Thurmes, Paul J.

PURPOSE To evaluate the use of early assessment of chemotherapy responsiveness by positron emission tomography (PET) imaging to tailor therapy in patients with esophageal and esophagogastric junction adenocarcinoma. METHODS After baseline PET, patients were randomly assigned to an induction chemotherapy regimen: modified oxaliplatin, leucovorin, and fluorouracil (FOLFOX) or carboplatin-paclitaxel (CP). Repeat PET was performed after induction; change in maximum standardized uptake value (SUV) from baseline was assessed. PET nonresponders (< 35% decrease in SUV) crossed over to the alternative chemotherapy during chemoradiation (50.4 Gy/28 fractions). PET responders (≥ 35% decrease in SUV) continued on the same chemotherapy during chemoradiation. Patients underwent surgery at 6 weeks postchemoradiation. Primary end point was pathologic complete response (pCR) rate in nonresponders after switching chemotherapy. RESULTS Two hundred forty-one eligible patients received Protocol treatment, of whom 225 had an evaluable repeat PET. The pCR rates for PET nonresponders after induction FOLFOX who crossed over to CP (n = 39) or after induction CP who changed to FOLFOX (n = 50) was 18.0% (95% CI, 7.5 to 33.5) and 20% (95% CI, 10 to 33.7), respectively. The pCR rate in responders who received induction FOLFOX was 40.3% (95% CI, 28.9 to 52.5) and 14.1% (95% CI, 6.6 to 25.0) in responders to CP. With a median follow-up of 5.2 years, median overall survival was 48.8 months (95% CI, 33.2 months to not estimable) for PET responders and 27.4 months (95% CI, 19.4 months to not estimable) for nonresponders. For induction FOLFOX patients who were PET responders, median survival was not reached. CONCLUSION Early response assessment using PET imaging as a biomarker to individualize therapy for patients with esophageal and esophagogastric junction adenocarcinoma was effective, improving pCR rates in PET nonresponders. PET responders to induction FOLFOX who continued on FOLFOX during chemoradiation achieved a promising 5-year overall survival of 53%.

Abstract BACKGROUND: Acquired hemoglobin disorders including α thalassemia (hemoglobin H (HbH) disease) can complicate neoplastic myeloid disorders, especially myelodysplastic syndrome (MDS). Acquired α thalassemia in MDS has recently been linked to mutations in the trans-acting ATRX at Xq13, a gene previously associated with inherited ATR-X (α thalassemia-mental retardation-X linked) syndrome. In the 3 instances where acquired ATRX point mutations predicted to affect splicing have been encountered, mRNA has been unavailable for assessment or unexpressed; in the 3 cases where ATRX splicing changes were described in MDS, the responsible genomic DNA mutation could not be located. Here we report a novel genomic point mutation in an ATRX splice donor motif and define the mutation’s splicing and expression consequences. METHODS AND RESULTS: A 59 year old anemic Swiss man with chronic myelomonocytic leukemia (CMML) and multiple autoimmune complications including granulomatous uveitis, diabetes insipidus, and pleuropericarditis, developed anemia (Hb 10 g/dL) with MCH of 16.7 pg and MCV of 54 fL. We detected 50% HbH-containing cells by supravital staining and 7.5% HbH by weak cation chromatography, confirmed as HbH by isoelectric focusing. Peripheral blood granulocytes, mononuclear cells and bone marrow mononuclear cells were isolated using dual density Ficoll-Hypaque, and DNA and RNA extracted for mutation screening. ATRX coding region (35 exons) and splice site DNA from mononuclear cells was amplified and subjected to denaturing high performance liquid chromatography (DHPLC). A heteroduplex peak was encountered in the amplicon centered on exon 4. Sequencing demonstrated thymidine to cytidine point mutation with high proportional clonality in the canonical GT splice donor motif 3′ to ATRX exon 4 (IVS 4+2 T&gt;C), also detectable in patient granulocytes. Electrophoresis of raw PCR product from the 5′ end of ATRX cDNA demonstrated multiple abnormal bands. Subcloning and sequencing of cDNA demonstrated 2 splicing consequences of this mutation: exon 4 skipping (i.e., exon 3 was spliced directly to exon 5), and a second isoform with intact exons 3, 4, and 5 but retaining 43 base pairs of intron 4 (including the T&gt;C point mutation in the splice donor site), activating a previously unrecognized cryptic splice site. Since exon 4 is 53 base pairs in length, both aberrant splicing consequences result in a frameshift and generation of a premature stop codon in the 5′ end of exon 5. Exon 6 is normally alternatively spliced, and this variation was retained, so there were two subforms of each aberrant spliceoform. Expression of ATRX mRNA in the patient’s monocytes, measured by RT-PCR probing the 3′ helicase domain, was 11.1% of healthy controls; in the granulocytes, expression was 15.3% of normal. CONCLUSION: Pre-mRNA splicing abnormalities such as that described here are increasingly recognized in association with human disease and particularly neoplasia, and may represent a potential therapeutic target. Point mutations in general are sought in myeloid disorders as they can provide proof of clonality in ambiguous cases and allow tracking of clonality with therapy and disease evolution. Germline 5′ ATRX splicing abnormalities are only rarely associated with inherited ATR-X syndrome, presumably because of the devastating consequences on gene expression and fetal development, but may be more common in acquired thalassemia. In future analyses of acquired HbH disease, when seeking ATRX mutations, splicing abnormalities should be routinely considered.

