Real-Time polymerase chain reaction of immunoglobulin rearrangements for quantitative evaluation of minimal residual disease in multiple myeloma.

The majority of patients with multiple myeloma (MM) have persistence of minimal residual disease (MRD), as determined by polymerase chain reaction (PCR) detection of clonal immunoglobulin H (IgH) gene rearrangements. As a result, PCR analysis has not provided clinically useful prognostic information in myeloma patients. Instead, quantitative PCR approaches are required to predict patient outcomes and assess response to novel treatment strategies. We adapted real-time PCR technology to quantify myeloma cells using the IgH rearrangement and then assessed the utility of this approach in 29 patients with myeloma who had undergone autologous stem cell transplantation. Because of the high cost of producing a specific reporting probe for each patient, H-chain V-region family-specific consensus probes were used in association with allele-specific oligonucleotides for PCR amplification. Because of the high frequency with which somatic hypermutation at the immunoglobulin locus occurs in MM, a number of mismatches occurred between the patient sequences and the consensus probe. However, construction of a limited number of probes allowed real-time PCR with a sensitivity of 10(-4) to 10(-5). To validate this method, we extensively evaluated assay accuracy and reproducibility. Results indicate that real-time PCR using consensus probes provides a feasible, accurate, and reproducible method for evaluating MRD in M M and possibly in other differentiated B-cell malignancies, and one that is less expensive than the use of patient-specific probes. This technique is being used to assess tumor depletion after immunologic purging and changes in tumor burden in patients undergoing stem cell transplantation and novel treatment approaches.

Complete remission of pulmonary spindle cell carcinoma after treatment with oral germanium sesquioxide.

Spindle cell carcinoma (SCC) is a rare form of lung cancer representing 0.2 to 0.3% of all primary pulmonary malignancies. Even with combined surgery, chemotherapy, and radiation therapy, these tumors are associated with a poor prognosis and only 10% of patients survive 2 years after diagnosis. We describe a patient with an unresectable SCC who, following no response to conventional treatment with combined modality therapy, chose to medicate herself with daily doses of germanium obtained in a health food store. She noted prompt symptomatic improvement and remains clinically and radiographically free of disease 42 months after starting her alternative therapy.

Immunoglobulin heavy-chain consensus probes for real-time PCR quantification of residual disease in acute lymphoblastic leukemia.

Tumor-related immunoglobulin heavy-chain (IgH) rearrangements are markers for polymerase chain reaction (PCR) detection of minimal residual disease (MRD) in B-cell malignancies. Nested PCR with patient IgH allele-specific oligonucleotide primers can detect 1 tumor cell in 10(4) to 10(6) normal cells. In childhood acute lymphoblastic leukemia (ALL), persistence of PCR-detectable disease is associated with increased risk of relapse. The clinical significance of qualitative PCR data can be limited, however, because patients can harbor detectable MRD for prolonged periods without relapse. Recent studies indicate that a quantitative rise in tumor burden identifies patients who are at high risk for relapse. Therefore, an efficient and reliable PCR method for MRD quantification is needed for ALL patients. We have developed a real-time PCR method to quantify MRD with IgH V(H) gene family consensus fluorogenically labeled probes. With this method, a small number of probes can be used to quantify MRD in a large number of different patients. The assay was found to be both accurate and reproducible over a wide range and capable of detecting approximately 1 tumor cell in 5 x 10(4) normal cells. We demonstrate that this methodology can discriminate between patients with persistence of MRD who relapse and those who do not. This technique is generally applicable to B-cell malignancies and is currently being used to quantify MRD in a number of prospective clinical studies at our institution. (Blood. 2000;95:2651-2658)

Failure of immunologic purging in mantle cell lymphoma assessed by polymerase chain reaction detection of minimal residual disease.

