Identification Of Minimal Residual Disease Research Articles

Value of Immunohistochemistry-Based Direct Visualization for Localization, Lineage Determination and Monitoring of IDH1 p.R132H Mutant Clones in AML

Abstract Background Isocitrate dehydrogenase 1 (IDH1) and IDH2 mutations are important prognostic biomarkers in acute myeloid leukemia (AML). Although the clinicopathologic correlates of IDH mutations have been extensively studied, the distribution of abnormal myeloid cells carrying these mutations has not been studied. Specific localization of cells carrying IDH mutations will be useful in further understanding the pathophysiology and post-treatment biology of IDH mutant cases of AML. This characterization is becoming particularly relevant for identification of minimal residual disease, especially for patients treated with novel IDHinhibitors. In this study, we characterized IDH1 p.R132H clones in bone marrow specimens involved by AML using a mutation specific antibody. Materials and Methods Bone marrow tissue sections (biopsy or clot specimens) from 32 AML cases with IDH1 p.R132H mutation were stained with IDH1 p.R132H-mutation specific antibody. These cases include 20 de novoAML and 12 cases of AML with myelodysplasia-related changes (AML-MRC). We also included 10 AML cases with wild-type IDH1 as a control. After confirmation of the positive IDH1 immunohistochemical (IHC) signal in the primary specimens, follow up bone marrow specimens (n=67) including (a) persistent disease, (b) minimal residual disease by flow cytometry, (3) complete remission by morphology and flow cytometry, but, positive for mutation by PCR, as well as (4) relapsed cases after complete remission were included in the study (in progress). We also included pre- and post-treatment (unresponsive with increasing blast counts, stable disease, persistent disease with decreasing blast counts, complete remission, and relapse) bone marrow specimens (n=72) from 16 patients treated with IDH inhibitors (in progress). Results All the IDH1 wild type AML cases were negative for IDH1 IHC stain showing 100% specificity. Positive signal was detected in all de novo AML and AML-MRC (allelic frequency ranges from 1.8% to 47% by PCR) except one AML case with 8.9% allele burden which was a limited sample; overall sensitivity was 96%. The IHC signal was detected in the cytoplasm of myelomonocytic cells, their precursors, and megakaryocytes. Erythroid precursors, lymphoid cells, endothelial cells, and osteoblasts were consistently negative. The signal intensity ranged from weak (n=10) to moderate (n=9), to strong (n=13). The positive cells predominantly showed an interstitial distribution in the bone marrow. In the de novo AML group, only the immature cells were positive in 100% of pre-treatment AML cases. However, both mature and immature cells were positive in 7/13 (54%) post-treatment AML cases (6 cases treated with hypomethylating agents). One case was transformed from MPN which also showed positivity in mature and immature cells. In two cases with complete morphologic remission and one case with minimal residual disease detected by flow cytometry, IHC signal was detected in both mature and immature cells; both patients relapsed in 8 and 11 months. In the AML-MRC group, both immature and mature cells were positive in 11/12 (92%) cases of which 2 were not previously treated indicating the possibility that IDH1 mutation is an early event. Since the remaining 9 patients were treated with hypomethylating agents, the positivity of both mature and immature cells as a result of maturation effect versus an early event cannot be assessed. Additional studies for follow-up AML cases, including cases on an IDH inhibitor clinical trial are in progress. Conclusions Our preliminary data indicate that IDH1 IHC is a highly specific and sensitive tool to detect IDH1 R132H mutated cases and can be used as a primary method to localize the population of mutation-bearing cells in the bone marrow. IHC also allows determination of whether the IDH1 mutation in the post-treatment setting is arising from immature or mature cells. IHC provides an opportunity to understand the difference between these two populations and, based on characterization of cell type and distribution, may be helpful to predict whether the risk of relapse is high. Disclosures DiNardo: Agios: Other: advisory board, Research Funding; Novartis: Other: advisory board, Research Funding; Daiichi Sankyo: Other: advisory board, Research Funding; Celgene: Research Funding; Abbvie: Research Funding.

