Disease Monitoring In Patients Research Articles

Variation in medical practice: getting the balance right

Contemporary clinical practice is characterized by its complexity as the volume and diversity of medical interventions, whether they are drugs, procedures or diagnostic tests, are increasing and threaten to overwhelm our capacity to deliver patient-centred care. Consider some statistics: the average American citizen can expect to undergo seven operations in their lifetime, 10% will undergo an MRI scan annually (three times higher than the rate in neighbouring Canada) and 50% of Medicare beneficiaries are prescribed five or more medications. In Ireland, one-fifth of the whole population aged over 70 years are taking long-term Proton Pump Inhibitor (PPI) therapy.1–3 The consequences of this phenomenon for patients in terms of benefit (increase quantity and quality of life) versus harm (medicalization of a person, side effects of therapies and costs to the health service budget) give rise to questions concerning the epidemiology of health care utilization and how it differs between and within countries. Seminal work carried out by John Wennberg, a health services researcher and epidemiologist who developed the Dartmouth Atlas Health Project (www.dartmouthatlas. org), has produced an emerging science that examines variation in medical practice and raises important questions about what constitutes ‘appropriate’ health care. This editorial outlines the taxonomy of medical practice variation with clinical examples showing how it relates to family medicine. Medical practice variation may be grouped into three categories each with different implications for patients, clinicians and policy makers.4 Effective care is defined as interventions for which the benefits far outweigh the risks; in this case the ‘right’ rate of treatment is 100% of patients defined by evidence-based guidelines to be in need, and unwarranted variation is generally a matter of underuse or ineffective care delivery. An example of this form of practice includes the prescription of statins for the secondary prevention of cardiovascular disease.5 Preference sensitive care is when more than one generally accepted treatment option is available, such as watchful waiting, medical treatment or surgery. In this situation, the right rate should depend on informed patient choice, but treatment rates can vary because of differences in professional opinion and their subsequent impact on patient decision making. An example of preference sensitive care would be the management of menorrhagia, where informed patient preference may drive treatment rates.6 There can be an overlap between effective care and preference sensitive care, particularly in the area of preventive medicine. For instance in stroke prevention in patients with non-valvular atrial fibrillation, patients judged to have an intermediate risk, choosing between aspirin and warfarin is dependent on individual preference in terms of the risk and benefits of these alternative drug treatments.7 Supply sensitive care comprises the frequency of clinical activities such as doctor visits, diagnostic tests and hospital admissions, which relate to the capacity of the local health care system. Increased availability of a health care resource will be associated with increased utilization. Supply of more specialists will result in more visits to specialists, increased numbers of hospital beds per capita will result in higher rates of hospital admissions and greater availability of imaging will result in higher imaging rates.4 In the USA, supply sensitive care is thought to account for up to 50% of Medicare health care spending. Supply sensitive care, and how it varies, remains a major consideration in terms of the organization and delivery of cost-effective chronic disease management. This lack of evidence and consensus in terms of the most appropriate form of care delivery for chronic disease management results in practice variation in terms of the number and timing of return visits, frequency of diagnostic testing and disease monitoring in patients. In the USA, regions with high rates of chronic disease care utilization do not have better patient outcomes as measured by mortality and indicators of the quality of care.8 This variation suggests that the problem is overutilization of this category of care in some regions. Evidence about supply sensitive care is emerging from other forms of health care systems that have different models of funding, for example, the UK’s National Health Service Health Atlas (www. rightcare.nhs.uk/) and the social insurance system of the Netherlands (www.healthcareperformance.nl). A relatively recent development has seen health care organizations drawing attention to ineffective care—care which lacks an evidence base and should not be part of routine clinical practice. The process of highlighting this form of clinical practices is gaining Family Practice 2012; 29:501–502 doi:10.1093/fampra/cms061

Clinical outcome and monitoring of minimal residual disease in patients with acute lymphoblastic leukemia expressing the MLL/ENL fusion gene

