# Briefing Information -- BRMAC February 27, 2002

Unrelated allogeneic hematopoietic stem/progenitor cells from
placental/umbilical cord blood for hematopoietic reconstitution

## **Introduction**

Health care professionals have had extensive clinical and non-clinical
laboratory experience with placental/umbilical cord blood for
hematopoietic reconstitution (UCB) since the first reported transplant
of UCB in a child with Fanconi anemia in 1988. By 1993, large public
repositories of UCB were established, in New York, Milan, and
Dusseldorf^1^. The functionality of long-term cryopreserved UCB cells
was demonstrated by Broxmeyer et al. in their in vitro studies using CFU
assays and, more recently, by the transplantation of CBU stored frozen
for 15 years into NOD/SCID mice^2^. A landmark clinical study of the
efficacy of UCB as an alternative source of hematopoietic
stem/progenitor cells (HSPCs), published by Rubinstein et al. in 1998,
describes outcomes of a large cohort of recipients of UCB transplants
from unrelated donors^3^. Over 2000 cord blood transplants have been
performed worldwide, according to recent estimates. Many published
reports support the use of UCB as an acceptable source of HSPCs for
transplantation for selected recipients^1^.

Currently, it is estimated that there are 70,000 cryopreserved,
HLA-typed UCB products stored in cord banks worldwide^1^. Many of the US
establishments that collect, process, and store UCB products follow
voluntary standards published by the AABB, FACT/NETCORD, and the NMDP.
Despite the availability of published standards, the quality and
characteristics of these stored products vary markedly due to several
controllable and uncontrollable factors. First, the medical technology
is rapidly evolving, resulting in the use of diverse collection and
processing techniques at different facilities. Also, there is inherent
variability in product volume due to uncontrollable donor factors. In
addition, there is variability in the tests used for determining
sterility and potency of the final product. Finally, the criteria for
acceptability of a UCB product into an inventory vary among facilities,
and the criteria for selection of a particular UCB product, such as
extent of HLA match and total nucleated cell dose, are determined
locally by transplant practitioners.

## **CBER issues for consideration**

FDA seeks further advice and comments from the Advisory Committee about:
(1) efficacy data derived from clinical studies of cord blood
transplantation in recipients of particular age groups, and (2) assays
for UCB potency; specifically, the utility of CD34+ cell count for
predicting engraftment after cord blood transplant.

[Efficacy of UCB from unrelated allogeneic donors for hematopoietic
reconstitution]{.underline}

The major concerns of CBER, with respect to the use of UCB as an
alternative source of HSPCs, are similar to those for HSPCs derived from
peripheral blood (PB) or bone marrow (BM). They include: cell dose and
how to measure it; safety, purity and potency of the product; donor
safety; clinical outcome; and adverse reactions.

In addition, there are concerns and considerations that are particularly
relevant to UCB. First, there are a limited number of HSPCs obtainable
from a single donor. Thus, the dose of cells from one donor may be lower
than necessary for engraftment. One consequence may be a higher rate of
delayed engraftment or engraftment failure in adult recipients of UCB
transplants. A balanced comparison to BM or PB is difficult due to
confounding effects in much of the reported data, including different
degrees of HLA disparity and differences in the disease entities and/or
stage of disease being treated.

Second, UCB may have different properties or may contain different
populations of primitive cells or lineages when compared with HSPCs
derived from BM and PB ^4^. There is suggestive evidence for a lower
frequency of severe GVHD after UCB transplants, although direct
controlled comparisons are not yet available. A major function of
transplanted allogeneic HSPC products is to supplement intensive
chemotherapy in removing tumor cells. This so-called graft versus
leukemia/tumor effect is uncertain with UCB transplants.

Finally, all HPSC infusions provide a mixed population of early cells,
some subsets of which may be relatively more mature and capable of
providing cells for controlling infection rather than repopulating
hematological lineages in a continuous manner. Once again there is
little available data with which to evaluate different sources of HSPCs,
with regard to subsets of true progenitor cells and more mature
hematopoietic precursors.

Today we are focusing on the efficacy of UCB transplants from unrelated
allogeneic donors and assays for potency testing of UCB products. The
four questions posed to the committee and the advice being sought from
the committee relate to these issues. The three invited guest speakers
will address critical aspects of the efficacy of UCB and review updated
UCB transplant experience.

[CBER analysis of clinical outcome data]{.underline}

Data on clinical outcomes of UCB transplantation made available to CBER
consisted of summary data. However, CBER recognized the importance of
using primary data as the basis for analyzing clinical outcome. For this
reason a series of analyses were performed by CBER staff on primary data
provided by Drs Pablo Rubinstein and Cladd Stevens of the NY Blood
Center. This data was selected as the most suitable data to analyze
because it was the most extensive.

