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Micrometastatic Tumor Detection in Patients With Head and Neck Cancer
A Preliminary Report
Ari Wirtschafter, MD;
Michael S. Benninger, MD;
Thomas J. Moss, MD;
Tehila Umiel, PhD;
Kathleen Blazoff, MSN, RN;
Maria J. Worsham, PhD
Arch Otolaryngol Head Neck Surg. 2002;128:40-43.
ABSTRACT
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Objective To apply a new immunocytochemistry (ICC) assay to peripheral blood samples
for micrometastatic circulating tumor cell detection in patients with head
and neck squamous cell cancer (HNSCC).
Design The ICC assay uses established monoclonal antibodies that bind to tumor-associated
antigens combined with an enrichment system that uses positive selection with
anti-human epithelial antigen (EpCAM antibody) to detect circulating tumor
cells.
Subjects Eighteen consecutive patients newly diagnosed as having HNSCC are described.
Results Of the 18 patients, 8 (44%) demonstrated circulating tumor cells using
the ICC assay. The numbers of patients positive for circulating tumor cells
per stage are as follows: stage I, 1 of 1; stage II, 0 of 2; stage III, 2
of 5; stage IV, 5 of 6; and unknown stage, 0 of 4. The numbers of patients
positive for circulating tumor cells per location are as follows: oral cavity,
1 of 2; oropharynx, 3 of 4; glottic area, 3 of 5; supraglottic area, 1 of
3; and unknown primary 0 of 4.
Conclusions Circulating tumor cells were identified in almost half of the patients
using the ICC assay. In a literature review, we were not able to identify
previous reports of circulating tumor cell detection in patients with HNSCC
from peripheral blood samples using ICC or identify any study that has attempted
to quantify circulating tumor cell levels. Although the clinical implications
of circulating tumor cells in micrometastatic tumor detection in patients
with HNSCC are still unknown, they may be significant. Long-term follow-up
may help elucidate the patients in whom conventional treatment may fail and,
thus, those who may benefit from different treatment; it may also assist with
the detection of recurrence with a simple blood collection.
INTRODUCTION
THERE ARE 42 000 new cases of head and neck cancer and 12 000
associated deaths in the United States each year.1
Worldwide, there are close to three quarters of a million new cases each year,
with incidence rates varying on the distribution of risk factors, notably,
tobacco chewing, smoking, and alcohol consumption.2
Of these cases, 95% are identified as squamous cell carcinoma. The 5-year
survival averages 50%, depending on the origin and the extent of the tumor.
Despite advances in technology and technique, morbidity and mortality rates
have changed little during the past 30 years. Treatment failures are determined
by the development of local and/or regional recurrence, distant metastasis,
or a second primary tumor.1
Why have morbidity and mortality rates not decreased significantly during
the past 30 years? Local recurrence rates are as high as 50% to 60% for stage
III and stage IV cancers, and 15% to 25% of those patients will develop distant
metastases. Furthermore, almost one third of the patients with stage III and
stage IV cancers who have tumor-free resection margins will develop a local
recurrence.3 Is there a gap in the TNM staging
system? Perhaps micrometastasis or minimal residual cancer has not been detected
or represented by the current assessment standards. The presence of minimal
residual cancer may correlate with a poor clinical outcome.
This study applies a new immunocytochemistry (ICC) assay, developed
by IMPATH/BIS Laboratories, to peripheral blood samples for micrometastatic
circulating tumor cell detection in patients with head and neck squamous cell
cancer (HNSCC). The information generated by this study may help identify
those patients in whom conventional treatment may fail and, thus, those who
may benefit from different treatment; it may also assist with the detection
of recurrence with a simple blood collection. By comparing and levels of circulating
tumor cells with those of other malignancies, such as breast, colon, or lung
cancer, insight may be gained into why head and neck cancer tends to be locally
and/or regionally aggressive.
MATERIALS AND METHODS
Institutional review board approval for the study was obtained through
the Henry Ford Hospital, Detroit, Mich, while informed consent was also obtained
from patients and/or guardians before initiating the study. Eighteen consecutive
patients newly diagnosed as having HNSCC have been enrolled into this study
to date. Patients were identified through Henry Ford Hospital's interdisciplinary
head and neck tumor board. The study is approved to examine 75 patients. Profiles
of the 18 patients enrolled in the study are listed in Table 1.
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Table 1. Patient Profiles
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Approximately 30 mL of blood was drawn from each patient during the
head and neck tumor board examination or on the morning of surgery. The blood
was collected using standard phlebotomy techniques and placed into 3 green-top
tubes (a preservative, heparin sodium, was added to the vacucontainer). The
green-top tubes were sent via Federal Express overnight to IMPATH/BIS Laboratories
for ICC analysis.