We analyzed 12 MLL/ENL positive ALL patients consecutively diagnosed between 1999 and 2009. The MLL/ENL fusion was identified in 4/150 (2.6%), 8/993 (0.8%), and 0/70 of pediatric, adult, and elderly patients, respectively. Eight patients had a WBC count >50 × 10(9) /L. Ten cases had an evaluable immunophenotyping. A B or T precursor ALL occurred in 7 and 3 patients, respectively. Eleven/12 patients (92%) achieved CR. At 48 months, overall survival and event-free survival rates were 73.3% and 67%, respectively. At CR, a parallel RT-PCR evaluation of the MLL/ENL expression was available in 5 cases. Of these latter, 2 tested MLL/ENL-negative and 3 positive. The minimal residual disease molecular monitoring showed that MLL/ENL status did not correlate with outcome. In fact, all the 2 PCR-negative and 1 of the 3 PCR-positive cases relapsed. Further, a MLL/ENL expression, not preceding a relapse, was detected several times during the follow-up of five long-survivors. In conclusion, also in adults, the MLL/ENL fusion identifies a rare leukemic entity with a favorable prognosis. The observed inconsistency between the clinical cure and the presence of detectable MLL/ENL transcript suggests the existence of a MLL/ENL-expressing "preleukemia" stem cells, similar to what demonstrated for the AML1/ETO-positive leukemia setting.

Development of an ELISA for sensitive and specific detection of IgA autoantibodies against BP180 in pemphigoid diseases

BackgroundPemphigoids are rare diseases associated with IgG, IgE and IgA autoantibodies against collagen XVII/BP180. An entity of the pemphigoid group is the lamina lucida-type of linear IgA disease (IgA pemphigoid) characterized by IgA autoantibodies against BP180. While for the detection of IgG and IgE autoantibodies specific to collagen XVII several ELISA systems have been established, no quantitative immunoassay has been yet developed for IgA autoantibodies. Therefore, the aim of the present study was to develop an ELISA to detect IgA autoantibodies against collagen XVII in the sera of patients with pemphigoids.MethodsWe expressed a soluble recombinant form of the collagen XVII ectodomain in mammalian cells. Reactivity of IgA autoantibodies from patients with IgA pemphigoid was assessed by immunofluorescence microscopy and immunoblot analysis. ELISA test conditions were determined by chessboard titration experiments. The sensitivity, specificity and the cut-off were determined by receiver-operating characteristics analysis.ResultsThe optimized assay was carried out using sera from patients with IgA pemphigoid (n = 30) and healthy donors (n = 105). By receiver operating characteristics (ROC) analysis, an area under the curve of 0.993 was calculated, indicating an excellent discriminatory capacity. Thus, a sensitivity and specificity of 83.3% and 100%, respectively, was determined for a cut-off point of 0.48. As additional control groups, sera from patients with bullous pemphigoid (n = 31) and dermatitis herpetiformis (n = 50), a disease associated with IgA autoantibodies against epidermal transglutaminase, were tested. In 26% of bullous pemphigoid patients, IgA autoantibodies recognized the ectodomain of collagen XVII. One of 50 (2%) of dermatitis herpetiformis patients sera slightly topped the cut-off value.ConclusionsWe developed the first ELISA for the specific and sensitive detection of serum IgA autoantibodies specific to collagen XVII in patients with pemphigoids. This immunoassay should prove a useful tool for clinical and translational research and should essentially improve the diagnosis and disease monitoring of patients with IgA pemphigoid. Moreover, our findings strongly suggest that IgA pemphigoid and IgG bullous pemphigoid represent two ends of the clinical spectrum of an immunological loss of tolerance against components of hemidesmosomes, which is mediated by both IgG and IgA autoantibodies.

Abstract 310: BCR-ABL1 transcript detection in patients with Philadelphia-positive Acute Lymphoblastic Leukemia (ALL) using a new and high sensitive nanofluidic method

