Association of Broad-Based Genomic Sequencing With Survival Among Patients With Advanced Non-Small Cell Lung Cancer in the Community Oncology Setting.

Importance: Broad-based genomic sequencing is being used more frequently for patients with advanced non-small cell lung cancer (NSCLC). However, little is known about the association between broad-based genomic sequencing and treatment selection or survival among patients with advanced NSCLC in a community oncology setting. Objective: To compare clinical outcomes between patients with advanced NSCLC who received broad-based genomic sequencing vs a control group of patients who received routine testing for EGFR mutations and/or ALK rearrangements alone. Design, Setting, and Participants: Retrospective cohort study of patients with chart-confirmed advanced NSCLC between January 1, 2011, and July 31, 2016, and who received care at 1 of 191 oncology practices across the United States using the Flatiron Health Database. Patients were diagnosed with stage IIIB/IV or unresectable nonsquamous NSCLC who received at least 1 line of antineoplastic treatment. Exposures: Receipt of either broad-based genomic sequencing or routine testing (EGFR and/or ALK only). Broad-based genomic sequencing included any multigene panel sequencing assay examining more than 30 genes prior to third-line treatment. Main Outcomes and Measures: Primary outcomes were 12-month mortality and overall survival from the start of first-line treatment. Secondary outcomes included frequency of genetic alterations and treatments received. Results: Among 5688 individuals with advanced NSCLC (median age, 67 years [interquartile range, 41-85], 63.6% white, 80% with a history of smoking); 875 (15.4%) received broad-based genomic sequencing and 4813 (84.6%) received routine testing. Among patients who received broad-based genomic sequencing, 4.5% received targeted treatment based on testing results, 9.8% received routine EGFR/ALK targeted treatment, and 85.1% received no targeted treatment. Unadjusted mortality rates at 12 months were 49.2% for patients undergoing broad-based genomic sequencing and 35.9% for patients undergoing routine testing. Using an instrumental variable analysis, there was no significant association between broad-based genomic sequencing and 12-month mortality (predicted probability of death at 12 months, 41.1% for broad-based genomic sequencing vs 44.4% for routine testing; difference -3.6% [95% CI, -18.4% to 11.1%]; P = .63). The results were consistent in the propensity score-matched survival analysis (42.0% vs 45.1%; hazard ratio, 0.92 [95% CI, 0.73 to 1.11]; P = .40) vs unmatched cohort (hazard ratio, 0.69 [95% CI, 0.62 to 0.77]; log-rank P < .001). Conclusions and Relevance: Among patients with advanced non-small cell lung cancer receiving care in the community oncology setting, broad-based genomic sequencing directly informed treatment in a minority of patients and was not independently associated with better survival.

Human papillomavirus vaccination and cervical cytology in young minority women.

BACKGROUND: Continued surveillance of human papillomavirus (HPV) vaccination is necessary to identify clinical benefits, particularly given the low rate of vaccine uptake and completion and vaccination of many young women after sexual debut. We studied the effect of catch-up HPV vaccination on cervical cytology and HPV infection in sexually active, low-income and minority young women. METHODS: We conducted a cross-sectional study of 235 women aged 21 to 30 years undergoing routine cervical cytology testing. Demographic and behavioral characteristics were self-reported. HPV vaccination status was determined by self-report and verified with electronic medical records. Liquid-based cytology samples were tested for HPV genotypes. Adjusted prevalence ratios between HPV vaccination and (1) abnormal cervical cytology result and (2) HPV genotype were estimated. FINDINGS: At the time of the study, 96 women (41%) had received at least 1 HPV vaccination, 54 of whom had completed the series; 97% of women were vaccinated after sexual debut. Twenty-four (10%) women had an abnormal cervical cytology result. The prevalence of abnormal cytology was 65% lower in women who received at least 1 HPV vaccination versus unvaccinated women (adjusted prevalence ratio, 0.35; 95% confidence interval, 0.14-0.89). Among 232 women with genotype results, 46 (20%; 95% confidence interval, 15%-26%) had HPV infection detected. HPV types 16, 18, 45, 53, and 66 were most prevalent. CONCLUSIONS: The prevalence of abnormal cytology was lower in vaccinated versus unvaccinated women, despite receipt of vaccination after sexual debut. Continued assessment of HPV vaccine effectiveness before and after sexual debut on HPV infection and cervical dysplasia is needed.

