ABSTRACT
Undifferentiated carcinoma with osteoclast-like giant cells (UCOGC) of the pancreas is a rare malignancy regarded as a subvariant of pancreatic ductal carcinoma (PDAC) characterized by variable prognosis. UCOGC shows a strikingly similar spectrum of oncogenic DNA mutations to PDAC. In the current work, we analyzed the landscape of somatic mutations in a set of 13 UCOGC cases via next-generation sequencing (NGS). We detected a spectrum of pathogenic or likely pathogenic mutations similar to those observed in PDAC following previously published results (10 KRAS, 9 TP53, 4 CDKN2A, and 1 SMAD4, CIC, GNAS, APC, ATM, NF1, FBXW7, ATR, and FGFR3). Our results support the theory that UCOGC is a variant of PDAC, despite its unique morphology; however, a UCOGC-specific genomic signature as well as predictive markers remain mainly unknown. Programmed death ligand 1 (PD-L1) status remains an important predictive marker based on previous studies.
Subject(s)
Carcinoma, Pancreatic Ductal , Pancreatic Neoplasms , Humans , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/pathology , Osteoclasts/pathology , Pancreas/pathology , Carcinoma, Pancreatic Ductal/pathology , Giant Cells/pathology , Mutation , Molecular BiologyABSTRACT
Frequency, distribution and prognostic meaning of ALK-partner genes other than NPM1 in ALK-positive anaplastic large-cell lymphoma (ALCL) are unknown. Forty-nine of 316 ALCL diagnosed in the NHL-BFM study group showed no nuclear ALK expression suggestive of a variant ALK-partner; 41 were analysed by genomic capture high-throughput sequencing or specific RT-PCRs. NPM1::ALK was detected in 13 cases. Among the 28 patients with a non-NPM1::ALK-fusion partner, ATIC (n = 8; 29%) and TPM3 (n = 9; 32%) were the most common. Five of eight patients with ATIC::ALK-positive ALCL relapsed, none of nine with TPM3::ALK. Variant ALK-partners are rare and potentially associated with different prognoses.
Subject(s)
Anaplastic Lymphoma Kinase , Lymphoma, Large-Cell, Anaplastic , Nucleophosmin , Humans , Lymphoma, Large-Cell, Anaplastic/genetics , Lymphoma, Large-Cell, Anaplastic/pathology , Child , Male , Female , Anaplastic Lymphoma Kinase/genetics , Anaplastic Lymphoma Kinase/analysis , Adolescent , Child, Preschool , Oncogene Proteins, Fusion/genetics , Prognosis , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Infant , TropomyosinABSTRACT
Anaplastic lymphoma kinase-positive anaplastic large cell lymphoma (ALK+ ALCL) originates from the T-lineage and is marked by rearrangements of the ALK gene. More than 10 fusion partners with the ALK gene are known, with the most common being the t(2;5)(p23;q35) translocation resulting in the NPM1::ALK fusion. In 10% to 20% of the ALK+ ALCL cases, the ALK gene fuses with various other partners. Modern molecular techniques, especially next-generation sequencing (NGS), have eased the identification of ALK gene fusion partners and have allowed in-depth characterization of the T-cell receptor (TCR) repertoire. We devised a real-time quantitative reverse-transcription polymerase chain reaction to measure the expression of the translocated portion of the ALK gene. Fusion partners for the ALK gene were analyzed using rapid amplification of 5'cDNA ends (RACE) method or NGS. TCR immunoprofiling was performed by amplicon NGS. We studied 96 ALK+ ALCL patients. NPM1::ALK fusion gene was observed in 71 patients, ATIC::ALK in 9, and TPM3::ALK in 3. CLTC::ALK, MYH9::ALK, and RNF213::ALK fusions were identified in 2 patients each. We also discovered the TPM4::ALK and SATB1::ALK fusion genes, plus the following 2 previously unidentified ALK+ ALCL fusions: SQSTM1::ALK and CAPRIN1::ALK. High expression of the translocated ALK gene segment was observed in all 93 analyzed samples. TCR testing was conducted on 23 patients with available DNA. In 18 (78%) patients, we discerned at least one (ranging from 1 to 4) clonal TCR rearrangement. In 59% of the patients, clonal TCR beta junctions corresponded with sequences previously observed in both healthy donors and under various pathological conditions. Reverse-transcriptase quantitative detection of ALK expression is a fast and reliable method for both diagnosing and monitoring treatment response in ALK+ ALCL patients, irrespective of the ALK gene translocation. NGS reveals new ALK translocation partners. Both malignant and reactive TCR repertoires in ALK+ ALCL patients are unique and do not consistently occur among different patients.