Peripheral blood was shipped at room temperature to IMPATH/BIS Laboratories
for enriched ICC analysis. Mononuclear cell fractions were isolated by gradient
separation at 1500 rpm for 20 minutes (Ficoll-Hypaque; Pharmacia, Uppsala,
Sweden), and washed twice in a medium of assorted salts, amino acids, vitamins,
and D-galactose (Leibovitz L-15; GIBCO/BRL, Grand Island, NY) supplemented
with 10% fetal bovine serum (GIBCO/BRL). The mononuclear cell fraction was
isolated using gradient separation (Ficoll-Hypaque). The mononuclear cells
were then placed in phosphate-buffered saline containing 0.2% sodium citrate
and washed twice at 1000 rpm for 10 minutes each. Following washing, up to
5 x 107 cells were incubated with blocking reagent and anti–cancer-conjugated
microbeads (anti-human epithelial antigen; Miltenyi Biotech, Inc, Auburn,
Calif) at 4°C for 30 minutes. The cells were washed twice with phosphate-buffered
saline and 0.2% sodium citrate to remove unbound beads and placed in a separation
column along with a magnet for 2 minutes at room temperature to bind cells.
Bound cells were removed by removing the column from the magnet followed by
gently flushing the column into a fresh tube with 1 mL of buffer using a plunger.
All bead-cell conjugates recovered from the magnet were used in the cytopreparations
and immunostained. About 2 to 4 cytopreparations were typically made and stored
for immunostaining at 4°C. Figure 1
represents an abridged schematic of this procedure.
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Figure 1. Schematic representation of tumor
cell identification and isolation. Tumor cell enrichment is accomplished by
incubation with anti-human epithelial antigen-125 (EpCAM antibody) bound to
magnetic beads and then separated using a magnetic column, thereby isolating
a positive and a negative fraction. MACS indicates magnetic-activated cell
sorting.
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Cytopreparations were fixed in 4% paraformaldehyde fixative, washed
thoroughly in Dulbecco-modified phosphate-buffered saline (GIBCO/BRL) with
detergent (1% Triton X; Sigma-Aldrich Co, St Louis, Mo), and placed on an
automated immunostainer (TechMate; Ventana Medical Systems, Inc, Tucson, Ariz).
Alkaline phosphatase immunostaining was performed, per protocol, as previously
described. Briefly, slides were incubated in blocking solution and anticancer
monoclonal antibody cocktail (anticytokeratin types 8 and 18, TFS-2 [a surface
glycoprotein derived from breast cancer cells], and antikeratin). This step
was followed by staining with alkaline phosphatase, chromogen, and finally
hematoxylin. Buffer washes were performed between each step. All slides were
then reviewed by a board-certified pathologist for tumor cell identification. Figure 2 demonstrates a hematoxylin-stained
slide identifying tumor cells (arrow) along with several mononuclear cells
that bypassed the magnetic separation process.
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Figure 2. Tumor cell identification using
immunocytochemistry. The positive fraction undergoes immunostaining with cytokeratin
markers followed by tumor cell identification (arrow) (hematoxylin, high-power
magnification).
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The number of tumor cells stained by the anticancer antibodies was recorded.
The tumor concentration was determined by dividing the number of cancer cells
detected by the number of cells enriched (the denominator being 10 000 000).
The final recorded value was the number of tumor cells detected per 10 000 000
cells enriched. The sensitivity is 1 per 10 000 000, and the specificity
is greater than 99%.4
RESULTS
The number of circulating tumor cells identified per patient is listed
in Table 2. Specifically, the
number of cells enriched per 10 000 000, the positive fraction,
and the positive fraction concentration are displayed. Only the positive fraction
results are displayed, as there were no cells detected in any of the negative
fractions collected.
To date, 8 (44%) of the 18 patients demonstrated circulating tumor cells
using the ICC assay. The percentage of patients positive for circulating tumor
cells per stage is illustrated in Figure 3, and the corresponding numbers are as follows: stage I, 1 of 1;
stage II, 0 of 2; stage III, 2 of 5; stage IV, 5 of 6; and unknown stage,
0 of 4. The percentage of patients positive for circulating tumor cells per
location is illustrated in Figure 4,
and the corresponding numbers are as follows: oral cavity, 1 of 2; oropharynx,
3 of 4; glottic area, 3 of 5; supraglottic area, 1 of 3; and unknown area,
0 of 4.
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Figure 3. Results are presented as the percentage
of patients positive for circulating tumor cells per stage.
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Figure 4. Results are presented as the percentage
of patients positive for circulating tumor cells per location.
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COMMENT
Micrometastatic tumor cell detection in patients with head and neck
cancer is a relatively uncharted field of study when compared with other cancers,
such as breast, prostate, and colorectal cancer and neuroblastoma. Previous
efforts in patients with head and neck cancer have also focused primarily
on bone marrow specimens. Wollenberg et al,5
using ICC directed against epithelial cell marker cytokeratin 19, demonstrated
micrometastasis in bone marrow specimens in 41 of 108 patients with HNSCC.