Dosing clopidogrel based on CYP2C19 genotype and the effect on platelet reactivity in patients with stable cardiovascular disease.

CONTEXT: Variants in the CYP2C19 gene influence the pharmacologic and clinical response to the standard 75-mg daily maintenance dose of the antiplatelet drug clopidogrel. OBJECTIVE: To test whether higher doses (up to 300 mg daily) improve the response to clopidogrel in the setting of loss-of-function CYP2C19 genotypes. DESIGN, SETTING, AND PATIENTS: ELEVATE-TIMI 56 was a multicenter, randomized, double-blind trial that enrolled and genotyped 333 patients with cardiovascular disease across 32 sites from October 2010 until September 2011. INTERVENTIONS: Maintenance doses of clopidogrel for 4 treatment periods, each lasting approximately 14 days, based on genotype. In total, 247 noncarriers of a CYP2C19*2 loss-of-function allele were to receive 75 and 150 mg daily of clopidogrel (2 periods each), whereas 86 carriers (80 heterozygotes, 6 homozygotes) were to receive 75, 150, 225, and 300 mg daily. MAIN OUTCOME MEASURES: Platelet function test results (vasodilator-stimulated phosphoprotein [VASP] phosphorylation and VerifyNow P2Y(12) assays) and adverse events. RESULTS: With 75 mg daily, CYP2C19*2 heterozygotes had significantly higher on-treatment platelet reactivity than did noncarriers (VASP platelet reactivity index [PRI]: mean, 70.0%; 95% CI, 66.0%-74.0%, vs 57.5%; 95% CI, 55.1%-59.9%, and VerifyNow P2Y(12) reaction units [PRU]: mean, 225.6; 95% CI, 207.7-243.4, vs 163.6; 95% CI, 154.4-173.9; P < .001 for both comparisons). Among CYP2C19*2 heterozygotes, doses up to 300 mg daily significantly reduced platelet reactivity, with VASP PRI decreasing to 48.9% (95% CI, 44.6%-53.2%) and PRU to 127.5 (95% CI, 109.9-145.2) (P < .001 for trend across doses for both). Whereas 52% of CYP2C19*2 heterozygotes were nonresponders (≥230 PRU) with 75 mg of clopidogrel, only 10% were nonresponders with 225 or 300 mg (P < .001 for both). Clopidogrel, 225 mg daily, reduced platelet reactivity in CYP2C19*2 heterozygotes to levels achieved with standard clopidogrel, 75 mg, in noncarriers (mean ratios of platelet reactivity, VASP PRI, 0.92; 90% CI, 0.85-0.99, and PRU, 0.94; 90% CI, 0.84-1.04). In CYP2C19*2 homozygotes, even with 300 mg daily of clopidogrel, mean VASP PRI was 68.3% (95% CI, 44.9%-91.6%) and mean PRU, 287.0 (95% CI, 170.2-403.8). CONCLUSION: Among patients with stable cardiovascular disease, tripling the maintenance dose of clopidogrel to 225 mg daily in CYP2C19*2 heterozygotes achieved levels of platelet reactivity similar to that seen with the standard 75-mg dose in noncarriers; in contrast, for CYP2C19*2 homozygotes, doses as high as 300 mg daily did not result in comparable degrees of platelet inhibition. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT01235351.

Rchimerism: An R Package for Automated Chimerism Data Analysis.

A quantitative chimerism test monitors engraftment of donor hematopoietic stem cells or relapse of leukemias or lymphomas in hematopoietic stem cell transplantation patients. The most common method used for chimerism testing is PCR amplification of short tandem repeat loci, followed by capillary gel electrophoresis. Manual data analysis is tedious and time consuming, as it involves the selection of informative loci and the repetition of quantifying chimerism percentage for multiple loci from multiple cell types. It is also susceptible to human errors. Currently, there is no free software to fully automate chimerism data analysis. Rchimerism, an R shiny package, was developed to automatically pick informative loci, calculate chimerism percentage, and display the results through a user-friendly interface. The accuracy of the program was compared with manual calculation on 60 patient samples with 100% concordance. Compared with manual calculation, Rchimerism drastically reduces analysis time from 20 to 40 minutes for single donor transplantation samples and from 40 to 80 minutes for double donor transplantation samples to >1 minute. Rchimerism can be downloaded and used freely by noncommercial laboratories.