Subject(s)
Lymphoma, Large-Cell, Anaplastic , Matrix Attachment Region Binding Proteins , Ubiquitin-Protein Ligases , Humans , Anaplastic Lymphoma Kinase/genetics , Lymphoma, Large-Cell, Anaplastic/genetics , Lymphoma, Large-Cell, Anaplastic/pathology , Receptor Protein-Tyrosine Kinases/genetics , Protein-Tyrosine Kinases/genetics , Translocation, Genetic , Transcription Factors/genetics , Nuclear Proteins/genetics , Receptors, Antigen, T-Cell/genetics , High-Throughput Nucleotide Sequencing , Cell Cycle Proteins/genetics , Adenosine Triphosphatases/geneticsABSTRACT
Focal cortical dysplasia (FCD) represents the most common cause of drug-resistant epilepsy in adult and pediatric surgical series. However, genetic factors contributing to severe phenotypes of FCD remain unknown. We present a patient with an exceptionally rapid development of drug-resistant epilepsy evolving in super-refractory status epilepticus. We performed multiple clinical (serial EEG, MRI), biochemical (metabolic and immunological screening), genetic (WES from blood- and brain-derived DNA), and histopathological investigations. The patient presented 1 month after an uncomplicated varicella infection. MRI was negative, as well as other biochemical and immunological examinations. Whole-exome sequencing of blood-derived DNA detected a heterozygous paternally inherited variant NM_006267.4(RANBP2):c.5233A>G p.(Ile1745Val) (Chr2[GRCh37]:g.109382228A>G), a gene associated with a susceptibility to infection-induced acute necrotizing encephalopathy. No combination of anti-seizure medication led to a sustained seizure freedom and the patient warranted induction of propofol anesthesia with high-dose intravenous midazolam and continuous respiratory support that however failed to abort seizure activity. Brain biopsy revealed FCD type IIa; this finding led to the indication of an emergency right-sided hemispherotomy that rendered the patient temporarily seizure-free. Postsurgically, he remains on antiseizure medication and experiences rare nondisabling seizures. This report highlights a uniquely severe clinical course of FCD putatively modified by the RANBP2 variant. PLAIN LANGUAGE SUMMARY: We report a case summary of a patient who came to our attention for epilepsy that could not be controlled with medication. His clinical course progressed rapidly to life-threatening status epilepticus with other unusual neurological findings. Therefore, we decided to surgically remove a piece of brain tissue in order to clarify the diagnosis that showed features of a structural brain abnormality associated with severe epilepsy, the focal cortical dysplasia. Later, a genetic variant in a gene associated with another condition, was found, and we hypothesize that this genetic variant could have contributed to this severe clinical course of our patient.
Subject(s)
Brain Diseases , Drug Resistant Epilepsy , Epilepsy , Focal Cortical Dysplasia , Molecular Chaperones , Nuclear Pore Complex Proteins , Status Epilepticus , Child , Child, Preschool , Humans , Male , Disease Progression , DNA , Drug Resistant Epilepsy/genetics , Drug Resistant Epilepsy/surgery , Epilepsy/complications , Midazolam , Status Epilepticus/genetics , Status Epilepticus/surgeryABSTRACT
Molecular assays for translocation detection in different tumors have gradually been incorporated into routine diagnostics. However, conventional methods such as fluorescence in situ hybridization (FISH) and reverse transcriptase-PCR come with several drawbacks. Next-generation sequencing (NGS) can provide in-depth detection of numerous gene alterations. The anchored multiplex PCR assay proved to be a fast and easy-to-analyze approach for routine diagnostics laboratories. Next-generation sequencing-based anchored multiplex PCR technique (Archer FusionPlex Panels) is beneficial in both diagnosis for patient care and in identification of a novel fusion breakpoint in tumors. NGS is useful in identifying targetable molecular changes (point mutations, fusion genes, etc.) in tumors that can serve as a rationale for inclusion of patients with advanced disease in ongoing clinical trials and allow for better risk stratification.
