1. Different strategies for identifying residual disease in the setting of B-Lymphoblastic Leukemia (B-LL) post anti-CD19 therapy Sa A.Wang1 Theodore Laetsch2 “Buddy” Frank Fuda2 and Sindhu Cherian3
1 MD Anderson Cancer Center; 2 University of Texas Southwestern Medical Center; 3 University of Washington Medical Center

2. Introduction
The presence of residual disease post therapy is an important prognostic factor for B cell lymphoblastic leukemia (B-LL)
Minimal residual disease in B-LL can be identified immunophenotypically through flow cytometry (the focus of this presentation) or genetically through molecular studies

3. Identification of residual B-LL by flow cytometry
A “fingerprint” or leukemia associated immunophenotype will be identified for the B-lymphoblast population on the original diagnostic material
This neoplastic fingerprint will show an expression pattern for several key markers that deviates from the normal expression pattern of a hematogone population
Identifying residual disease on follow up cases involves recognizing this neoplastic fingerprint on any remaining B-LL population
Historically, minimal residual disease (MRD) was defined as identification of residual disease by flow cytometry or molecular studies when the blast count was below 5% of total nucleated cells on morphologic examination. This antiquated definition has little value in current clinical practice. Rather, the actual percentage of residual disease should be reported. MRD can be identified by flow cytometry down to 0.01% depending on the sensitivity of the particular flow cytometric testing system. When performing MRD analysis, the sensitivity of the validated test should be clearly indicated on the test report.

4. Approach to B-LL MRD assessment
CD19
B-lymphoblast
The B-lineage marker CD19 is an ideal marker for identifying B-lineage cell populations including B-LL
CD19 is almost universally expressed on B-LL
CD19 is relatively bright on most B-LL
Bright CD19 is typically preserved in post therapy B-LL

5. Therefore, most strategies for detection of minimal residual disease (MRD) begin with gating CD19+ populations to identify the abnormal blast population. PE
Anti-CD19 Antibody
CD19
B-lymphoblast

6. CD19 is also ideal as a tumor target for therapy in B-LL
B-lymphoblast
CD19 is also ideal as a tumor target for therapy in B-LL
CD19 is typically expressed strongly on all B-lymphoblasts
CD19 is easily accessible since it is expressed on the surface of B-lymphoblasts
CD19 is absent on indispensible normal cells
CD19 is not expressed on pluripotential bone marrow stem cells
CD19 is restricted to B-cells and possibly follicular dendritic cells
Purified intravenous immunoglobulin therapy (IVIG) can be given to correct iatric induced B-cell immunodeficiency

7. Chimeric Antigen Receptor T-cell (CART)-19 Therapy for B-Lymphoblastic Leukemia
CART-19 Cell
Anti-CD19 Receptor
CD19
B-lymphoblast
CART-19 are T-cells genetically engineered to express a chimeric T-cell receptor directed against CD19
These T-cells have cancer killing ability and can expand and persist after infusion
CART-19 therapy is currently under investigation and is currently not FDA approved
CART-19 therapy is showing significant efficacy in the treatment of B-LL patients with refractory disease
See ICCS newsletter for more information at the following link
A third generation of CART-19 therapy is currently being evaluated

8. Uses of flow cytometry in B-LL treated with CART-19 therapy
1. Identify persistence of CART-19 cells
Efficacy depends upon the persistence of CART-19 cells
2. Assessing for MRD
The focus of this presentation

9. Flow cytometric MRD assessment following CART-19 B-LL therapy
Objectives
A). Identify persistent B-LL
B). Identify and monitor for re-emergence of normal CD19+ B-lineage cells
You should not only look for MRD but also look for CD19(+) B-lymphocytes and CD19(+) Hematogones.
The significance of identifying minute populations of normal CD19(+) B-cells is currently unclear
Identifying larger CD19(+) B-cells populations suggests that the CART-19 cells have significantly diminished or are now absent.

10. Diagnostic challenges following anti-CD19 therapy
In the setting of prior anti CD19 therapy, a CD19 negative B-LL clone may emerge and be missed by standard CD19 gating strategies
Therefore, the approach to B-LL MRD assessment should be adjusted in this setting

11. Approaches to MRD detection in B-LL post anti CD19 therapy
Several strategies may be considered in approaching MRD detection in patients post anti-CD19 therapies
We describe in this presentation 3 approaches
Use of an alternative antigen that have built in the MRD panel for MRD detection
Sa Wang, MDACC, Houston
Use of B cell markers other than CD19 for B cell detection
Sindhu Cherian, University of Washington, Seattle
B-LL MRD detection following CART-19 therapy
“Buddy” Frank Fuda, UT Southwestern, Dallas

12. Use of an alternative blast antigen for MRD detection Sa Wang MDACC
2 cases demonstrate the use of CD10 and CD34 to detect MRD post anti-CD19 therapy