Abstract Background: Detection of residual leukemic cells by measuring BCR-ABL1 transcript level with assays based on quantitative polymerase chain reaction (qPCR) is necessary in the monitoring of minimal residual disease in patients with BCR-ABL1 positive ALL. Aim: To investigate the efficacy of a high sensitive method based on nanofluidic platform (Fluidigm Corporation, South San Francisco, CA) to detect and quantify residual and rare BCR-ABL1 copies, we compared results obtained by conventional qPCR with those obtained by the nanofluidic approach. Patients and methods: We analyzed 60 BCR-ABL1 positive ALL samples expressing p190 (42) or p210 (18) isoform, first by TaqMan absolute qPCR (Applied Biosystems 7900HT Fast Real-Time PCR System). BCR-ABL1 target gene and ABL1 control gene copies were derived by the interpolation of cycle threshold values to the appropriate standard curve obtained using serial plasmid dilutions containing known BCR-ABL1 or ABL1 gene copies. Results: Samples resulted in complete (22) or major (38) molecular response (BCR-ABL1/ABL1 ratio ≪ 0.001 or < 0.1, respectively; 3,8 BCR-ABL1 mean copy number copies). Subsequently we reanalyzed the samples using the 12.765 Digital array (Fluidigm) and the integrated BioMark Digital PCR Analysis software (Fluidigm). The nanofluidic biochip consists in twelve panels, each containing 765 individual reaction chambers where samples are portioned prior to qPCR. As fluorescent signal is produced only in chambers containing copies of the target sequence, digital array provides an absolute quantification by counting the number of positive reactions. Sensitivity and reproducibility of the assay were assessed using six serial dilutions of plasmids (Ipsogen) expressing known BCR-ABL1 p190 transcript copy number (10000; 1000; 100; 50; 10; 1 copies). A 2 µl volume of input cDNA was loaded and two panel for each dilution were used. Results showed a detection rate until a copy of target sequence and a pairing significantly effective between replicates. We then analyzed duplicates of BCR-ABL1 ALL samples: digital array resulted positive in 21 of major molecular response samples (2,13 BCR-ABL1 mean copy number copies) and in any complete molecular response samples. In conclusion, the Fluidigm nanofluidic platform provides a high sensitive assay, able to detect until a single copy of BCR-ABL1 transcript, and it could provide an accurate monitoring method for BCR-ABL1-positive ALL CR patients. Supported by European LeukemiaNet, AIL, AIRC, FIRB 2006, PRIN 2008, Ateneo RFO grants, Project of integreted program (PIO), Programma di Ricerca Regione – Università 2007 – 2009. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 310. doi:10.1158/1538-7445.AM2011-310

Development of a dual‐color, double fusion FISH assay to detect RPN1/EVI1 gene fusion associated with inv(3), t(3;3), and ins(3;3) in patients with myelodysplasia and acute myeloid leukemia

Approximately 2-3% of adult patients with acute myeloid leukemia harbor a rearrangement of RPN1 (at 3q21) and EVI1 (at 3q26.2) as inv(3)(q21q26.2), t(3;3)(q21;q26.2), or ins(3;3)(q26.2;q21q26.2). The most recent World Health Organization (WHO) classification has designated AML with inv(3) or t(3;3) and associated RPN1/EVI1 fusion, as a distinct AML subgroup associated with an unfavorable prognosis. We have created a dual color, double fusion fluorescence in situ hybridization (D-FISH) assay to detect fusion of the RPN1 and EVI1 genes. A blinded investigation was performed using 30 normal bone marrow samples and 51 bone marrow samples from 17 patients with inv(3)(q21q26.2), 11 patients with t(3;3)(q21;q26.2), and one patient with ins(3;3)(q26.2;q21q26.2) previously defined by chromosome analysis. The unblinded results indicated abnormal RPN1/EVI1 fusion results in 30 (97%) of 31 samples from the inv(3)(q21q26.2) group including seven bone marrow samples for which chromosome analysis was unsuccessful or failed to detect an inv(3)(q21q26.2). Abnormal FISH results were detected in 14 (88%) of 16 samples with t(3;3)(q21;q26.2) and in the sole sample with an ins(3;3)(q26.2;q21q26.2). All 30 negative controls were normal and were used to establish a normal cutoff of 0.6% for the typical abnormal D-FISH signal pattern. Overall, this D-FISH assay was more accurate than chromosome analysis and based on the normal cutoff of 0.6%, this assay can be used for minimal residual disease detection and disease monitoring in patients with RPN1/EVI1 fusion.

RNA extracted from blood samples with a rapid automated procedure is fit for molecular diagnosis or minimal residual disease monitoring in patients with a variety of malignant blood disorders.

Scientific studies in oncology, cancer diagnosis, and monitoring tumor response to therapeutics currently rely on a growing number of clinico-pathological information. These often include molecular analyses. The quality of these analyses depends on both pre-analytical and analytical information and often includes the extraction of DNA and/or RNA from human tissues and cells. The quality and quantity of obtained nucleic acids are of utmost importance. The use of automated techniques presents several advantages over manual techniques, such as reducing technical time and thus cost, and facilitating standardization. The purpose of this study was to validate an automated technique for RNA extraction from cells of patients treated for various malignant blood diseases. A well-established manual technique was compared to an automated technique, in order to extract RNA from blood samples drawn for the molecular diagnosis of a variety of leukemic diseases or monitoring of minimal residual disease. The quality of the RNA was evaluated by real-time quantitative RT-PCR (RQ-PCR) analyses of the Abelson gene transcript. The results show that both techniques produce RNA with comparable quality and quantity, thus suggesting that an automated technique can be substituted for the reference and manual technique used in the daily routine of a molecular pathology laboratory involved in minimal residual disease monitoring. Increased costs of reagents and disposables used for automated techniques can be compensated by a decrease in human resource.

