Observational study of subclinical diabetic macular edema.

PURPOSE: To determine the rate of progression of eyes with subclinical diabetic macular edema (DME) to clinically apparent DME or DME necessitating treatment during a 2-year period. METHODS: In all, 43 eyes from 39 study participants with subclinical DME, defined as absence of foveal center edema as determined with slit lamp biomicroscopy but a center point thickness (CPT) between 225 and 299 µm on time domain (Stratus, Carl Zeiss Meditec) optical coherence tomography (OCT) scan, were enrolled from 891 eyes of 582 subjects screened. Eyes were evaluated annually for up to 2 years for the primary outcome, which was an increase in OCT CPT of at least 50 µm from baseline and a CPT of at least 300 µm, or treatment for DME (performed at the discretion of the investigator). RESULTS: The cumulative probability of meeting an increase in OCT CPT of at least 50 µm from baseline and a CPT of at least 300 µm, or treatment for DME was 27% (95% confidence interval (CI): 14%, 38%) by 1 year and 38% (95% CI: 23%, 50%) by 2 years. CONCLUSIONS: Although subclinical DME may be uncommon, this study suggests that between approximately one-quarter and one-half of eyes with subclinical DME will progress to more definite thickening or be judged to need treatment for DME within 2 years after its identification.

Randomized trial of peribulbar triamcinolone acetonide with and without focal photocoagulation for mild diabetic macular edema: a pilot study.

OBJECTIVE: To provide pilot data on the safety and efficacy of anterior and posterior sub-Tenon injections of triamcinolone either alone or in combination with focal photocoagulation in the treatment of mild diabetic macular edema (DME). DESIGN: Prospective, phase II, multicenter, randomized clinical trial. PARTICIPANTS: One hundred nine patients (129 eyes) with mild DME and visual acuity 20/40 or better. METHODS: The participants were assigned randomly to receive either focal photocoagulation (n = 38), a 20-mg anterior sub-Tenon injection of triamcinolone (n = 23), a 20-mg anterior sub-Tenon injection followed by focal photocoagulation after 4 weeks (n = 25), a 40-mg posterior sub-Tenon injection of triamcinolone (n = 21), or a 40-mg posterior sub-Tenon injection followed by focal photocoagulation after 4 weeks (n = 22). Follow-up visits were performed at 4, 8, 17, and 34 weeks. MAIN OUTCOME MEASURES: Change in visual acuity and retinal thickness measured with optical coherence tomography (OCT). RESULTS: At baseline, mean visual acuity in the study eyes was 20/25 and mean OCT central subfield thickness was 328 mum. Changes in retinal thickening and in visual acuity were not significantly different among the 5 groups at 34 weeks (P = 0.46 and P = 0.94, respectively). There was a suggestion of a greater proportion of eyes having a central subfield thickness less than 250 mum at 17 weeks when the peribulbar triamcinolone was combined with focal photocoagulation. Elevated intraocular pressure and ptosis were adverse effects attributable to the injections. CONCLUSIONS: In cases of DME with good visual acuity, peribulbar triamcinolone, with or without focal photocoagulation, is unlikely to be of substantial benefit. Based on these results, a phase III trial to evaluate the benefit of these treatments for mild DME is not warranted.

Comparison of competitive-nested PCR and real-time PCR in detecting BCR-ABL fusion transcripts in chronic myeloid leukemia patients.