13. Case 1-MDACC
27 year old with Ph+ B-ALL, treated with hyper-CVAD plus dasatinib, relapsed.
The patient was treated with CAR-T at outside hospital, relapsed, came to us.
Original B-ALL are CD19+, CD10partial+, CD20partial+, CD22 partial, CD25subset+, CD45-neg, CD38neg

14. Flow cytometry MRD, gating
No obvious blast population on CD45/SSC
There are no CD19 positive cells
Change the CD19 gating to CD34 gating. A total of 0.168% CD34+ cells seen
Further showing no CD19 expression on CD34+ cells

15. These cells are partial CD10, similar to pretreatment B-ALL
CD34+ cells
These cells express CD22, partial CD20, and are negative for CD13/CD33 (not shown) confirming these to be B cell lineage and not myeloid precursors
These cells are partial CD10, similar to pretreatment B-ALL
These immature cells show a number of abnormalities, with partial CD25 expression, negative/low CD38 and negative CD45, similar to pretreatment B-ALL

16. Case 2-MDACC
25 year old with Ph-negative B-ALL, treated with hyper-CVAD, recurred; treated with salvage chemotherapy, again recurred; then treated with Blinatumomab
Monoclonal antibodies, bi-specific T-cell engagers (BiTEs), that exert action selectively and direct the human immune system to act against tumor cells. Blinatumomab specifically targets the CD19 antigen present on B cells
The pretreatment B-ALL were CD19+, CD34+, CD20+, CD22+, CD10bright+, CD45-neg/very dim, CD38low, CD58bright

17. Flow cytometry MRD, gating
Change the CD19 gating to CD10/low SSC gating. A total of 0.275% CD10/low SSC+ cells seen
There are no CD19 positive cells

18. CD10/low SSC cells
These cells express CD34, CD22, and bright CD20, consistent with B-lymphoblasts
These lymphoblasts show increased CD58expression, and decreased CD45 and CD38 expression

19. Case 2 The patient continued the treatment, this is the follow-up MRD study
There are no CD19 positive cells
There are also no CD10/low SSC cells
There are a small number of CD34+cells

20. CD34+ cells are normal myeloid precursors
Unlike B-ALL cells, CD34+ cells have no CD10 expression
Unlike B-ALL cells, CD34+ cells have no CD20 expression
Partial/small subset of CD22 expression likely corresponds to plasmacytoid dendritic cell precursors
Unlike B-ALL cells, CD34+ cells have moderate CD45 and bright CD38 expression
CD34+ cells are CD13+, CD33+, consistent with myeloid precursors

21. Use of alternative B cell markers for MRD detection Sindhu Cherian, University of Washington, Seattle
A description of an assay using expression of CD22 or CD24 for B cell and MRD detection follows

22. 21 year old male, bone marrow aspirate 8 months post CART-19 therapy
* Genetically modified T cells expressing a chimeric antigen receptor with specificity for CD19
However, an abnormal blast population is seen on a plot of CD45 vs SSC (red oval) and blasts are seen on the aspirate smear
No CD19 positive cells are present

23. The abnormal population is clearly seen in other projections
Normal for comparison: plot shows all normal CD19+ B cells
Patient: Plot shows all viable cells
For example, in a plot of CD10 versus CD38, an abnormal population can be seen expressing bright CD10 with low to absent CD38. This population is absent in the example in plot 2 which shows CD19+ normal B cells (see arrow). This abnormal population is highlighted in orange in the next slide.

24. The abnormal population is highlighted in orange
The abnormal cells are highlighted in orange and express bright CD10 and low CD45 without CD34 or CD19. This patient has relapsed post CART-19 therapy with a CD19 negative clone.
Although this population was not detected using CD19, alternative B cell markers can be used to identify the population (see next slide).

25. Alternative B cell markers may be used to identify CD19 negative neoplastic B cell populations
In this case, the CD19 negative blasts expressed additional B cell markers CD22, CD24, and cCD79a. Any of these markers could be used for B cell identification in this case

26. Standardize your approach
For MRD detection, pattern recognition is critical.
For this reason in our laboratory we have adopted a standardized approach using expression of CD22 or CD24 without CD66b (as CD24 is also expressed on neutrophils) for initial B cell identification in samples submitted for MRD detection post anti-CD19 therapy
Once we identify the B cell population we use a difference from normal approach to identify the an abnormal population based on aberrant expression of markers expressed at varying levels during B cell maturation (including CD10, CD20, CD38, CD45)

27. Standardize your approach1 2
Step 2: After B cells are defined using expression of CD22 or CD24 without CD66b, a variety of combinations are evaluated for evidence of aberrant antigen expression. This example shows normal hematogones and mature B cells with no abnormal blast population.
Of note, this strategy allows identification of B cells (shown in pink) at all stages of maturation but also includes some cells that are not B cells. For instance, the population shown in orange largely consists of CD22 positive, CD24 negative basophils
Step 1. B cells are defined by expression of CD22 or CD24 without CD66b
Preliminary data presented as a poster presentation at ICCS 2014, Seattle WA.