To assess the clinical significance of minimal residual disease (MRD) detection by polymerase chain reaction (PCR) we analyzed samples from 26 patients with mantle cell lymphoma (MCL) who had undergone bone marrow transplantation (BMT) at the Dana-Farber Cancer Institute. The BCL-1/IgH translocation and clonally rearranged Ig heavy chain genes (IgH) provided molecular markers for detection and follow-up of MRD by polymerase chain reaction (PCR) amplification in 19 of the 26 (73%) MCL patients studied. IgH gene sequencing analysis showed somatic mutations in MCL that are characteristic of an antigen driven process suggesting that, in MCL, the final malignant transformation occurs in a mature B cell. Of the 19 patients with a PCR amplifiable marker, 17 underwent autologous, 1 an allogeneic, and 1 a syngeneic bone marrow transplantation (BMT). All patients had PCR-detectable MRD in the bone marrow (BM) at the time of BMT, irrespective of any history of histological BM involvement. In contrast to other B-cell malignancies, we found that immunological purging with complement-mediated lysis eradicated PCR-detectable MCL in only two patients. Moreover reinfusion of MRD was associated with a poor outcome. More than half of the patients undergoing autologous BMT had relapsed by the time of restaging at 2 years after autologous BMT. In four MCL patients in whom no residual lymphoma was reinfused, including the allogeneic and the syngeneic BMT, only one patient relapsed. Persistence of MRD detection after BMT was also associated with a high probability of relapse, although one patient did not have PCR-detectable MRD in peripheral blood or BM before relapse at nodal sites. We conclude that PCR amplification of disease-specific markers is a feasible and sensitive method to assess MRD and its clinical significance in patients with MCL. Moreover, PCR amplification provides a tool to evaluate modifications of purging and stem cell collection procedures that may be required for the management of this otherwise incurable disease.

The accumulation of alpha- and beta-carotene in serum and tissues of preruminant calves fed raw and steamed carrot slurries.

The preruminant calf model was used to evaluate the effects of mild heat treatment on the serum and tissue accumulation of alpha- and beta-carotene from carrots. Twenty-four 1-wk-old Holstein male calves were assigned to one of four groups and fed a milk replacer diet. Negative control animals received no additional supplement. The three remaining groups received an additional 20 mg beta-carotene/d from either water-soluble beadlets, homogenized raw carrots or homogenized steamed carrots. Serum samples were obtained daily, and calves were killed after 7 d and samples of serum, liver and adrenal collected. Tissue and serum alpha- and beta-carotene concentrations were not significantly higher in steamed carrot-fed animals than in raw carrot-fed animals. The molar ratios of beta-carotene to alpha-carotene in both raw carrot-fed and steamed carrot-fed groups were highest in adrenal tissues, intermediate in serum and lowest in the diets. None of these differences were statistically significant. When the serum, liver and adrenal beta-carotene data were pooled, the mean relative accumulation of beta-carotene, expressed as a percentage of the mean response of calves receiving water-soluble beadlets, was 46.8% for calves fed raw carrots and 74.0% for calves fed steamed carrots. These results suggest a small enhancing effect of mild heat treatment of carrots on the serum and tissue accumulation of carotenoids.

Evaluation of the preruminant calf as a model for the study of human carotenoid metabolism.

This study evaluated the preruminant calf as an animal model for the study of human carotenoid metabolism. Fifteen newborn male Holstein calves were fed a carotenoid-free milk replacer diet to maintain them in the preruminant state. After a 7-d adjustment period, three calves were killed and 12 calves received a single oral dose (20 mg) of beta-carotene in the form of water-soluble beadlets. Blood samples were collected periodically and samples of various tissues were collected when the calves were killed. Three animals each were killed by exsanguination at 1, 3, 6 and 11 d post-dosing. Serum beta-carotene concentrations peaked between 12 and 30 h post-dosing and declined slowly afterwards. Serum data were fitted to a two-compartment model and yielded an elimination constant (k2) that was similar to reported human values. Adrenal tissue showed significant concentrations of beta-carotene by 24 h post-dosing, and levels were still elevated at 264 h. Liver, spleen and lung beta-carotene concentrations were significantly elevated by 24 h and rapidly declined thereafter. Adipose and kidney peak beta-carotene concentrations were observed at 72 and 144 h, respectively. Heart and muscle did not display significant changes in beta-carotene concentrations. The preruminant calf shows promise as an animal model for the study of absorption and metabolism of carotenoids by humans.

Concentrations of selected carotenoids and vitamin A in human liver, kidney and lung tissue.