Subject(s)
High-Throughput Nucleotide Sequencing , Neoplasms , Humans , In Situ Hybridization, Fluorescence , Multiplex Polymerase Chain Reaction , Neoplasms/diagnosis , Neoplasms/genetics , Translocation, GeneticABSTRACT
The most important findings revealing pathogenesis, molecular characteristics, genotyping and targeted therapy of gastrointestinal stromal tumors (GISTs) are activated oncogenic mutations in KIT and PDGFRA genes. Imatinib mesylate (IM), which inhibits both KIT and PDGFRA receptors, significantly improved treatment of advanced (metastatic, recurrent, and/or inoperable) GISTs. However, in a significant number of patients the treatment fails due to the primary or secondary resistance to targeted therapy. Most common cause of secondary resistance is a presence of secondary mutations. Approximately 15% of adult patients with GISTs are negative for mutations in KIT or PDGFRA genes. These so-called wild-type GISTs appear to be characterized by other oncogenetic drivers, including mutations in BRAF, RAS, NF1 genes, and subunits of succinate dehydrogenase (SDH) complex. In the present study we investigated 261 tumour specimens from 239 patients with GIST. Primary mutations were detected in 82 % tumor specimens. 66 of them were in KIT, and 16 % in PDGFRA genes. Remaining 18 % were KIT/PDGFRA wild-type. Secondary KIT mutations were detected in 10 from 133 (7 %) patients treated with IM. We examined secondary KIT mutations in exons 13 and 17 and secondary PDGFRA mutation in exon 18 in sixteen progressive tumors and/or metastasis (from overall 22 samples). We identified BRAF V600E point mutation in 4 % of KIT/PDGFRA wild-type GIST patients. Moreover, we analysed SDH complex mutations in 4 younger patients (15, 33, 37, and 45 years old) from 44 patients without KIT, PDGFRA, and BRAF mutations. Two patients (a 37-year old man, and a 33-year old woman) had defects of the SDH complex. Our findings of mutational status of the primary and secondary KIT/PDGFRA mutations in patients with GIST confirm mechanisms of primary and secondary resistance, and also intralesional and interlesional heterogeneity of secondary mutations within and between progressive lesions. Moreover, detection of V600E BRAF mutation and defects of SDH complex in KIT/PDGFRA wild-type GISTs confirm their activation and allow for a selection of targeted therapy.
Subject(s)
Biomarkers, Tumor/genetics , Gastrointestinal Neoplasms/genetics , Gastrointestinal Stromal Tumors/genetics , Mutation , Proto-Oncogene Proteins B-raf/genetics , Proto-Oncogene Proteins c-kit/genetics , Receptor, Platelet-Derived Growth Factor alpha/genetics , Succinate Dehydrogenase/genetics , Adolescent , Adult , Aged , Aged, 80 and over , Antineoplastic Agents/therapeutic use , DNA Mutational Analysis , Drug Resistance, Neoplasm/genetics , Female , Gastrointestinal Neoplasms/drug therapy , Gastrointestinal Neoplasms/pathology , Gastrointestinal Stromal Tumors/drug therapy , Gastrointestinal Stromal Tumors/pathology , Genetic Heterogeneity , Genetic Predisposition to Disease , Humans , Male , Middle Aged , Young AdultABSTRACT
Rituximab maintenance (RM) prolongs survival of elderly patients with mantle cell lymphoma (MCL). Persistent minimal residual disease (MRD) after induction repeatedly correlated with shorter progression-free survival (PFS). However, none of the published studies analyzed patients treated with RM. The main purpose was to analyze prognostic significance of MRD in the elderly patients with newly diagnosed MCL treated according to the recently published observational trial protocol (alternation of R-CHOP and R-cytarabine, 3 + 3 cycles, GovTrial number NCT03054883) at the centers that implemented RM. Minimal residual disease was evaluated by a EuroMRD standardized real-time PCR approach after 3 and 6 cycles of the induction therapy. Prognostic significance of MRD was analyzed in a subcohort of patients treated at the centers that implemented RM as a standard approach. Bone marrow proved to be a significantly more sensitive source for MRD detection than peripheral blood. In either compartment MRD (positive versus negative) after 3 or 6 cycles of the induction therapy did not correlate with PFS. The observed loss of prognostic significance of MRD after the R-CHOP-based induction appears to be a consequence of RM immune control over the residual lymphoma.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/administration & dosage , Lymphoma, Mantle-Cell , Maintenance Chemotherapy , Rituximab/administration & dosage , Aged , Aged, 80 and over , Antibodies, Monoclonal, Murine-Derived/administration & dosage , Cyclophosphamide/administration & dosage , Disease-Free Survival , Doxorubicin/administration & dosage , Female , Follow-Up Studies , Humans , Lymphoma, Mantle-Cell/blood , Lymphoma, Mantle-Cell/drug therapy , Lymphoma, Mantle-Cell/mortality , Male , Middle Aged , Neoplasm, Residual , Prednisone/administration & dosage , Survival Rate , Vincristine/administration & dosageABSTRACT
