Polymerase Chain Reaction In Patients Research Articles

Primary Effusion Lymphoma: Cytopathologic Diagnosis Using In Situ Molecular Genetic Analysis for Human Herpesvirus 8

Primary effusion lymphoma is a form of diffuse large B-cell lymphoma with neoplastic cells largely limited to proliferation within major body cavities. Human herpes virus-8 is both integral to and required for an unequivocal diagnosis of primary effusion lymphoma. Prior methods for virus identification include DNA extraction with Southern blot analysis or in situ hybridization from paraffin-embedded samples. Our aim is to examine the utility of human herpesvirus-8 identification performed directly on smears from effusion samples by reverse transcriptase in situ polymerase chain reaction in patients with primary effusion lymphoma. Smears and cell block of body cavity fluids from five patients with effusions (three pleural, one peritoneal, and one both pleural and peritoneal) were examined microscopically by conventional Papanicolaou and Romanowsky (Diff-Quik) staining, and by reverse transcriptase in situ polymerase chain reaction for human herpesvirus-8 detection. In situ hybridization was performed also for Epstein-Barr virus (EBER-1, -2), T-cell receptor-β, and kappa (κ) and lambda (λ) mRNA in all cases. Five adults ranged from 40–81 years of age. Three adults were HIV positive, one was a renal transplant recipient, and the oldest patient (Case 3) had the unusual distinction of a normal immune status. Two of three HIV-seropositive patients had concurrent Kaposi sarcoma. All samples were cytologically similar with lymphocytes having large-cell, plasmablastic, and immunoblastic morphology. Malignant cells from effusions were as follows: human herpesvirus-8 positive (all five cases), exhibited κ monoclonal light chain (five cases), Epstein-Barr virus positive (three cases), and T-cell β-gene receptor positive (two cases). Diffuse large B-cell lymphoma was evident in one peritoneal nodule (<10% human herpesvirus-8 positive cells in contrast to >90% positive in effusions, all κ positive). Six other tissue specimens (lung, bone marrow, spleen, lymph node) were human herpesvirus-8 negative, and showed no evidence of lymphoma. Reverse transcriptase in situ polymerase chain reaction demonstrated near-complete restriction of human herpesvirus-8–infected malignant lymphoid cells to those in body cavities. Definitive diagnosis of primary effusion lymphoma is possible directly from cytologic smears/cell block by combining cytologic morphology with reverse transcriptase in situ polymerase chain reaction detection of human herpesvirus-8.

Primary Effusion Lymphoma: Cytopathologic Diagnosis Using In Situ Molecular Genetic Analysis for Human Herpesvirus 8

Primary effusion lymphoma is a form of diffuse large B-cell lymphoma with neoplastic cells largely limited to proliferation within major body cavities. Human herpes virus-8 is both integral to and required for an unequivocal diagnosis of primary effusion lymphoma. Prior methods for virus identification include DNA extraction with Southern blot analysis or in situ hybridization from paraffin-embedded samples. Our aim is to examine the utility of human herpesvirus-8 identification performed directly on smears from effusion samples by reverse transcriptase in situ polymerase chain reaction in patients with primary effusion lymphoma. Smears and cell block of body cavity fluids from five patients with effusions (three pleural, one peritoneal, and one both pleural and peritoneal) were examined microscopically by conventional Papanicolaou and Romanowsky (Diff-Quik) staining, and by reverse transcriptase in situ polymerase chain reaction for human herpesvirus-8 detection. In situ hybridization was performed also for Epstein-Barr virus (EBER-1, -2), T-cell receptor-β, and kappa (κ) and lambda (λ) mRNA in all cases. Five adults ranged from 40–81 years of age. Three adults were HIV positive, one was a renal transplant recipient, and the oldest patient (Case 3) had the unusual distinction of a normal immune status. Two of three HIV-seropositive patients had concurrent Kaposi sarcoma. All samples were cytologically similar with lymphocytes having large-cell, plasmablastic, and immunoblastic morphology. Malignant cells from effusions were as follows: human herpesvirus-8 positive (all five cases), exhibited κ monoclonal light chain (five cases), Epstein-Barr virus positive (three cases), and T-cell β-gene receptor positive (two cases). Diffuse large B-cell lymphoma was evident in one peritoneal nodule (<10% human herpesvirus-8 positive cells in contrast to >90% positive in effusions, all κ positive). Six other tissue specimens (lung, bone marrow, spleen, lymph node) were human herpesvirus-8 negative, and showed no evidence of lymphoma. Reverse transcriptase in situ polymerase chain reaction demonstrated near-complete restriction of human herpesvirus-8–infected malignant lymphoid cells to those in body cavities. Definitive diagnosis of primary effusion lymphoma is possible directly from cytologic smears/cell block by combining cytologic morphology with reverse transcriptase in situ polymerase chain reaction detection of human herpesvirus-8.