Abstract Background: It is widely accepted that an elevated serum endogenous erythropoietin (EPO) level in a patient presenting with erythrocytosis and a genuinely increased red cell mass makes a diagnosis of polycythemia vera (PV) extremely unlikely (Mossuz et al Haematologica2004:1194; Tefferi and Gilliland Mayo Clin Proc2005:947.) However, in the absence of a definitive molecular marker for PV, there is often uncertainty, especially when PV-associated clinical features are present. Here we describe 4 patients presenting with Budd-Chiari syndrome (BCS) and erythrocytosis who had EPO levels well above the normal reference range (Levy et al Hepatology1985:858), yet all had the newly described JAK2 V617F point mutation in myeloid cells, confirming PV. Methods and Results: A 30-year-old man presented in March 2005 with ascites, mild splenomegaly, abnormal liver tests, and erythrocytosis (hemoglobin (Hb) 18.6 g/dL) with a normal platelet count (187 x 10(9)/L). BCS was diagnosed by Doppler ultrasonography, transjugular venogram, and liver biopsy. While bone marrow examination showed hypercellularity and megakaryocyte clustering consistent with chronic myeloproliferative disorder (CMPD), his EPO level was elevated to 32 U/L (normal 4–16 U/L), suggesting secondary erythrocytosis. Arterial oxygen saturation, Hb oxygen-dissociation curves (P50), molecular studies of the globin loci, Hb chromatography, and CT scans of the body were all normal. Granulocyte DNA demonstrated the JAK2 V617F mutation (heterozygous/mixed clonality), absent in buccal cells, confirming PV. Karyotype was normal and BCR/ABL was not present. The patient was treated with phlebotomy, warfarin, and hydroxyurea. One month later, his EPO level was markedly reduced at 13 U/L. We hypothesized that since hepatocytes express low levels of EPO, a remnant of fetal erythropoiesis, acute necrosis from BCS could lead to transient EPO surge even in the presence of chronic EPO suppression from PV. We then reviewed clinical data from all 33 patients at our institution since 1995 with BCS that was ultimately believed to be associated with PV. We identified 13 such patients in whom endogenous EPO levels were measured. Most had a low EPO level at the time of diagnosis (range of &lt;1.5 to 9 U/L), but 3 of these patients were found to have an elevated EPO level. DNA was extracted from archival samples from these 3 patients; JAK2 V617F mutation was detected in all 3. Clinical presentations were as follows: Patient 1: 21-year-old woman with BCS and pulmonary embolus, Hb 15.9 mg/dL, EPO 28 U/L. Bone marrow biopsy was consistent with PV. Subsequent EPO level was 19U/L in the setting of progressive liver failure. Patient 2: 48-year-old woman with known PV receiving intermittent phlebotomy presented with BCS, Hb 17.2 mg/dL, EPO 25 U/L. Marrow was consistent with CMPD. Patient 3: 33-year-old woman presented with BCS, Hb 18.8 mg/dL, EPO 53 U/L. Bone marrow biopsy showed early CMPD. Conclusion: Elevated EPO levels do not exclude the diagnosis of PV, especially in the presence of BCS with hepatic necrosis. These cases highlight the continuing challenges in accurate PV and CMPD diagnosis and emphasize the importance of complementing clinical assessment with molecular genetic testing, including JAK2 mutation analysis.