Real-time RT-PCR has great advantages for estimating transcript levels in a variety of situations. These include relative rapid assay times (hours), reliability and ease of performing replicate analyses. In contrast, competitive PCR is a very labor-intensive procedure requiring a few days to generate useful data. We compared the same samples from CML patients by both methods. Importantly, we used the Bcr-Abl junction plasmid DNA, which is used as a competitor in the manual competitive PCR assay, to generate a standard curve for the real-time assay. This permitted reporting the real-time data as the number of BCR-ABL transcripts per microg of total RNA, which is the same format used for the competitive PCR assay. In this study, a total of 435 peripheral blood and marrow samples from 285 CML patients were analyzed by RT-PCR; these patients were undergoing therapy by STI-571, interferon, and bone marrow transplantation treatment. Most samples also had assay values for the Philadelphia chromosome (Ph), FISH and Western blotting for the Bcr-Abl oncoprotein. Our findings indicated that the real-time assay was less sensitive than the manual competitive RT-PCR assay (t = 5.118; P < 0.001). Of interest, the transcript levels in cell line mixtures with various ratios of K562/KG-1 (BCR-ABL positive/negative) cells were also significantly higher with the competitive RT-PCR assays than real-time RT-PCR, except for levels of BCR-ABL below 200 transcripts per microg of RNA. In both patient and cell line experiments, dividing the BCR-ABL transcripts by the total ABL transcripts virtually eliminated the difference between real-time BCR-ABL transcript values and quantitative competitive BCR-ABL transcript values, indicating that both BCR-ABL and ABL transcripts were underestimated by the real-time assay. In addition, the increased sensitivity of the nested, competitive RT-PCR was readily apparent in patients with minimal residual disease, which by the real-time were negative in the majority of patients but were positive by nested, competitive RT-PCR in 44.6% (n = 29) of samples analyzed (n = 65). These findings indicate that real-time RT-PCR, when normalized for the total ABL transcripts, can be used to monitor CML patients during therapy, but we suggest that nested, competitive RT-PCR be used to determine BCR-ABL/ABL transcript ratios at low transcript values or especially when real-time analyses are negative.

Quantitative measurement of BCR/abl transcripts using real-time polymerase chain reaction.

BACKGROUND: Quantitative real-time polymerase chain reaction (Q-Rt-PCR) is a new tool in the detection and quantification of the BCR/abl fusion transcripts in chronic myelogenous leukemia (CML). This study investigates its specificity, sensitivity and potential clinical usefulness. PATIENTS AND METHODS: Parallel analysis of Q-Rt-PCR and the conventional reverse transcription-mediated PCR (RT-PCR) were performed on 567 samples from 481 patients. Treatment response was monitored by Q-Rt-PCR at 6 and 12 months of 61 patients on STI-571 and 103 patients on interferon. RESULTS: The concordance rate between Q-Rt-PCR and RT-PCR was 96.3% (546/567), with 341 positives and 205 negatives. The positive equivalents ranged from 2 x 10(-6) to 1.2 microg of K562 cell RNA. Karyotyping in 372 samples revealed excellent correlation with Q-Rt-PCR measurements (P < 0.001). Setting residual BCR/abl < 0.01 as an early goal of molecular response, we observed that STI-571 induced a better response than interferon: 49% (20 of 41 patients) versus 35% (15 of 62 patients) at 6 months (P = 0.025) and 52% (32 of 61 patients) versus 34% (35 of 103 patients) at 12 months (P = 0.01), respectively. CONCLUSIONS: Q-Rt-PCR provides reliable measurements of BCR/abl fusion transcripts. It is potentially useful in assessing molecular residual disease after therapy.

Depression and nicotine: preclinical and clinical evidence for common mechanisms.

Updated findings on the relationship between nicotine and depression are presented. Clinical and preclinical research on nicotine use and depression suggests that nicotine may have some properties in common with antidepressants. Updated findings involve the comorbidity of smoking and major depressive disorder (MDD), the influence of depression during withdrawal on failure to quit smoking, the course of MDD without nicotine and the neurobiology of smoking and depression.

Cytogenetic findings in lymphoplasmacytic lymphoma/Waldenström macroglobulinemia. Chromosomal abnormalities are associated with the polymorphous subtype and an aggressive clinical course.

We correlated bone marrow cytogenetic findings with morphologic and immunophenotypic data in 37 patients with lymphoplasmacytic lymphoma (LPL)/Waldenström macroglobulinemia (WM). Each LPL/WM case was classified as lymphoplasmacytoid (n = 18), lymphoplasmacytic (n = 10), or polymorphous (n = 9) using the Kiel criteria. Of 12 cases with chromosomal abnormalities, a single numeric abnormality was present in 4 and a complex karyotype in 8. The most common numeric abnormalities were and -8 in 3 cases each; the most common structural abnormality was del(6q) in 6 cases. Cytogenetic abnormalities were significantly less common in the lymphoplasmacytic and lymphoplasmacytoid groups (5/28 [18%]) compared with the polymorphous group (7/9 [78%]). Clinical follow-up was available for 28 patients for a median of 36 months. Six (67%) of 9 patients with aneuploid tumors, including 4 with polymorphous subtype, subsequently had clinical progression or developed high-grade lymphoma. In contrast, 4 (21%) of 19 patients with diploid tumors, including 1 of polymorphous type, developed clinical progression or high-grade lymphoma. We conclude that abnormal cytogenetic findings in LPL/WM correlate with the polymorphous subtype and poor prognosis.