HHV8-positive, EBV-positive Hodgkin lymphoma-like large B-cell lymphoma and HHV8-positive intravascular large B-cell lymphoma.

Human herpesvirus type 8 (HHV8), also known as Kaposi's sarcoma-associated herpesvirus, is a human gamma herpesvirus that underlies the pathogenesis of Kaposi's sarcoma, primary effusion lymphoma and multicentric Castleman's disease. We recently encountered two cases of HHV8-positive large B-cell lymphoma with features not previously described. The first patient was a 61-year-old immunocompetent man with an enlarged cervical lymph node containing scattered large, bizarre cells in a reactive background of lymphocytes, plasma cells and scattered regressed follicles resembling those of hyaline-vascular Castleman's disease. The appearance suggested classical Hodgkin's lymphoma, but the large cells were negative for CD15, CD30, CD20 and CD3, and positive for MUM1/IRF4, EMA, HHV8, EBER and dim IgM lambda. The second patient was a 59-year-old HIV-positive man who died after several weeks of fever, night sweats, anemia, thrombocytopenia, hepatosplenomegaly and multiorgan failure. At autopsy an intravascular large B-cell lymphoma that was positive for MUM1/IRF4, HHV8 and IgM lambda, and negative for CD20 and EBER involved multiple organs, including lung, heart, kidney, liver and spleen. On the basis of the histologic features in these two cases, the presence of HHV8 was unexpected. These cases expand the spectrum of lymphoproliferative disorders that can be associated with HHV8.

Phase II study of temozolomide, thalidomide, and celecoxib for newly diagnosed glioblastoma in adults.

We conducted a phase II study of the combination of temozolomide and angiogenesis inhibitors for treating adult patients with newly diagnosed glioblastoma. Patients who had stable disease following standard radiation therapy received temozolomide for 5 days in 28-day cycles, in combination with daily thalidomide and celecoxib. Patients were treated until tumor progression or development of unacceptable toxicity. Four-month progression-free survival (PFS) from study enrollment was the primary end point, and overall survival (OS) was the secondary end point. In addition, we sought to correlate response with O(6)-methylguanine-DNA methyltransferase promoter methylation status and serum levels of angiogenic peptides. Fifty patients with glioblastoma were enrolled (18 women, 32 men). Median age was 54 years (range, 29-78) and median KPS score was 90 (range, 70-100). From study enrollment, median PFS was 5.9 months (95% confidence interval [CI]: 4.2-8.0) and 4-month PFS was 63% (95% CI: 46%-75%). Median OS was 12.6 months (95% CI: 8.5-16.4) and 1-year OS was 47%. Of the 47 patients evaluable for best response, none had a complete response, five (11%) had partial response, four (9%) had minor response, 22 (47%) had stable disease, and 16 (34%) had progressive disease. Analysis of serial serum samples obtained from 47 patients for four angiogenic peptides failed to show a significant correlation with response or survival for three of the peptides; higher vascular endothelial growth factor levels showed a trend toward correlation with decreased OS (p=0.07) and PFS (p=0.09). The addition of celecoxib and thalidomide to adjuvant temozolomide was well tolerated but did not meet the primary end point of improvement of 4-month PFS from study enrollment.

Gender differences in research grant applications and funding outcomes for medical school faculty.

PURPOSE: To evaluate whether there were differences in acquisition of research grant support between male and female faculty at eight Harvard Medical School-affiliated institutions. METHODS: Data were obtained from the participating institutions on all research grant applications submitted by full-time faculty from 2001 through 2003. Data were analyzed by gender and faculty rank of applicant, source of support (federal or nonfederal), funding outcome, amount of funding requested, and amount of funding awarded. RESULTS: Data on 6319 grant applications submitted by 2480 faculty applicants were analyzed. Women represented 29% of investigators and submitted 26% of all grant requests. There were significant gender differences in the mean number of submissions per applicant (women 2.3, men 2.7), success rate (women 41%, men 45%), number of years requested (women 3.1, men 3.4), median annual amount requested (women $115,325, men $150,000), mean number of years awarded (women 2.9, men 3.2), and median annual amount awarded (women $98,094, men $125,000). After controlling for academic rank, grant success rates were not significantly different between women and men, although submission rates by women were significantly lower at the lowest faculty rank. Although there was no difference in the proportion of money awarded to money requested, women were awarded significantly less money than men at the ranks of instructor and associate professor. More men than women applied to the National Institutes of Health, which awarded higher dollar amounts than other funding sources. CONCLUSIONS: Gender disparity in grant funding is largely explained by gender disparities in academic rank. Controlling for rank, women and men were equally successful in acquiring grants. However, gender differences in grant application behavior at lower academic ranks also contribute to gender disparity in grant funding for medical science.

