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An improved quantitative assay to measure cytokeratin 19 (CK19) expression has been developed. The assay utilizes reverse transcription and a one-step polymerase chain reaction (PCR), with capillary electrophoresis and fluorescent labelling, to separate and detect the PCR products. Calibration curves were constructed from a serial dilution of CK19 cDNA coamplified with a fixed amount of CK19 internal standard, which was found to be linear between 10 and 500 molecules. Quantitative measurement of CK19 in samples was carried out by coamplifying the cDNA with a fixed amount of internal standard. The values were calculated from the calibration curve. The integrity of RNA and cDNA synthesis was checked by quantitative measurement of the breakpoint cluster region (BCR) gene expression. The assay is sensitive, detecting<10 CK19 transcripts, and reproducible with a coefficient of variation of approximately 10%. CK19 expression showed overlapping values when measured in samples from peripheral blood and bone marrow in operable breast cancer patients, in healthy volunteers or patients without epithelial cancer and in blood samples from patients with metastatic breast cancer. As the assay is easier to perform than traditional quantitative competitive PCR assays, it might be useful for quantitative measurement of other specific transcripts. [ABSTRACT FROM AUTHOR]

Abstract: Fitusiran, a subcutaneous investigational small interfering RNA therapeutic, targets antithrombin to rebalance hemostasis in people with hemophilia A or B (PwHA/B), irrespective of inhibitor status. This phase 3, open-label study evaluated the efficacy and safety of fitusiran prophylaxis in males aged ≥12 years with hemophilia A or B, with or without inhibitors, who received prior bypassing agent (BPA)/clotting factor concentrate (CFC) prophylaxis. Participants continued their prior BPA/CFC prophylaxis for 6 months before switching to once-monthly 80 mg fitusiran prophylaxis for 7 months (onset and efficacy periods). Primary end point was annualized bleeding rate (ABR) in the BPA/CFC prophylaxis and fitusiran efficacy period. Secondary end points included spontaneous ABR (AsBR) and joint ABR (AjBR). Safety and tolerability were assessed. Of 80 enrolled participants, 65 (inhibitor, n = 19; noninhibitor, n = 46) were eligible for ABR analyses. Observed median ABRs were 6.5 (interquartile range [IQR], 2.2-19.6)/4.4 (IQR, 2.2-8.7) with BPA/CFC prophylaxis vs 0.0 (IQR, 0.0-0.0)/0.0 (IQR, 0.0-2.7) in the corresponding fitusiran efficacy period. Estimated mean ABRs were substantially reduced with fitusiran by 79.7% (P = .0021) and 46.4% (P = .0598) vs BPA/CFC prophylaxis, respectively. Forty-one participants (63.1%) experienced 0 treated bleeds with fitusiran vs 11 (16.9%) with BPAs/CFCs. Median AsBR and AjBR were both 2.2 with BPA/CFC prophylaxis and 0.0 in the fitusiran efficacy period. Two participants (3.0%) experienced suspected or confirmed thromboembolic events with fitusiran. Once-monthly fitusiran prophylaxis significantly reduced bleeding events vs BPA/CFC prophylaxis in PwHA/B, with or without inhibitors, and reported adverse events were generally consistent with previously identified risks of fitusiran. This trial was registered at www.ClinicalTrials.gov as #NCT03549871., (© 2024 American Society of Hematology. Published by Elsevier Inc. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

In a clinical non-trial setting, the efficacy and safety of caspofungin was compared with liposomal amphotericin B for the management of febrile neutropenia or invasive fungal infections in 73 episodes in patients with haematological malignancy. There were fewer episodes of drug toxicity with caspofungin than liposomal amphotericin B (58.3 vs 83.7 %, P=0.02). The favourable response rate for episodes of febrile neutropenia treated with caspofungin or liposomal amphotericin B was similar at 37.5 and 53.8 %, respectively, but more breakthrough fungal infections occurred with caspofungin than with liposomal amphotericin B (33.3 vs 0 %, P<0.05) in these patients who did not receive antifungal prophylaxis. None of four episodes of candidaemia or hepatosplenic candidiasis responded to caspofungin compared with three of four episodes treated with liposomal amphotericin B. Mortality was significantly higher with caspofungin treatment compared with liposomal amphotericin B (6/24 vs 2/49, P=0.01), mainly due to an excess of fungal infections (P=0.04). Caspofungin treatment was a significant independent predictor of mortality [odds ratio=7.6 (95 % confidence interval 1.2-45.5)] when sepsis severity, prolonged neutropenia and length of antifungal therapy were considered in a multiple logistic regression model. In clinical practice, there is a suggestion that caspofungin may not be as effective as liposomal amphotericin B in preventing breakthrough invasive fungal infections in febrile neutropenia or in preventing fungus-related deaths. Because of the potential biases in this observational study, these preliminary findings should be interpreted with caution and clarified with a larger cohort of patients.