Implementation of cytarabine into induction therapy became standard of care for younger patients with mantle cell lymphoma (MCL). On the basis of its beneficial impact, many centers incorporated cytarabine at lower doses also into first-line treatments of elderly patients. We conducted a multicenter observational study that prospectively analyzed safety and efficacy of alternating 3 + 3 cycles of R-CHOP and R-cytarabine for newly diagnosed transplant-ineligible MCL patients. A total of 73 patients were enrolled with median age 70 years. Most patients had intermediate (39.7%) and high-risk (50.7%) disease according to MCL international prognostic index. Rituximab maintenance was initiated in 58 patients. Overall response rate reached 89% by positron emission tomography-computed tomography, including 75.3% complete remissions. Two patients (2.7%) did not complete the induction therapy because of toxicity. Three patients (4.1%) were considered nonresponders, which led to therapy change before completion of induction. Estimated progression-free survival and overall survival were 51.3% and 68.6% at 4 years, respectively. Mantle cell lymphoma international prognostic index, bulky disease (≥ 5 cm), and achievement of positron emission tomography-negativity independently correlated with progression-free survival. Grade 3 to 4 hematologic and nonhematologic toxicity was documented in 48% and 20.5% patients, respectively. Alternation of R-CHOP and R-cytarabine represents feasible and very effective regimen for elderly/comorbid MCL patients. This study was registered at GovTrial (clinicaltrials.gov) NCT03054883.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Cytarabine/therapeutic use , Lymphoma, Mantle-Cell/drug therapy , Aged , Aged, 80 and over , Antibodies, Monoclonal, Murine-Derived/pharmacology , Antibodies, Monoclonal, Murine-Derived/therapeutic use , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Cyclophosphamide/pharmacology , Cyclophosphamide/therapeutic use , Cytarabine/pharmacology , Doxorubicin/pharmacology , Doxorubicin/therapeutic use , Female , Humans , Lymphoma, Mantle-Cell/pathology , Male , Middle Aged , Prednisone/pharmacology , Prednisone/therapeutic use , Rituximab , Vincristine/pharmacology , Vincristine/therapeutic useABSTRACT
Chromosomal translocation t(11;14)(q13;q32) is a characteristic molecular marker of mantle cell lymphoma (MCL) and leads to the fusion of the immunoglobulin heavy chain enhancer-promoter with the cyclin D1 gene. Both aberrant cyclin D1 expression and underlying chromosomal aberration may be used as molecular targets for monitoring minimal residual disease (MRD). The present study aims to assess the usefulness of quantitative cyclin D1 gene expression compared to the standardised but more technologically demanding DNA-based method for immunoglobulin heavy chain (IGH) or t(11;14) clone-specific gene rearrangement quantification in a cohort of bone marrow (BM) and peripheral blood (PB) samples from patients with MCL. We simultaneously evaluated DNA-MRD and cyclin D1 expression levels in 234 samples from 57 patients. We observed that both in DNA-MRD positive and negative BM/PB pairs from the same time points the expression levels of cyclin D1 are lower in PB than in BM (median 19×, BM/PB range 0.41-352). The correlation of cyclin D1 transcript levels with DNA-MRD or with flow cytometry was good only in samples with a very high infiltration. In DNA-MRD-negative BM samples, we observed a significant heterogeneity of cyclin D1 expression (in the range of more than three orders of magnitude). This is in contrast to previous reports demonstrating the usefulness of cyclin D1 for MRD monitoring that did not use DNA-based method as a reference. In PB, the specificity of cyclin D1 expression was better due to a lower physiological background. In conclusion, we show that cyclin D1 is unsuitable for MRD monitoring in BM.
