For mutated cases and non-mutated cases, complete initial response to chemoradiation therapy was 71 and 73% (P=0.32). LRC was respectively 32 and 83% (P=0.03), DFS was 27 and 68% (P=0.12), distant metastasis-free survival was 100 and 81% (P=0.30) and OS was 57 and 65% (P=0.14) at three years. K-Ras codon 13 analysis revealed no mutation.Conclusion. K-RAS codon 12 mutational status, although not associated with a difference in response rate, may influence the failure pattern and the type of therapy offered to patients with HNSCC. Our study did not reveal any mutation of K-RAS codon 13.

Background: Non-small-cell lung cancer (NSCLC) tumours with activating mutations of the epidermal growth factor receptor (EFGR) tyrosine kinase are highly sensitized to the effects of oral tyrosine kinase inhibitors such as gefitinib and erlotinib, suggesting the possibility of targeted treatment of NSCLC based on EFGR mutation status. However, no standardized method exists for assessing the EGFR mutation status of tumours. Also, it is not known if available methods are feasible for routine screening. To address that question, we conducted a validation study of methods used for detecting EGFR mutations in exons 19 and 21 at molecular laboratories located in five specialized Canadian cancer centres. Methods: The screening methods were first optimized using cell lines harbouring the mutations in question. A validation phase using anonymized patient samples followed. Results: The methods used at the sites were highly specific and sensitive in detecting both mutations in cell-line DNA (specificity of 100% and sensitivity of at least 1% across all centres). In the validation phase, we observed excellent concordance between the laboratories for detecting mutations in the patient samples. Concordant results were obtained in 26 of 30 samples (approximately 87%). In general, the samples for which results were discordant were also less optimal, containing small amounts of tumour. Conclusions: Our results suggest that currently available methods are capable of reliably detecting exon 19 and exon 21 mutations of EFGR in tumour samples (provided that sufficient tumour material is available) and that routine screening for those mutations is feasible in clinical practice.

Introduction. The mechanisms of haemoglobin (Hb) subtype switch remain largely unknown even though it has a therapeutic potential (Bauer et al., 2012). In fact, reactivation of fetal haemoglobin (HbF) production in adult erythrocytes (F-cells) is protective in many Hb disorders. Pregnancy may represent the only physiological condition where HbF production increases transiently (Ibrahim et al, 2009; Yamada et al., 2012). Moreover, flow cytometry (FC), by intracellular staining using anti-HbF antibodies, has improved the evaluation of maternal F-cells (Corcoran et al., 2014; Chen et al., 2000). In this study, the objectives are: 1) to document HbF expression prospectively peri- and post-partum and confirm its maternal origin; 2) to assess potential causes of HbF modulation throughout pregnancy (i.e. β-hCG, EPO) and; 3) to identify gene differences between HbF-positive and HbF-negative pregnant women. Methods. In this observational, single institution, prospective study, 879 pregnant women were screened. A total of 345 participants were included in the study: the first consecutive 176 women with negative total HbF expression ( Results. At the time of analysis, 93 HbF-positive and 147 HbF-negative women had completed follow-up. Results revealed that, among all pregnant women screened, 22.07% (n = 194) had positive HbF expression. Of this percentage, 90.72% (n = 176) filled the inclusion criteria. Of all women with positive HbF expression during 1 or more of the first 3 visits, 72.04% showed a post-partum decrease of HbF below 1%. Furthermore, FC analyses in HbF-positive women confirmed the maternal origin of HbF in all cases and revealed an average of 13.28% (sd = 5.38%) F-cells when total HbF was ≥ 1%. Only modest correlations could be found so far between HbF levels and β-hCG (ρ = 0.25) or EPO (ρ = 0.07). Comparative DNA analyses of HbF-positive and HbF-negative women are currently underway and will be presented at the meeting. Conclusions. To our knowledge, this represents the largest prospective study evaluating HbF expression throughout pregnancy, as well as the first to evaluate post-partum HbF dynamics and the genetic background of HbF-negative and HbF-positive pregnant women. Preliminary data shows that: 1) a significant percentage of pregnant women have a transient elevation of HbF which cannot be explained by fetomaternal hemorrhage nor hereditary persistence of HbF; 2) the proportion of F-cells in women remains constant peri- and post-partum when HbF ≥ 1%; 3) a large proportion of maternal erythrocytes express HbF at low levels and; 4) HbF modulation during pregnancy is only modestly correlated to β-hCG and EPO. These data may lead to the study of a new paradigm of Hb subtype switch as well as HbF-induction therapeutics for the benefit of Hb disorders. Disclosures No relevant conflicts of interest to declare.