There are multiple interacting factors that are considered in the
selection of an optimal UCB product for transplantation and prediction
of transplant outcomes. These factors include age and weight of the
recipient, cell dose, HLA parity or disparity, among others. The
following analyses focus on the age of the recipient and the number of
cells transplanted as points of central interest. The results of our
analysis are displayed in the four attachments to this briefing
document.

Table 1 shows each of the four clinical outcomes which were examined;
time (days) to achieve neutrophil engraftment, \[\> 500 Absolute
Nucleated Cells /mm3\], time (days) to achieve platelet engraftment
\[\>20,000 platelets /mm3\], incidence of severe acute Graft Versus Host
Disease \[aGVHD grade III and IV\], and disease free survival. For
purposes of analysis age ranges were grouped into three-year cohorts
from newborn to age 29. There are sharp differences for all four
outcomes between the youngest and oldest age groups. A stepwise analysis
shows that the trend toward poorer outcomes becomes most marked in the
middle range of cohorts for platelet engraftment and disease free
survival, less so for ANC engraftment and even less clearly present for
aGVHD.

Figure 1 graphically shows the same data. The numbers of patients in
each cohort are shown below the abscissa.

Figure 2 shows disease free survival. It compares successive age
cohorts. The most substantial difference between cohorts is between the
cohort 10 (ages 9-11 years) and cohort 13 (ages 12-14 years). The
numbers of transplants available for analysis were relatively lower and
therefore less informative in the older age groups. Figure 1 shows the
numbers of patients in each cohort.

Table 2 and figure 3 compare the 0-12 years to the 13-29 year old
patients for the percentage achieving each outcome as well as the odds
ratios. There is again a sharp difference in outcomes, with the older
group having the poorer outcomes. Figure 3 shows disease free survival
for the two groups compared above. The difference is statistically
significant.

In the cord blood transplant setting the age-related outcomes are, in
part, widely believed to be related to the number of nucleated cells in
a single unit of cord blood, as mentioned earlier ^5^. Adult recipients
generally weigh more than children and the number of UCB nucleated cells
expressed per kg body weight of recipient will be less in older
recipients. Rubinstein et al. in their analysis of data on UCB
transplants emphasize a close relationship between the number of cells
and engraftment success^3^. BM and peripheral blood HSPC transplant
outcomes also demonstrate age-related increases in transplant related
events.

The results of CBER analyses can be interpreted as showing that:

\(i\) Older UCB recipients have a poorer outcome.

\(ii\) The poorer outcomes extend over the entire chronological age
range of data but appear to be marked at or around ages 12-13.

[Characterization and assays for potency testing of UCB]{.underline}

Although the only true measure of the potency of a HSPC graft is
hematopoietic reconstitution in an ablated recipient, there are a
variety of *in vitro* surrogates that have been used to attempt to
predict engraftment potential. In order to be useful as clinical
correlates, *in vitro* assessment of the viability, composition and
function of these surrogates must be able to predict the product's
capacity to engraft *in vivo*.

While human self-renewing pluripotential marrow-repopulating stem cells
cannot be directly enumerated, their existence can be inferred by
analysis of their progeny. Numerous studies in murine and canine models
indicate a strong correlation between the transplanted dose of
colony-forming progenitor cells and time to sustained hematopoietic
reconstitution^6;\ 7^. Other reports from clinical trials in human
subjects demonstrate a correlation between transplanted CFU-GM and days
to neutrophil recovery ^8^.

Complex *in vitro* systems for culturing progenitor cells at various
levels of commitment have been developed to predict the type and number
of cells necessary for engraftment. The earlier, less committed
progenitors, such as cobblestone area-forming cells (CAFC), high
proliferative potential colony-forming cells (HPP-CFC) and long term
culture-initiating cells (LTC-IC) are present in low numbers and require
long culture periods, so their utility is confined to experimental
situations. As the level of commitment increases, the more committed
progenitors such as CFU-GEMM, CFU-GM, and BFU-E are present in
increasing numbers, with colonies apparent in 10 to 14 days. This may be
enough time for evaluating products collected and stored for later
infusion, but is not appropriate for assessing products collected for
immediate transplantation or for monitoring circulating progenitor cell
levels in peripheral blood of progenitor cell donors.

Furthermore, although there are data suggesting that the number of
CFU-GM may be predictive of hematopoietic reconstitution, these culture
assays are not standardized and the wide variety of colony shapes and
sizes makes interpretation difficult and subjective. Because of these
difficulties, correlation between colony number and days to engraftment
varies widely in the published literature, suggesting the need for an
alternative indicator of progenitor cell content for more accurate
assessment of engraftment potential of progenitor cell grafts.