Potentials, challenges and limitations of a molecular characterization of circulating tumor DNA for the management of cancer patients

Abstract: A liquid profiling, i.e. the analysis of cell-free circulating tumor DNA (ctDNA), enables a continuous non-invasive monitoring of tumor-specific changes during the entire course of the disease with respect to early detection, identification of minimal residual disease, assessment of treatment response and monitoring tumor evolution. Technological improvements, advances in understanding the nature of ctDNA, the implementation of ctDNA analyses in clinical trials as well as efforts for the establishment of benchmarks, will bring an actual widespread clinic use within reach in the near future. However, despite this progress there are still hurdles that have to be overcome, which are discussed in this review. Moreover, present knowledge and new findings about the biology of ctDNA as well as selected potential clinical applications for metastatic cancer patients are pointed out.

Clinical Utility of Liquid Diagnostic Platforms in Non-Small Cell Lung Cancer.

: A firmer understanding of the genomic landscape of lung cancer has recently led to targeted, therapeutic advances in non-small cell lung cancer. Historically, the reference standard for the diagnosis and genetic interrogation for advanced-stage patients has been tissue acquisition via computed tomography-guided core or fine needle aspiration biopsy. However, this process can frequently put the patient at risk and remains complicated by sample availability and tumor heterogeneity. In addition, the time required to complete the diagnostic assays can negatively affect clinical care. Technological advances in recent years have led to the development of blood-based diagnostics or "liquid biopsies" with great potential to quickly diagnose and genotype lung cancer using a minimally invasive technique. Recent studies have suggested that molecular alterations identified in cell-free DNA (cfDNA) or circulating tumor DNA can serve as an accurate molecular proxy of tumor biology and reliably predict the response to tyrosine kinase therapy. In addition, several trials have demonstrated the high accuracy of microRNA (miRNA) platforms in discerning cancerous versus benign nodules in high-risk, screened patients. Despite the promise of these platforms, issues remain, including varying sensitivities and specificities between competing platforms and a lack of standardization of techniques and downstream processing. In the present report, the clinical applications of liquid biopsy technologies, including circulating tumor cells, proteomics, miRNA, and cfDNA for NSCLC, are reviewed and insight is provided into the diagnostic and therapeutic implications and challenges of these platforms. Although tumor biopsies remain the reference standard for the diagnosis and genotyping of non-small cell lung cancer, they remain fraught with logistical complexities that can delay treatment decisions and affect clinical care. Liquid diagnostic platforms, including cell-free DNA, proteomic signatures, RNA (mRNA and microRNA), and circulating tumor cells, have the potential to overcome many of these barriers, including rapid and accurate identification of de novo and resistant genetic alterations, real-time monitoring of treatment responses, prognosis of outcomes, and identification of minimal residual disease. The present report provides insights into new liquid diagnostic platforms in non-small cell lung cancer and discusses the promise and challenges of their current and future clinical use.

VDJ-Seq: Deep Sequencing Analysis of Rearranged Immunoglobulin Heavy Chain Gene to Reveal Clonal Evolution Patterns of B Cell Lymphoma.

Understanding tumor clonality is critical to understanding the mechanisms involved in tumorigenesis and disease progression. In addition, understanding the clonal composition changes that occur within a tumor in response to certain micro-environment or treatments may lead to the design of more sophisticated and effective approaches to eradicate tumor cells. However, tracking tumor clonal sub-populations has been challenging due to the lack of distinguishable markers. To address this problem, a VDJ-seq protocol was created to trace the clonal evolution patterns of diffuse large B cell lymphoma (DLBCL) relapse by exploiting VDJ recombination and somatic hypermutation (SHM), two unique features of B cell lymphomas. In this protocol, Next-Generation sequencing (NGS) libraries with indexing potential were constructed from amplified rearranged immunoglobulin heavy chain (IgH) VDJ region from pairs of primary diagnosis and relapse DLBCL samples. On average more than half million VDJ sequences per sample were obtained after sequencing, which contain both VDJ rearrangement and SHM information. In addition, customized bioinformatics pipelines were developed to fully utilize sequence information for the characterization of IgH-VDJ repertoire within these samples. Furthermore, the pipeline allows the reconstruction and comparison of the clonal architecture of individual tumors, which enables the examination of the clonal heterogeneity within the diagnosis tumors and deduction of clonal evolution patterns between diagnosis and relapse tumor pairs. When applying this analysis to several diagnosis-relapse pairs, we uncovered key evidence that multiple distinctive tumor evolutionary patterns could lead to DLBCL relapse. Additionally, this approach can be expanded into other clinical aspects, such as identification of minimal residual disease, monitoring relapse progress and treatment response, and investigation of immune repertoires in non-lymphoma contexts.

