The National Cancer Institute (NCI)-sponsored Chronic Lymphocytic Leukemia (CLL) Working Group was convened to develop a set of standardized eligibility, response, and toxicity criteria for clinical trials. We recognized the previous efforts in 1967 (published again in 1973 as the report of the Chronic Leukemia-Myeloma Task Force [1] and 1978 of Cancer and Leukemia Group B (CALGB) [2]). We have used these reports for guidance during the current effort. Several noteworthy developments in the past few years have made it necessary to modify the previous guidelines. First, the diagnostic criteria for CLL and its clinical staging have been developed and well defined. Second, although staging systems facilitated entry of comparable and relatively homogeneous groups of patients in clinical trials, the definitions of response (CR) and partial response (PR) were not uniformly adopted from the previous guidelines in the clinical trials (Tables IA, IB); therefore, comparisons of results obtained in different studies became difficult. Third, there has been an improvement in our understanding of the immunology and biology of CLL. Finally, we are witnessing the emergence of several chemotherapy agents that promise impressive activity in CLL (e.g., 2'-deoxycoformycin [3], fludarabine monophosphate [4, 5]), and thereby offer the potential for improving survival time in this disease. To best identify regimens worthy of continued pursuit in large comparative trials, standardized guidelines for evaluation are essential. A number of laboratory investigations are also presented for which scientific interest is high yet relevance remains to be determined; thus, they are presented as companion studies to the clinical trials. This mechanism allows for flexibility in the testing of these questions and for additional ideas in the future without requiring modification of an entire treatment protocol. The following guidelines were developed to be used as a form of standardization for clinical trials, incorporating current technologies, yet remaining relevant to the general hematology/oncology community. Based on the membership of the Working Group, it is expected that these guidelines will serve as the criteria for most clinical trials in the near future.

When human lymphocytes are cultured in the presence of phytomitogens, 70-90% of the cells undergo blast transformation and synthesize DNA. However, less than 40% of these lymphocytes actually undergo Although phytohemagglutinin (PHA) is known to be a mitogen, the number of mitoses actually counted after addition of PHA to lymphocyte cultures is relatively small. For example, Nowell found only 10% after 18 hr of incubation with colchicine at the peak of DNA synthesis despite morphologic transformation of up to 90% of these cells (1). Such PHAtransformed lymphocytes are known to be synthesizing DNA, since they are labeled when pulsed with radioactive thymidine and studied by autoradiography. The discrepancy between many cells making DNA and few caught dividing has been attributed to asynchrony of the cultured cells (2).In PHA-stimulated lymphocyte cultures, one might expect to find a significant increase in cell number resulting from mitosis, as well as a concomitant increase in the total DNA content of the culture. The reports dealing with this question do not consistently support this expectation. Schellekens and Eijsvoogels noted that total cell number declined 17% the first day, while DNA content dropped 25%; by day 3, the cell count was still only 89% of the initial values, and the DNA content was 96% (3). Hirschhorn et al. reported a decline in DNA content during a 48-hr culture of PHA-stimulated lymphocytes to 75% of the initial value (4). Clearly, if most PHA-transformed lymphocytes in these experiments underwent mitosis, a large number of daughter cells must have died and lysed. In contrast, Loeb and Agarwal measured a PHA-induced increase in DNA to nearly twice that of unstimulated cultures by 72 hr (5). Their results could be explained if each stimulated lymphocyte underwent a single mitosis and suggest that lymphocyte cultures reported in the former papers were complicated for some reason by excessive cell lysis. However, this simple explanation is confounded by the data of Polgar and Kibrick (6). These authors report experiments in which greater than 90% of lymphocytes stimulated for 24 hr with PHA ultimately became blasts, as judged morphologically; when grown continuously in the presence of ['H]thymidine, a maximum of 63% of the total number of lymphocytes present were labeled, as revealed by autoradiography on day 5, while on day 10 only 6% of the lymphocytes were labeled, yet the number of viable cells on day 10 was 57% of the original number. Cumulative in- Quadruplicate cultures of 3.3 X 106 lymphocytes in 2 ml of medium were stimulated with 24 pg of E-PHA. On day 3, 3 pCi ['Hjthymidine (6.7 Ci/mmol) was added; at the end of a 4-hr pulse, 8 ml of minimal essential medium was added, the cultures were centrifuged at 500 X g for 5 min, the cell pellets were suspended in 2 ml of medium, and grown for up to 3 more days.On the days noted above, duplicate tubes were harvested. Culture tubes were centrifuged at 500 X g for 10 min. On one set, DNA content was determined by the diphenylamine reac...