28. Abnormal: Data from patient post CART 19 therapy
The upper plots show a normal sample containing hematogones and mature B cells while the lower plots are from a patient with history of B-LL, now post CART 19 therapy. An abnormal population (highlighted in red) is present at 0.06% of the white blood cells. Note, this population has expression of bright CD10 and CD34 without CD38 and with low to absent CD45, an immunophenotype not seen in normal B cell maturation.
Abnormal: Data from patient post CART 19 therapy
Hp16-29

29. Impact of changes to your gating strategy
When you alter your gating strategy and use a different marker to identify your B cells other than CD19, it is critical to re-establish what represents normal
This requires a thorough validation including steps to:
Confirm that you can detect both normal and abnormal B cell populations seen in a CD19 gate using your alternative B cell gate
Describe populations included using your alternative B cell gate that would not be seen in a CD19 defined B cell gate
For instance, basophlis are CD22 positive and will be included using this gating strategy.

30. B-LL MRD detection following CART-19 therapy Buddy Fuda, University of Texas Southwestern Medical Center, Dallas

31. B-LL MRD detection following CART-19 therapy
Analysis strategies differ between different flow cytometry laboratories
Regardless of the analysis approach, identifying MRD in B-LL following CART-19 therapy remains similar to the assessment in non-CART-19 treated cases
As mentioned previously, the key to identifying minimal residual B-LL in post CART-19 therapy is still identifying deviation from the normal expression pattern of hematogones
However, in B-LL following CART-19 therapy, CD19 may be absent from a residual B-LL clone, and therefore, a combination of alternative markers may be helpful to identify the disease
Excellent examples of different approaches for CART-19 treated B-LL are presented in the preceding slides from MDAAC and the University of Washington

32. Our laboratory does not employ a rigid sequential gating strategy but rather allows the analyst to “color and clean” clusters of cells in any manner that they choose
As such, the most important step in our approach to B-LL MRD assessment, post CART-19 therapy is the panel of markers chosen
On these cases, we perform our routine B-LL follow up panel, which includes B-lineage markers CD19, CD20 and surface Igs, but we also add additional B-lineage markers or other markers depending on the original immunophenotype
If the original B-LL immunophenotype is not available, we begin by adding several additional B-lineage markers not included in our routine panel
These B-lineage markers may include CD22, cCD79a, and/or CD24
We will include at least one tube that has CD19 in combination with one of these other B-lineage markers in addition to markers that enable us to identify deviation from normal B-lineage cells including CD10, CD13, CD15, CD20, CD33, CD34, CD38, CD45, surface Igs, and/or TdT
It is important to remember, that B-LL MRD, post CART-19 therapy may or may not express CD19. Furthermore, the persistence or recovery of normal CD19(+) B-cells has clinical implications. Therefore, assessment of CD19 remains an important step in the analysis

33. CD34(predominantly +/few -)
The following is an example of a case of a patient with relapsed B-LL followed by CART-CD19 therapy
Originally diagnosed at outside institution as B-LL
The immunophenotype was not available
The patient achieved remission but had a relapse of B-LL with the following immunophenotype:
The patient was placed on CART-19 therapy
CD10(bright +)
CD13(partial +)
CD15(predominantly -)
CD19(+)
CD20(-)
CD22(+)
CD25(-)
CD34(predominantly +/few -)
CD38(partial dim +)
CD45(-)
cCD79a(+)
surface Ig(-)
HLA-DR(partial +)
TdT(-)
MPO(-)

34. Follow up Flow Cytometry
The patient had 3 post CART-19 follow ups that showed disease remission
Follow up Flow Cytometry
Our standard 4 color B-LL follow up panel includes B-lineage markers
CD19
CD20
CD22
(notably, CD22 was included in our 4-color B-LL follow up panel but is not in our current 10-color panel and would need to be added separately in a custom tube)
Surface Igs

35. Our Standard Four Color B-LL Follow up panel
FITC PE
PerCP
APC 10 22 20 34 14 45 38 pL pK 19 36 64 16 56
11b 8 5 3 4 15 33 13 DR