Evaluating the use of Multiparametric Flow Cytometry in establishing the presence of Minimal Residual Disease in systemic AL amyloidosis: a report of eight patients

The identification of Minimal Residual Disease (MRD) is becoming increasingly important for defining outcomes in haematological malignancies; particularly in independently predicting prognosis in myeloma. The role of MRD analysis in systemic AL amyloidosis is less well established. Patients with systemic AL amyloidosis at diagnosis have a lower clonal burden than that seen in myeloma. Moreover, upfront autologous transplantation even in the era of novel therapies continues to provide a more durable remission than that seen in myeloma. Here, as a proof of principle, eight patients with systemic AL amyloidosis in complete response (CR) underwent MRD analysis. MRD analysis was completed if CR was achieved, usually six months from induction treatment. Induction treatments included two patients treated with cyclophosphamide, thalidomide, dexamethasone and six patients with cyclophosphamide, bortezomib and dexamethasone. Two of the six patients that had received CVD based treatment underwent consolidation autologous transplantation. Bone marrow samples were immunophenotyped with eightcolour multiparametric flow cytometry (MFC). The expression of CD138 and CD38 was used to gate the plasma cell population. Patients were identified as having residual disease if a discreet population of phenotypically aberrant plasma cells comprising 50 events were identified in the 500, 000 event file (0.01% limit of detection). An aberrant phenotype was defined as a lack of CD19 expression, strong CD138 expression, weak CD27 expression, and or weak CD45 expression. Interestingly, none of the eight patients demonstrated MRD negativity. All patients demonstrated a measurable clone despite evidence of CR, including two patients who had an autologous bone marrow transplantation. CD117 and CD81 expression failed to provide any additional discrimination between polyclonal and clonal plasma cells. Moreover, the spread between CD81 positive and negative populations was difficult to distinguish in certain patients. In conclusion, this proof of principle study supports MFC as an effective measurement of MRD in patients with systemic AL amyloidosis. The finding that all patients demonstrate MRD positivity raises difficulties in rationalizing if MRD negativity is necessary for a durable remission in systemic AL amyloidosis. To address this further, an adequately powered study randomizing MRD positive patients to further treatment or no treatment may be necessary. PO-179 Hospitalization Rates for Newly Diagnosed Multiple Myeloma Patients in the United States Medicare Database A.A. Yusuf, T. Bovitz, W. Werther, D. Felici, M. Mahue, K. Bridges, Y. Peng Chronic Disease Research Group, Minneapolis, MN, USA; University of Minnesota, Minneapolis, MN, USA; Onyx Pharmaceuticals Inc., an Amgen Subsidiary, South San Francisco, CA, USA

Hospitalization Rates for Newly Diagnosed Multiple Myeloma Patients in the United States Medicare Database