Subject(s)
Cyclin D1/genetics , Lymphoma, Mantle-Cell/diagnosis , Lymphoma, Mantle-Cell/pathology , Monitoring, Physiologic/methods , Biomarkers, Tumor/analysis , Biomarkers, Tumor/genetics , Bone Marrow/metabolism , Bone Marrow/pathology , Cyclin D1/metabolism , Female , Gene Expression Regulation, Neoplastic , Humans , Lymphoma, Mantle-Cell/genetics , Male , Middle Aged , Neoplasm, Residual , RNA, Messenger/analysisABSTRACT
Complex laboratory investigation is necessary for the diagnosis and relevant classification of lymphomas. The classical histopathological morphology and cytology investigation is essential, but further investigations such as immunohistochemistry and fluorescence in situ hybridization are necessary. It is also important to employ flow cytometry as a method of investigation running synchronously or preceding the histopathological approach. Last but not least, the investigation of nucleic acids in lymphoma by molecular approaches is necessary and has become an everyday practice. Communication between pathologists and clinical colleagues (oncologists, hematologists, internal medicine specialists and radiologists) is very important. We demonstrate the necessity of a complex diagnostic approach to lymphomas and an appropriate interpretation of all laboratory investigations giving examples of eight patients with various types of lymphomas. In some cases, it is impossible to properly diagnose a lymphoma without molecular investigation. Occasionally, the results of the molecular investigation may be misleading and/or may be inaccurately interpreted, leading to an incorrect conclusion. For that reason, it is very important to incorporate all specialized laboratories and their teams under one roof (preferably that of pathology departments), enabling tight and daily cooperation between the specialists. This is the way to reach a precise diagnosis in a majority of cases, as well as how to comply with clinical expectations of properly classified lymphomas for a targeted therapy of patients.
Subject(s)
Lymphoma/diagnosis , Pathology, Clinical/methods , Flow Cytometry , Humans , Immunohistochemistry , In Situ Hybridization, Fluorescence , Male , Referral and ConsultationABSTRACT
Soft tissue tumors (SSTs) constitute a broad spectrum of neoplasms with diverse biological properties. Rare or unusual types are often difficult to classify. Recent studies show, that a significant subset of SSTs including many types of sarcomas are associated with specific genetic changes such as chromosomal translocations producing chimeric genes, which play a role in the pathogenesis of SSTs. Because SSTs represent a diagnostically challenging group of tumors, molecular-genetic techniques (FISH or PCR) are useful as supplementary and/or confirmatory diagnostic tools. In the present paper we demonstrate the usefulness of a combined diagnostic approach using the tools of classical histopathology and immunohistochemistry together with the molecular diagnostic approach in selected nosologic entites.
Subject(s)
Pathology, Molecular/methods , Soft Tissue Neoplasms/diagnosis , Soft Tissue Neoplasms/genetics , Humans , Immunohistochemistry , In Situ Hybridization, Fluorescence , Polymerase Chain Reaction , Sarcoma/diagnosis , Sarcoma/geneticsABSTRACT
Minimal residual disease (MRD) monitoring is of a great importance for the patients and often determines their future therapy. At present, MRD detection in patients suffering from hematologic malignancies is included in most of treatment protocols. However, it appears that the MRD detection, sometimes described as circulating tumor cells or minimal disseminated disease, is important in patients with solid tumors as well. Molecular MRD monitoring principles include detection of specific DNA, RNA or protein markers of tumour cells, which are not present in bone marrow and/or peripheral blood cells. High specificity and sensitivity of this specific molecular tumour marker is necessary. Quantitative MRD monitoring by means of molecular methods can determine the decrease or increase of MRD level. In this review, we describe different molecular methods used, an overview on their advantages, limitations, and the sample quality and processing requirements. A summary of molecular markers employed in hematological and non-hematological diseases is also presented.