Ovarian tumor cell detection in peripheral blood progenitor cells harvests by RT-PCR

Background. To evaluate the frequency of tumor cell contamination in autologous peripheral-blood progenitor cells from patients with ovarian cancer, and to determine the impact of infusing such cells on relapses after high-dose chemotherapy. Methods. Seventy-three samples of peripheral-blood progenitor cells from 24 ovarian cancer patients were studied for contaminated tumor cells by cytokeratin 7 and cytokeratin 20 reverse transcriptase polymerase chain reaction. Results. Tumor cell contamination in peripheral-blood progenitor cells was detected in 11 of 24 patients (46%) and, among these, in four of 11 patients who received transplantations of peripheral-blood progenitor cells. There was no trend towards longer relapse-free survival in patients infused with cytokeratin-negative peripheral-blood progenitor cells as compared with positive ones. Interestingly, two of four patients who received transplantations of peripheral-blood progenitor cells containing tumor cells were free from progression at 20 and 41 months after transplantation. Conclusion. Tumor cell contamination of peripheral-blood progenitor cells was frequently noted by transcriptase polymerase chain reaction in patients with ovarian cancer. The biological and clinical significance of this finding remains to be elucidated.

Detection of Mycobacterium Tuberculosis in Bronchial Specimens Using a Polymerase Chain Reaction in Patients with Bronchial Anthracofibrosis

Background : To Investigate the association between bronchial anthracofibrosis (AF) and tuberculosis (TB), and the clinical utility of a polymerase chain reaction (PCR) on bronchial specimens for rapid diagno-sis of active pulmonary TB in patients with bronchial AF. Method : Thirty patients (25 women and 5 men ranging in age from 53 to 88), who were diagnosed with bronchial AF by a bronchoscopic exami-nation, were enrolled in this study. PCR targeting the IS6110 segment of Mycobacterium tuberculosis was performed on the bronchial wash fluid and anthracofibrotic bronchial tissue. The PCR results were compared with the bacteriological, histological, and clinical findings. Results : Eighteen of the 30 patients (60%) were associated with TB, nine of whom were confirmed as having active TB. The remaining 9 had a past history of TB. The sputum or bronchial aspirate AFB smear, culture, and histological findings were positive in 4 (13%), 9 (30%), and 5 (17%) patients, respectively. PCR of the AF tissue and bronchial wash fluid was positive in 5 (17%) and 11 (37%) of the 30 patients, respectively. PCR was more sensitive than the AFB smears for diagnosing pulmonary TB (22 % us 89 %, respectively, p

Decreased expression of transcription factor GATA-2 in haematopoietic stem cells in patients with aplastic anaemia.

Aplastic anaemia is characterized by reduced haematopoiesis resulting in pancytopenia. It has been speculated that there is an injury in haematopoietic stem cells in the bone marrow; however, the precise nature of the injury has not been elucidated. In this study, the levels of expression of mRNAs for three transcription factors, GATA-2, SCL and AML1, which function in the early stages of haematopoiesis, were examined by quantitative polymerase chain reaction in patients with aplastic anaemia, idiopathic thrombocytopenic purpura (ITP) and normal subjects. Among these factors, expression of GATA-2 mRNA in purified CD34-positive cells was markedly decreased in aplastic anaemia compared with that in ITP and in normal subjects. The expression levels of SCL and AML1 mRNA in CD34-positive cells in aplastic anaemia were not different from those in normal subjects. When the expression of GATA-2 protein in CD34-positive cells was examined by immunocytochemical analysis, the percentage of GATA-2-positive cells in aplastic anaemia was lower than that in normal subjects. These findings strongly suggest that there is an aberrant expression of transcription factors in stem cells in aplastic anaemia, which may be responsible for the development of the disease.