The identification of Minimal Residual Disease (MRD) is becoming increasingly important for defining outcomes in haematological malignancies; particularly in independently predicting prognosis in myeloma. The role of MRD analysis in systemic AL amyloidosis is less well established. Patients with systemic AL amyloidosis at diagnosis have a lower clonal burden than that seen in myeloma. Moreover, upfront autologous transplantation even in the era of novel therapies continues to provide a more durable remission than that seen in myeloma. Here, as a proof of principle, eight patients with systemic AL amyloidosis in complete response (CR) underwent MRD analysis. MRD analysis was completed if CR was achieved, usually six months from induction treatment. Induction treatments included two patients treated with cyclophosphamide, thalidomide, dexamethasone and six patients with cyclophosphamide, bortezomib and dexamethasone. Two of the six patients that had received CVD based treatment underwent consolidation autologous transplantation. Bone marrow samples were immunophenotyped with eightcolour multiparametric flow cytometry (MFC). The expression of CD138 and CD38 was used to gate the plasma cell population. Patients were identified as having residual disease if a discreet population of phenotypically aberrant plasma cells comprising 50 events were identified in the 500, 000 event file (0.01% limit of detection). An aberrant phenotype was defined as a lack of CD19 expression, strong CD138 expression, weak CD27 expression, and or weak CD45 expression. Interestingly, none of the eight patients demonstrated MRD negativity. All patients demonstrated a measurable clone despite evidence of CR, including two patients who had an autologous bone marrow transplantation. CD117 and CD81 expression failed to provide any additional discrimination between polyclonal and clonal plasma cells. Moreover, the spread between CD81 positive and negative populations was difficult to distinguish in certain patients. In conclusion, this proof of principle study supports MFC as an effective measurement of MRD in patients with systemic AL amyloidosis. The finding that all patients demonstrate MRD positivity raises difficulties in rationalizing if MRD negativity is necessary for a durable remission in systemic AL amyloidosis. To address this further, an adequately powered study randomizing MRD positive patients to further treatment or no treatment may be necessary. PO-179 Hospitalization Rates for Newly Diagnosed Multiple Myeloma Patients in the United States Medicare Database A.A. Yusuf, T. Bovitz, W. Werther, D. Felici, M. Mahue, K. Bridges, Y. Peng Chronic Disease Research Group, Minneapolis, MN, USA; University of Minnesota, Minneapolis, MN, USA; Onyx Pharmaceuticals Inc., an Amgen Subsidiary, South San Francisco, CA, USA

Detection of Minimal Residual Disease by Flow Cytometry for Patients with Multiple Myeloma Submitted to Autologous Hematopoietic Stem Cell Transplantation

The treatment strategy in multiple myeloma (MM) is to get complete remission followed by high-dose chemotherapy and autologous Hematopoietic Stem Cell Transplantation (HSCT). Neoplastic Plasma Cells (NPCs) are CD45−/dim, CD38+high, CD138+, CD19−, and CD56+high in most cases. The description of this immunophenotype is of major importance as it leads to the correct identification of minimal residual disease (MRD). Samples from 44 Patients were analyzed prospectively in this study. We analyzed if the presence of MRD at three months after HSCT was predictive of relapse or death. There were 40 evaluable patients of whom 16/40 patients had MRD at three moths after HSCT and there were none in cytological relapse. The mean overall survival (OS) was 34 months and disease-free survival (RFS) was 28 months after HSCT. There was no significant difference in the log rank analysis comparing OS and the presence of MRD (P = 0,611) and RFS (P = 0,3106). Here, we demonstrate that three color flow cytometry (FCM) is more sensitive for MDR evaluation than cytological analyzes. However, based in our data we can not affirm that MRD is a good predictor of MM relapse or death. In conclusion, our results could be attributed to a short followup, small sample size, and over most to the inability of a three-color FCM to detect the NPC population.

Allogeneic Hematopoietic Stem Cell Transplantation Overcomes the Adverse Prognostic Impact of Minimal Residual Disease in Acute Myeloid Leukemia