A novel method for interpretation of T-cell receptor gamma gene rearrangement assay by capillary gel electrophoresis based on normal distribution.

T-cell receptor gamma (TRG) gene rearrangement status is useful for the differential diagnosis of T-cell lesions. The BIOMED-2 protocol that uses two sets of Jgamma and four sets of Vgamma primers in a multiplex, two-tube reaction followed by capillary gel electrophoresis is emerging as a standard assay for this application. Here, we report a computer-aided method to evaluate the significance of a peak in this TRG clonality assay. A best-fit normal distribution (ND) curve and the chi(2) error for each peak are used to determine whether a peak is significantly taller than the background (cutoff for Vgamma(1-8) is 1). Eighty clinical samples that have been previously analyzed by a GC-clamped primer polymerase chain reaction/denaturing gradient gel electrophoresis assay were reanalyzed with the BIOMED-2 assay and scored by the ND method and four previously published methods: relative peak height (RPH), relative peak ratio (RPR), height ratio (HR), and peak height ratio (Rn). A greater than 90% concordance rate was observed between RPH and ND analysis, whereas RPR, Rn, and HR had a lower threshold to call a peak positive. The advantage of the ND method is that it is more objective, reproducible, and can be automated.

HHV-8+, EBV+ multicentric plasmablastic microlymphoma in an HIV+ Man: the spectrum of HHV-8+ lymphoproliferative disorders expands.

Human herpesvirus-8 (HHV-8) is associated with several distinct lymphoproliferative disorders: primary effusion lymphoma, multicentric Castleman disease (MCD), MCD-associated plasmablastic lymphoma and HHV-8+, Epstein-Barr virus (EBV)+ germinotropic lymphoproliferative disorder. We report the case of a human immunodeficiency virus (HIV)+ male with fever, generalized lymphadenopathy, and splenomegaly. Two peripheral lymph nodes were excised and showed features of MCD and a prominent proliferation of HHV-8+, EBV+, CD20, CD138, MUM1+, lambda dim+, Ig heavy chain plasmablasts and immunoblasts replacing some follicles. Subsequently, a splenectomy and biopsy of retroperitoneal lymph nodes were performed; the retroperitoneal and splenic hilar lymph nodes showed changes similar to those in the peripheral lymph nodes while the markedly enlarged spleen showed replacement of occasional white pulp by the HHV-8+, EBV+ large cells. The histologic features and coinfection by EBV and HHV-8 suggested a diagnosis of HHV-8+ germinotropic lymphoproliferative disorder. However, the occurrence in an HIV+ individual, the background of MCD, the widespread anatomic distribution and the aggressive clinical course tended to exclude germinotropic lymphoproliferative disorder, and to favor multifocal plasmablastic microlymphoma. The patient died shortly after surgery; postmortem examination showed progression to overt lymphoma. The marrow showed extensive hemophagocytosis, consistent with development of a hemophagocytic syndrome. This unique case has clinical features compatible with a MCD-associated plasmablastic lymphoproliferative disorder, with pathologic features intermediate between HHV-8+ plasmablastic microlymphoma, and HHV-8+ germinotropic lymphoproliferative disorder, although in contrast to both of these, in our case, light chain expression was dim and heavy chain was not detected.

Epidermal growth factor receptor mutation testing in the care of lung cancer patients.

As the literature about epidermal growth factor receptor (EGFR) mutations grows and screening for mutations becomes increasingly integrated into clinical care, it is important to examine how best to do somatic mutational analyses and how best to use the test results in clinical decision making. We began offering mutation screening by comprehensive direct sequence analysis of exons 18 to 24 of the tyrosine kinase domain of EGFR in August 2004 as part of clinical cancer care and protocol therapy at our institutions. All identified potential mutations are confirmed with three to five independent PCRs of the original genomic DNA sample and, if not previously noted in the literature, are compared with the patient's germ-line DNA to ensure the finding is somatic. We formally analyzed the first 100 patients to undergo EGFR sequence analysis and found that testing was feasible and significantly affected the treatment of patients with non-small cell lung cancer (NSCLC). Patients harboring EGFR mutations were significantly more likely to receive recommendations for therapy with EGFR tyrosine kinase inhibitors (i.e., gefitinib or erlotinib) than patients without mutations. However, negative EGFR test results did not prevent physicians from administering these agents to selected patients. Ideally, a standardized technique for mutation testing could be developed, with demonstrated reproducibility and validity. Clinical trials incorporating molecular diagnostics are ongoing to assess the efficacy of EGFR tyrosine kinase inhibitors as first-line therapy for metastatic NSCLC and as adjuvant therapy for early-stage resected NSCLC. It is likely that mutation testing and other molecular analyses will be most useful in these two clinical situations.