Prevalence of circulating Trypanosoma cruzi detected by polymerase chain reaction in patients with Chagas’ cardiomyopathy

In recent years, the polymerase chain reaction technique (PCR)1,2 has demonstrated higher sensitivity than microscopy3 and xenodiagnosis4 for the detection of Trypanosoma cruzi (T. cruzi), providing a novel tool for our understanding of Chagas’ disease. It has been reported that 81% to 100% of seropositive patients living in rural endemic areas have circulating T. cruzi detected by PCR.5–8 Also, it has been observed in patients with Chagas’ disease that the parasite deoxyribonucleic acid (DNA) sequence is amplified more frequently from tissues showing microscopic evidence of inflammation.9,10 The detection of T. cruzi by PCR from blood samples or cardiac tissue of patients with Chagas’ disease has revived the old controversy regarding the role of the parasite in the mechanisms leading to chronic cardiomyopathy.

Fibromyalgia, chronic fatigue syndrome, and myofascial pain syndrome.

Fibromyalgia and widespread pain were common in Gulf War veterans with unexplained illness referred to a rheumatology clinic. Increased tenderness was demonstrated in the postmenstrual phase of the cycle compared with the intermenstrual phase in normally cycling women but not in users of oral contraceptives. Patients with fibromyalgia had high levels of symptoms that have been used to define silicone implant-associated syndrome. Tender points were found to be a common transient finding associated with acute infectious mononucleosis, but fibromyalgia was an unusual long-term outcome. The common association of fibromyalgia with other rheumatic and systemic illnesses was further explored. A preliminary study revealed a possible linkage of fibromyalgia to the HLA region. Patients with fibromyalgia were found to have an impaired ability to activate the hypothalamic pituitary portion of the hypothalamic pituitary adrenal axis as well as the sympathoadrenal system, leading to reduced corticotropin and epinephrine response to hypoglycemia. Much interest has been expressed in the literature on the possible role of autonomic dysfunction in the development or exacerbation of fatigue and other symptoms in chronic fatigue syndrome. Mycoplasma genus and mycoplasma fermentans were detected by polymerase chain reaction in patients with chronic fatigue syndrome. It was reported that myofascial temporomandibular disorder does not run in families. No major therapeutic trials in fibromyalgia, chronic fatigue syndrome, or myofascial pain syndrome were reported over the past year. The effectiveness of cognitive behavioral therapy and behavior therapy for chronic pain in adults was emphasized. A favorable outcome of fibromyalgia and chronic fatigue syndrome in children and adolescents was reported.

Amplicor enterovirus polymerase chain reaction in patients with aseptic meningitis: a sensitive test limited by amplification inhibitors.

To evaluate the sensitivity and specificity of reverse-transcription polymerase chain reaction (RT-PCR) and routine cell culture for the detection of enterovirus in cerebrospinal fluid. Thirty-eight cerebrospinal fluid specimens were included. Cell culture was inoculated immediately and incubated for 14 days. An aliquot was kept frozen for Amplicor RT-PCR. Chart review was performed to determine the validity of the results. Nine of 38 specimens were positive for enterovirus by culture, and 14 were positive by RT-PCR. There were 7 discrepancies between the 2 methods. Six specimens were positive by RT-PCR and negative by the culture method. The 1 culture-positive but RT-PCR--negative specimen was determined to contain PCR inhibitors. All discrepant results were confirmed as true positives by chart review. Patients whose cerebrospinal fluid was negative by both methods had a final diagnosis other than enterovirus infection. Amplicor PCR is more sensitive than cell culture (93.3% vs. 60%) and is very specific. With the incorporation of appropriate controls for the detection of amplification inhibitors, RT-PCR could be a valuable tool in the diagnosis of enteroviral meningitis.

