Response to Rossi et al.

To the Editor:

THANK you for providing us with the opportunity to respond

to the Communication regarding our published, peer-

reviewed Cytometry Part A paper entitled ‘‘User-defined pro-

tein marker assay development for characterization of circulat-

ing tumor cells using the CellSearch� system’’ [Cytometry A

2012;81A:983–995; doi: cyto.a.22158]. We appreciate the inter-

est in our work and will address the three specific concerns of

Rossi and colleagues in this response.

(1) Optimization conducted on spiked samples only,

without further validation in ex vivo samples:

Rossi et al. express concern regarding our use of only

spiked samples throughout the study and question the sound-

ness of our approach based on lack of validation in clinical

samples (1). As is clearly stated in the Introduction of the ori-

ginal article, the primary objective of our study was not to

demonstrate the clinical significance of our developed assays

but instead to provide a detailed description of the process of

user-defined protein marker development and optimization

using the CellSearch system such that it may act as a reference

guide for other users of this platform. Rossi et al. subsequently

state that in order to prove the clinical significance of a new

method, optimization must be undertaken in the setting of a

pilot set of patients. We could not agree more with this state-

ment, and as we move forward with implementing these assays

in our own lab, this is exactly our course of action.

Our interpretation of our study outcomes was based

solely on the assays’ expected performance when using highly

controllable models (cell lines with known expression of the

user-defined marker). We hypothesized that if optimized

results could not be obtained in these ‘‘best case scenario’’

model systems, then the assays would be unlikely to work with

a high degree of efficiency in more variable samples obtained

from patients. At no point do we assert that our model system

replicates the heterogeneous nature of patient samples but

instead that our findings be utilized as a starting point for

developing user-defined markers on the CellSearch that will

subsequently be considered for use in a clinical setting. This is

evidenced by our use of multiple cell lines when optimizing

the CD44 protocol. This approach allowed us to examine dif-

ferences in assay performance when presented with varying

degrees of marker expression (high/low/negative). These

results could not have been obtained from patient samples

since the level of marker expression and the presence or ab-

sence of a marker would not be known. We, therefore strongly

agree with Rossi et al. that clinical validation of all developed

assays of this nature, regardless of the platform utilized, is

absolutely necessary. However, initial assay validation with

known controls (as is described in our article) is always

required first in order to ensure appropriate quality control

and the sensitivity/specificity of the assay before implementing

it for use in patient samples.

(2) The choice of FCM as a reference method:

The second concern of Rossi et al. related to our use of

flow cytometry (FCM) as a reference method with which to

assess the ‘‘suitability’’ of our user-defined markers. As stated

in the Introduction of the original article, we are very aware

and agree with Rossi et al. that these systems should not be

directly compared with regards to their capacity for circulating

tumor cell (CTC) analysis. As described in the Methods and

Results section of our article, the FCM method was not used

to determine the ‘‘suitability’’ of user-defined markers but

rather to accurately determine the percentage of cells expres-

sing our protein of interest (i.e., EGFR, CD44, or M-30) in

samples consisting of tumor cells only (i.e., not in spiked

blood samples). All FCM experiments were done using

unspiked tumor cells in order to eliminate any confounding

sensitivity in CTC detection between the two platforms. Once

the percentage of cells expressing each marker was determined

by FCM, we could then take on the task of optimizing our

CellSearch protocols and ensure that these expression percen-

Received 21 March 2013; Accepted 26 March 2013

*Correspondence to: Alison L. Allan.

E-mail: Alison.Allan@lhsc.on.ca

Published online 23 April 2013 in Wiley Online Library(wileyonlinelibrary.com)

DOI: 10.1002/cyto.a.22298

© 2013 International Society for Advancement of Cytometry

Communication to the Editor

Cytometry Part A • 83A: 599�601, 2013

tages could also be obtained on the CellSearch. Without such

a comparison, it would not have been possible to determine if

the integrated assays were efficiently identifying all cells

expressing our markers of interest. The validity of this

approach is demonstrated in Figure 1 of the original article,

which shows that when using a Veridex-validated marker,

EGFR, there is no significant difference in marker expression

percentages observed using FCM in unspiked tumor cells vs.

spiked samples analyzed on the CellSearch system.

In addition, Rossi et al. question our conclusion that con-

taminating leukocytes could contribute to CD44 antigen

‘‘loss’’ and our subsequent decision to utilize the CellSearch

CXC kit (optimized for use with lower antigen density mar-

kers) for CD44 optimization instead of the traditional Cell-

Search CTC kit. Rossi et al. reference the mean fluorescence

intensity values obtained by FCM for fluorescein isothiocya-

nate (FITC) and phycoerythrin (PE) conjugated CD44 as evi-

dence that antigen density values were comparably high

regardless of which fluorophore was utilized. However, we

contend that direct comparison of flow cytometry detection

and the Veridex system was not the intention and is not

appropriate. The data presented clearly show a difference in

detection efficiency between FITC and PE using CD44 and the

Veridex system, on paired sample that were clearly strongly

positive for both of these fluorochromes using flow cytometry.

Whether this discrepancy is due to detection system differ-

ences or processing on the Veridex system was not explored.