Molecular Analysis of Gene Rearrangements and Mutations in Acute Leukemias and Myeloid Neoplasms.

A subset of acute leukemias and other myeloid neoplasms contains specific genetic alterations, many of which are associated with unique clinical and pathologic features. These alterations include chromosomal rearrangements leading to oncogenic fusion proteins or alteration of gene expression by juxtaposing oncogenes to enhancer elements, as well as mutations leading to aberrant activation of a variety of proteins critical to hematopoietic progenitor cell proliferation and differentiation. Molecular analysis is central to diagnosis and clinical management of leukemias, permitting genetic confirmation of a clinical and histologic impression, providing prognostic and predictive information, and facilitating detection of minimal residual disease. This unit will outline approaches to the molecular diagnosis of the most frequent and clinically relevant genetic alterations in acute leukemias and myeloid neoplasms. © 2017 by John Wiley & Sons, Inc.

Molecular Analysis of Genetic Markers for Non-Hodgkin Lymphomas.

Molecular analysis complements the clinical and histopathologic tools used to diagnose and subclassify hematologic malignancies. The presence of clonal antigen-receptor gene rearrangements can help to confirm the diagnosis of a B or T cell lymphoma and can serve as a fingerprint of that neoplasm to be used in identifying concurrent disease at disparate sites or recurrence at future time points. Certain lymphoid malignancies harbor a characteristic chromosomal translocation, a finding that may have significant implications for an individual's prognosis or response to therapy. The polymerase chain reaction (PCR) is typically used to detect antigen-receptor gene rearrangements as well as specific translocations that can be supplemented by fluorescence in situ hybridization (FISH) and karyotype analysis. © 2017 by John Wiley & Sons, Inc.

Immunoglobulin gene rearrangement analysis in cerebrospinal fluid of patients with lymphoproliferative processes.

OBJECTIVE: To determine the sensitivity and specificity of clonal immunoglobulin heavy chain gene rearrangement (IGHR) analysis in the distinction of benign and malignant lymphoproliferative diseases. METHODS: A retrospective analysis was conducted of patients in whom a malignant lymphoproliferative process was suspected. Cells of CSF samples were collected by centrifugation, resuspended in 100 microl of the supernatant and boiled. A 10 microl aliquot of this lysate served as template for semi-nested polymerase chain reaction using variable and joining region consensus primers. PCR products were analyzed by polyacrylamide gel electrophoresis. Cytopathological diagnosis and flow cytometry results were recorded. Sensitivity and specificity of IGHR analysis, cytopathology and flow cytometry were calculated. RESULTS: Eleven patients (12 specimens) had involvement of leptomeninges at the time of lumbar puncture. Another 25 cases (27 specimens) had normal CSF findings or were diagnosed with benign lymphoproliferative conditions. Sensitivity of CSF cytopathology, flow cytometry and IGHR analysis were 0.27 [95% confidence interval 0.06, 0.61], 0.1 [0.003, 0.45] and 0.58 [0.28, 0.85]. Specificity was 1 [0.86, 1], 0.95 [0.77, 1.0] and 0.85 [0.66, 0.96]. INTERPRETATION: IGHR analysis appears to be a useful addition to morphological and flow cytometry analysis of cerebrospinal fluid in the evaluation of CNS lymphoproliferative processes.

Development and clinical application of an integrative genomic approach to personalized cancer therapy.

