Enhanced Ex vivo drug sensitivity testing of chronic lymphocytic leukaemia using refined DiSC assay methodology

Bosanquet, Andrew G.; Bell, Philip B.

doi:10.1016/0145-2126(95)00127-1

Cited by 37 publications

(46 citation statements)

References 26 publications

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“…11 Briefly, mononuclear cells were isolated from blood over FicollHypaque, washed, counted and incubated for 94 h with seven to 10 standard CLL drugs: chlorambucil, cyclophosphamide (as mafosfamide in vitro), prednisolone, vincristine, doxorubicin, epirubicin, fludarabine, cladribine, pentostatin and methylprednisolone. At the end of incubation, fixed duck erythrocytes (as an internal standard) and fast green/nigrosin (to stain dead cells black) were added and the cells cytocentrifuged.…”

Section: Methodsmentioning

confidence: 99%

Ex vivo assessment of drug response by differential staining cytotoxicity (DiSC) assay suggests a biological basis for equality of chemotherapy irrespective of age for patients with chronic lymphocytic leukaemia

Bosanquet

2000

Leukemia

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With a mean age at diagnosis for chronic lymphocytic leukaemia (CLL) of 65 years, development of optimal therapeutic regimens has been hampered by the advanced age of patients. In general, because of comorbidity older patients are not treated with the intent of achieving a complete response and so do not attain the quality of response of younger patients and do not survive as long. We have investigated whether or not ex vivo cellular sensitivity to cytotoxic drugs could be an underlying biological basis for this age differential in response and survival by comparing ex vivo drug response with age in untreated CLL patients. Cells from 365 untreated CLL patients aged 31.1-87.1 years (average 65.3 years) were tested for drug response by differential staining cytotoxicity (DiSC) assay with a panel of 10 drugs. An average of 280 results (range 196-361) obtained for each drug was compared with patient age. For chlorambucil, cyclophosphamide, prednisolone, vincristine, doxorubicin, epirubicin, fludarabine, cladribine and methylprednisolone, no relationship was found between ex vivo drug response and age (r Ͻ 0.12). For pentostatin, a possible but very weak relationship (r = 0.18; n = 210; P = 0.06) was found. We conclude that cellular sensitivity to cytotoxic drugs does not support the differential treatment of older and younger CLL patients. Leukemia (2000) 14, 712-715.

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Section: Methodsmentioning

confidence: 99%

Ex vivo assessment of drug response by differential staining cytotoxicity (DiSC) assay suggests a biological basis for equality of chemotherapy irrespective of age for patients with chronic lymphocytic leukaemia

Bosanquet

2000

Leukemia

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“…Subsequent morphological evaluation (ie of cell shrinkage and other typical morphological signs of apoptosis) of slides by light microscopy facilitated the determination of drug efficacy at each concentration; to obtain the net effect of druginduced apoptosis, live lymphocytes in the drug-treated samples were determined as a percentage of live lymphocytes in control cultures as described. 18 …”

Section: Chemosensitivity Assaysmentioning

confidence: 99%

“…18 In culture, B-CLL cells remain in G0/G1 phase non-proliferative state and begin to die by apoptosis over time, 4 and all drugs tested induced measurable drug induced apoptosis within 4 days. 18 A mixture of fast green and nigrosin dyes in 10 l PBS, and including fixed duck erythrocytes (50 000) as an internal standard, was added to the cell suspensions to stain dead cells black before cytocentrifugation on to microscope slides. The slides were air-dried and counter-stained with Romanowsky stain.…”

Section: Chemosensitivity Assaysmentioning

confidence: 99%

“…For drug sensitivity testing, fresh cells (8 × 10 4 ) were incubated in duplicate in 0.6 ml polypropylene tubes in wash medium plus 10% fetal bovine serum (FBS) (Gibco, Paisley, UK) or UltraCulture (BioWhittaker, Walkersville, MD, USA). Final volume of 90 l included 10% phosphate-buffered saline (PBS) either without (control samples) or with added 18 Cells were incubated at 37°C in a humidified atmosphere containing 5% CO 2 for 92 h, allowing time for all drugs studied to exert their apoptotic effect. 18 In culture, B-CLL cells remain in G0/G1 phase non-proliferative state and begin to die by apoptosis over time, 4 and all drugs tested induced measurable drug induced apoptosis within 4 days.…”