Bone marrow specimens positive for tumor cells also correlated with higher
rates of local and distant recurrences and with shorter disease-free intervals.5 Gath et al,6 using
monoclonal antibody A45-B/B3 directed at oral cavity tumors, demonstrated
that 10 (32%) of 31 patients had tumor cells in bone marrow specimens. van
Dongen et al3 demonstrated the use of monoclonal
antibodies E48 and U36 directed against HNSCC-associated antigens for the
detection and treatment of minimal residual cancer. According to the study,
polymerase chain reaction and ICC were used successfully in the detection
of cells in bone marrow and peripheral blood; however, no specific data or
patient profiles were presented for review. Most recently, Kawamata et al,7 using reverse transcription polymerase chain reaction,
demonstrated the expression of cytokeratin 20 in the peripheral blood samples
of 10 of 11 patients with oral squamous cell carcinoma. In summary, a literature
review has not identified previous reports of circulating tumor cell detection
in peripheral blood samples of patients with HNSCC using ICC and has not found
any study that has attempted to quantify circulating tumor cell levels.
The uniqueness of this study is that it not only uses advanced ICC techniques
to analyze peripheral blood but also seeks to quantify tumor load. The ICC
assay used (IMPATH/BIS Laboratories) is capable of reliably detecting a single
tumor cell among 10 000 000 mononuclear cells. This technique also
has the added advantage of actually being able to see the tumor cell, while
reverse transcription polymerase chain reaction techniques may be amplifying
some other undesirable product, most notably skin cells. The timing for the
blood collection for analysis was scheduled during the head and neck tumor
board examination or on the morning of surgery. The presumption is that the
number of circulating tumor cells will decrease after treatment; however,
this was not addressed in this study. Documenting this reduction will be essential
in future studies so that this assay might be used for tumor surveillance
following definitive treatment.
Of all 18 patients studied thus far, 8 (44%) demonstrated circulating
tumor cells using the ICC assay. If the 4 patients who had primary tumors
of unknown origin were discarded (as all 4 were negative for circulating tumor
cells), then 8 (57%) of 14 patients would have been positive for circulating
tumor cells. Although lesions of the oropharynx had the highest percentage
of positive results (3 [75%] of 4), there are too few patients enrolled in
the study to statistically conclude a higher rate of micrometastasis. Similarly,
although 100% (1/1) of the patients with stage I cancer and 0% (0/2) of the
patients with stage II cancer were positive for circulating tumor cells, the
number of patients for these 2 stages is too few to draw definitive conclusions.
To draw statistically significant conclusions, a total of 75 patients, including
all stages, will ultimately need to be examined.
What is the clinical significance of these findings? The presence of
minimal residual disease in bone marrow specimens provides prognostic information,
most notably in patients with neuroblastoma.8
More recently, however, the presence of circulating tumor cells in peripheral
blood in patients with prostate cancer has also been shown to affect overall
survival and disease-free days. Specifically, patients with circulating tumor
cells had decreased disease-free and overall survival (49 and 122 days, respectively)
when compared with patients with no circulating cells (251 and 347 days, respectively). 9 There are also some preliminary data in patients with
breast and colorectal cancer.
Our hope is that by demonstrating circulating tumor cells in the blood
and by quantifying that tumor load, we will be able to stratify patients and,
thus, tailor their treatment. Perhaps those patients who present with circulating
tumor cells will need more aggressive or neoadjuvant therapy to effectively
treat the tumor. It will also be interesting to compare circulating tumor
cell loads between different types of cancers to better understand why certain
tumors tend to be more locally and/or regionally aggressive vs those that
tend to metastasize early. Finally, we hope to use this blood test in patient
follow-up care for recurrence detection, as this is a simple and minimally
invasive technique.
CONCLUSIONS
The presence of circulating tumor cells in peripheral blood samples
was successfully demonstrated in 8 (44%) of the 18 patients studied with HNSCC.
Although the clinical implications of circulating tumor cells in micrometastatic
tumor detection in patients with HNSCC are still unknown, they may be significant.
Long-term follow-up may help elucidate the patients in whom conventional treatment
is more likely to fail and, thus, those who may benefit from different treatment;
it may also assist with the detection of recurrence with a simple blood collection.
AUTHOR INFORMATION
Accepted for publication August 16, 2001.
This study was supported in part by the Cummings-Brush Chair in Surgical
Education, Henry Ford Health System, Detroit.
Presented as a poster at the Fifth International Conference on Head
and Neck Cancer, San Francisco, Calif, July 29-August 2, 2000.
Corresponding author and reprints: Michael S. Benninger, MD, Department
of Otolaryngology–Head and Neck Surgery (Floor K-8), Henry Ford Hospital,
2799 W Grand Blvd, Detroit, MI 48202 (e-mail: Mbenning{at}HFHS.org).
From the Department of Otolaryngology–Head and Neck Surgery (Drs
Wirtschafter and Benninger and Ms Blazoff) and Research Division, Department
of Otolaryngology–Head and Neck Surgery (Dr Worsham), Henry Ford Hospital,
Detroit, Mich; and Clinical Protocol Development (Dr Moss) and Research and
Development (Dr Umiel), IMPATH/BIS Laboratories, Los Angeles, Calif.
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