Increasing multi-ethnicity is likely to make alpha-thalassemia (alpha-thal) more prevalent in Western metropolitan areas. Multiplex polymerase chain reaction (m-PCR) allows rapid and precise identification of most of alpha-thal carriers. With this method, we sought to determine the prevalence of alpha-thal and the corresponding genotype, among all non repetitive consecutive blood samples that had an unexplained microcytosis. These specimens had been sent to the hematology laboratory for a blood count analysis, found to be microcytic, and secondarily tested for ferritin level and hemoglobin (Hb) high performance liquid chromatography (HPLC) profile. Five hundred and sixteen microcytic blood samples were evaluated and 197 samples with normal ferritin and Hb HPLC were studied by m-PCR. Among 196 interpretable PCRs, 48 alpha-thal cases (24.5%) were identified: 28 with a single alpha-globin gene deletion and 20 with two alpha-globin gene deletions. Of these 20 cases, six showed two deletions in cis. None of the erythrocytic parameters studied predicted the presence of alpha-thal deletions. We conclude that a significant proportion (24.5%) of blood counts with microcytosis not explained by an iron deficiency, an inflammatory state or an abnormal Hb on HPLC, are caused by an alpha-globin gene deletion. The pertinence of genetic counseling for alpha-thal based on molecular diagnosis should be evaluated more formally in urban centers where this genetic condition is likely to have an increasing prevalence and clinical relevance.

The human immunodeficiency virus type 1 (HIV-1) Nef protein is an important determinant of AIDS pathogenesis. We have previously reported that HIV-1 Nef is responsible for the induction of a severe AIDS-like disease in CD4C/HIV transgenic (Tg) mice. To understand the molecular mechanisms of this Nef-induced disease, we generated Tg mice expressing a mutated Nef protein in which the SH3 ligand-binding domain (P 72 XXP 75 XXP 78 ) was mutated to A 72 XXA 75 XXQ 78 . This mutation completely abolished the pathogenic potential of Nef, although a partial downregulation of the CD4 cell surface expression was still observed in these Tg mice. We also studied whether Hck, one of the effectors previously found to bind to this PXXP motif of Nef, was involved in disease development. Breeding of Tg mice expressing wild-type Nef on an hck −/− (knockout) background did not abolish any of the pathological phenotypes. However, the latency of disease development was prolonged. These data indicate that an intact PXXP domain is essential for inducing an AIDS-like disease in CD4C/HIV Tg mice and suggest that interaction of a cellular effector(s) with this domain is required for the induction of this multiorgan disease. Our findings indicate that Hck is an important, but not an essential, effector of Nef and suggest that another factor(s), yet to be identified, may be more critical for disease development.

e22159 Background: Recent advances in lung cancer treatment embrace the recognition of molecular pathways implicated in its pathogenicity, paving the path to personalized therapies. We conducted a retrospective analysis to characterize the molecular features of the population treated for non-squamous non-small cell lung cancer (NS-NSCLC) in the province of Quebec. Methods: 622 patients with NS-NSCLC and adequate tumor blocks, treated at the CHUM between 2006 and 2008, were included. All samples were tested for ALK translocations (by IHC and FISH), EGFR classical exon 19 and 21 mutations by PCR (fragment analysis and qPCR) and for KRAS codon 12 and 13 mutations by mismatch PCR-RFLP. Molecular features were matched to demographic characteristics and clinical outcomes. Results: So far, complete results are available for 153 patients. Considering the amount of tumor tissue available, this population is largely represented by patients with local or loco-regional disease (n= 140, 91.5%). A minority of patients (10.3%) was never or light smokers (< 10 pack-yrs). Only 2 patients (1.3%) were of Asian descent. The following table depicts the outcomes of this cohort of patients segregated according to mutation status and extent of disease. Conclusions: ALK rearrangements were not identified in this unselected NS-NSCLC population characterized by localized disease and strong smoking history. ALK translocation prevalence in different populations is likely to be largely influenced by its tumor stage distribution, tobacco exposure and the use of selection criteria for molecular testing. An expanded cohort of patients will be presented at the meeting. [Table: see text]