Reticulum cell sarcoma of lymph node with mixed dendritic and fibroblastic features.

We report a case of clinically aggressive reticulum cell sarcoma with mixed follicular dendritic cell (FDC) and fibroblastic reticular cell (FRC) features. Histologically, the tumor was confined to lymph nodes occurring as a multifocal epithelioid and spindle cell proliferation with appreciable mitotic rate and numerous admixed non-neoplastic B-cells. Ultrastructural examination revealed elongated cells with prominent nucleoli, interdigitating cell processes and frequent desmosomes. These features are typical of FDC sarcoma. However, immunohistochemical stains showed no expression of antigens characteristic of FDCs, including CD21, CD23 and CD35. Cytogenetic characterization of this tumor, by conventional G-banding and multicolor spectral karyotyping, revealed multiple clonal chromosomal aberrations, including del(X)(p11.4) and add (21)(p11.2). Gene expression analysis by cDNA microarray of RNA obtained from short-term tumor cultures revealed high-level expression of a set of genes (including PDGF receptor-alpha and -beta, certain metalloproteinases, and CD105) that were also highly expressed in cultures of nodal FRC cultured from non-neoplastic lymph nodes. We propose that this tumor represents a nodal sarcoma with intermediate differentiation between FDCs and FRCs. This case adds to the diversity of tumors that may arise from lymph node stroma and supports a possible relationship between the FDC and FRC lineages.

Smoking cessation and the course of major depression: a follow-up study.

BACKGROUND: Smokers with a history of major depression who attempt to stop smoking have a higher risk of failure than non-depressed smokers. Anecdotal and post-hoc data suggest that those who successfully abstain are at increased risk of depression compared with individuals who continue to smoke. However, these studies confound effects of abstinence and history of depression. We aimed to assess whether there is an increased risk of depression and for how long that increase lasts. METHODS: We enrolled 100 smokers (>1 pack per day) with a history of major depression, but who were currently free from major depression and had not been on antidepressant medicine for at least 6 months, in a 2-month smoking-cessation trial. The primary outcome was recurrence of major depression, which we assessed by structured clinical interviews 3 and 6 months after the end of treatment. We verified smoking status by serum-sample cotinine concentrations. FINDINGS: 76 participants (42 successful abstainers, 34 smokers) were followed up. 13 abstainers and two smokers had an episode of major depression (odds ratio 7.17 [95% CI 1.5-34.5]; Kaplan-Meier survival curve, log-rank statistic 9.11 [p=003]). Risk of major depression was similar between the first and second 3 months of follow-up. INTERPRETATIONS: Smokers with a history of depression who abstain from smoking are at significantly increased risk of developing a new episode of major depression. This risk remains high for at least 6 months.

Spectral karyotyping study of chromosome abnormalities in human leukemia.

Chromosomal analysis plays an important role in the diagnosis, treatment and prognosis of human leukemia. Currently, the GTG-banding technique (G-banding) is the most commonly used diagnostic method in clinical cytogenetics. G-banding analysis of subtle chromosomal rearrangements or complex karyotypes with multiple markers can be inadequate because of poor chromosome morphology and/or an insufficient yield of analyzable metaphases. Fluorescence in situ hybridization (FISH) is a highly sensitive and specific method to detect chromosomal alterations. Conventional FISH is used optimally in instances where only one or a few abnormalities are investigated. Spectral karyotyping (SKY), a novel cytogenetic technique, has been developed to unambiguously display and identify all chromosomes at one time using a spectrum of 24 different colors. This report presents the use of SKY for examination of the entire karyotype in specimens with complex chromosomal abnormalities from three leukemia patients. Conventional cytogenetic analysis (G-banding) showed complex hyperdiploid clones with multiple markers in each case. SKY was able to clarify and identify additional cryptic chromosomal translocations [e.g., t(2;10), t(3;10), t(5;7), t(7;18), t(9;17), t(10;12), t(13;16)] insertions [e.g., ins(17;9), ins(20;Y)], duplications [e.g., i(8)(q10), dup(4)(q31q35)] and marker chromosomes in each case. This study demonstrates that the combination of SKY and G-band techniques results in a more complete characterization of the complex chromosomal aberrations seen in leukemia.