Molecular diagnosis of thiopurine S-methyltransferase deficiency: genetic basis for azathioprine and mercaptopurine intolerance.

Thiopurine S-methyltransferase (TPMT) catalyzes the S-methylation (that is, inactivation) of mercaptopurine, azathioprine, and thioguanine and exhibits genetic polymorphism. About 10% of patients have intermediate TPMT activity because of heterozygosity, and about 1 in 300 inherit TPMT deficiency as an autosomal recessive trait. If they receive standard doses of thiopurine medications (for example, 75 mg/m2 body surface area per day), TPMT-deficient patients accumulate excessive thioguanine nucleotides in hematopoietic tissues, which leads to severe and possibly fatal myelosuppression. To elucidate the genetic basis and develop molecular methods for the diagnosis of TPMT deficiency and heterozygosity. Diagnostic test evaluation. Research hospital. The TPMT phenotype was determined in 282 unrelated white persons, and TPMT genotype was determined in all persons who had intermediate TPMT activity (heterozygotes) and a randomly selected, equal number of persons who had high activity. In addition, genotype was determined in 6 TPMT-deficient patients. Polymerase chain reaction (PCR) assays were developed to detect the G238C transversion in TPMT*2 and the G460A and A719G transitions in TPMT*3 alleles. Radiochemical assay was used to measure TPMT activity. Mutations of TPMT were identified in genomic DNA, and the concordance of TPMT genotype and phenotype was determined. 21 patients who had a heterozygous phenotype were identified (7.4% of sample [95% CI, 4.7% to 11.2%]). TPMT*3A was the most prevalent mutant allele (18 of 21 mutant alleles in heterozygotes; 85%); TPMT*2 and TPMT*3C were more rare (about 5% each). All 6 patients who had TPMT deficiency had two mutant alleles, 20 of 21 patients (95% [CI, 76% to 99.9%]) who had intermediate TPMT activity had one mutant allele, and 21 of 21 patients (100% [CI, 83% to 100%]) who had high activity had no known TPMT mutation. Detection of TPMT mutations in genomic DNA by PCR coincided perfectly with genotypes detected by complementary DNA sequencing. The major inactivating mutations at the human TPMT locus have been identified and can be reliably detected by PCR-based methods, which show an excellent concordance between genotype and phenotype. The detection of TPMT mutations provides a molecular diagnostic method for prospectively identifying TPMT-deficient and heterozygous patients.

Prognosis Value of Residual Disease Monitoring by Polymerase Chain Reaction in Patients With CBFβ/MYH11-Positive Acute Myeloblastic Leukemia

To the Editor: Pericentric inversion of chromosome 16, translocation (16; 16), and del(16q), resulting in a chimeric fusion of the CBFβ and MYH11 genes, are typically seen in the M4Eo French-American-British (FAB) subset of acute myelogenous leukemias (AML). Karyotypic detection of these

Detection of submicroscopic lymph node metastases with polymerase chain reaction in patients with malignant melanoma.

Detection of submicroscopic lymph node metastases with polymerase chain reaction in patients with malignant melanoma.

Detection of EBV in tumor tissue and peripheral blood by polymerase chain reaction in patients with nasopharyngeal carcinoma.

Sixty-nine untreated patients with a pathologically verified nasopharyngeal carcinoma were selected for the study of detection of EBV in nasopharyngeal tumor and peripheral blood by polymerase chain reaction (PCR). Primers were directed to conserved regions of EBV genome encoding capsid protein gp 220 (Bam HI L region). A distinct 239 bp band of the PCR products indicated the presence of EBV. Results showed that EBV DNA was obtained in 91.3% of 69 NPC patients and 16.7% of 18 healthy individuals on nasopharyngeal tissue, and the difference was statistically significant between the above two groups. Nevertheless, no EBV DNA was verified from the mononuclear cells of the peripheral blood of the two groups. There was no relationship between the positive EBV DNA and the titer of serological markers. Meanwhile, the positive EBV DNA did not show any relationship with the histology type, tumor and nodal bulk, or even metastasis. Although a high positive rate of EBV DNA was detected in nasopharyngeal tumor of patients, additional environmental and genetic factors must still be considered.