Concentrations of preformed vitamin A and five individual carotenoids (alpha-carotene, beta-carotene, cryptoxanthin, lutein and lycopene) were determined in liver, kidney and lung tissue from 20 autopsies of subjects ranging in age from 4 mo to 86 y. Total carotenoid concentrations in liver tissue were always greater than in kidney or lung tissue within the same patient. Total carotenoid concentration in adult subjects was 2.5-77.1 nmol/g tissue (mean 21.0 nmol/g tissue) in liver tissue (n = 14), 0.2-12.7 nmol/g tissue (mean 3.1 nmol/g tissue) in kidney tissue (n = 13) and 0.1-8.4 nmol/g tissue (mean 1.9 nmol/g tissue) in lung tissue (n = 13). Carotenoid content in tissue samples from two infants was low, ranging from 0 to 1.0 nmol/g tissue. beta-Carotene and lycopene were almost always the predominant carotenoids found in liver, kidney and lung tissue. beta-Carotene was positively correlated (P less than 0.05) with alpha-carotene, lycopene and total carotenoids in all of the tissues examined. In addition, beta-carotene and total carotenoids from liver tissue were positively correlated with the same carotenoids in both kidney and lung tissue within each patient. Total vitamin A (free plus esterified) concentration was 8.7-1102.2 nmol/g tissue in liver (n = 17), 3.5-343.9 nmol/g tissue in kidney (n = 14) and 0.7-404.6 nmol/g tissue in lung (n = 14). Vitamin A concentrations were significantly correlated with both beta-carotene and total provitamin A carotenoid concentrations in liver tissue, but not in kidney or lung tissue.

High-performance liquid chromatography and capillary supercritical-fluid chromatography separation of vegetable carotenoids and carotenoid isomers.

Carotenoids from carrots and tomatoes were separated with high-performance liquid chromatography (HPLC) and capillary supercritical fluid chromatography (SFC). All trans alpha- and beta-carotene were separated from their respective cis-isomers with capillary SFC. Carotenoids extracted from tomatoes included xanthophyll, lycopene and beta-carotene, while alpha- and beta-carotene were extracted from carrots. The HPLC separations were accomplished isocratically with a 25-cm column containing 5-microns ODS and methanol-acetonitrile-chloroform (47:47:6) or acetonitrile-dichloromethane (80:20). beta-Carotene cis-isomers were separated with SFC with a SB-cyanopropyl-25-polymethylsiloxane column, while alpha-carotene isomers were separated with two SB-cyanopropyl-50-polymethylsiloxane columns. Carotenoids from carrots and tomatoes were separated with a SB-phenyl-50-polymethylsiloxane column. Carbon dioxide with 1% ethanol was the SFC mobile phase. The eluent was monitored at 461 nm for HPLC and either 453 or 461 nm for SFC.

The radiologic diagnosis of complications following gynecologic surgery: radiography, computed tomography, sonography, and scintigraphy.

673,000 hysterectomies were done in the United States in 1981. The mortality rate of 16.4/100,000 was less than that for appendectomy. Gynecologic surgery, however, is the most common surgical cause of complications related to the urinary tract with the reported incidence varying from less than 1 to 10%. We reviewed the charts of 1403 patients who had 736 abdominal, 315 vaginal and 143 radical or modified radical hysterectomies. Seventy (5%) patients had complications. Thirty-two (2.2%) patients had 35 complications related to the urinary tract. Complications directly related to surgery were grouped into ureteral injuries (13), fistulas (10), post surgical fluid collections (13), and hydronephrosis following radical hysterectomy. Characteristic images obtained by radiography, sonography (US), computed tomography (CT), and nuclear medicine (NM) are presented, and their use in each group of complications is summarized in the conclusion.

Vocational potential assessment.

Determination of employment potential will become increasingly important in the foreseeable future, largely due to pending Federal legislation which relates to welfare reform. The heretofore "permanently and totally disabled versus able-bodied" principle in welfare reforms is being abbandoned. Pending legislative proposals dealing with welfare reform provide for considering physically impaired persons as partially disabled and partially employable simultaneously. Thus, the need to systematically and effectively assess physically impaired citizens' capacities to participate in the job market will increase. Unquestionably, rehabilitation medicine in general, and the emerging art and science of vocational evaluation in particular, will contribute much to supplying these services. While it is widely acknowledged that the vocational potential of physically impaired persons should be evaluated in an organized manner, there are differences of opinion among professional evaluators as to which approach, or approaches, are the most meritorious; The four principal approaches are: (1) mental testing, (2) work sampling, (3) situational analysis, and (4) job tryouts. Each of these approaches is explained, contrasted, and evaluated in this paper.