Detection of trisomy 8 in philadelphia chromosome-positive CML patients using conventional cytogenetic and interphase fluorescence in situ hybridization techniques and its relation to c-myc involvement.

Trisomy 8 (+8) is a common clonal evolution marker for progression in chronic myelogenous leukemia. The relationship of +8 to various stages of t(9;22) leukemias is not firmly established. To explore this association we examined bone marrow (BM) cells from 10 Philadelphia chromosome positive (Ph+) chronic myeloid leukemia (CML) patients in different stages of the disease, using conventional cytogenetic technique(CCT) and interphase fluorescence in situ hybridization (FISH). FISH detection of chromosome 8 was accomplished using the D8Z2 (Oncor) probe specific for the centrometric region of chromosome 8. Five hundred interphase nuclei were counted for each patient. Three of the 10 patients were selected for detection of c-myc gene locus located in the 8q24.2-24.3 region using the L

Mature B-cell leukemias with more than 55% prolymphocytes. A heterogeneous group that includes an unusual variant of mantle cell lymphoma.

We studied 20 cases of mature B-cell leukemia with more than 55% prolymphocytes in peripheral blood or bone marrow, fulfilling the French-American-British criteria for B-cell prolymphocytic leukemia (PLL). Cases segregated into 3 groups: de novo PLL, 6; PLL occurring in patients with a previous well-established diagnosis of chronic lymphocytic leukemia (PLL-HxCLL), 10; and t(11;14)(q13;q32)-positive neoplasms, 4. All cases expressed monotypic immunoglobulin light chain, and most were positive for CD5. All t(11;14)-positive neoplasms were CD23- and uniquely positive for cyclin D1. Cytogenetic abnormalities were present in 19; in all 19, the karyotype was complex, indicating clonal evolution and genomic instability. The most frequent cytogenetic abnormality in de novo PLL involved chromosome 7 in 4 cases. Trisomy 12 or add(12p) was present in 4 cases of PLL-HxCLL. We conclude that mature B-cell leukemias with more than 55% prolymphocytes are a heterogeneous group that includes t(11;14)-positive neoplasms, which we suggest are best classified as mantle cell lymphoma. We also suggest that prolymphocytic morphologic features are a common end-stage of transformation for several B-cell neoplasms.

A unique, complex variant philadelphia chromosome translocation in a patient with typical chronic myelogenous leukemia.

The Philadelphia (Ph) chromosome [der(22) t(9;22)(q34;q11)] is the characteristic chromosomal abnormality found in chronic myelogenous leukemia (CML). This chromosome has been reported in patients with other chromosomal abnormalities. In this study, we describe a patient with hematologically typical chronic-phase CML with an unusual and complex translocation involving chromosomes 9, 11, and 22. These complex translocations were identified by G-banded conventional cytogenetics and confirmed by fluorescence in situ hybridization (FISH) using whole chromosome painting probes (wcp). To the best of our knowledge, these are unique translocations involving the short and the long arms of chromosome 9 in 4 different translocations with the short arm of chromosome 11 and the long arm of chromosome 22.

11q23 abnormalities in patients with acute myelogenous leukemia and myelodysplastic syndrome as detected by molecular and cytogenetic analyses.

11q23 chromosomal abnormalities and rearrangement of the mixed lineage leukemia (MLL) gene are important prognostic factors in acute myelogenous leukemia (AML) and myelodysplastic syndrome (MDS). However, the presence of 11q23 abnormalities does not always correlate with that of MLL gene rearrangement. We retrospectively compared the occurrence of 11q23 abnormalities (measured by karyotyping) and MLL gene rearrangement (measured by Southern blotting) in bone marrow from 311 consecutive adult patients with AML or MDS. 11q23 abnormalities were found in 18 patients (5.8%), of whom 7 (39%) did not have the MLL gene rearrangement. MLL gene rearrangement was detected in 35 patients (11.2%). Of these 35 patients, only 11 (31%) had cytogenetic evidence of 11q23 abnormalities. None of the 21 patients with chronic myelomonocytic leukemia had 11q23 abnormalities or MLL gene rearrangement. 11q23 abnormalities were associated with shorter survival than was a diploid karyotype. Both cytogenetic and molecular studies should be performed to detect 11q23 abnormalities in patients with AML or MDS.