We compared the gene expression profile of adult acute lymphoblastic leukemia (ALL) to normal hematopoietic and non-ALL samples using oligonucleotide arrays. Connective tissue growth factor (CTGF) was the highest overexpressed gene in B-cell ALL compared with the other groups, and displayed heterogeneous expression, suggesting it might have prognostic relevance. CTGF expression was examined by quantitative reverse transcriptase-polymerase chain reaction (QRT-PCR) on 79 adult ALL specimens. CTGF expression levels were significantly increased in ALL cases with B-lineage (P < .001), unfavorable cytogenetics (P < .001), and blasts expressing CD34 (P < .001). In a multivariate proportional hazards model, higher CTGF expression levels corresponded to worsening of overall survival (OS; hazard ratio 1.36, for each 10-fold increase in expression; P ‫؍‬ .019). Further studies are ongoing to confirm the prognostic value of CTGF expression in ALL and to investigate its role in normal and abnormal lymphocyte biology. IntroductionThe prognosis of adults with acute lymphoblastic leukemia (ALL) is poor, especially when contrasted with the impressive progress made in curing pediatric ALL. Even with increasingly intensive chemotherapy regimens, relapse rates remain high, and long-term survival is 40%. 1-5 Transplantation regimens can be curative, but it remains challenging to identify high-risk patients suitable for early transplantation. Age, cytogenetic abnormalities, WBC count, and time to achieve complete remission (CR) are risk factors in adult ALL. [4][5][6] New prognostic biomarkers may fine-tune risk assessment in adult ALL.We compared the gene expression profile of adult ALL to control samples, and found that connective tissue growth factor (CTGF) had the highest expression in B-cell ALL, with heterogeneous expression within B-ALL specimens. Quantitative reverse transcriptase-polymerase chain reaction (Q-RT-PCR) in adult patients with ALL showed that high CTGF expression correlated with specific biologic features and poor outcome. Patients, materials, and methodsBone marrow (BM) and/or peripheral blood (PB) specimens containing at least 50% blasts from 43 adult ALL cases were included in the microarray experiments, together with 26 acute myeloid leukemia (AML) cases, 10 normal bone marrows (NBMs), 10 normal peripheral bloods (NPBs), 9 CD34-enriched cells from granulocyte colonystimulating factor (G-CSF) mobilized NPBs, 7 CD34-enriched cells from NBMs, and 2 CD22 ϩ selected B cells from NPBs. Q-RT-PCR experiments were conducted using all 79 patients with available specimens (28 BM and 60 PB) enrolled on SWOG S9400, a study for treating adult non-L3 ALL. Outcomes of the patients included in our study did not differ significantly from those of the remaining patients in S9400. 7 Twenty-six patients were included in both microarray and Q-RT-PCR experiments. Approval was obtained from the FHCRC institutional review board for this study. Informed consent was provided in accordance with the Declaration of Helsinki. Table 1 disp...

Thirty-six patients with metastatic melanoma were entered into a study of the therapeutic efficacy of adoptive immunotherapy with high-dose interleukin-2 (IL-2) and lymphokine-activated killer (LAK) cells. Thirty-two patients who received all components of the therapy are evaluable for response, and all patients are evaluable for toxicity. Sites of disease included lung, liver, subcutaneous nodules, and intra-abdominal metastases. One complete response (CR) and five partial responses (PRs) resulted from treatment (19% response rate). The median response duration was 5 months, with the durable CR continuing at 31+ months and one durable PR continuing for 13 months. Sites of response included lung, liver, subcutaneous nodules, and lymph nodes. Response, response duration, or site of response did not correlate with the total dose of IL-2 administered, rebound lymphocytosis, or the number of LAK cells infused. Toxicity included hypotension, fluid retention with a "capillary leak syndrome" in most patients, and transient multiorgan dysfunction that resolved promptly after the completion of therapy. Adverse cardiac events occurred in 16% of patients, with one myocardial infarction leading to a death. This study confirms the activity of the initial IL-2/LAK cell regimen in metastatic melanoma reported by Rosenberg et al, supporting the concept of adoptive immunotherapy as an important new treatment approach for this disease.

NF-kappaB/Rel transcription factors have been implicated in the differentiation of monocytes to either dendritic cells (DCs) or macrophages, as well as in the maturation of DCs from antigen-processing to antigen-presenting cells. Recent studies of the expression pattern of Rel proteins and their inhibitors (IkappaBs) suggest that their regulation during this differentiation process is transcriptional. To investigate differential gene expression between macrophages and DCs, we used commercially available gene microarrays (GEArray KIT), which included four of the NF-kappaB/Rel family genes (p50/p105, p52/p100, RelB, and c-rel) and 32 additional genes either in the NF-kappaB signal transduction pathway or under transcriptional control of NF-kappaB/Rel factors. To generate macrophages and DCs, human adherent peripheral blood monocytes were cultured with M-CSF or GM-CSF + IL-4 respectively for up to 8 days. DCs (and in some experiments, macrophages) were treated with lipopolysaccharide (LPS) for the last 48 h of culture to induce maturation. Cells were harvested after 7 days, cDNA was prepared and radiolabeled with alpha-(32)P-dCTP, then hybridized to gene arrays containing specific gene probes. beta-actin and GAPDH or PUC18 oligonucleotides served as positive or negative controls, respectively. The expression of all four NF-kappaB/Rel family genes examined was significantly upregulated in maturing DCs compared to macrophages. The strongest difference was observed for c-rel. RT-PCR determinations of c-rel, RelB, and p105 mRNAs confirmed these observations. Among the 32 NF-kappaB/Rel pathway genes, 14 were upregulated in mature DCs compared to macrophages. These genes were IkappaBalpha, IKK-beta, NIK, ICAM-1, P-selectin, E-selectin, TNF-alpha, TNFR2, TNFAIP3, IL-1alpha, IL-1R1, IL-1R2, IRAK, and TANK. By contrast, only mcp-1 (monocyte chemotactic protein 1) was upregulated in macrophages compared to DCs. NF-kappaB pathway genes upregulated in DCs compared to macrophages were constitutively expressed in monocytes then selectively downregulated during macrophage but not DC differentiation. LPS did not induce expression of most of these genes in macrophages but LPS did induce upregulation of IL-8 in mature macrophages. We conclude that NF-kappaB/Rel family genes, especially c-rel, are selectively expressed during differentiation of monocytes towards DCs. Moreover, this differential expression is associated both with activation of different NF-kappaB signal transduction pathways in DCs and macrophages and with expression of a unique subset of genes in DCs that are transcriptionally targeted by NF-kappaB/Rel factors. The results illustrate the ability of the NF-kappaB pathway to respond to differentiation stimuli by activating in a cell-specific manner unique signalling pathways and subsets of NF-kappaB target genes.