Lung cancer susceptibility and prognosis associated with polymorphisms in the nonhomologous end‐joining pathway genes

Nonsmall cell lung cancer (NSCLC) frequently exhibits genomic instability, such as high fractional allelic loss (FAL). Genomic instability may result from unrepaired or misrepaired double-strand breaks (DSBs). The authors of this report postulated that polymorphisms in genes of the nonhomologous end-joining (NHEJ) pathway, which is the major DSB repair pathway in mammalian cells, may modulate lung cancer susceptibility and prognosis. Patients with NSCLC (n = 152) and a group of appropriate age-matched and sex-matched controls (n = 162) were subjected to genotype analysis of the NHEJ pathway genes x-ray repair complementing defective repair in Chinese hamster cells 6 (Ku70) (reference single nucleotide polymorphism number [rs] 2267437), x-ray repair complementing defective repair in Chinese hamster cells 5 (Ku80) (rs3835), x-ray repair complementing defective repair in Chinese hamster cells 4 (XRCC4) (rs1805377), and DNA ligase IV (LIG4) (rs1805388). The gene-gene interaction (joint effect), genotype-environmental (ie, smoking) correlation, and genotype-phenotype (ie, FAL) correlation were examined. The Kaplan-Meier method and log-rank tests were used to assess the prognostic effect. There was a significant association between the XRCC4 and LIG4 genotypes with NSCLC risk in an analysis of individual polymorphism associations, and the risk of NSCLC increased further in a combined analysis of multiple polymorphisms (adjusted odds ratio [OR], 8.74). The patients who had a homozygous variant guanine/guanine genotype of the XRCC4 gene had a poorer prognosis compared with other patients (P = .015). There was a significant difference between the patient smokers and controls for XRCC4 (adjusted OR, 2.67) and LIG4 (adjusted OR, 2.04). In addition, polymorphisms in XRCC4 and LIG4 were linked significantly with patients who had high FAL (adjusted OR, 2.03-3.84). To the authors' knowledge, this is the first nested case-control study to demonstrate a significant association between the polymorphisms of genes in the NHEJ pathway and lung cancer susceptibility and prognosis. The results may be useful for risk assessment and disease monitoring of patients with NSCLC.

Monitoring of minimal residual disease in patients with core binding factor acute myeloid leukemia and the impact of C-KIT, FLT3, and JAK2 mutations on clinical outcome

Mutational analysis of C-KIT, fms-like tyrosine kinase 3 (FLT3), and JAK2 genes was performed in 60 patients with core binding factor acute myeloid leukemia (CBF-AML). Patients reaching molecular remission had lower incidence of relapse and better overall survival (OS) than those not achieving molecular remission (p = 0.008 and 0.044, respectively). The overall incidence of C-KIT mutations was 33.3%, FLT3/internal tandem duplication (ITD) 6.6%, FLT3D835 10.0% and JAK2V617F mutations 3.3%. C-KIT mutations did not predict for clinical/molecular relapse (p = 0.33). OS of patients with C-KIT mutations was identical to patients without them when all patients with CBF-AML were analyzed together (p = 0.58). When AML1/ETO-positive patients were evaluated separately, OS in C-KIT-mutated patients was slightly inferior to unmutated ones (p = 0.14). Patients with CBF-AML with a mutated C-KIT gene were also more prone to extramedullary disease (p = 0.08). Of six patients harboring various FLT3D835 mutations, four (66.7%) relapsed, whereas among 43 cases without these mutations, 16 relapses (37%) were observed (p = 0.08). Our results on minimal residual disease, C-KIT, and FLT3/ITDs are in line with previous studies. Surprisingly, a possible role for FLT3D835 mutations was noted in addition. These results need validation in even larger patient cohorts than ours. For routine clinical practice, it may be meaningful to screen for C-KIT mutations in AML1/ETO-positive patients, as well as for FLT3D835 mutations in CBF-AML.