Detection of chromosome 11q13 breakpoints by interphase fluorescence in situ hybridization. A useful ancillary method for the diagnosis of mantle cell lymphoma.

We assessed cytologic specimens from 11 mantle cell lymphomas (MCLs) and 32 other B-cell non-Hodgkin lymphomas (NHLs) for 11q13 breakpoints using a 2-color fluorescence in situ hybridization (FISH) assay that uses an 11q13 probe centered on the CCND1 gene and a centromeric chromosome 11 probe (CEP11). The number of nuclei in 200 cells were counted, and results were expressed as an 11q13/CEP11 ratio. All MCLs showed a high percentage of interphase nuclei with 3 or more 11q13 signals (mean, 74.8%; range 57%-90%). In contrast, in other B-cell NHLs the mean percentage of cells with 3 or more 11q13 signals was 9.2%. All MCLs had an elevated 11q13/CEP11 ratio (mean, 1.38). The mean ratio for other B-cell NHLs was 0.99. Two non-MCL cases, 1 large B-cell and 1 B-cell unclassified NHL, had high 11q13/CEP11 ratios of 1.15 and 1.30, respectively. Conventional cytogenetic analysis performed on the former case revealed a t(5;11)(q31;q13). Interphase FISH analysis using 11q13 and CEP11 probes is a convenient ancillary method for assisting in the diagnosis of MCL. This commercially available assay is simple to use on cytology or imprint specimens, and results can be obtained within 24 hours.

Numeric chromosomal abnormalities in small lymphocytic and transformed large cell lymphomas detected by fluorescence in situ hybridization of fine-needle aspiration biopsies.

BACKGROUND: Chromosomal abnormalities in some lymphomas are associated with a poor prognosis. Trisomy 12 and deletions of chromosome 13 have been described in small lymphocytic lymphoma (SLL). To determine whether chromosomal aberrations could be detected by fluorescence in situ hybridization (FISH) on fine-needle aspiration biopsies (FNABs), we analyzed and compared specimens from seven patients with SLL and nine patients with a history of SLL that had transformed to large cell lymphoma. METHODS: DNA probes specific to chromosome 12 and the chromosome 13/RB-1 gene locus were used for in situ hybridization on interphase cell nuclei. The cytologic features, ploidy, proliferation index, and conventional cytogenetic analysis findings were correlated with the FISH results. RESULTS: Trisomy 12 was detected in 2 (29%) of the 7 cases of SLL and 2 (22%) of the 9 cases of transformed large cell lymphoma. Deletion of chromosome 13/RB-1 was present in 3 (43%) of 7 cases of SLL and 3 (33%) of 9 cases of transformed large cell lymphoma. CONCLUSIONS: This study showed that interphase cytogenetic analysis by FISH is feasible on FNAB specimens. Trisomy 12 and deletions of chromosome 13/RB-1 were present in some cases of SLL and transformed large cell lymphoma, and the presence of these chromosomal abnormalities did not correlate with transformation to a higher grade of lymphoma.

Enhanced detection of chromosomal abnormalities with the use of RxFISH multicolor banding technique.

Hematological disorders often have complex karyotypes with multiple markers. Proper assignment of chromosome number or aberration or both can be difficult. Specific identification of chromosomal abnormalities aids in the diagnosis and selection of treatment of patients. Fluorescence in situ hybridization (FISH) has been applied to the identification of translocations, markers, and other chromosomal abnormalities in clinical cytogenetics. However, the standard FISH technique is unable to detect the entire genome in a single experiment. This report presents the use of a cross-species comparative genomic hybridization color-banding technique (RxFISH) that permits examination of an entire karyotype at one time. Specimens from two patients, one with acute lymphocytic leukemia (ALL) and the other with multiple myeloma (MM), were studied. Metaphases were prepared by standard culture techniques. Conventional cytogenetic analysis (GTG banding) showed multiple clones in each of the cases. These clones were hyperdiploid metaphases with complex chromosomal abnormalities and multiple markers. The slides were then hybridized with FITC-, Cy-3-, and Cy-5-labeled RxFISH probes; the results were analyzed by a digital imaging system. The RxFISH color banding confirmed the hyperdiploid metaphases and identified multiple chromosomal abnormalities. In the specimen from the patient with ALL, several chromosomes, which had been classified as markers by G-banding, were found to be specific chromosomes. This study suggests that RxFISH can provide more accurate and specific identification of complex chromosomal abnormalities. RxFISH is a useful complement to the clinical cytogenetic laboratory armamentarium.

