Bone marrow core biopsy (Case HS-10-10547), 5 months prior to CART-19 therapy, H&E, 10x: The bone marrow biopsy obtained at 5 months prior to CART-19 therapy (submitted for review) demonstrates a hypercellular marrow (~60%) involved by a nodular interstitial expansion of small mature lymphocytes (~40% of total cellularity). Bone marrow core biopsy (Case HS-10-10547), 5 months prior to CART-19 therapy, H&E, 10x: The bone marrow biopsy obtained at 5 months prior to CART-19 therapy (submitted for review) demonstrates a hypercellular marrow (~60%) involved by a nodular interstitial expansion of small mature lymphocytes (~40% of total cellularity).
Bone marrow aspirate, Day 0 prior to CART-19 infusion, flow cytometry: Flow cytometry performed on the bone marrow aspirate obtained immediately prior to CART-19 therapy reveals an expansion (65% of total cellularity) of variably-sized bright surface kappa restricted CD5+ CD10- CD19+ CD20+ CD23+ CD38- IgM+ B cells.Bone marrow aspirate, Day 0 prior to CART-19 infusion, flow cytometry: Flow cytometry performed on the bone marrow aspirate obtained immediately prior to CART-19 therapy reveals an expansion (65% of total cellularity) of variably-sized bright surface kappa restricted CD5+ CD10- CD19+ CD20+ CD23+ CD38- IgM+ B cells.
Bone marrow aspirate, Day 0 prior to CART-19 infusion, cytogenetics: Cytogenetics performed at this time shows an abnormal karyotype 46,XY,del(17)(p12)[5]/46,XY,der(17)t(17;21)(q10;q10)[5]/46,XY[14]. Shown here is the deletion of the short arm of chromosome 17 at p12, which includes the TP53 locus.Bone marrow aspirate, Day 0 prior to CART-19 infusion, cytogenetics: Cytogenetics performed at this time shows an abnormal karyotype 46,XY,del(17)(p12)[5]/46,XY,der(17)t(17;21)(q10;q10)[5]/46,XY[14]. Shown here is the deletion of the short arm of chromosome 17 at p12, which includes the TP53 locus.
Bone marrow aspirate, Day 0 prior to CART-19 infusion, cytogenetics: Additionally, a derivative chromosome 17 is identified, which is composed of the long arm of chromosome 21 replacing the short arm of chromosome 17. This rearrangement leads to monosomy 17 from 17p10 through 17pter and trisomy 21, and loss of the TP53 locus.Bone marrow aspirate, Day 0 prior to CART-19 infusion, cytogenetics: Additionally, a derivative chromosome 17 is identified, which is composed of the long arm of chromosome 21 replacing the short arm of chromosome 17. This rearrangement leads to monosomy 17 from 17p10 through 17pter and trisomy 21, and loss of the TP53 locus.
The day -270 bone marrow core demonstrates a markedly hypercellular bone marrow (90%) composed of a dense lymphocytic infiltrate. The day 0 specimen is also hypercellular (60%), and shows a mostly interstitial expansion of lymphocytes. The day +23 bone marrow biopsy continues to be hypercellular, now with occasional interstitial aggregates of lymphocytes accounting for 10% of cellularity. The day +180 bone marrow is normocellular and contains scattered groups of small lymphocytes (<5% of cellularity).The day -270 bone marrow core demonstrates a markedly hypercellular bone marrow (90%) composed of a dense lymphocytic infiltrate. The day 0 specimen is also hypercellular (60%), and shows a mostly interstitial expansion of lymphocytes. The day +23 bone marrow biopsy continues to be hypercellular, now with occasional interstitial aggregates of lymphocytes accounting for 10% of cellularity. The day +180 bone marrow is normocellular and contains scattered groups of small lymphocytes (<5% of cellularity).