6041 Background: Dacomitinib is an irreversible pan-HER TKI with preclinical (EGFRvIII+ cell lines, SCCHN xenografts) and clinical activity (phase II recurrent/metastatic SCCHN; Razak et al, Ann Oncol 2012). However, little is known about predictive markers of efficacy related to EGFR signalling in this setting. Methods: Of69 pts treated with 1st-line dacomitinib in a phase II trial for recurrent/metastatic SCCHN, 48 pts had archival tumor specimens obtained before treatment and 13 had paired biopsies (days 0 and 7 of therapy, FFPE and snap frozen). EGFRvIII and PTEN (IHC), HPV genotyping and human genomic mutations (Sequenom OncoCarta Panel – 19 genes, 238 mutations) were evaluated on archival tissue. IHC expression of AKT, CC3, EGFR, ERK, HER2, HER3, MET, Ki67, pAKT, pEGFR, pERK, pHER2 and pMET was evaluated in paired specimens. The presence/absence or expression level of these markers was correlated with response (RR)/clinical benefit (CB), PFS and OS. Results: In pts with archival tissue, no statistically significant difference was found in RR/CB or PFS based on HPV, EGFRvIII, PTEN or presence of mutation. There was a trend to increased OS in HPV+ pts (HR 0.47, 95% CI 0.21–1.07, P=0.068). In paired biopsies, some expression variation was seen for cytoplasm AKT, membrane EGFR, nuclear ERK and pAKT. There was no correlation between basal expression of these markers and RR/CB or PFS. Variations in ratio to baseline of EGFR, pAKT, pERK and MET were qualitatively associated with RR/CB. No statistically significant correlations could be established for PFS, but there were interesting qualitative variations in the levels of expression of some molecules, eg, EGFR, pAKT. Conclusions: No predictive efficacy marker was identified. It cannot be determined if increased OS in HPV+ cases is due to prognostic or predictive effects. Paired biopsies demonstrated that dacomitinib was associated with variation in expression of multiple elements in signalling pathways linked to EGFR. Given the small number of paired biopsies, and large amount of data generated, descriptive study of cases is required. Further data will be presented at the meeting. Clinical trial information: 00768664.

5574 Background: HPV has recently been recognised as a good prognostic factor in head and neck (H&N) cancer. However, most of the data is derived from randomised trials with different treatment opt...

6032 Background: Excision repair cross-complementation group 1 (ERCC1) is a gene coding for the nucleotide excision repair complex. Its increased expression and polymorphism at codon 118 have been linked to poor response to chemotherapy or chemoradiation in several types of cancer. ERCC1 removes the cisplatin adducts on the DNA of cells and its polymorphism appears to be a marker of chemotherapeutic resistance to platinum-based therapy. Objectives: To determine the prevalence of the polymorphism of ERCC1 (codon 118) in patients with locally advanced HNSCC treated with concomitant platinum-based chemoradiation therapy with or without prior surgery, and its effect on eficacy evaluated by locoregional control, disease-free survival and overall survival. Methods: Prospective data on efficacy was available on 460 consecutive patients treated with concomitant chemoradiation in our institution with a minimal follow-up of 2 years. Of these, 230 fixed and paraffin embedded biopsies or surgical specimens were collected. DNA was extracted from specimens and polymorphism of codon 118 was determined using a PCR technique. All analysis were performed using Kaplan-Meier survival curves, Fisher's test for categorical data and log-rank statistics for failure times. Results: DNA extraction was successful in 222 patients. Polymorphism mapping was possible in 178 specimens. Genotypic distribution in the population was the following : AAT/AAT:40% (Gr1), AAC/AAT: 48%(Gr2), AAC/AAC: 12% (Gr3). At 3 years, evaluation of efficacy for Gr1, Gr2, and Gr3 was determined. Locoregional control was respectively 77%, 83%, and 67% (p = NS), DFS was 58%, 68% and 55% (p=NS), and OS was 70%, 69%, and 70% (p = NS). Conclusions: ERCC1 polymorphism did not have an impact in our population on response to chemoradiation therapy. It can be postulated that ERCC1 does not seem to discriminate patients for whom another treatment option should be sought for patients with locally advanced SCCHN. Further data will be presented at the meeting including a multivariate analysis using different markers tested in our laboratory (KRAS, MGMT methylation, etc). No significant financial relationships to disclose.