Minimal Residual Disease Monitoring in Patients with Newly Diagnosed Acute Promyelocytic Leukemia Treated with Arsenic Trioxide

There is limited data on the optimal timing and predictive value of minimal residual disease (MRD) detection in newly diagnosed patients with acute promyelocytic leukemia (APL) treated with arsenic trioxide (ATO). Between January 1998 and December 2007, 120 patients with newly diagnosed APL were treated with a single agent ATO based regimen (Mathews V et al. Blood 2006). Sample for MRD assessment was taken at the end of induction, end of consolidation, once in 3 months during maintenance and subsequently once in 3 months for two years followed by once in 6 months for the next 3 years. Qualitative nested RT-PCR was performed prospectively for all patients using recommendations of BIOMED-1 Concerted Action (1999). Real-time quantitative RT-PCR (RQ-PCR) was done retrospectively on archived MRD samples 3–6 months prior to documented relapse, using ABI Prism 7000 sequence detection system based on the EAC protocol. PML-RARa copy number was normalized to the expression of the Abelson gene and normalized copy number (NCN) given as ratio of PML-RARa CN/ABL CN X 100. The NCN 30.001 was considered as positive RQ-PCR value. During this period there were 18 haematological relapses (HRel), 2 transient molecular relapses (MRel). Prospective RT-PCR and retrospective RQ-PCR from the relapsed cases prior to either HRel or MRel were analyzed and compared with 18 patients (included two cases who had transient MRel) in continuous complete remission (CCR).Among 105 patients who achieved hematological remission, an RT-PCR at the end of induction was positive in 71/105 (68%) and at the end of consolidation in 2/105 (1.9%). One of these post consolidation positive cases subsequently had a HRel. Retrospective RQ-PCR analysis at the end of consolidation was positive in 6/18 (33%) cases who had a HRel. RQ-PCR was also positive at the same time point in 2/18 (11%) of the non relapsed controls who are in continuous complete remission (CCR) at 44 and 35 months respectively.Eight of the 18 cases with HRel were RT-PCR positive in the 3–6 month period prior to HRel and progressed from MRel to HRel at a median of 3 months (range: 1–4 months). Ten cases had HRel without a prior documented MRel. A positive RQ-PCR result was detected in 2 of these 10 cases which were not picked up by RT-PCR. Among cases in CCR, there were 2 patients who had a transient RQ-PCR positivity but did not progress to a HRel with a follow up period of 30.3 and 39.5 months respectively. Among the 10 MRel cases an RQ-PCR was positive 3 months prior to detection of MRel by RT-PCR in 5 cases (PML-RARa NCN range: 0.02–3.9), all these subsequently had a HRel. This analysis suggests that with the use of ATO in induction and consolidation, RT-PCR for MRD at the end of consolidation would not predict relapse in the majority of cases. Also post consolidation, the current RT-PCR based MRD assessment would not predict the majority of HRel and if RQ-PCR was utilized at the same time intervals it would have a higher positive predictive value of 87.5% but would continue to have a low sensitivity (44%). More frequent MRD monitoring than the currently used time points may be required to improve the sensitivity.

Flow cytometric minimal residual disease monitoring in patients with multiple myeloma undergoing autologous stem cell transplantation: A retrospective study

We retrospectively reviewed the flow cytometry (FCM) data from bone marrow aspirates of multiple myeloma (MM) patients before and after autologous stem cell transplantation (ASCT). Light scatter properties and CD38 expression were used to identify plasma cells, and CD19/CD45/CD56 further distinguished normal from abnormal plasma cells (NPC and APC, respectively), the latter defined as plasma cells with aberrant CD56, decreased or absent CD19 and/or CD45 expression. Forty-seven patients were screened. After ASCT, 66% (31/47) patients achieved complete remission (CR)/very good partial remission (VGPR), as compared to only 40% (19/47) prior to ASCT (P = 0.01). In 39 patients with data before and after ASCT, all 39 (100%) had a detectable APC population prior to ASCT. Twenty-six out of 39 patients (67%) also had detectable NPC. Following ASCT, the number of patients with detectable NPC increased to 35/39 (89%), while 3/39 (8%) had no detectable NPC and 1/39 (3%) had neither NPC nor APC. The proportion of APC decreased significantly after transplant from a median of 79% to 52% post-transplant (P < 0.001). The APC/total events ratio decreased significantly (P < 0.0001) after transplant. Prior to transplant, patients with less than 1.8% APC had significantly longer progression free survival (PFS) than patients with greater than 1.8% (P = 0.017, by log rank test), while the difference in overall survival did not achieve statistical significance (P = 0.081). Patients who achieved CR/VGPR after transplant had significantly longer PFS in comparison to all others (26 months vs. 11 months, P = 0.004). In conclusion, the detection of a significant population of APC by FCM prior to ASCT significantly correlates with poorer PFS in MM patients.