Clinical significance of bone marrow metastases as detected using the polymerase chain reaction in patients with breast cancer undergoing high-dose chemotherapy and autologous bone marrow transplantation.

The present study evaluates the clinical significance of detection of cytokeratin 19 (K19) in the bone marrow of patients with breast cancer undergoing high-dose chemotherapy (HDCT) and autologous bone marrow transplantation (ABMT). We studied retrospectively cryopreserved bone marrow aspirates from 83 patients with high-risk stage II, III, and IV breast cancer obtained before bone marrow harvest but after induction chemotherapy. All samples were histologically negative for metastases. Polymerase chain reaction (PCR) for K19 was performed according to methods described previously and results were correlated with the probability of relapse following HDCT and ABMT. The incidence of occult metastases as defined by PCR for K19 message was 52% for 19 stage II, 57% for 14 stage III, and 82% for 50 stage IV patients (two-tailed P = .0075, chi 2 test). The probability of relapse at 3 years after ABMT was 32% and 94% for K19-positive stage II/III and stage IV patients, respectively, versus 10% and 14% for K19-negative stage II/III and stage IV patients, respectively. The difference was significant for stage IV patients (two-tailed P = .0002). It has been shown that PCR is a highly sensitive method to detect K19 message in the bone marrow. The incidence of K19 positivity in bone marrow increases significantly with advancing stage. In patients with breast cancer, especially metastatic breast cancer, undergoing HDCT and ABMT, the presence of K19 is associated with a poor prognosis.

Clinical relevance of residual disease monitoring by polymerase chain reaction in patients with ALL‐1/AF‐4 positive‐acute lymphoblastic leukaemia

In this study we used reverse transcriptase-polymerase chain reaction (RT-PCR) for the longitudinal monitoring of minimal residual disease in 12 patients with All-1/AF-4 positive ALL. Of these, seven also showed at presentation a typical t(4;11) cytogenetic translocation. Seven patients were infants <18 months of age and five were adults. Eleven patients were treated with high-dose intensive induction and consolidation chemotherapy without bone marrow transplantation and one received conservative treatment due to poor performance status. Three had resistant disease, four relapsed within 12 months after achieving complete remission, and five are in continuous complete remission (CCR) at 32, 39, 52, 53 and 61 months from diagnosis, respectively. The sequential analysis of the ALL-1/AF-4 hybrid transcript showed a persistently negative RT-PCR in the five CCR long-term survivors. The PCR analysis resulted persistently positive in the remaining seven cases, including the four cases who relapsed after the achievement of clinical CR. These data emphasize the clinical relevance of PCR monitoring analysis in t(4;11) ALL patients and should be considered in order to better determine variable post-remission treatment according to risk prediction.

Detection of Serum HCV RNA by Polymerase Chain Reaction in Patients with Chronic Hepatitis C

Hepatitis C virus (HCV) RNA was isolated from the sera of 15 anti-HCV positive patients. cDNAs were synthesized by reverse transcription and amplified by two round nested polymerase chain reaction (PCR). HCV infection was confirmed by the detection of 256 base pairs long DNA fragments on the 2% agarose gels in the electrophoresis of PCR products with the positive and negative controls. 10 of 15 infection were confirmed by this molecular technique.

Persistence of RAR alpha-PML fusion mRNA detected by reverse transcriptase polymerase chain reaction in patients in long-term remission of acute promyelocytic leukaemia.

Acute promyelocytic leukaemia (APL) is characterized by t(15;17), which results in the formation of two chimaeric genes, PML-RAR alpha and RAR alpha-PML. PML-RAR alpha transcripts have been detected in all cases of APL whilst those of RAR alpha-PML have been detected in only about 67% of cases. We have used reverse transcriptase polymerase chain reaction (RT-PCR) to detect both fusion transcripts serially in 18 patients in remission of APL after chemotherapy and bone marrow transplantation. All patients were negative for PML-RAR alpha, whereas in six patients (remission 3-9 years) RAR alpha-PML was consistently detected. Only one patient at remission showed the 5' breakpoint RAR alpha-PML, with the rest showing the 3' breakpoint 144 bp RAR alpha-PML. The level of sensitivity for detecting RAR alpha-PML was some 10-fold higher than that for PML-RAR alpha. Serial negative tests for PML-RAR alpha have been correlated with durable remissions, suggesting possible eradication of residual leukaemia in APL. Our results, however, show persistence of t(15;17) cells expressing RAR alpha-PML fusion mRNA in patients in long-term remission of APL. They indicate that patients considered clinically 'cured' of APL still have molecular evidence of minimal residual disease and also provide further insight into the biology of acute myeloid leukaemia.

