# Presentation: 663345

## Bcl-2 is Expressed in CLL Patients 

- ASchena et al. Blood 1992;79:2981, BHanada et al. Blood 1993;15:1820, CRobertson et al. Leukemia 1996;10:456, DZaja F, Leuk Lymphoma 1998;28:567, EAviram et al. Eur J Haematol 2000;64:80, FKlobusicka et al. Neoplasma 2002;49:387, GMenendez et al. Leukemia 2004;18:491, HTracey et al. J Pathology 2005;206:123

**MA-9**

## Intracellular Uptake after Systemic Treatment

**MA-19**

- Dai et al., Clin Cancer Res 2005;11:2998

## Suppression of Bcl-2 Correlates with Intracellular Levels of Genasense

**K562 leukemia cell line, Dai et al. Clin Cancer Res. 2005;11:2998**

**Bcl-2 mRNA**
**(% of control)**

- Dose (μM)

**Intracellular Genasense**
**(pmol/mg protein)**

**MA-22**

**Suppression of Bcl-2 Correlates ****with Intracellular Levels of Genasense**

**Notes:**
The left axis shows the level of Bcl-2 RNA relative to untreated controls.  The right axis shows the measured intracellular level of Genasense.  

Note the extraordinary steepness of the reduction in Bcl-2 RNA at very low levels of Genasense.  Thus, low levels of Genasense are capable of causing significant reductions in Bcl-2 expression.  

Expression of the molecular target of Genasense, Bcl-2, correlates with Genasense activity, as shown in the next slide.

The IC50 occurred  0.29 micromoles/L – this is approximately the plasma steady state concentration of FULL LENGTH in patients treated at 3 mg/kg/day, and the N-1 and N-2 add together to about 60% of the full length oligo, so the total concentration of active oligos in plasma is approx 0.46 micromoles/L, above the IC50, and TISSUE concentrations are HIGHER.

Bcl-2 RNA by RT-PCR, Oblimersen by ELISA assay of Dai et al.

K562 cells were derived from a CML patient in blast cell crisis by Klein et al. Int J Cancer. 1976 Oct 15;18(4):421-31 

## Down Regulation of Bcl-2 in AML Patient Marrow

**0**

**5**

**11**

**0**

**70**

**60**

**50**

**40**

**30**

**20**

**10**

**Time (days)**

**Bcl-2 to Actin Ratio **
**(chemiluminescence)**

**Genasense 7 mg/kg/d qdx10 CIVI, Marcucci et al. Blood 2003; 15:425 **

**28**

**MA-23**

**Down Regulation of Bcl-2 ****in AML Patient Marrow**

**Notes:**
Marrow taken from an AML patient treated with Genasense for ten days and analyzed for Bcl-2 protein expression. In this study, Genasense was administered for ten days, with FLAG given from days 6-10. 

In the inset panel you can see the relative quantity of Bcl-2 to actin by western blot and the quantitation of the results as the Bcl-2 to actin ratio by chemiluniescent detection.

You can see a significant decrease in Bcl-2 protein protein by day 5, before the administration of any FLAG components.   (((This patient achieved a complete response.  Platelet and neutophil counts were recovered by the 28-day timepoint.)))  

Reduction of Bcl-2 RNA and protein in small groups of patients treated with Genasense has been published by independent research labs, and these reports include mononuclear cells from peripheral blood and bone marrow, AML cells, and solid tumor samples.  Another example is shown in the next slide.

Dai thesis, Figure 4.5, page 162.  Genasense was administered Days 1 through 10, see Figure 4.1 for regimen.  FLAG is fludarabine, cytarabine, and G-CSF.  See page 143 for discussion.  

## Down Regulation of Bcl-2 by GenasensePBMC from Pediatric Solid Tumor Patients

**Rheingold S et al., in preparation,  M. Hogarty laboratory, COG-ADVL0211 Study**

**MA-24**

**Notes:**
Rheingold et al. has analyzed lymphoid cells for Bcl-2 protein expression after treatment of pediatric patients across a range of Genasense doses.  

While this is an early result, you can see that the majority of the patients were treated with Genasense at 3 milligrams per kilogram per day, and the majority of patient samples showed a clear reduction in Bcl-2 protein at this dose.  

These analyses are continuing in Genasense sponsored trials that are underway at this time.

In summary...

## Age Impact on Response – Overall

|  | <75 year old
(N=214)
n (%) | > 75 year old
(N=27)
n (%) |
| --- | --- | --- |
| CRnPRPR, n % | 95 (44.4) | 8 (29.6) |
| CRnPR, n % | 26 (12) | 2 (7) |

## Age Impact on Response by Arm

|  | GFC |  | FC |  |
| --- | --- | --- | --- | --- |
|  | < 75
(N=103)
n (%) | ≥ 75
(N=17)
n (%) | < 75
(N=111)
n (%) | ≥ 75
(N=10)
n (%) |
| CR/nPR/PR | 45 (44) | 4 (24) | 50 (45) | 4 (40) |
| CR/nPR | 18 (18) | 2 (12) | 8 (7) | 0 |

## Reasons for Response Rate Difference in Study GL303

- Randomized
- Multicenter
- Blinded histopathologic review
- Blinded clinical review
- More heavily pretreated (≥ 4 prior regimens) (29% vs 9%)
- More Fludarabine Refractory (59% vs. 31%)
- CT/US scans used to determine response