HLA-B27 is a strong diagnostic biomarker as it is found in 90– 95% of ankylosing spondylitis. Routinely, the false-positive results generated by cross-reactivity of HLA-B27 monoclonal antibodies (mAbs) with some other type of HLA-B antigens are problematic. The present study aims at improving the sensitivity and specificity of typing for HLA-B27 by using a more accurate DNA assay. A real time sequence specific primer polymerase chain reaction (PCR-SSP) is performed by using MGB TaqMan oligoprobe and an ABI PRISM™ 7500 sequence detection system. The TaqMan PCR-SSP assay is compared with Immunophenotyping by flow cytometric antigen assay (BD HLA-B27-Kit, clone GS145.2). The results are finally confirmed by sequencing (ABI PRISM ® 3100). As summarised in the table, three methods are identical for clearly positive and negative results. There is 90% concordance for borderline negative results obtained by immunophenotyping with TaqMan PCR-SSC and/or sequencing. However, only 15.4% of borderline positives obtained by immunophenotyping are confirmed as true HLA-B27 positives by sequencing as well as TaqMan PCR-SSC. TaqMan PCR-SSP DNA assay completely correlates with sequencing (gold standard) with 100% PPV and NPV, compared to a PPV of 15% and a NPV of 98% for borderline results by immunophenotyping. Conclusion: when compared with flow cytometric antigen assay, typing HLA-B27 by TaqMan PCR-SSP clearly demonstrates an advantage to better establish the genotype of the patient. Specifically, there is a marked difference for ambitious positive results from immunophenotyping. Moreover, compared to immunophenotyping, there is no need of fresh samples, can test a larger number of samples concomitantly, and reduces technical time as well as cost. N = 52 Immunophenotyping TaqMan PCR-SSP Sequencing Immuno vs Sequencing TaqMan PCR-SSP vs Sequencing Positive 11 11 11 100% 100% Negative 17 17 17 100% 100% Positive (borderline by Immunophenotyping) 13 2 2 15% 100% Negative (borderline ) by Immunophenotyping) 10 9 9 98% 100%

Introduction: α-Thalassemia results from an heterogeneous group of disorders involving the α-Globin genes. Common deletions are responsible for 95% of cases of α-thalassemia, whereas Constant Spring mutation (Hb CS), one of the known mutations involving the termination codon of α2-globin gene, is the most common non-deletional cause of α-thalassemia worldwide. Its prevalence has never been studied in occidental countries. Patients and methods: as part of our neonatal hemoglobinopathy screening program, fetal cord blood samples are systematically tested by HPLC. When an abnormal Hb or HbH is found, α-Globin deletion is carried by multiplex-PCR. A two part observational study was done at our center based on the available data from our hemoglobinopathy screening program available since 2002. Firstly, we aimed to determine the level of Hb H present and to correlate it to a specific type of α-Globin gene deletion by m-PCR sequencing (table 1). 306 samples were analyzed and demonstrated a statistically significant difference between categories of α-globin gene deletions: 2 del cis vs 1 del, 2 del trans vs 1 del, 2 del cis vs 2 del trans (difference of means of 8.07, 4.33 and 3.75 respectively, P Table 1 Deletion/allelic distribution Hb H level (range) Hb H level (means) Std Dev 1 deletion 1.0 – 12.4 2.03 1.12 2 deletions cis - 10.1 2.16 2 deletions trans - 6.35 1.5 Our second goal was to establish the prevalence of α-Globin termination codon mutation in the fetal blood sampling containing ≥1% of Hb H in absence of α-Globin gene deletion by m-PCR. 395 fetal blood samples were analysed. Of these, 59 lacked α-Globin gene deletions. 17 were excluded from our analysis for lack of personal data or DNA. The 42 remaining samples were studied by amplification of the termination codon followed by an enzymatic digestion for the recognition site TTAA. 2 samples (5%) were found to be heterozygotes for α2-globin mutation, presumably CS mutation. The small number of positive cases does not permit the conduct of a powerful statistical analysis, however the two mutated samples had a high level of Hb H (2,9%) whereas only 21% of the 42 studied samples had an Hb H ≥2%. Conclusion: These results represent a step forward in diagnosis of α-thalassemia and in identification of patients requiring particular genetic counselling based on HbH level at birth in cord blood. These results also indicate that 5% of Hb H positive/α-Globin deletion negative samples were caused by termination codon mutation in an occidental urban based population, and that it could be of interest to search for these mutations, especially when the Hb H level is high.