Biomarkers for mesothelioma

Mesothelioma is an incurable cancer and its global incidence continues to increase. There has been strong interest in the search for a biomarker that would be of value for the diagnosis, prognosis and disease monitoring of mesothelioma. Large series evaluating the use of novel candidate markers have recently been published. To date, global gene profiling studies have failed to find a molecule that reliably captures all subtypes of mesothelioma, and differentiates it from benign pathologies and metastatic carcinomas. Soluble mesothelin-related peptide (SMRP), osteopontin and megakaryocyte potentiating factor have been assessed as markers. SMRP testing is clinically available and provides reasonable diagnostic sensitivity and specificity when applied to serum or pleural fluid. Elevated SMRP levels can occur in metastatic, especially ovarian and pancreatic, adenocarcinomas. False negatives are common with sarcomatoid mesothelioma. SMRP levels may reflect tumor load and disease progression. The role of SMRP in predicting mesothelioma development in subjects exposed to asbestos has raised interest. Osteopontin lacks specificity as a diagnostic marker for mesothelioma but may have value in disease monitoring. The proposed markers have insufficient accuracy to replace cytohistology as the gold standard for diagnosis for mesothelioma. Elevated SMRP levels raise suspicion of mesothelioma although negative values do not exclude disease. Its role in disease monitoring in patients and in predicting disease development in at-risk individuals warrant further study.

Flow Cytometric Disease Monitoring in Patients with Multiple Myeloma Undergoing Autologous Stem Cell Transplantation: A Retrospective Study.

The persistence of abnormal neoplastic plasma cells (APC) detectable in the bone marrow by flow cytometry at more than 3 months after autografting for multiple myeloma (MM) has been reported to predict early disease progression. In this study, we retrospectively reviewed the flow cytometric data from bone marrow aspirates of MM patients before and after autologous stem cell transplantation (ASCT). Light scatter properties and CD38 expression were used to identify plasma cells, and CD19/CD45/CD56 further distinguished normal plasma cells (NPC) from APC. Conventional response criteria (Blade criteria) and survival data were also collected. Forty-seven (47) patients treated with the same conditioning regimen were screened. Median follow up from ASCT was 19 months. After ASCT, 66% (31/47) patients achieved complete remission (CR)/very good partial remission (VGPR), as compared to only 36% (17/47) prior to ASCT. In 39 patients with data before and after ASCT, all 39 (100%) had a detectable abnormal plasma cell population identified phenotypically by flow cytometry prior to ASCT. Of these patient, 18/39 (46%) had greater than 30 APC and these patients had significantly shorter PFS independent of other covariates (1-sided P=0.036, 2 sided P=0.072, logrank). Twenty-six out of 39 patients (67%) also had detectable NPC. Following ASCT, the number of patients with detectable NPC increased to 35/39 (89%), while 3/39 (8%) had no detectable NPC and 1/39 (3%) had neither NPC or APC. The proportion of APC decreased significantly after transplant (81% prior to transplant vs. 59% post-transplant, P=0.008, 2 tailed t-test). Patients with a APC to NPC ratio < 1 post transplant has higher PFS rate at 2 year (54%) when compared to patients with higher APC/NPC ratio (29% PFS at 2 year), however, the difference is not statistically significant. In addition to the presence of APC, the ratio of APC to NPC, age, beta-2 microglobulin levels, and the presence of normal immunoglobulin levels were analyzed. Patients who achieved CR/VGPR after transplant had significantly longer PFS (23 months vs. 11 months, P=0.03). All other covariates were not found to be significant. Because only 10 deaths were observed, covariate analysis for OS was not feasible. In conclusion, the recovery of NPC after ASCT is seen in a substantial propotion of patients with a trend towards better PFS in patients with low APC/NPC ratio. On the other hand, the presence of a significant population of APC (> 30) prior to transplant appears to correlate with poorer PFS in MM patients.