Detection of mycobacterium paratuberculosis by polymerase chain reaction in patients with Crohn's disease versus controls: An updated report

Detection of mycobacterium paratuberculosis by polymerase chain reaction in patients with Crohn's disease versus controls: An updated report

Detection of t(8;21) by reverse transcriptase polymerase chain reaction in patients in remission of acute myeloid leukaemia type M2 after chemotherapy or bone marrow transplantation

Seven patients with acute myeloid leukaemia (AML) type M2 and t(8;21), treated with intensive chemotherapy followed in two cases by allogeneic or autologous bone marrow transplant (BMT), were tested for the presence of transcripts of the characteristic chimaeric gene AML1/ETO by reverse transcriptase polymerase chain reaction (RT-PCR) at serial intervals during remission. Six patients, including both BMT patients, demonstrated persistence of t(8;21), one being consistently negative in peripheral blood but bone marrow positive. Bone marrow may, therefore, be more reliable than peripheral blood for detecting residual t(8;21) cells. Our results show persistence of t(8;21) cells in AML-M2 patients following chemotherapy or BMT, some of whom were in long-term remission.

Detection of Submicroscopic Lymph Node Metastases with Polymerase Chain Reaction in Patients with Malignant Melanoma

The presence or absence of lymph node metastases in patients with malignant melanoma is the most powerful prognostic factor for predicting survival. If regional nodal metastases are found, the 5-year survival for the patient decreases approximately 50%. If the presence or absence of regional nodal metastases will determine which patients receive formal dissections or which patients enter adjuvant trials, then a technique is needed to accurately screen lymph node samples for occult disease. Routine histopathologic examination routinely underestimates the number of patients with metastases. This study was initiated to develop a highly sensitive clinically applicable method to detect micrometastases by examining lymph nodes for the presence of tyrosinase messenger RNA (mRNA). The hypothesis was that if mRNA for tyrosinase is found in the lymph node preparation, that finding is good evidence that metastatic melanoma cells are present. The assay is accomplished using the combination of reverse transcription and double-round polymerase chain reaction (RT-PCR). The amplified samples are examined on a 2% agarose gel and tyrosinase cDNA is seen as a 207 base pair fragment. Lymph node preparations from 29 patients who were clinically stage I and II and undergoing elective node dissections were analyzed both by standard pathologic staining and RT-PCR. Eleven of 29 lymph node (38%) samples from 29 patients with intermediate thickness melanoma were pathologically positive. Nineteen of the 29 lymph node preparations (66%) were RT-PCR-positive, and these included all of the pathologically positive samples, so that the false-negative rate was 0. In a spiking experiment, one SK-Mel-28 melanoma cell in a background of one million normal lymphocytes could be detected, thus indicating the sensitivity of this method. In addition, analysis by restriction enzyme mapping showed that the amplified 207-bp PCR product produced is part of the tyrosinase gene sequence. The RT-PCR method is an extremely sensitive, reproducible, and efficient technique for the identification of micrometastases in patients with melanoma and could be widely applicable. If clinical correlation is obtained, staging of the melanoma patient becomes more accurate, and treatment becomes more standardized and rational, because all those patients who have evidence of nodal disease can be identified so that they may benefit from more extensive surgery (formal node dissections) or adjuvant therapies. Based on these results, RT-PCR could be a powerful tool to detect micrometastatic melanoma.

HCV-RNA detected by polymerase chain reaction in patients with chronic hepatitis C treated with recombinant α interferon

HCV-RNA detected by polymerase chain reaction in patients with chronic hepatitis C treated with recombinant α interferon