Section: Chemosensitivity Assaysmentioning

confidence: 99%

“…Final volume of 90 l included 10% phosphate-buffered saline (PBS) either without (control samples) or with added 18 Cells were incubated at 37°C in a humidified atmosphere containing 5% CO 2 for 92 h, allowing time for all drugs studied to exert their apoptotic effect. 18 In culture, B-CLL cells remain in G0/G1 phase non-proliferative state and begin to die by apoptosis over time, 4 and all drugs tested induced measurable drug induced apoptosis within 4 days. 18 A mixture of fast green and nigrosin dyes in 10 l PBS, and including fixed duck erythrocytes (50 000) as an internal standard, was added to the cell suspensions to stain dead cells black before cytocentrifugation on to microscope slides.…”

Section: Chemosensitivity Assaysmentioning

confidence: 99%

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Bax expression correlates with cellular drug sensitivity to doxorubicin, cyclophosphamide and chlorambucil but not fludarabine, cladribine or corticosteroids in B cell chronic lymphocytic leukemia

et al. 2002

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In B-CLL, non-proliferating B cells accumulate due to defective apoptosis. Cytotoxic therapies trigger apoptosis and deregulation of apoptotic pathways contributes to chemoresistance. Loss of the apoptosis-promoting Bax has been implicated in resistance to cytotoxic therapy. We therefore evaluated ex vivo drug sensitivity of CLL, producing chemoresponse data which are prognostic indicators for B-CLL, in particular in the case of purine nucleoside analogs. To analyze the underlying mechanisms of drug resistance, we compared endogenous Bax and Bcl-2 expression to ex vivo response to eight drugs, and to survival in 39 B-CLL patients. We found that reduced Bax levels correlated well with ex vivo resistance to traditional B-CLL therapies -anthracyclines, alkylating agents and vincristine (all P Ͻ 0.04). Surprisingly, no such relationship was observed for the purine nucleoside analogs or corticosteroids (all P Ͼ 0.5). Mutational analysis of p53 could not explain the loss of Bax protein expression. Levels of Bcl-2 were not associated with sensitivity to any drug. In contrast to the ex vivo data, neither Bax or Bcl-2 expression nor doxorubicin sensitivity were associated with increased survival whereas sensitivity to fludarabine correlated with better overall survival (P = 0.031). These findings suggest that the resistance to purine nucleoside analogs and corticosteroids in B-CLL is due to inactivation of pathways different from those activated by anthracyclines, vinca alkaloids and alkylating agents and may be the molecular rationale for the efficacy of purine analogs in this disease.

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CD79a detected by ZL7.4 separates chronic lymphocytic leukemia from mantle cell lymphoma in the leukemic phase

1999

Self Cite

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Both B‐cell chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL) are characterized by a lymphoproliferation of neoplastic CD5+ B‐cells, but an accurate differential diagnosis between these two malignancies is vitally important for guiding treatment options. Because CD79a has been identified as a pan‐B marker, we intended to use it in place of CD19 to identify B‐cells and to use CD23 to distinguish between CLL and MCL in the leukemic phase. Anti‐CD79a (clone ZL7.4) was used to detect the Igα/mb1 protein in fresh CD5+ B‐lymphocytes by dual‐channel flow cytometry. Expression of CD19 and CD23 were similarly assessed. As expected, CD19 was expressed in all specimens, whereas CD23 expression was zero in 3/4 MCLs, weak in 1/4 MCLs, and 2/8 CLLs (10–19%) and stronger in 6/8 CLLs (≥45%). However, although all the CD19+/CD5+ cells of MCL expressed high CD79a levels, CD79a expression was negligible or absent in 8/8 CLL specimens (mean positivity for CD79a = 2.41 ± 2.71%). CD79a (ZL7.4) levels may provide a more reliable distinction than CD23 levels between CLL and MCL. If these results hold up in a larger series, we recommend that the ZL7.4 antibody should be considered in routine marker panels for CLL and low‐grade lymphoma. Cytometry (Comm. Clin. Cytometry) 38: 102–105, 1999. © 1999 Wiley‐Liss, Inc.

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Enhanced Ex vivo drug sensitivity testing of chronic lymphocytic leukaemia using refined DiSC assay methodology

Cited by 37 publications

References 26 publications

Ex vivo assessment of drug response by differential staining cytotoxicity (DiSC) assay suggests a biological basis for equality of chemotherapy irrespective of age for patients with chronic lymphocytic leukaemia

Ex vivo assessment of drug response by differential staining cytotoxicity (DiSC) assay suggests a biological basis for equality of chemotherapy irrespective of age for patients with chronic lymphocytic leukaemia

Bax expression correlates with cellular drug sensitivity to doxorubicin, cyclophosphamide and chlorambucil but not fludarabine, cladribine or corticosteroids in B cell chronic lymphocytic leukemia

CD79a detected by ZL7.4 separates chronic lymphocytic leukemia from mantle cell lymphoma in the leukemic phase

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