The bcl-2/IgH rearrangement in a population of 204 healthy individuals: Occurrence, age and gender distribution, breakpoints, and detection method validity

This study assessed prevalence, frequency, age and gender distribution and breakpoint locations, and detection method validity for the bcl-2/IgH rearrangement in 204 healthy individuals. For this purpose, both classic two-step, nested, semi-quantitative PCR as well as a newly established sequence-specific, hybridization probe-based real-time quantitative PCR (RQ-PCR) were employed and tested for their sensitivity and specificity for detecting t(14;18) positive cells in healthy blood donors. Interestingly, almost a quarter (24%; 39/204) of all healthy individuals carried the translocation, confirming data of one large prior report [Summers KE, Goff LK, Wilson AG, Gupta RK, Lister TA, Fitzgibbon J. Frequency of the Bcl-2/IgH rearrangement in normal individuals: implications for the monitoring of disease in patients with follicular lymphoma. J Clin Oncol 2001;19(2):420–4]. Regarding presence as well as frequency of the translocation, no correlation to age (mean frequency 2.0:10 4, with a median of <l:10 4, for <40 years, and mean frequency 1.9:10 4, with a median of <l:10 4 for individuals ≥40 years) nor gender was detected. Comparing the two PCR approaches, a 95.1% concordance (194/204) regarding t(14;18) detection was determined for nested and RQ-PCR, with nested PCR being slightly more sensitive (reproducible detection limit l:10 5 cells versus 1:10 4; maximum detection limit l:10 6 versus 1:10 5). Sequence analysis confirmed individual breakpoints for all samples analyzed (29/29), indicating detection validity for both PCR approaches and ruling out contamination. The breakpoint location distribution pattern appeared to be comparable to the pattern seen with follicular lymphoma (FL) patient collectives. In conclusion, clonal bcl-2/IgH rearrangements are indeed a very frequent observation in healthy individuals, and appear to be independent of age and gender in regard to presence and frequency. This represents a conflicting finding in context of potential biological significance, and presents a potential disruptive factor for minimal residual disease (MRD) monitoring in FL patients. Prospective future trials will have to clarify the biological significance of this important observation.

Cerebrovascular reactivity and dynamic autoregulation in nondemented patients with CADASIL (cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy)

Reduced cerebrovascular reactivity has been reported in patients with cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), and the measurement has been suggested as a useful surrogate marker of disease progression. Previous studies have not determined whether cerebral autoregulation is also impaired. We measured dynamic cerebral autoregulation and carbon dioxide reactivity in 24 nondemented CADASIL patients and 20 controls, using transcranial Doppler ultrasound (TCD). No impairment in either measure was found in the CADASIL group. We conclude that either cerebrovascular reactivity and autoregulation are not impaired in early disease, or that TCD may not be a sufficiently sensitive tool to detect haemodynamic changes in early disease. TCD is unlikely to be useful for disease monitoring in patients without advanced disease.

Molecular Monitoring of Minimal Residual Disease in Patients with Multiple Myeloma

Improvement of transplantation strategies and a multitude of emerging novel therapies result in a better treatment outcome in patients with multiple myeloma (MM). This gives rise to the need for sensitive methods to detect minimal residual disease (MRD) in MM. Qualitative molecular monitoring using allele-specific oligonucleotide PCR for the immunoglobulin heavy chain (IgH) is well established to detect clonotypic cells after therapy or in stem cell harvests. Recently, real-time IgH PCR or limiting dilution based PCR assays offer the possibility to quantify the amount of residual tumour cells. In this review, different qualitative and quantitative IgH PCR techniques will be discussed as well as the current clinical role of molecular monitoring of MRD in patients with MM.

Polarity of helper T cell subsets represents disease nature and clinical course of experimental autoimmune myocarditis in rats.

The mechanisms of progression, remission and relapse of myocarditis remain unclear. To clarify these mechanisms, we focused on T helper-1 (Th1)/T helper-2 (Th2) subsets balance of peripheral lymphocytes and serum cytokine levels during disease progression in rats with experimental autoimmune myocarditis (EAM). Lewis rats were immunized with cardiac myosin on day 0. Blood samples were collected on days 0, 7, 15, 18, 21, 28, 35, 42, 49 and 56 following immunization. We examined percentages of interferon (IFN)-gamma and/or interleukin (IL)-4 producing cells in stimulated peripheral CD4-positive lymphocytes using flow cytometry analysis. Serum IFN-gamma, IL-2, IL-6 and IL-10 levels were measured by enzyme-linked immunosorbent assay (ELISA). The percentage of Th1/Th2 subsets in EAM on days 0, 15, 28 and 56 were 2.5 +/- 0.5/0.5 +/- 0.1%, 19.4 +/- 3.2/1.6 +/- 0.3%, 2.0 +/- 0.5/22.1 +/- 5.7% and 3.0 +/- 0.4/1.7 +/- 0.3%, respectively. Serum levels of Th1 cytokines, IFN-gamma and IL-2 significantly increased in the acute phase (from day 15-18) and immediately decreased in the early recovery phase. On the other hand, serum levels of Th2 cytokine, IL-10 significantly increased in the early recovery phase (from day 24-30). These results suggest that induction of acute myocarditis might be associated with systemic Th1 dominance, while recovery is related to systemic Th2 polarity. Thus, analysis of Th1/Th2 balance in peripheral T cells may be useful in disease monitoring in patients with myocarditis and postmyocarditic dilated cardiomyopathy.