Background: We have previously developed a quantitative calibrated PCR assay to measure cytokeratin 19 (CK19) expression in haematopoietic tissue in order to detect systemic micrometastases., Patients and Methods: Serial measurements of CK19 expression in bone marrow of patients with primary breast cancer were performed at operation, at 3 weeks and 6 months postoperatively., Results: Reference values for CK19 expression were established by analysing bone marrow samples from 48 healthy female volunteers or patients without epithelial cancer. Samples from breast cancer patients with CK19 values above the upper reference limit were considered positive. Bone marrow samples taken at operation were positive in 29 out of 141 patients (20.6%) and remained positive in 12, turned negative in 4 and were unavailable in 13 at 6 months postoperatively., Conclusion: Serial measurements increase the reliability of detecting micrometastases perioperatively. Further studies are in progress to evaluate the relationship between elevated CK19 values and clinical outcome.

Objective: We developed a quantitative reverse-transcription polymerase chain reaction (RT-PCR) to determine CK20 expression in colorectal tumor and hematopoietic tissue., Design and Methods: Our method incorporates a calibrated PCR with an internal competitor and an external standard., Results: The RT-PCR assay is sensitive detecting 10 target molecules of CK20 in solution with one round of 38 amplification cycles. Genomic DNA contamination was eliminated by Dnase I digestion of total RNA. The inclusion of a calibrator in the quantitative RT-PCR analysis allowed for a high throughput of unknown samples within the same assay improving comparative analysis between the samples tested. Analysis of peripheral blood and bone marrow from 20 healthy volunteers revealed a low level of CK20 expression in all samples., Conclusion: To study the clinical significance of CK20 expression as a marker of systemic metastatic disease it is essential to measure CK20 mRNA levels in hematopoietic tissue with sensitive quantitative RT-PCR. A sensitive and reproducible method, which is easily performed, is described.

We studied clinical and immunological characteristics of 15 patients with chronic cold agglutinin disease (CAD). Mean age at disease debut was 68 years for female and 67 years for male patients. The patients had no signs of other autoimmune diseases. All patients had V(H)4-34 encoded IgM kappa cold agglutinins (CA) in high titre. In five patients IgM increased significantly with advancing disease. Seven patients had reduced concentrations of lymphocytes, largely of CD4 and CD8 T cells. Percentages of NK cells (CD56) and B cells (CD19) were increased in seven and three patients, respectively. In six out of nine patients a clonal expansion of kappa positive B cells was found. Serum C3 was decreased in nine patients and C4 was decreased in 11 patients, six of whom had reduced CH50. Such data indicate that patients with CAD experience a continuous low-grade complement consumption. Five patients had experienced increased haemolysis during infections. After addition of active complement to patient sera in vitro, six sera showed increased haemolytic activity. Our results indicate that some patients with CAD have a relative deficit of complement in their serum and that an increase of complement production occurs during an acute phase reaction which enhances haemolysis. Our data also indicate that both CA titre and thermal amplitude are important characteristics when predicting complement activation and clinical course in CAD.

The aim of the present investigation was to obtain information about treatment, clinical course and outcome for all patients with chronic myeloid leukaemia through a six-year period in a defined part of Norway. A total number of 141 patients fulfilled the diagnostic criteria. This is equivalent to 0.9 patients per 100,000 per year. The median age was 62 years. More than 70% of the patients were primarily treated with hydroxyurea, either alone or combined with interferon. 40 out of 57 patients younger than 55 years underwent allogeneic stem cell transplantation. Median survival for all patients was 36 months with an estimated five-year survival rate of 33%. Patients older than 55 years had a median survival of 30 months with 16% alive after five years. The five-year survival rate for patients younger than 55 years was 56%, for transplanted patients 72%. 60 of 84 patients older than 55 years have died after 4 1/2 years median observation time. Two thirds of those died of leukaemia; one third of other causes. 23 of 57 patients younger than 55 years have died. 11 of them had had transplantations and most of them died from transplantation-related causes, while leukaemia was the dominating cause of death in the others.

Secondary or therapy related myelodysplastic syndrome may develop following treatment with alkylating agents and radiotherapy. The condition may also follow high dose therapy for malignant lymphomas. We describe two patients who developed secondary myelodysplasia. The first is a 76-year old female with a low grade lymphoma who developed pancytopenia with monosomy 7. Secondary myelodysplasia was diagnosed 24 months after treatment with chlorambucil (total dose of 900 mg) and 12 months after treatment with trophosphamide (total dose of 24 g). The second patient was a 48-year old male who underwent autologous bone marrow transplantation for a relapsed low grade lymphoma. Following autografting he had persistent anemia and trombocytopenia. Secondary myelodysplastic syndrome with complex karyotype was diagnosed 54 months after high dose therapy. He died nine months later of bone marrow failure.