6014 Background: The O6-methylguanine-DNA methyltransferase (MGMT) gene encodes a specific DNA repair enzyme that protects cells from toxicity of alkylating agents. Thus, MGMT activity is a major m...

17005 Background: RAS gene mutations have been shown to occur in certain malignancies and have an impact on response to treatment and overall prognosis for certain types of cancer, more specificall...

Introduction: The usefulness of performing a cytocentrifuge preparation (CCP) analysis on cerebrospinal fluid (CSF) containing less than 5 cells has never been studied. To determine if the CCP analysis has a clinical impact on central nervous system (CNS) management of patients with CSF samples containing less than five cells, we correlated the result of CCP to cytomorphology and flow cytometry. Methods: From November 20th 2005 to July 18th 2007, CCP analysis were performed on 105 CSF consecutive samples containing less than 5 cells from patients with various cancers. For each of these samples, a research via computerized data was conducted to obtain cytomorphology and flow cytometry results. If results were positive or inconclusive, the clinical history and treatment were reviewed. Results: All 105 samples had negative CCP results. Of the 105 samples, 74 (70,5%) had available cytomorphologies, with 5 (6,8%) being positive, confirming central nervous system infiltration. All 5 patients received treatment for their CNS involvement. The sensitivity of cytomorphology was 83,3%(5/6) and its specificity 100% (68/68).Of the 105 samples, 23 (21,9%) had available flow cytometries: 4 of the 23 (17,4%) cytometries were positive, 2 correlated by positive cytomorphology and treated. 2 had negative cytomorphology with one patient receiving treatment. The sensitivity of cytometry was 75% (3/4) and the specificity 94,7% (19/20). Conclusion: We found no advantage to performing CCP analysis on CSF samples of less than 5 cells. However, this suggests that performing both cytomorphology and flow cytometry allows a better detection and management of CNS infiltration.

Introduction: Increasing multiethnicity is likely to make α-thalassemia minor more prevalent in Western metropolitan areas. It also increases the probability of finding newborns with Hemoglobin H disease or hydrops foetalis. Therefore, there is a need to implement screening methods to detect α-thalassemia at birth. Screening should also identify patients and families with specific requirements in genetic counselling. The goal of the present study is to evaluate the value of HbH measurement in cord blood to identify patients with α-globin deletions and patients with genotypes at risk that should be directed towards genetic counselling. Methods: A universal neonatal screening for hemoglobinopathies and thalassemia has been performed at our institution since 2002. Between October 2002 and July 2005, hemoglobin HPLC (High Performance Liquid Chromatography, Bio-Rad Variant II)) analysis performed on ombilical cord blood identified 123 newborns with hemoglobin H (HbH). Subsequently, DNA samples were analysed by m-PCR to detect the most common alpha-globin gene deletions:-α 3.7, -α 4.2, -α 20.5, -- SEA,-- FIL, -- THAI, -- MED. Groups were formed based on the genotype (1 deletion, 2 deletions, 2 deletions in cis, 2 deletions in trans) The mean level of HbH was calculated for each genotype and group. Student t-test was used to compare the level of HbH between the different groups. Due to the heterogeneity of the group populations, a Mann-Whitney test was also used to corroborate results obtained with t-test. Results: At least one α-globin gene deletion was observed in 90.2% of cases.(95%I.C: 83.5%-95.0%). Comparison between the groups based on the number of α-globin gene deletion and their allelic distribution allowed identification of significant differences in the HbH levels. Results were similar with Mann-Whitney (data not shown).Comparisons are presented in Table 1. HbH levels for the specific genotypes are presented in Figure 1. Conclusion: Hemoglobin HPLC analysis performed on umbilical cord blood allows rapid and efficient detection of HbH and highly predicts the presence of common α-globin gene deletions. Moreover a significant correlation is demonstrated between the levels of HbH and the presence of an α-thalassemic genotype. HbH level seems to correlate positively with the clinical severity of the genotype. However, a discriminate level of HbH could not be established to identify patients specifically requiring genotyping and genetic counselling. Further testing is necessary to evaluate the causes of HbH expression when there is no α-globin gene deletion. Genotype and HbH level Genotype and HbH level Mean HbH level (% of total hemoglobin) deletions/allelic distribution HbH level (means) HbH level (range) level of significance 1 deletion 2.10 1.4–5.9 2 deletions 6.96 4.0–12.6 p