Subject(s)
Biomarkers, Tumor/analysis , Neoplasm, Residual/diagnosis , Neoplastic Cells, Circulating/pathology , Female , HumansSubject(s)
Oncogene Proteins, Fusion/genetics , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , Core Binding Factor Alpha 2 Subunit , Humans , In Situ Hybridization, Fluorescence , Infant, Newborn , Oncogene Proteins, Fusion/blood , Precursor Cell Lymphoblastic Leukemia-Lymphoma/blood , RNA, Messenger/blood , RNA, Messenger/genetics , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
A clinical course of patients with mantle cell lymphoma (MCL) is aggressive, and the disease is rarely curable. Proliferation rate is the most important prognostic factor. We developed a novel, reliable, rapid, and routinely applicable approach allowing a precise quantitative assessment of three proliferation markers, Ki-67, topoisomerase IIalpha, and TPX2. A total of 95 lymphoma specimens were measured in the study by real-time reverse transcription PCR (RQ-RT-PCR). We tested the reproducibility and accuracy of the assay and correlated the results with the immunohistochemical staining of the corresponding proteins. The results obtained indicated individual variability of the mRNA expression levels, reflecting heterogeneity of the proliferation rate in individual patients. In general, we observed the highest mRNA expression in the group of Burkitt lymphomas and the lowest in patients with reactive lymphadenopathies. We found increased proliferation rate in MCLs with high cyclin D1 mRNA, indicating a quantitative control of the cell cycle. We observed a correlation between mRNA expression level and the immunohistochemical staining of corresponding proteins, which significantly argues for the prognostic significance of the mRNA expression measuring. We confirmed the accuracy of the current assay for a precise quantitative examination of the proliferation activity. Real-time RT-PCR provides a novel approach applicable for clinical trials, and it represents a potent approach allowing to stratify MCL patients for entry into clinical trials according to the expression of the proliferation signature genes in their tumors. This approach may contribute to improved and individualized therapeutic options respecting the individual progression risk of patients with MCL.
Subject(s)
Antigens, Neoplasm/analysis , Cell Cycle Proteins/analysis , DNA Topoisomerases, Type II/analysis , DNA-Binding Proteins/analysis , Ki-67 Antigen/analysis , Lymphoma, Mantle-Cell/pathology , Microtubule-Associated Proteins/analysis , Nuclear Proteins/analysis , Polymerase Chain Reaction/methods , Antigens, Neoplasm/genetics , Cell Cycle Proteins/genetics , Cell Proliferation , Cyclin D1/analysis , Cyclin D1/genetics , DNA Topoisomerases, Type II/genetics , DNA-Binding Proteins/genetics , Humans , Immunohistochemistry , Ki-67 Antigen/genetics , Lymphoma, Mantle-Cell/chemistry , Microtubule-Associated Proteins/genetics , Nuclear Proteins/genetics , Reproducibility of ResultsABSTRACT
Synovial sarcoma (SS) is a highly aggressive soft tissue sarcoma that causes death in more than half of the affected patients. An immunohistochemical and molecular study of the BCL2, MKI67, and CCND1 genes (expressing the BCL2, KI-67, and cyclin D1 proteins, respectively) was performed to determine the expression profiles in correlation with mRNA levels, and to assess the possible utility of these genes as a potential target for the treatment. Cyclin D1 staining was identified in 18 of 30 cases (60%), and CCND1 mRNA was overexpressed in 15 of 32 cases (47%). KI-67 nuclear immunoreactivity was found in 14 of 29 cases (48%), and MKI67 mRNA was overexpressed in 12 of 32 cases (37.5%). The high level of MKI67 mRNA was observed predominantly in monophasic SS. BCL2, a negative regulator of apoptosis, was expressed in all 32 cases. The intensity of the BCL2 protein expression correlated well with the mRNA level (P<0.0001). The high level of BCL2 mRNA correlated with a high level of CCND1 mRNA, but not with MKI67 mRNA level. Despite advances in therapy of sarcomas, the prognosis of patients with SS remains unfavorable, and a search for an improved therapy approach remains necessary. The strong immunopositivity of BCL2 in SS correlates well with a high level of BCL2 mRNA. Treatment with antisense BCL2 (G3139) may therefore represent an appropriate alternative therapy for patients with BCL2-positive synovial sarcomas.