BACKGROUND: Personalized therapy provides the best outcome of cancer care and its implementation in the clinic has been greatly facilitated by recent convergence of enormous progress in basic cancer research, rapid advancement of new tumor profiling technologies, and an expanding compendium of targeted cancer therapeutics. METHODS: We developed a personalized cancer therapy (PCT) program in a clinical setting, using an integrative genomics approach to fully characterize the complexity of each tumor. We carried out whole exome sequencing (WES) and single-nucleotide polymorphism (SNP) microarray genotyping on DNA from tumor and patient-matched normal specimens, as well as RNA sequencing (RNA-Seq) on available frozen specimens, to identify somatic (tumor-specific) mutations, copy number alterations (CNAs), gene expression changes, gene fusions, and also germline variants. To provide high sensitivity in known cancer mutation hotspots, Ion AmpliSeq Cancer Hotspot Panel v2 (CHPv2) was also employed. We integrated the resulting data with cancer knowledge bases and developed a specific workflow for each cancer type to improve interpretation of genomic data. RESULTS: We returned genomics findings to 46 patients and their physicians describing somatic alterations and predicting drug response, toxicity, and prognosis. Mean 17.3 cancer-relevant somatic mutations per patient were identified, 13.3-fold, 6.9-fold, and 4.7-fold more than could have been detected using CHPv2, Oncomine Cancer Panel (OCP), and FoundationOne, respectively. Our approach delineated the underlying genetic drivers at the pathway level and provided meaningful predictions of therapeutic efficacy and toxicity. Actionable alterations were found in 91 % of patients (mean 4.9 per patient, including somatic mutations, copy number alterations, gene expression alterations, and germline variants), a 7.5-fold, 2.0-fold, and 1.9-fold increase over what could have been uncovered by CHPv2, OCP, and FoundationOne, respectively. The findings altered the course of treatment in four cases. CONCLUSIONS: These results show that a comprehensive, integrative genomic approach as outlined above significantly enhanced genomics-based PCT strategies.

Follicular lymphoma with bilateral testicular and epididymal involvement: case report and review of the literature.

Testicular involvement with indolent lymphoma is extremely rare, particularly in the absence of transformation to an aggressive histology. We report a case of a 64-year-old man who presented with cervical lymphadenopathy. Staging CT scans revealed extensive lymphadenopathy as well as bilateral testicular and epididymal masses. Histologic examination of lymph node, bone marrow, and testicular/epididymal biopsies revealed involvement with grade I follicular lymphoma. The patient was started on chemotherapy with cyclophosphamide, vincristine, prednisone, and rituximab in addition to intrathecal methotrexate and testicular radiation. He is now 6 months into therapy and responding well. A review of the literature demonstrated this to be the first confirmed case of testicular and epididymal involvement with grade I follicular lymphoma.

Extended follow-up of patients treated with imatinib mesylate (gleevec) for chronic myelogenous leukemia relapse after allogeneic transplantation: durable cytogenetic remission and conversion to complete donor chimerism without graft-versus-host disease.

PURPOSE: Over the last several years, donor lymphocyte infusions have become the standard approach for patients with chronic myelogenous leukemia (CML) who relapse after allogeneic stem cell transplantation (SCT). Recent reports indicate that imatinib mesylate (Gleevec) can induce remissions in these patients as well. Less is known about the extent and durability of these responses. EXPERIMENTAL DESIGN: We studied 15 patients treated with imatinib for recurrent CML after SCT, 10 patients with stable phase CML (SP-CML), 1 with accelerated phase (AP-CML), and 4 with blast phase (BP-CML). The dose of imatinib was 600 mg (n = 10) or 400 mg (n = 5) daily. Patients were followed for hematological, cytogenetic, and molecular response. A susbset of responders was followed for changes in donor-derived hematopoietic chimerism. RESULTS: Of the 10 patients with SP-CML, all achieved a hematological response. Within 3 months, five of these patients had achieved a complete cytogenetic response (CCR). By six months, 9 of 10 patients had achieved CCR. The BCR-ABL transcript could not be detected by reverse transcription-PCR in 7 of these 9 patients. Patients who achieved CCR showed evidence of conversion to complete donor chimerism. No patient developed graft-versus-host disease. With a median follow up of 25 months, all patients are alive and 9 of 10 patients remain in CCR. Only one of the 5 patients with AP/BP-CML achieved a complete cytogenetic response. CONCLUSIONS: We find that imatinib is well tolerated in patients with SP-CML who relapse after SCT. Responses were rapid, durable, and associated with conversion to full donor chimerism without graft-versus-host disease. Imantinib was far less effective in patients who relapsed with AP/BP-CML. Imatinib should be evaluated as either an alternative or as an adjunct to donor lymphocyte infusions for patients with SP-CML who relapse after SCT.