Introduction: Thalassemia minor is a prevalent genetic disorder around the world. A large number of mutations have been implicated in the development of a β-thalassemia minor phenotype. The diagnosis of β-thalassemia minor is made based on phenotypic parameters: microcytosis and elevated HbA2, with or without elevated HbF. We previously reported (ASH 2004, abstract #3761) that erythrocytic parameters of β-thalassemia minor are significantly different in patients with concomitant α-globin deletions, suggesting a reduction of ineffective erythropoiesis. The goal of the present study is to evaluate the effect of α and β-globin mutations on erythrocytic parameters in β-thalassemia minor patients. Methods: Diagnosis of β-thalassemia minor was established using Hb HPLC analysis (Variant II, Bio-Rad) with increased HbA2 +/− increased HbF without expression of an abnormal Hb in cases of microcytosis. The DNA of consecutive cases with newly diagnosed β-thalassemia minor was extracted from leucocytes. A m-PCR was used to detect the presence of 7 α-globin gene deletions (-α3.7, -α4.2, --SEA, --FIL, --MED, --THAI, -α20.5). PCR and automated sequencing using fluorescence-based capillary electrophoresis (ABI PRISM 3100 Genetic Analyser) was performed to identify β-globin mutations causing the thalassemia phenotype. Data on age, sex and erythrocyte indices (Hb, MCV, RBC and RDW) was recorded. Student t-test was used to compare groups on the erythrocytic parameters. Results: At the time of analysis, 175 samples were fully studied. At least one mutation in the β-globin gene was identified in 172 cases (98.3%, 95%IC: 95–99.5%). Groups were defined for comparison of erythrocytic parameters: mutations causing β+ phenotype, mutations causing β0 phenotype. Sub-groups were also analysed based on the presence or absence of α-globin gene deletions. Results are presented in Table 1 and 2. Conclusion: The correlation between β-globin mutation and a β-thalasemia minor phenotype indicates that the phenotypic parameters correctly diagnoses β-thalasemia minor in the great majority of cases. Mutations classified as β0 affect more negatively the MCV, probably as a consequence of a reduced β-globin production and a greater level of ineffective erythropoiesis than in the β+ group. Higher MCV values in both β+ and β0 mutations when paired with an α-globin deletion compared with cases without an α-globin gene deletion could be explained by the concomitant reduction of functional α and β globin chain production, therefore causing less ineffective erythropoiesis. Differences in erytrocytic parameters between β° and β+ genotypes Erythrocytic parameter β+ β° P Hb 122.3 118.4 NS RBC 5.70 5.78 NS MCV 67.2 64.5

3149 Background: CLL is associated with loss of normal cellular apoptotic function. Apoptosis in cell lines has been identified with GX015–070 (GX), a small molecule antagonist of the BH3-binding groove of the BCL-2 family of proteins which is frequently overexpressed in cancers including CLL. This study evaluated GX induced apoptosis in CLL cells ex vivo correlating it with prognostic factors. Methods: Circulating lymphocytes were collected from patients (pts) with ≥ 20x109/L lymphocytes with no CLL treatment or red cell transfusions within 4 wks. Medical history was recorded. The diagnosis was confirmed with immunophenotyping by flow cytometry. CD38 was assessed in this analysis. The cytogenetic abnormalities del(11q), +12, del(13)(q14.3), del(13)(q34) & del(17p) were established by fluorescent in situ hybridization. Apoptosis was assessed with surface annexin V immunophenotyping in B cell enriched samples with positive glucocorticoid and negative DMSO controls. Results: 30 pts were recruited including ...