Abstract 2027 BACKGROUND:The identification of minimal residual disease (MRD) can predict impending relapse in acute myeloid leukemia (AML) patients. Little data exists evaluating the prognostic impact of MRD, as determined by multiparametric flow cytometry (MFC), at the time of allogeneic (allo-HCT). Although disease outcomes may be worse for MRD positive (MRD+) patients, MRD is often associated with other adverse risk factors leaving it unclear whether MRD is an independent risk factor for relapse or a surrogate marker for underlying poor risk disease features. METHODS:We retrospectively analyzed 97 consecutive AML patients in complete morphological remission (CR) who underwent allo-HCT with a matched related donor (MRD, n=30, 31%), matched unrelated donor (MUD, n=4, 4%) or umbilical cord blood (UCB, n=63, 65%) at the University of Minnesota between January 2005 and June 2009. Presence of MRD at allo-HCT was determined by MFC. Analyses were done separately for myeloablative (MAC) and reduced intensity conditioning (RIC) patients, testing the impact of MRD along with conditioning intensity, age, donor type and disease status on allo-HCT outcomes. RESULTS:Of 97 patients, 41 (42%) had MAC; 57 (58%) had RIC. Sixty-six were in first CR (CR1) with the rest in CR2 or later remission (CR2+). MRD at allo-HCT was detected in 7 patients after MAC (17%) and 7 after RIC (12.5%); and this frequency was similar in patients in CR1 and CR2+ (13% vs. 16%, p=0.7). MRD+ and MRD- patients had similar median age (40 vs. 44 yrs), gender, donor source (MSD or MUD vs. UCB), CMV serostatus and diagnostic cytogenetic risk group. Six of 40 (15%) intermediate and 5 of 39 (13%) high risk cytogenetics patients had MRD+ (p=0.8). Only 3 of 53 patients with a cytogenetic abnormality at diagnosis had it detected prior allo-HCT and 1 of 3 had MRD.The median follow-up of survivors was 25 months. Two-year probabilities for MAC and RIC patients were similar: Overall survival (OS), 48% and 47 % and leukemia free survival (LFS) 43% and 41% respectively. When disease outcomes were analyzed separately by MRD status (table), OS and LFS were markedly worse in MRD+ patients receiving RIC, but this difference was not statistically significant. In multivariate analysis, MRD+ was not an independent prognostic factor for OS and LFS. Although we identified no adverse prognostic factors for MAC patients, patient with RIC in CR2+ had worse OS and LFS vs. CR1 (HR 2.6, p=0.04 and HR 2.7, p=0.04 respectively). CONCLUSION:The negative prognostic impact of MRD was overcome by allo-HCT with MAC, but outcomes with MRD+ were suggestively inferior after RIC. However due to limited sample size, MRD in patients with RIC should be further investigated.2 year outcomesMRD+ (95% CI)MRD− (95% CI)pMACOS43% (10%–73%)49% (32%–65%)0.7LFS43% (10%–73%)44% (27%–60%)0.6RICOS14% (7%–46%)51% (36%–65%)0.1LFS14% (7%–46%)44% (29%–58%)0.1 Disclosures:Weisdorf:Genzyme: Consultancy, Research Funding.

Chemo-immunotherapy for hairy cell leukemia

With the introduction of the nucleoside analogs cladribine and pentostatin the treatment of patients with hairy cell leukemia (HCL) has been revolutionized, and the majority of patients achieve a long-lasting complete remission with rare patients ever needing the traditional treatment strategies such as splenectomy. However, in the studies employing either of these nucleoside analogs, a proportion of patients do not respond to the initial therapy and the event-free survival curve does not reach a plateau, with up to 40% of patients relapsing within a few years. New therapeutic modalities including monoclonal antibodies such as rituximab and immunotoxins such as BL22 are now available. Furthermore, progress in the identification of minimal residual disease (MRD) using consensus primer or patient specific polymerase chain reaction for the immunoglobulin heavy chain variable region gene (IGHV), as well as multiparameter flow cytometry, allows for the detection and eradication of MRD. Whether this will translate to a reduction in the rate of relapse will require large prospective randomized trials. Alternatively, it may be possible to identify patients who are more likely to relapse using pretreatment characteristics such as the mutational status of IGHV and apply these strategies solely to them.