Subject(s)
Cyclin D1/metabolism , Ki-67 Antigen/metabolism , Proto-Oncogene Proteins c-bcl-2/metabolism , Sarcoma, Synovial/metabolism , Soft Tissue Neoplasms/metabolism , Adolescent , Adult , Aged , Analysis of Variance , Cell Nucleus/chemistry , Cell Nucleus/metabolism , Child , Child, Preschool , Cyclin D1/genetics , Female , Humans , Immunohistochemistry , Infant , Ki-67 Antigen/genetics , Male , Middle Aged , Proto-Oncogene Proteins c-bcl-2/genetics , RNA, Messenger/analysis , RNA, Messenger/genetics , RNA, Neoplasm/analysis , Reverse Transcriptase Polymerase Chain Reaction , Sarcoma, Synovial/genetics , Soft Tissue Neoplasms/genetics , Statistics, NonparametricABSTRACT
In mantle cell lymphoma (MCL), minimal residual disease (MRD) is an indicator of the disease outcome. Quantitative methods used so far do not provide a suitable molecular marker in 30-70% patients with MCL (depending on the technique used). We tested cyclin D1 as a marker for quantitative MRD monitoring. The real-time PCR of cyclin D1 mRNA was performed in 144 bone marrow (BM) specimens including 95 BMs from MCL patients, 39 BMs from patients with other B-cell non-Hodgkin's lymphomas and 10 BMs from healthy volunteer donors. In 73 BMs obtained from 20 MCL patients we examined the cyclin D1 level during the treatment and follow-up period. We detected a cyclin D1 overexpression exclusively in BMs infiltrated with MCL, including minimal residual infiltration. Dynamics of cyclin D1 correlated with the patient's clinical status in 69/73 BMs. Individual monitoring of patients during the disease course showed cyclin D1 quantitative changes accompanying either the disease relapse or a successful treatment response or the disease-free survival (remission) and it showed a predictive significance. Cyclin D1 detection is a promising approach for the quantitative MRD monitoring in MCL patients, and the individual monitoring of the cyclin D1 dynamics represents a suitable indicator of the disease course.
Subject(s)
Biomarkers, Tumor/metabolism , Bone Marrow/metabolism , Cyclin D1/metabolism , Lymphoma, Mantle-Cell/diagnosis , Lymphoma, Mantle-Cell/metabolism , Adult , Aged , Case-Control Studies , Cyclin D1/genetics , Disease-Free Survival , Female , Gene Expression Regulation, Neoplastic , Humans , Male , Middle Aged , Neoplasm, Residual/diagnosis , Neoplasm, Residual/metabolism , Polymerase Chain Reaction , Predictive Value of Tests , Prognosis , RNA, Messenger/metabolism , Sensitivity and SpecificityABSTRACT
Wilms' tumor gene 1 (WT1) is overexpressed in various hematological malignancies and has been proposed as a target for minimal residual disease (MRD) detection and for immunotherapy. Although WT1 is known as a key molecule for tumor cell proliferation, the expression pattern of WT1 in leukemic cells in dependency of proliferation has not yet been investigated. Furthermore, WT1 expression was mostly studied by reverse transcriptase PCR and the expression of WT1 protein has not been extensively studied. Here, we analyzed WT1 protein expression in the human myeloid leukemia cell lines K562 and HL-60 by indirect immunofluorescence and flow cytometry. Both cell lines exhibited varying nuclear WT1 immunoreactivity pointing to a cell cycle-dependent and/or proliferation-dependent WT1 expression. In rapidly proliferating cells high levels of WT1 protein were detected by flow cytometry. A reduced proliferation rate was associated with a low WT1 protein expression and an accumulation of cells in G(0)/G(1) phase. During G(0)/G(1) phase cells expressed WT1 at a lower level than in S or G(2)/M phase. Moreover, WT1 expression was diminished in all cell cycle phases in slowly proliferating cells. We conclude that WT1 protein expression is dependent on the cell cycle phase as well as on the proliferation rate. This finding might be relevant for MRD studies and immunotherapeutic strategies targeting WT1.