Introduction: Increasing multi-ethnicity is likely to make α-thalassemia more prevalent in western metropolitan areas. The current prevalence of α-thalassemia in regions outside the traditional thalassemia areas with the associated risk for HbH disease and hydrops foetalis is unknown. Genetic counseling for reproductive risks due to α-globin gene mutations requires adequate and precise genotyping. Objectives: To determine by multiplex polymerase chain reaction (m-PCR) the prevalence and genotypes of α-thalassemia among non selected, consecutive cases of unexplained microcytosis identified in a clinical hematology laboratory. To evaluate if differences or variations in erythrocytic indices are indicative of α-thalassemia. Methods: During a period of 3 months, α-globin genotype was determined by m-PCR on all non repetitive microcytic (MCV Results: 516 microcytic blood samples were evaluated of which 197 had normal ferritin and Hb HPLC analysis and were submitted for m-PCR. Among 196 interpretable m-PCRs, 148 did not have α-globin gene deletion(s) (group 1) and 48 α-thalassemia cases (24,5%) were identified: 28 cases of single α-globin gene deletion (group 2) and 20 cases of 2 α-globin gene deletions (group 3). In group 3, 6 cases showed deletions in cis. Results on differences between the groups are presented in Table 1. Even though some differences were statistically different, none was clinically useful and diagnostic. Conclusion: A significant proportion (24,5%) of cases with microcytosis not likely explained by iron deficiency, inflammation or a hemoglobinopathy are caused by α-globin gene deletion(s). This laboratory survey, despite a short period of sample collection (3 months) identified 6 carriers of a 2 gene deletion in cis that may have a severe impact on reproductive decisions and potential future utilisation of resources. These findings are likely to be reproducible in other North American cosmopolitan cities where α-thalassemia is not endemic but likely to be of increasing prevalence due to immigration. The availability, precision and reliability of diagnostic methods such as m-PCR and the increasing ethnic diversity of North-American cities point to an emerging need to develop genetic counseling programs based on the molecular diagnosis of α-thalassemia. Erythrocytic parameters with their standard deviation in the different α-globin genotype groups RBC (x1012/L) MCH (pg) Hb (g/dL) Mentzer† RDW MCV (fL) † calculated for samples with Hb>90 g/L *indicates values that are significantly different from data of other groups. RBC: Red Blood Cells, MCH: Mean Corpuscular Hemoglobin, Hb: Hemoglobin, RDW: Red Cells Distribution Width, MCV: Mean Corpuscular Volume group 1 (normal genotype) 4,27±0,75 * 25±1,5 107±20 * 17,4±2,4 * 17,0±3,1 * 76,6±3,2 group 2 (single α-globin gene deletion) 4,70±0,85 25,2±1,5 118±22 16,2±2,9 * 15,1±2,1 76,9±3,5 group 3 (double α-globin gene deletion) 5,20±0,57 23,2±1,3 * 120±9 14,1±2,1 * 14,8±2,8 72,4±3,0 *

Introduction: β-thalassemia (thal) minor is caused by multiple β-globin (gb) gene mutations, most being located in the promotor and IVS-1. Large variations in erythrocyte indices between β-thal minor patients have been reported and are thought to be caused by the type of β-gb gene mutation. α-thal minor is caused by partial or total α-gb gene deletions. α-gb gene deletions have been reported in all racial origins. It is therefore likely that α-gb gene deletions occur in β-thal minor patients.This combination of gb genes mutations could also have an impact on erythrocyte indices. Objectives: The trial objectives were to determine the prevalence of α-gb gene deletions in β-thal minor patients and to compare erythrocyte indices between 3 groups. Group 1: no α-gb gene deletion, Group 2: deletion of 1 α-gb gene, Group 3: deletion of 2 α-gb genes. Methods: Diagnosis of β-thal minor was established using hemoglobin HPLC analysis (Variant II, Bio-Rad) with increased HbA2 +/− increased HbF without mutant Hb. The DNA of consecutive cases with newly diagnosed β-thal minor was extracted from leucocytes. A multiplex PCR assay was used to detect the presence of 7 α-globin gene deletions: −α3.7,− α4.2, −− SEA, −− FIL, −− MED, −− THAI, − α20.5. Data on age, sex and erythrocyte indices (Hb, MCV, RBC and RDW) was recorded. An ANOVA was used to compare groups. P significance was established at 0.01 to adjust for multiple analysis. Results: 300 specimen were collected in 9 months. 34 were excluded because of poor DNA quality or presence of a mutant Hb. 25 patients (9.4%) had at least 1 α-globin gene deletion. Group1 included 241 patients, Group 2: 20 patients and Group 3: 5 patients. No differences for age or sex were present between the groups. Differences in erythrocyte indices are reported in Table 1. LogMCV was used to adjust for variance heterogeneity. Conclusions: 9.4% of β-thal minor have α-gb gene deletions. The only parameter that is significantly different between groups is MCV (Hb not reaching the pre-specified level of 0.01). It is therefore possible that differences in MCV between patients with β-thal minor can be explained by α-gb gene deletions. The concommittant presence of β-gb and α-gb mutations might improve the β-gb/α-gb imbalance implicated in ineffective erythropoiesis and a significant increase in MCV (and Hb but not reaching statistical significance in this study due to the sample size). However, this parameter does not have sufficient power to differenciate or identify β-thal minor patients with α-globin gene deletions. Other factors certainly influence MVC variability in β-thal minor patients and the independant influence of this variable (α-gb gene deletions) will require further investigation. Erythrocyte indices between groups Parameter Group Mean Std Deviation 95% CI P MCV 1 65.8657 3.9845 65.3580–66.3734