Additionally, Rossi et al. suggest that leukocyte contam-

ination following enrichment would be equivalent to only

approximately 1000 leukocytes in a 7.5 mL blood volume

(2) and thus would not impact antibody staining. Our ini-

tial assumption (in agreement with Rossi et al) was that the

CellSearch system would provide a highly purified specimen

post-enrichment, with very few remaining leukocytes. How-

ever, our post-CellSearch analysis by FCM demonstrated a

high level of contaminating leukocytes in a spiked sample

that had been processed on the CellSearch system (Fig. 1A).

In fact, in this particular spiked sample, it was shown that

only 10% of the cells analyzed were CTCs, in a sample

spiked with 10,000 cells, 103 greater than the number of

cells utilized in our experiments. In addition, we have pro-

vided a CellSearch galley image in which a contaminating

leukocyte was captured within the same frame as a CTC, to

demonstrate that these cells can also stain positively for

CD44 (Fig. 1B). Based upon these results, we are confident

that our use of the CXC kit for CD44 optimization was

appropriate.

(3) M-30 as an apoptotic marker and its utility as an

indicator of tumor response:

Rossi et al. suggest that our manuscript criticizes the

choice of M-30 as an apoptosis marker and on its potential as

an indicator of tumor response. We would like to clarify any

misunderstanding with regards to our belief about the clinical

utility of M-30 vs. our interpretation of the results in terms of

Figure 1. Samples processed on the CellSearch system show blood cell contamination. (A) Flow cytometry dot-plot images of 10,000

EGFR-FITC1 MDA-MB-468 tumor cells spiked into 7.5 mL of blood and processed on the CellSearch system. These samples demonstrate

that post-CellSearch enrichment there is significant blood cell contamination, with <10% of the remaining cells classified as tumor cells.

Tumor cells were identif ied as EGFR-FITC1. (B) CellSearch gallery image of a white blood cell captured in the same frame as a CTC,

demonstrating that white blood cells will also stain positively for CD44-FITC. [Color figure can be viewed in the online issue, which is avail-

able at wileyonlinelibrary.com.]

COMMUNICATION TO THE EDITOR

600 Communication to the Editor

assay optimization. At several points throughout the manu-

script, we do state that the M-30 assay may not be ideally suited

for use in conjunction with the CellSearch system. Due to the

complexity of the apoptotic process and its many phases, we

submit that it may be difficult to optimize a single marker of

cell death on the CellSearch system (i.e., one that would capture

all dying/dead cells). This statement should not be misinter-

preted as a challenge to the clinical relevance and/or significance

of apoptotic assays and their use in conjunction with the Cell-

Search system. We recognize and acknowledge in the manu-

script that detection of all apoptotic CTCs may not be necessary

to demonstrate clinical response to therapy. Instead we propose,

as previously stated in the original manuscript, that the detec-

tion of any apoptotic cells within the CTC population is a

potential indicator of therapy response, as has been elegantly

demonstrated by previous work by Rossi et al. (3,4). Building

on their previous studies, this Communication has afforded

Rossi et al. the opportunity to publish a small amount of addi-

tional data that demonstrates (not surprisingly) that a 2-marker

apoptosis assay (M-30/gH2AX) may have more utility than M-

30 alone on the CellSearch, at least in spiked samples.

In conclusion, we would like to reaffirm our agreement

with many of the statements set forth in this Communication

and thank the authors and the editors for their continued in-

terest in our work.

Sincerely,

Lori E. Lowes

London Regional Cancer Program, London Health

Sciences Centre, London, Ontario, Canada

Department of Anatomy & Cell Biology, Schulich

School of Medicine and Dentistry, Western University,

London, Ontario, Canada

Benjamin D. Hedley

Special Hematology/Flow Cytometry, London Health

Sciences Centre, London, Ontario, Canada

Mike KeeneySpecial Hematology/Flow Cytometry, London Health

Sciences Centre, London, Ontario, Canada

Lawson Health Research Institute, London, Ontario,

Canada

Alison L. Allan*London Regional Cancer Program, London Health

Sciences Centre, London, Ontario, Canada

Lawson Health Research Institute, London, Ontario,

Canada

Department of Anatomy & Cell Biology, Schulich

School of Medicine and Dentistry, Western University,

London, Ontario, Canada

Department of Oncology, Schulich School of

Medicine and Dentistry, Western University, London

Ontario, Canada

LITERATURE CITED

1. Rossi E, Facchinetti A, Zamarchi R. Customizing CellSearch platform. Cytometry A2013;83A:595–598.

2. Sieuwerts AM, Kraan J, Bolt-de Vries J, van der Spoel P, Mostert B, Martens JW, Gra-tama JW, Sleijfer S, Foekens JA. Molecular characterization of circulating tumor cellsin large quantities of contaminating leukocytes by a multiplex real-time PCR. BreastCancer Res Treat 2009;118:455–468.

3. Rossi E, Basso U, Celadin R, Zilio F, Pucciarelli S, Aieta M, Barile C, Sava T, Bonciar-elli G, Tumolo S, et al. M30 neoepitope expression in epithelial cancer: Quantifica-tion of apoptosis in circulating tumor cells by CellSearch analysis. Clin Cancer Res2010;16:5233–5243.

4. Rossi E, Fassan M, Aieta M, Zilio F, Celadin R, Borin M, Grassi A, Troiani L, Basso U,Barile C, et al. Dynamic changes of live/apoptotic circulating tumour cells as predic-tive marker of response to Sunitinib in metastatic renal cancer. Br J Cancer2012;107:1286–1294.

COMMUNICATION TO THE EDITOR

Cytometry Part A � 83A: 599�601, 2013 601