Modern Strategies for Hairy Cell Leukemia

Enormous progress in the treatment of hairy cell leukemia over the last five decades has emerged as a result of organized clinical investigations. Although interferon represented one of the initial major therapeutic advances in the management of this disease in 1984, the subsequent introduction of purine nucleoside analogs (pentostatin and cladribine) changed the natural history of this rare disease by achieving a high rate of complete and durable remissions. The disease-free survival after effective therapy has not reached a plateau, suggesting control but not cure of the disease. Identification of minimal residual disease in patients achieving a complete hematologic remission provides insight into the potential source for predicting eventual relapse. Modern strategies of targeted therapies directed against immunophenotypic markers on the leukemic cells provide hope that improved long-term control of the disease is possible. Combined chemoimmunotherapy may hold the highest promise for disease eradication, but the optimal strategy for using this approach is under active investigation. Despite the perception by hematologists that this disease has already been conquered, there are critically important unanswered questions that remain. Investigation of the bone marrow microenvironment and its impact on minimal residual disease may ultimately prevent relapse. Consideration of the median age of patients at diagnosis combined with a substantial relapse rate mandates continued pursuit of improved therapy. The ultimate goal will be to achieve cure rather than simple control of the disease.

Detection of ultrarare somatic mutation in the humanTP53gene by bidirectional pyrophosphorolysis-activated polymerization allele-specific amplification

The detection of ultra-rare mutation in the presence of excess amounts of normal genomic DNA is highly advantageous in a number of circumstances, including: 1) identification of minimal residual disease for improved cancer chemotherapy; 2) measurement of mutation load to assess environmental mutagen exposure or endogenous DNA repair; and 3) prenatal diagnosis of paternally-derived mutations within fetal cells in the maternal circulation. Bidirectional pyrophosphorolysis activated polymerization allele-specific amplification (Bi-PAP-A) utilizes two opposing 3'-terminal blocked oligonucleotides (P(*)s) with 1 nucleotide overlap at their 3' termini. The selectivity of Bi-PAP-A derives from the serial coupling of pyrophosphorolysis and DNA polymerization. A total of 13 Bi-PAP-A assays were developed and validated for the human p53 gene (TP53). The sensitivity and specificity of each assay were determined with mutated and wild-type DNA templates, respectively. Bi-PAP-A has a sensitivity of one molecule for most assays and a selectivity (sensitivity:specificity) greater than 1:10(7)-1:10(9) for four of all six mutation types. Four assays with high selectivity were used to detect rare somatic mutations in blood white cells. The silent g.13147C>G (p.R156) mutation was present at an estimated frequency of 1.1 x 10(-7). The g.14523A>T (p.E285V), g.14487G>C (p.R273P), and g.14060G>C (p.G245R) mutations were undetectable with frequencies less than 2.0 x 10(-8). We conclude that Bi-PAP-A is a general and rapid method for detecting ultra-rare mutations.

PCR-based detection of minority point mutations.

The need for detection of minority mutations (i.e., a few mutants within a high excess of wild-type alleles) arises frequently in the field of cancer and molecular genetics. Current mutation detection technologies are limited by several technical factors when it comes to the detection of minority point mutations, including generation of misincorporations by the DNA polymerase during PCR amplification. Primer ligation-mediated PCR methodologies for detection of mutations in an excess wild-type sequences are described, that can be applied for detection of both known and unknown minority point mutations. Furthermore, a new methodology is described, hairpin-PCR, which has the potential to completely eliminate PCR errors from amplified sequences, prior to minority mutation detection. Combination of these technologies can effectively tackle the problem of minority mutation detection, in order to pursue demanding applications such as identification of cancer cells at an early stage, detection of mutations in single cells, identification of minimal residual disease, or investigation of mechanisms of spontaneous mutagenesis.

Does sentinel lymph node biopsy have a role in colorectal cancer?

Sentinel lymph node (SLN) biopsy in colorectal cancer is feasible, and may offer a means of reducing histopathology workload while maintaining staging accuracy. Although SLN biopsy allows use of sensitive molecular markers that can identify isolated tumour cells and micrometastases, these findings require more extensive clinical evaluation before they can be used to guide patient management. The application of molecular markers to identification of minimal residual disease after primary tumour resection, shows promise as a means of indicating patients requiring chemotherapy.