The purification of monoclonal antibodies to the CD34 antigen, a
transmembrane glycoprotein present on a diverse population of
multipotential and lineage-committed myeloid and lymphoid
progenitors^9^, permitted the development of a dual-color direct
immunofluorescence assay for rapid and reproducible enumeration of cells
expressing this marker^10^. A positive correlation between total CD34+
cell count and circulating day-14 CFU-GM has been demonstrated ^11^,
providing evidence that enumeration of CD34+ cells is a reliable
indicator of progenitor cell content and, by extension, hematopoietic
potential^12^. Although the function of CD34 is not known, experimental
evidence indicates a relationship with marrow localization and adhesion
of progenitor cells. For optimal specificity in identifying and
quantitating these rare cells (0.1 -- 0.4% of cord blood cells) (cite),
any immunophenotypic assay for CD34+ cells should include the use of a
vital nucleic acid dye such as propidium iodide or 7-amino-actinomycin D
(7-AAD)^13^. Addition of this parameter permits exclusion of dead cells
and debris.

Extensive clinical experience demonstrates that the CD34+ cell content
is an accurate predictor of neutrophil and platelet engraftment after
myeloablative chemotherapy in recipients of peripheral blood progenitor
cell transplants^12;\ 14^. In addition, recent data from Wagner et al.
from 102 patients receiving umbilical cord blood grafts identified CD34+
cell dose as the only factor among a number of variables that correlated
with rate of engraftment^15^. We anticipate further advances in the
available technology to assess the potency of hematopoietic
stem/progenitor cell products using new methodologies and yet to be
identified cell surface and bioactivity markers.

#### *[Table I]{.underline}*

####  *Clinical Outcomes for Different Age Groups*

+-------------+------------+------------+------------+----------------+
| **Age       | **A        | **A        | **AGVHD**  | **Disease      |
| Group**     | chieving** | chieving** |            | Free**         |
|             |            |            | **Grade**  |                |
|             | **ANC      | **Platelet |            | **Survival**   |
|             | 500**      | 20,000**   | **3/4**    |                |
+-------------+------------+------------+------------+----------------+
| **(0, 1,    | 122/148    | 80/137     | 21/124     | 78/151         |
| 2)**        |            |            |            |                |
|             | (82.4%)    | (58.4%)    | (16.9%)    | (51.7%)        |
+-------------+------------+------------+------------+----------------+
| **(3, 4,    | 63/87      | 35/81      | 15/68      | 32/89          |
| 5)**        |            |            |            |                |
|             | (72.4%)    | (43.2%)    | (22.1%)    | (36.0%)        |
+-------------+------------+------------+------------+----------------+
| **(6, 7,    | 52/75      | 33/69      | 12/54      | 27/75          |
| 8)**        |            |            |            |                |
|             | (69.3%)    | (47.8%)    | (22.2%)    | (36.0%)        |
+-------------+------------+------------+------------+----------------+
| **(9, 10,   | 50/63      | 34/62      | 11/52      | 25/64          |
| 11)**       |            |            |            |                |
|             | (79.4%)    | (54.8%)    | (21.2%)    | (39.1%)        |
+-------------+------------+------------+------------+----------------+
| **(12, 13,  | 26/45      | 10/39      | 9/24       | 11/45          |
| 14)**       |            |            |            |                |
|             | (57.8%)    | (25.6%)    | (37.5%)    | (24.4%)        |
+-------------+------------+------------+------------+----------------+
| **(15, 16,  | 25/36      | 10/35      | 6/25       | 9/36           |
| 17)**       |            |            |            |                |
|             | (69.4%)    | (28.6%)    | (24.0%)    | (25.0%)        |
+-------------+------------+------------+------------+----------------+
| **(18, 19,  | 6/12       | 4/12       | 1/6        | 4/12           |
| 20)**       |            |            |            |                |
|             | (50.0%)    | (33.3%)    | (16.7%)    | (33.3%)        |
+-------------+------------+------------+------------+----------------+
| **(21-29)** | 18/37      | 11/36      | 8/20       | 5/37           |
|             |            |            |            |                |
|             | (48.7%)    | (30.6%)    | (40.0%)    | (13.5%)        |
+-------------+------------+------------+------------+----------------+
| Total       | 362/503    | 217/471    | 83/373     | 191/509        |
+-------------+------------+------------+------------+----------------+