CD20 staining shows an overwhelming B cell infiltrate in the bone marrow specimen obtained 9 months prior to CART-19 therapy. CD20 also highlights the interstitial aggregates of B cells in the day 0 specimen. The day +23 bone marrow contains scattered lymphoid aggregates that are partially composed of CD20+ B cells. Finally, the day +180 bone marrow shows rare B cells.CD20 staining shows an overwhelming B cell infiltrate in the bone marrow specimen obtained 9 months prior to CART-19 therapy. CD20 also highlights the interstitial aggregates of B cells in the day 0 specimen. The day +23 bone marrow contains scattered lymphoid aggregates that are partially composed of CD20+ B cells. Finally, the day +180 bone marrow shows rare B cells.
Background CD3+ T cells can be seen admixed within the dense B cell infiltrate in the bone marrow core biopsy obtained 9 months and day 0 prior to CART-19 infusion. CD3 staining confirms that the lymphoid aggregates seen on the day +23 bone marrow are composed of both B and T cells. Finally, the day +180 specimen shows scattered CD3+ T cells, likely accounting for the majority of the lymphocyte population at this time.Background CD3+ T cells can be seen admixed within the dense B cell infiltrate in the bone marrow core biopsy obtained 9 months and day 0 prior to CART-19 infusion. CD3 staining confirms that the lymphoid aggregates seen on the day +23 bone marrow are composed of both B and T cells. Finally, the day +180 specimen shows scattered CD3+ T cells, likely accounting for the majority of the lymphocyte population at this time.
Detection of modified antigen receptor transgene over time: Genomic DNA isolated from serial samplings of the patient’s whole blood (A), and bone marrow aspirate (B) was subjected to quantitative real-time polymerase-chain-reaction (PCR) analysis to detect transgenic DNA. Whole blood specimens were also subjected to flow cytometric analysis to detect the presence of CART-19 T cells (A). Both assays show that the modified T cells expanded to levels greater than 1000x as high as initial engraftment levels in the peripheral blood and bone marrow. Further, the number of copies of transgene stayed elevated even at day +180 post infusion.Detection of modified antigen receptor transgene over time: Genomic DNA isolated from serial samplings of the patient’s whole blood (A), and bone marrow aspirate (B) was subjected to quantitative real-time polymerase-chain-reaction (PCR) analysis to detect transgenic DNA. Whole blood specimens were also subjected to flow cytometric analysis to detect the presence of CART-19 T cells (A). Both assays show that the modified T cells expanded to levels greater than 1000x as high as initial engraftment levels in the peripheral blood and bone marrow. Further, the number of copies of transgene stayed elevated even at day +180 post infusion.
Bone marrow core biopsy (Case HS-11-6512), 6 months (day +180) post CART-19 therapy, H&E, 20x: Bone marrow biopsy obtained at six months post CART-19 therapy (submitted for review) shows normocellular marrow with trilineage hematopoiesis, and no morphologic evidence of CLL.Bone marrow core biopsy (Case HS-11-6512), 6 months (day +180) post CART-19 therapy, H&E, 20x: Bone marrow biopsy obtained at six months post CART-19 therapy (submitted for review) shows normocellular marrow with trilineage hematopoiesis, and no morphologic evidence of CLL.
Bone marrow aspirate, 1 year post CART-19 therapy, flow cytometry: Flow cytometry performed on the bone marrow aspirate obtained 1 year post CART-19 therapy shows CD5+ T cells and a virtual absence of mature B cells.Bone marrow aspirate, 1 year post CART-19 therapy, flow cytometry: Flow cytometry performed on the bone marrow aspirate obtained 1 year post CART-19 therapy shows CD5+ T cells and a virtual absence of mature B cells.
Bone marrow aspirate, 2 years post CART-19 therapy, cytogenetics: Cytogenetics performed on the bone marrow aspirate obtained 2 years post CART-19 therapy shows a normal male karyotype 46,XY[25]. FISH confirmed the absence of TP53.Bone marrow aspirate, 2 years post CART-19 therapy, cytogenetics: Cytogenetics performed on the bone marrow aspirate obtained 2 years post CART-19 therapy shows a normal male karyotype 46,XY[25]. FISH confirmed the absence of TP53.