We investigated the clinical, pathological, and immunological features of "idiopathic" cold agglutinin disease (CAD) in a population-based study. Fourteen patients were studied, giving a prevalence of about 14 per million with a mean age of 75 years. Haemolysis was present in all cases, but only eight patients had clinical symptoms of peripheral haemagglutination. Serum electrophoresis, immunofixation, morphological bone marrow evaluation, and flow cytometric immunophenotyping were used to detect any monoclonal lymphoproliferative disorder. Flow cytometry seemed to be a sensitive way to demonstrate a clonal B-cell proliferation. Some evidence of clonality was found in 13 patients, and a clonal lymphoproliferative disease was documented by flow cytometry or biopsy in 10 out of 11 patients. We conclude that CAD is a symptom-producing monoclonal lymphoproliferative disorder in nearly all patients.

We describe the case of a 57-year-old woman with chronic myelogenous leukemia who was on hydroxyurea and developed a fatal thrombotic microangiopathy with renal, retinal and central nervous system involvement. There was no evidence of medullary or extramedullary leukemia transformation. Repeated examinations of the peripheral blood film revealed only minimal morphological changes of microangiopathic hemolysis. The diagnosis was made by postmortem examination of the kidneys, brain, meninges and retina. The underlying etiology may have been a paraneoplastic phenomenon of the chronic phase of CML or may have indicated the beginning of transformation to an accelerated phase. A late side effect of hydroxyurea therapy cannot be excluded.

The Philadelphia chromosome in cells of patients with chronic myeloid leukemia and acute lymphoblastic leukemia can be detected by reverse transcriptase-polymerase chain reaction (RT-PCR). We have tested two new methods for this purpose. For diagnostic purposes, three different BCR-ABL translocations (b3a2, b2a2 and ela2) can be detected in a multiprimed, one step PCR reaction. By using a competitor DNA construct and a two-step, nested PCR reaction, a quantitative measure of the number of specific BCR-ABL transcripts can be estimated. We tested five patients with chronic myeloid leukemia. All of them showed positive BCR-ABL translocations in the diagnostic test. Patients with other myeloproliferative disorders, used as controls, were all negative. Quantitative measurements of specific BCR-ABL mRNA showed that as few as ten transcripts could be quantified in the assay. The analysis showed that coefficients of variation between 15% and 30% were obtained for specific transcripts per micrograms RNA, whereas specific BCR-ABL per normal ABL showed a coefficient of variation of 10%. These new methods to detect BCR-ABL translocation by RT-PCR should provide easy and sensitive diagnosis, and possibilities of monitoring residual disease or relapse.

A case of fibroepithelial polyp arising from the labium majus is described. The tumor measured 12 cm in its largest diameter and was connected to the left labium majus by a 12-cm pedicle. The microscopic examination showed hypocellular connective tissue and focal myxoid areas. One year following surgical excision, the patient did not manifest any signs of recurrence. This case is very unusual in that almost all reported vulvar fibroepithelial polyps are small and sessile.

A total of 8 patients with advanced squamous cell carcinoma of the penis (Jackson stages III and IV) received chemotherapy with 100 mg./m2. cisplatin intravenously on day 1 and a 24-hour infusion of 1,000 mg./m.2 5-fluorouracil on days 1 to 5. Of the patients 2 (25%) achieved a partial response: 1 required a further operation and 1 required surgery with radiotherapy to achieve a complete response. These 2 patients were disease-free at 32+ and 57+ months. Nonresponders had a survival range of 2+ to 28 months after chemotherapy. Nausea and vomiting were the most frequent side effects of chemotherapy. Chemotherapy-related increase in serum creatinine occurred in 3 patients. Two patients had septicemia and 1 complained of tinnitus. Poor tolerability especially in the elderly was the main reason for discontinuing chemotherapy. The combination of cisplatin and 5-fluorouracil may have a role in the management of advanced penile cancer together with surgery and radiotherapy.

The glycogen content of human megakaryocytes was studied using a quantitative method. Smears of bone marrow from 13 individuals were stained with the modified PAS reaction with and without prior treatment with alpha-amylase. The intensity of the reaction was determined by microspectrophotometry in 50 megakaryocytes from each individual. It was found that megakaryocytes are rich in glycogen which is not only confined to the intensely PAS-positive granules and inclusion bodies, but also makes up a good part of the diffuse cytoplasmic staining. In the diffusely stained megakaryocytes, glycogen makes up 32% of the intensity of the PAS reaction, while it reaches 47% in those with granules and up to 60% in those with inclusion bodies. The total extinction of the PAS-stained megakaryocytes is not only dependent on the morphological appearances of the cells, but is also positively correlated with their size.