Early detection of relapse by hypermetaphase fluorescence in situ hybridization after allogeneic bone marrow transplantation for chronic myeloid leukemia.

PURPOSE: Standard G-band cytogenetic analysis (CG) provides information on approximately 25 metaphases for monitoring the presence of Philadelphia chromosome positive (Ph+) cells in chronic myelogenous leukemia (CML) patients, making the detection of a low frequency of Ph+ cells problematic. The purpose of this study was to improve the detection of a low frequency of Ph+ cells. PATIENTS AND METHODS: We combined fluorescence in situ hybridization (FISH) with long-term colcemid exposure, capturing several hundred metaphases in bone marrow cultures (hypermetaphase FISH [HMF]). Using probes that identify Ph+ cells, HMF was compared with CG analysis in the follow-up evaluations of 51 patients with CML at various time points after allogeneic bone marrow transplant (BMT). RESULTS: Thirty-five patients never showed the presence of Ph+ cells by either method. In four patients, high frequencies of Ph+ cells were detected by both methods. In the remaining 12 patients, Ph+ cells were detected by HMF at time points after BMT when they were not detected by CG. In seven of the 12 patients, low but statistically significant frequencies of Ph+ cells (0.37% to 5.20%) were detected 3 months or later after BMT, and when no intervention was initiated, all seven patients later relapsed. Based on those data, an eighth patient with mixed chimerism and a similar HMF-detected Ph+ frequency (1.8% at 27 months after BMT) was reinfused with donor lymphocytes and achieved remission with 0% Ph+ cells studied by HMF (up to 50 months after BMT). Ph+ cells detected by HMF but not by CG less than 3 months after BMT disappeared on later examination in two of four patients. After detection of Ph+ cells by HMF only, the median time to cytogenetic progression (detection of Ph+ cells by CG) was 101 days. CONCLUSION: The results demonstrate the ability of HMF to detect low but clinically relevant levels of leukemic cells not detected by CG in transplant patients. The data indicate that HMF can detect low levels of Ph+ cells before standard cytogenetics at a time that may be useful in monitoring disease status and planning clinical interventions.

Chromosomal abnormalities in acute leukemias.

Conventional cytogenetic techniques are the standard for the diagnosis and follow-up of patients with AML and ALL. Some characteristic translocations associated with various groups of AML diagnoses, such as t(8;21), t(15;17), and inv(16) for M2, M3, and M4eo, respectively, have been recognized for years. The most common cytogenetic abnormality found in childhood ALL and hyperdiploid adult ALL is t(9;22). Future directions include increased use of FISH and molecular diagnostic techniques. The clinical cytogenetics laboratory plays a major role in the diagnosis and management of AML and ALL.

Advances in non-nicotine pharmacotherapy for smoking cessation.

Progress in understanding the pharmacological nature of tobacco addiction, along with the modest success rates achieved by the nicotine replacement therapies, has provided the major impetus for the development of non-nicotine drugs as smoking cessation aids. This article reviews evidence from controlled trials of several non-nicotine medications for the treatment of nicotine dependence. Clonidine was the first non-nicotine medication to show efficacy for smoking cessation in multiple studies, but its effect was found to be limited at best. Positive results across several trials have been consistently demonstrated for amfebutamone (bupropion). Encouraging results have also been observed for nortriptyline and moclobemide. Studies of combined treatments using non-nicotine medications (amfebutamone, mecamylamine, oral dextrose) with nicotine replacement therapy suggest increased efficacy relative to treatments using one or the other treatment strategy alone. Thus, available evidence indicates that non-nicotine drug treatments offer a promising panoply of therapeutic strategies for the addicted smoker.