Advocacy and perfect storms: an interview with Janina Longtine, President of the AMP.

Interview with Professor Janina Longtine PhD, by Claire Raison (Commissioning Editor) Janina Longtine is a professor of pathology at the Mount Sinai Hospital (New York, NY, USA) and currently serves as the President of the Association for Molecular Pathology (Bethesda, MD, USA). Prior to this, Professor Longtine was with the Brigham and Women's Hospital (Boston, MA, USA) for 30 years, working on understanding the molecular pathology of cancer and developing diagnostic procedures for cancers. In this interview article to mark 15 years of Expert Review of Molecular Diagnostics, she reflects on her experience as a molecular pathologist in US hospitals, and warns of the regulatory and reimbursement challenges that face laboratory-developed procedures today.

Filter Paper-based Nucleic Acid Storage in High-throughput Solid Tumor Genotyping.

Molecular testing of tumors from formalin-fixed paraffin-embedded (FFPE) tissue blocks is central to clinical practice; however, it requires histology support and increases test turnaround time. Prospective fresh frozen tissue collection requires special handling, additional storage space, and may not be feasible for small specimens. Filter paper-based collection of tumor DNA reduces the need for histology support, requires little storage space, and preserves high-quality nucleic acid. We investigated the performance of tumor smears on filter paper in solid tumor genotyping, as compared with paired FFPE samples. Whatman FTA Micro Card (FTA preps) smears were prepared from 21 fresh tumor samples. A corresponding cytology smear was used to assess tumor cellularity and necrosis. DNA was isolated from FTA preps and FFPE core samples using automated methods and quantified using SYBR green dsDNA detection. Samples were genotyped for 471 mutations on a mass spectrophotometry-based platform (Sequenom). DNA concentrations from FTA preps and FFPE correlated for untreated carcinomas but not for mesenchymal tumors (Spearman σ=0.39 and σ=-0.1, respectively). Average DNA concentrations were lower from FTA preps as compared with FFPE, but DNA quality was higher with less fragmentation. Seventy-six percent of FTA preps and 86% of FFPE samples generated adequate DNA for genotyping. FTA preps tended to perform poorly for collection of DNA from pretreated carcinomas and mesenchymal neoplasms. Of the 16 paired DNA samples that were genotyped, 15 (94%) gave entirely concordant results. Filter paper-based sample preservation is a feasible alternative to FFPE for use in automated, high-throughput genotyping of carcinomas.

Warfarin dosing and cytochrome P450 2C9 polymorphisms.

Two cytochrome P450 2C9 (CYP2C9) polymorphisms, CYP2C9*2 and *3, metabolize warfarin inefficiently. We assessed the extent to which these polymorphisms explain very low warfarin dose requirements and hemorrhagic complications after excluding non-genetic determinants of warfarin dosing. In this retrospective observational study, 73 patients with stable warfarin doses for > or =1 month and International Normalized Ratios (INR) of 2.0-3.0 were enrolled from our Anticoagulation Clinic. Seventeen patients required < or =2 mg (low-dose), 41 required 4-6 mg (moderate-dose), and 15 required > or =10 mg (high-dose) of daily warfarin. CYP2C9 genotyping was assessed by PCR amplification and restriction enzyme digestion analysis of DNA isolated from circulating leukocytes. The CYP2C9 polymorphisms independently predicted low warfarin requirements after adjusting for Body Mass Index, age, acetaminophen use, and race (OR 24.80; 95% CI 3.83-160.78). At least one polymorphism was present in every patient requiring < or =1.5 mg of daily warfarin, and 88%, 37%, and 7% of the low-, moderate-, and high-dose groups, respectively. All homozygotes and compound-heterozygotes for the variant alleles were in the low-dose group. Rates of excessive (INR>6.0) anticoagulation (and bleeding) were 4.5 (6.0), 7.9 (7.9), and 14.7 (0) per 100 patient-years in the wild-types, heterozygotes, and compound heterozygotes/homozygotes, respectively. In conclusion, CYP2C9*2 or *3 compound heterozygotes and homozygotes have low warfarin requirements even after excluding liver disease, excessive alcohol or acetaminophen consumption, low body weight, advancing age, and drug interactions. These polymorphisms increase the rate of excessive anticoagulation, but this risk does not appear to be associated with higher bleeding rates when anticoagulation status is closely monitored.