Introduction: A 38-year-old causasian male with hepatomegaly, splenomegaly and erythrocytosis (Ht 69.2%, Hb 217 g/L, MCV 76fl, normal WBC and platelets counts) presented with flank pain found to be a renal artery thrombosis. He had a history of increased Ht since birth without bone marrow (BM), cardiac, pulmonary, renal or cerebral anomalies and for which a diagnosis of a high oxygen affinity hemoglobinopathy was made. The disease had previously been uncomplicated without therapy. Initial evaluation in our center revealed a normal BM morphology, a normal karyotype and an abnormal Hb HPLC (elevated HbF (4.9%) and an abnormal Hb eluting after normal HbA1). The red cell mass was increased at 74.9 ml/kg (normal = 26.5 ml/kg). The oxygen (O2) P50 saturation determined from the Hb-O2 dissociation curve using an Hemox-Analyser was markedly decreased at 6 mmHg (normal = 27 mmHg). α and β globins (gb) HPLC demonstrated normal α, but 100% abnormal β-gb. A diagnosis of a double heterozygote for β-gb gene was established: an allele with mutation causing high affinity for O2 and an allele causing β-thalassemia (thal) minor. Anticoagulation and serial phlebotomies did not improve the erythrocytosis. Therapy with hydroxyurea (HU) was therefore proposed to the patient. Objectives: To determine the β-gb genotype and to evaluate the effect of HU therapy at maximally tolerated dose (MTD) on induction of HbF and its effect on Ht, P50, red cell mass, 2,3-DPG and total HbNO concentrations. Methods and results: Sequencing of the β-gb locus was done by RT-PCR amplified mRNA and by PCR amplified DNA, using primers spanning almost the entire gene (−450 to 601 bp, excluding a small portion of IVS2). Two mutations were identified: Leu96→Val (339C→G) in exon 2, producing Hb Regina, a high O2 affinity hemoglobin variant, and IVS1-110 G/A, a frequent mutation causing β-thal minor. Therapy with HU was initiated at 7 mg/kg/day. Dose was increased to MTD resulting in a dose of 25 mg/kg/day. Table 1 summarizes variations in relevant parameters while on HU therapy. Conclusion: HU rapidly induced HgF and improved measured parameters in this patient with a high O2 affinity Hb/β-thal minor. HU’s effect in this case did not seem to be strictly related to its anti-proliferation properties. Induction of HbF and subsequent increase in P50 probably reduced Epo production (data pending) and erythropoiesis. Modifications in other mediators of O2 release were also modified by HU. The changes in HbNO are not totally consistant with the rest of the data, being increased at 3 months but decreased at 6 months. While on HU therapy, the patient did not present any new complications (thrombotic or other) and clinically reported an improved exercise tolerance. Further evaluation will focus on epigenetic factors affecting HbF expression and correlation of NO level with plasma L-arginine concentration. | Time | HU dose (mg/kg) | Ht (%) | HbF (%) | P50 (mm/Hg) | 2,3-DPG (umol/g Hb) | Total HbNO (nM) | Red cell mass (ml/kg) | |:----------------------------------------:| --------------- | ------ | ------- | ----------- | ------------------- | --------------- | --------------------- | | NA: not available, TBD: to be determined | | Baseline | | 61.1 | 3.6 | 6 | 21.3 | 242.7 | 74.9 | | 3 months | 21 | 69.4 | 9.1 | 6 | 19.0 | 694.3 | NA | | 6 months | 25 | 56.9 | 15.1 | 9 | 21.4 | 105.8 | NA | | 8 months | 25 | 46.7 | 25.4 | TBD | TBD | TBD | 51.7