Immunologic monitoring in adults with acute lymphoblastic leukemia.

Investigation of minimal residual disease (MRD) by immunophenotyping and molecular techniques has proven to be a powerful approach for disease monitoring in patients with acute leukemia. Multiparameter flow cytometry, through the use of triple or quadruple marker combinations, identifies aberrant or uncommon phenotypic profiles in more than 90% of adult patients with acute lymphoblastic leukemia (ALL) at diagnosis. These profiles allow identification of residual leukemic cells in bone marrow or peripheral blood once morphologic complete remission is achieved. Until now, most immunophenotypic MRD studies in ALL have focused on children. In contrast, information on the value of MRD in adults with ALL is scanty and usually restricted to polymerase chain reaction studies. In this review, we focus on technical aspects of MRD detection by flow cytometry and on the clinical data concerning the value of immunologic MRD studies as a tool for relapse prediction in adult ALL. Although prospective studies are needed, we assert that immunophenotypic MRD studies are clinically useful. Such studies should be incorporated into the routine management of adult ALL patients for identification of those at high risk of relapse, who could benefit from new alternative therapeutic approaches, and to distinguish these patients from others who could be cured with more conventional approaches.

Circulating Cell‐Free Epstein‐Barr Virus DNA Levels in Patients with EBV‐Associated Lymphoid Malignancies

Epstein-Barr virus (EBV) is a human lymphotrophic herpesvirus implicated in the development of several lymphoid malignancies. Recently, detection of cell-free EBV DNA in sera or plasma from patients with EBV-associated malignancies has been reported. However, most published studies are qualitative in nature, with only a few providing quantitative data. We have described the temporal changes of plasma EBV DNA levels in patients with EBV-associated lymphoid malignancies during therapy using quantitative real-time PCR. A close correlation between plasma EBV DNA levels and therapeutic response was observed. Our aim was to update our results and to assess the role of the plasma EBV DNA levels as a molecular marker for disease monitoring in patients with EBV-associated lymphoid malignancies.

Frequency of the Bcl-2/IgH rearrangement in normal individuals: implications for the monitoring of disease in patients with follicular lymphoma.

To determine the incidence and frequency of the Bcl-2/IgH rearrangement in the peripheral blood of normal individuals to define the potential complication this may pose for the molecular monitoring of disease in patients with follicular lymphoma (FL). The incidence and frequency of the major breakpoint cluster region rearrangement in DNA extracted from peripheral blood or lymphoblastoid cell lines from 481 normal individuals was determined using a TaqMan real-time polymerase chain reaction assay (PE Applied Biosystems, Foster City, CA). Twenty three percent of samples were positive for the Bcl-2/IgH rearrangement, with approximately 3% of these at levels of more than 1 in 10(4) cells. The presence of circulating Bcl-2/IgH+ cells, other than those derived from the malignant clone, could confound the detection and quantitation of minimal residual disease in patients with FL, particularly at low levels of tumor burden.

Molecular monitoring of minimal residual disease in patients in long-term complete remission after allogeneic stem cell transplantation for multiple myeloma

In the present study, we used a polymerase chain reaction-based (PCR-based) strategy to retrospectively analyze the presence of residual myeloma cells in serial posttransplant bone marrow samples obtained from 13 patients in remission after allogeneic hemopoietic stem cell transplantation (allo SCT). For this purpose, patient-specific primers were generated from complementarity determining regions 2 and 3 of the rearranged IgH gene. The level of sensitivity of the PCR-based assay ranged from 1 in 10(5) to 1 in 10(6) normal marrow cells. Following transplantation, 9 of 12 patients who attained stringently defined complete remission (CR) remained persistently PCR(-) for a median of 36 months, and 4 of the patients remained PCR(-) up to the latest analysis, which was performed at 48, 72, 72, and 120 months, respectively, after allo SCT. None of the patients in the PCR(-) subgroup experienced a disease relapse, and only 1 of 4 PCR(+) patients experienced a relapse. It is concluded that allo SCT has the potential ability to induce sustained serological and molecular CR in selected patients with multiple myeloma.