***Figure 1. Clinical outcomes for different age groups***

![](media/image1.wmf){width="2.0833333333333335in"
height="2.7291666666666665in"}
![](media/image2.wmf){width="2.2118055555555554in"
height="2.8965277777777776in"}

<table>
<colgroup>
<col style="width: 3%" />
<col style="width: 6%" />
<col style="width: 4%" />
<col style="width: 4%" />
<col style="width: 4%" />
<col style="width: 4%" />
<col style="width: 4%" />
<col style="width: 4%" />
<col style="width: 4%" />
<col style="width: 9%" />
<col style="width: 5%" />
<col style="width: 5%" />
<col style="width: 5%" />
<col style="width: 5%" />
<col style="width: 5%" />
<col style="width: 5%" />
<col style="width: 5%" />
<col style="width: 5%" />
</colgroup>
<tbody>
<tr class="odd">
<td><p id="n" class="heading">N</p></td>
<td>148</td>
<td>87</td>
<td>75</td>
<td>63</td>
<td>45</td>
<td>36</td>
<td>12</td>
<td>37</td>
<td></td>
<td>124</td>
<td>68</td>
<td>54</td>
<td>52</td>
<td>24</td>
<td>25</td>
<td>6</td>
<td>20</td>
</tr>
</tbody>
</table>

![](media/image3.wmf){width="2.0104166666666665in"
height="2.6979166666666665in"}
![](media/image4.wmf){width="2.2604166666666665in"
height="2.6666666666666665in"}

<table>
<colgroup>
<col style="width: 3%" />
<col style="width: 6%" />
<col style="width: 4%" />
<col style="width: 4%" />
<col style="width: 4%" />
<col style="width: 4%" />
<col style="width: 4%" />
<col style="width: 4%" />
<col style="width: 4%" />
<col style="width: 9%" />
<col style="width: 5%" />
<col style="width: 5%" />
<col style="width: 5%" />
<col style="width: 5%" />
<col style="width: 5%" />
<col style="width: 5%" />
<col style="width: 5%" />
<col style="width: 5%" />
</colgroup>
<tbody>
<tr class="odd">
<td><p id="n-1" class="heading">N</p></td>
<td>137</td>
<td>81</td>
<td>69</td>
<td>62</td>
<td>39</td>
<td>35</td>
<td>12</td>
<td>36</td>
<td></td>
<td>151</td>
<td>89</td>
<td>75</td>
<td>64</td>
<td>45</td>
<td>36</td>
<td>12</td>
<td>37</td>
</tr>
</tbody>
</table>

> ***Figure 2. Disease Free Survival Curves for Two Consecutive Age
> Groups***

![](media/image5.wmf){width="2.7493055555555554in"
height="1.9333333333333333in"}![](media/image6.wmf){width="0.4791666666666667in"
height="0.4166666666666667in"}

![](media/image7.wmf){width="2.7493055555555554in"
height="1.9284722222222221in"}![](media/image8.wmf){width="0.5416666666666666in"
height="0.4166666666666667in"}

![](media/image9.wmf){width="2.7493055555555554in"
height="1.9284722222222221in"}![](media/image10.wmf){width="0.5416666666666666in"
height="0.4166666666666667in"}

1\--Age 0-2; 4\--Age 3-5; 7\--Age 6-8; 10\--Age 9-11;

13\--Age 12-14; 16\--Age 15-17; 19\--Age 19-21.

> ![](media/image7.wmf){width="2.875in"
> height="2.0097222222222224in"}![](media/image11.wmf){width="0.4791666666666667in"
> height="0.4166666666666667in"}

![](media/image12.wmf){width="2.7493055555555554in"
height="1.9284722222222221in"}

![](media/image13.wmf){width="0.5416666666666666in"
height="0.4166666666666667in"}

![](media/image14.wmf){width="2.7493055555555554in"
height="1.9284722222222221in"}

![](media/image15.wmf){width="0.5416666666666666in"
height="0.4166666666666667in"}

**Table 2. Odds ratios for outcomes**

> ***Figure 3. Survival plot for younger and older age groups***

*(Age-Cut include age 12)*

![](media/image16.wmf){width="3.4895833333333335in"
height="2.4479166666666665in"}![](media/image17.wmf){width="0.4791666666666667in"
height="0.4166666666666667in"}

\*\* 1\-- Age 0-12; 2\-- Age 13-29

Tests Between Groups

  -------------------------------------------------------------------------
  Test                ChiSquare               DF        Prob\>ChiSq
  ------------------- ----------------------- --------- -------------------
  Log-Rank            24.76                   1         \<.0001

  Wilcoxon            26.95                   1         \<.0001
  -------------------------------------------------------------------------