Minimal residual disease in hematologic disorders.

In almost no other area of medical oncology has the introduction of new drugs, combinations of chemotherapeutic agents, and novel biologic treatments caused such dramatic responses as it has in the treatment of malignant hematologic disorders. However, despite some therapeutic success, many patients relapse and die from recurrence of their disease. The implications of minimal residual disease (MRD), a term referring to disease that is undetectable by conventional morphologic methods, have therefore attracted increasing attention in recent years. New and powerful laboratory tools such as polymerase chain reaction assays have extraordinary sensitivity and provide exciting new insights into the detection, nature, quantification, and kinetics of MRD. This article summarizes methods used in the identification of MRD and its importance as exemplified in the case of acute leukemias and chronic myelogenous leukemia.

Clinical Significance of Cytogenetic Findings at Diagnosis and in Remission in Childhood and Adult Acute Lymphoblastic Leukemia: Experience from India

We report cytogenetic findings in 114 patients of acute lymphoblastic leukemia (ALL), which includes 78 children ( ≤15 years) and 36 adults (16–60 years). Chromosome aberrations were detected in 109 (95%) cases. A lower frequency of hyperdiploidy (15%) in children and a higher frequency of hypodiploidy both in children (38.4%) and adults (44.4%) were found, in contrast to literature. Translocations were detected in one third of adult and pediatric cases. The incidence of t(9;22) was comparatively low in adults (7.7%). Frequency of t(1;19) was also low in overall ALL cases. Various other recurrent abnormalities such as del(6q), abn(11q23), i(9p), abn(12p13), del(7q), and i(17q) were seen in our cases; a striking difference in the incidence of del(6q) (41%) and abn(11q23) (30%) was found in our series versus reported literature. Ploidy distribution indicated association of pseudo- and hypodiploidy with B-lineage, and hypodiploidy with T-lineage in children. The occurrence of del(6q) was more frequent in pediatric ALL with highly aberrant pattern and also with lymphadenopathy. Abn(11q23) was found to be early-B and pre-B specific. Kaplan-Meier analysis of overall survival revealed prognostic value of sex, FAB, immunophenotype, and cytogenetic findings. Females and T-ALL patients had a better prognosis, whereas males and B-ALL patients had poor outcome in overall and pediatric age groups. Prognostic evaluation of cytogenetics indicated translocations as an independent high-risk predictor in childhood ( P < 0.008) and adult ALL ( P < 0.01). Childhood ALL with t(8;14) and t(4;11) and adults with t(9;22) had poor survival. Cytogenetics of remission marrows demonstrated disappearance of abnormal clones in 31.4%, and expansion in normal clones in 50% of patients. Persistence of original clones and development of new clones were observed in 20% and 33% of patients, respectively; whereas karyotype evolution was identified in 10% of patients. The prognostic significance of cytogenetic findings at diagnosis, and differential cytogenetic response in so-called clinical remission in our study indicated the utmost need for more intensive therapy for eradication of resistant clones, and necessity of sequential cytogenetic follow-up in these patients for identification of minimal residual disease.

Minimal Residual Disease, Its Detection and Significance in Hairy-Cell Leukemia

As minimal residual disease (MRD) is considered the detection of hairy cells (HCs) in a patient with hairy cell leukemia (HCL) in complete remission with the absence of detectable HCs by routine morphology of peripheral blood, aspirates and bone marrow core sections, using more sensitive methods of identification as immunohistological staining or polymerase chain reaction (PCR) to detect immunoglobulin heavy chain genes rearrangement. Various monoclonal antibodies (MoAbs) as CD20, DBA.44, B ly-7, HC2, CD25 and CD11c have been applied using immunological staining. There is no standardized technique for identification of MRD. According to the technique used the MRD has been detected in 13% to 100% of patients in complete remission (CR). It may be concluded that many patients, if not all, in stable CR may have residual HCs. Whether MRD will have impact on early relapse or on long term outcome, or whether patients in CR with persistent MRD will remain so, is a matter of a longer follow-up.