Subject(s)
Cell Proliferation , Leukemia, Myeloid/metabolism , WT1 Proteins/metabolism , Cell Cycle , Flow Cytometry , Fluorescent Antibody Technique, Indirect , HL-60 Cells , Humans , K562 Cells , Leukemia, Myeloid/pathologyABSTRACT
Cyclin D1 overexpression as a result of t(11;14) is a specific marker for diagnosis of mantle cell lymphoma (MCL) and plays an important role in MCL pathogenesis. To set a highly reliable cutoff value that discriminates MCL from other B-cell lymphoproliferative disorders, we performed a retrospective study of cyclin D1 expression. We established cyclin D1 expression level in 116 frozen and formalin-fixed, paraffin-embedded primary tumors from patients diagnosed with a variety of B-cell lymphoproliferative disorders. We used real time quantitative reverse-transcription polymerase chain reaction to quantify levels of cyclin D1 mRNA. The range of cyclin D1 expression in MCLs exceeded the range found in other lymphomas and reactive lymph nodes by a considerable margin. Cyclin D1 overexpression was found in 60/61 MCLs and in none of the other lymphomas, except for 12/19 mucosa-associated lymphoid tissue lymphomas from the lungs and stomach, which also revealed cyclin D1 overexpression. As epithelial tissues are known to express cyclin D1, an admixture of non-neoplastic epithelial cells present in the extranodal specimens probably influenced the quantitative reverse-transcription polymerase chain reaction result. Quantitative cyclin D1 monitoring provides a diagnostic test and an approach for studying MCL pathogenesis and may be of clinical importance.
Subject(s)
Genes, bcl-1 , Lymphoma, Mantle-Cell/diagnosis , Lymphoproliferative Disorders/diagnosis , RNA, Messenger/analysis , Adult , Aged , Aged, 80 and over , Child , Diagnosis, Differential , Female , Gene Expression Regulation, Neoplastic , Humans , In Situ Hybridization, Fluorescence , Lymph Nodes/metabolism , Lymphoma, B-Cell, Marginal Zone/diagnosis , Lymphoma, B-Cell, Marginal Zone/genetics , Lymphoma, B-Cell, Marginal Zone/metabolism , Lymphoma, Mantle-Cell/genetics , Lymphoma, Mantle-Cell/metabolism , Lymphoproliferative Disorders/genetics , Lymphoproliferative Disorders/metabolism , Male , Middle Aged , Polymerase Chain Reaction , RNA, Messenger/metabolism , Sensitivity and SpecificityABSTRACT
Anaplastic large cell lymphoma (ALCL) represents a heterogeneous group of malignant lymphoproliferative diseases with a consistent expression of the cytokine receptor CD30. ALCL is frequently associated with a NPM/ALK fusion gene which is found in up to 75% of pediatric ALCLs. Real-time quantitative RT-PCR (RQ-RT-PCR) of NPM/ALK and CD30 gene expression was employed to analyze minimal residual disease (MRD) in 10 patients with NPM/ALK positive ALCL in 79 follow-up bone marrow (BM) and/or peripheral blood (PB) samples. In all BM samples from relapses and/or closely before a relapse, BM samples revealed NPM/ALK and CD30 positivity in at least one of the iliac BM trephines. Five out of nine relapses were preceded or were accompanied by minimally half log increased NPM/ALK levels in the BM. We found that RQ-RT-PCR of the CD30 expression is not suitable for MRD detection--only two relapses were accompanied by an increase of the CD30 level above a level which was detected in BM/PB samples from healthy individuals. RQ-RT-PCR of NPM/ALK expression is a promising and rapid approach for monitoring MRD.