36. Additional 4 color tubes were added to assess for a possible CD19(-) B-LL clone and included selected markers (colored blue) that were distinctly aberrant on the relapsed B-LL [i.e., CD45(-)/CD38(partial dim +)/CD33(+)]
1st follow up
10/22/45/38 and 10/79b*/45/38
*Note: While CD79b is a B-lineage marker, the vast majority of B-LL do not show surface CD79b expression. In our limited experience with this antibody, normal hematogones do not significantly express surface CD79b either. Hence, we do not recommend using surface CD79b as a alternative to CD19 in post CART-19 therapy cases. Cytoplasmic expression of CD79b is only seen in approximately 2/3rds of B-LL.1 Alternatively, cytoplasmic CD79a is commonly expressed in B-LL and would be a better choice as an alternative to CD19.
2nd follow up
10/22/45/38; 10/22/45/19; and 15/33/34/22
Astsaturov IA, Matutes E, Morilla R, Seon BK, Mason DY, Farahat N, Catovsky D. Differential expression of B29 (CD79b) and mb-1 (CD79a) proteins in
acute lymphoblastic leukaemia. Leukemia May;10(5):

37. Analysis was performed on ungated data
Example plots from selected tubes are presented in the following slides
CD19(+) cells were identified but showed a relatively normal expression pattern reminiscent of hematogones and lacked the distinct aberrancy seen on the B-LL
These cells were normal CD19(+) hematogones

38. Relapsed
B-LL
Follow up
CD19(+) Hematogones
Relapsed
B-LL
Follow up
CD19(+) Hematogones

39. At this point we can see that there is no residual CD19(+) B-LL clone
Now, we would focus on the remainder of the original B-LL immunophenotypic fingerprint to look for a CD19(-) B-LL clone
For instance, we know that the relapsed B-LL was negative for both CD45 and CD38 expression
So, we can focus on that area to see if there is CD19(-) MRD

40. Relapsed B-LL Could CD19(-) B-LL be Follow up hiding in this area
The B-lymphoblasts were CD45(-) and CD38(partial dim +)
The few black dots
in this area are
CD36(+) (not shown)
and represent
maturing RBCs
After coloring these events black,
it is apparent that they are
mostly debris and mature
red blood cells. These are
still present because the
analysis is performed on
ungated data.

41. Alternatively, the B-LL was CD22(+)
Alternatively, the B-LL was CD22(+). Coloring the CD22(+) cells blue shows that none of them fall in the CD38(-)/CD45(-) area
The CD22(+)/CD10(+) cells are
hematogones, and the
CD22(+)/CD10(-) cells are
mostly basophils with a few
plasmacytoid dendritic cells
CD123 and HLA-DR could be
added to confirm the
identity of these CD22(+)/CD10(-)
cells in difficult cases. Basophils
are CD22(+)/CD123(+)/HLA-DR(-)
and plasmacytoid dendritic cells
are CD22(+)/CD123(+)/HLA-DR(+)

42. The patient showed disease remission in both follow up cases
Similarly, other distinct aberrancies from the B-LL fingerprint [e.g., CD33(+) on CD22(+) lymphoid cells] were investigated and no abnormal CD19(-) B-LL clone was identified
The patient showed disease remission in both follow up cases
Follow up #1 was performed at an MRD test sensitivity of 0.01%
Follow up #2 was performed at an MRD test sensitivity 0.5%
There was re-emergence of normal CD19(+) B-lineage cells in both cases
<0.10% in the first follow up case
<0.20% in the second follow up case

43. - Approximately 5% hematogones and <0.50% B-lymphocytes
A third follow up case showed disease remission at a test sensitivity of 0.01% with a higher re-emergence of CD19(+) B-lineage cells
- Approximately 5% hematogones and <0.50% B-lymphocytes
Blue: hematogones
Black: Mature B-lymphocytes
Here, we have significant CD19(+) B-cell recovery, which suggests
that the CART-19 cells have diminished

44. Another case with a similar scenario showing normal CD19(+) B-cell recovery
Original diagnosis of B-LL in February 2015 with remission
Relapsed B-LL in September 2015
Remission achieved with CART-19 therapy
Follow up in early February 2016 showed disease remission but B-cell recovery with 13% hematogones and 0.95% mature B-lymphocytes.
This suggests that no CART-19 cells remained
In the presumed CART-19 depleted state, a large CD19(+) B-LL clone recurred in late February 2016

45. Summary
With the increased investigational use of anti-CD19 therapy in B-LL, alternatives to the traditional use of CD19 for B cell gating are required
Alternative strategies may include the following:
Use of a different marker to identify the neoplastic population
Bright CD10, CD34
Use of alternative markers for B cell identification
Options to consider include CD22, CD24, and cCD79a or some combination of these markers
When using an alternative gating strategy to identify B cells it is critical to do a thorough validation
In some cases, molecular methods may be considered to provide an adjunct or alternative to MRD testing in B-LL

46. Summary
Recovery of normal B-cell populations likely correlates to a significant decrease or absence of CART-19 cells
Efficacy of the therapy seems dependent on the persistence of CART-19 cells; however, since CART-19 studies have only been conducted over a relatively short period of time, the importance of long term persistence is unknown
Flow cytometry can also be used to directly assess for CART-19 cells