The effect of chemo- and chemoimmunotherapy on the presence of circulating melanoma cells in peripheral blood. Preliminary results.

Reverse transcription and polymerase chain reaction (RT/PCR) with primers specific for tyrosinase allow for a new method of early detection of individual melanoma cells in peripheral blood. Using this test the effect of chemo- and chemoimmunotherapy on the spread of early micrometastatic cancer cells has been evaluated. No significant correlations have been found between RT/PCR results on the one hand and stage of disease, a kind of the therapy protocol used and usage of the therapy as an adjuvant or palliative on the other hand. Thus, although the RT/PCR test for detection of circulating individual melanoma cells might help in identification of minimal residual disease in some patients, it has no application for routine staging of more advanced disease and in monitoring the response to therapy.

Sensitive Detection of Clonal Antigen Receptor Gene Rearrangements in Non-Hodgkin’s Malignant Lymphoma With an Anchored Polymerase Chain Reaction–Based Strategy

The identification of minimal residual disease (MRD) in non-Hodgkin's malignant lymphoma is of immediate clinical relevance. Although several polymerase chain reaction (PCR)-based strategies are currently available, they all share serious technical or theoretic limitations with respect to their ability to detect the broad spectrum of mature B-cell neoplasia. We report a new strategy based on anchored PCR that does not rely on a particular chromosomal translocation or VH consensus sequence. It can yield the complete DNA sequence of the immunoglobulin heavy chain variable region (VHDJH). The assay is capable of detecting one neoplastic cell among 10(3)-10(4) normal cells with high specificity and has the potential to greatly expand the variety of B-cell malignant neoplasms that can be studied.

Retrospective DNA analysis using fixed tissue specimens.

The recent explosion of scientific and technical knowledge in the field of molecular biology has allowed us to make important advances in our understanding of the molecular basis of many human diseases. This technology has now entered the clinical laboratory where identification of specific genetic sequences can aid in the precise diagnosis of hematologic and other malignancies, inherited diseases, specific infectious agents, and inherited predisposition to disease. In addition, it can be applied to prenatal diagnosis, paternity testing, identification of minimal residual disease following treatment, and assessment of drug sensitivity or resistance. In many cases in diagnostic pathology, the need for molecular analysis often is not realized until after a critical tissue specimen has been fixed, embedded, and examined microscopically. Thus, there is a clear need for development of techniques that would allow the retrospective study of archival tissues that have been fixed and embedded in paraffin. This review examines in depth those factors which influence the quality of the DNA available from fixed embedded tissues and discusses the usefulness of polymerase chain reaction amplification in obtaining sufficient diagnostically useful DNA from archival specimens. It is hoped that this review will aid the diagnostic pathologist interested in the application of molecular techniques in the retrospective study of fixed embedded tissues.

Treatment of childhood acute lymphoblastic leukaemia: Present issues and future prospects

Modern treatment for acute lymphoblastic leukaemia (ALL) achieves long term survival in two thirds of children, many of whom are truly cured. Recent improvements have occurred as a result of progressive intensification of treatment, particularly during the first 6 months from diagnosis. This article reviews the means of identifying children for whom standard therapy is not appropriate, and the aspects of standard therapy requiring further refinement, in particular central nervous system (CNS) directed treatment and continuing (maintenance) therapy, a concept unique to ALL. Modern methods for identification of minimal residual disease afford the hope that it may become possible to identify both children who have received sufficient treatment and, conversely, those at subsequent risk of relapse. Selection of patients for alternative therapy, for example marrow transplantation in first remission is difficult and this area needs further study. Treatment for the one third of children who relapse remains unsatisfactory and when successful is only achieved with significant morbidity. Most long-term survivors of childhood leukaemia are well and problem-free but a significant number have problems with learning, concentration, growth and development; the risk of second neoplasms remains unclear. Recent years have seen undeniable progress in the treatment of ALL but there is a continuing need to develop effective forms of treatment which have less potential for damaging late effects.