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Clinical Cancer Research Vol. 10, 7827-7833, December 1, 2004
© 2004 American Association for Cancer Research

Molecular Oncology, Markers, Clinical Correlates

Clinical Significance of Micrometastatic Cells Detected by E48 (Ly-6D) Reverse Transcription-Polymerase Chain Reaction in Bone Marrow of Head and Neck Cancer Patients

David R. Colnot1, Eline J. C. Nieuwenhuis1, Dirk J. Kuik2, C. René Leemans1, Janny Dijkstra1, Gordon B. Snow1, Guus A. M. S. van Dongen1 and Ruud H. Brakenhoff1

Departments of 1 Otolaryngology/Head and Neck Surgery and 2 Clinical Epidemiology and Biostatistics, VU University Medical Center, Amsterdam, the Netherlands


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Purpose: Despite improvements in locoregional treatment of head and neck squamous cell carcinoma (HNSCC), local and distant failure rates remain high. The strongest prognostic indicator of HNSCC is the presence of lymph node metastases in the neck, but the value of this indicator has limitations when using for the individual patient. The presence of micrometastatic cells in bone marrow has been shown to be a putative prognostic indicator in HNSCC and other epithelial malignancies, which might allow more accurate staging and selection of patients for whom adjuvant or experimental therapy is recommended. The gene encoding the E48 antigen is selectively expressed by HNSCC, and the detection of E48 transcripts in bone marrow by reverse transcription-polymerase chain reaction (RT-PCR) presumably represents the presence of micrometastatic cells. The purpose of this study was to determine the association between the presence of micrometastatic cells in bone marrow of HNSCC patients and clinical outcome.

Experimental Design: A total of 162 patients treated surgically for primary HNSCC underwent a single bone marrow aspiration from the upper iliac crest for detection of micrometastatic cells using E48 RT-PCR. In total, 139 patients were evaluable. The primary statistical endpoints were disease-free survival and distant metastasis-free survival. In addition, bone marrow samples of 30 noncancer controls were evaluated.

Results: E48 RT-PCR indicated the presence of micrometastatic cells in the bone marrow in 56 of 139 (40%) of the HNSCC patients and 0 of 30 of the noncancer controls (P < 0.0001). The presence of micrometastatic cells had no significant influence on disease-free survival or distant metastasis-free survival for the whole group of HNSCC patients (P = 0.1460 and P = 0.2912, respectively). For patients with ≥2 lymph node metastases, however, the presence of micrometastatic cells was associated with a poor distant metastasis-free survival (P = 0.0210).

Conclusions: The presence of micrometastatic cells in bone marrow of HNSCC patients with ≥2 lymph node metastases is correlated with a poor distant metastasis-free survival. In this subgroup of HNSCC patients, E48 RT-PCR seems to be a valuable tool to identify patients who are at increased risk for development of distant metastases and therefore might benefit from experimental adjuvant systemic therapy.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Head and neck squamous cell carcinoma (HNSCC) represents ~95% of head and neck tumors. The estimated incidence worldwide in 2000 was approximately 551,000 cases, and 217,000 died of the disease (1) . About one third of the patients present with early-stage disease (stages I and II; see Table 1Citation ), whereas two thirds of the patients present with advanced-stage disease (stages III and IV; ref. 2 ). Initial therapy of HNSCC is surgery and/or radiotherapy. Whereas early stages (stages I and II) generally have a good prognosis, the more advanced stages have a high failure rate. Despite improvements in locoregional control for patients with advanced HNSCC, the overall survival rates have not improved significantly over the last 25 years because the incidence of metastases at distant sites has increased (3) .


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  		Table 1 Relationship between stage and TNM classification of HNSCC

 
In a large series of patients, development of distant metastases is seen in 5% to 25% of patients and generally occurs within 2 years after locoregional treatment, whereas in subgroups of patients with advanced disease, the incidence of distant metastasis can be much higher (4, 5, 6, 7, 8, 9) . The most important prognostic indicator for relapse of HNSCC, both locoregionally and at distant sites, is the presence of metastatic spread to lymph nodes in the neck. When multiple lymph node metastases are present, the incidence of distant metastases can be as high as 50% (4 , 6 , 7 , 9 , 10) .

The presence of small tumor deposits or even single metastatic tumor cells in bone marrow of patients with various types of epithelial cancers can be detected by immunocytochemistry and molecular methods, notably the polymerase chain reaction (PCR). Both techniques mainly use tissue-specific marker antigens such as cytokeratins because these are expressed abundantly in the majority of epithelial tumors and homogeneously among the cells of these tumors (11) . The clinical relevance of micrometastatic cells in bone marrow has been illustrated convincingly for breast and colorectal cancer patients, in whom their presence correlates with poor prognosis (11, 12, 13, 14) . The early detection of these micrometastatic cells could thus contribute to a more accurate staging and the identification of patients who might benefit from adjuvant therapy.

Detection of single tumor cells in bone marrow of HNSCC patients with immunocytochemical techniques using monoclonal antibodies directed against cytokeratins seems a feasible approach (15 , 16) . These immunocytochemical methods are, however, difficult to standardize, and many researchers use subjective morphologic criteria to assign immunostained cells as cancer cells (17) . Reverse transcription-polymerase chain reaction (RT-PCR) methods are an alternative approach. For detection of micrometastatic cells in the bone marrow of HNSCC patients, we exploited the E48 antigen. The gene encoding the E48 antigen is selectively expressed in HNSCC, and we have shown previously that E48 RNA transcripts can be detected in bone marrow aspirates of 35% of HNSCC patients, suggesting the presence of micrometastatic cells, whereas samples of noncancer controls were negative (18) .

In this study, the clinical significance of micrometastatic cells in bone marrow as detected by E48 RT-PCR at initial work-up is assessed in HNSCC patients who were treated with primary surgery and postoperative radiotherapy and then followed for a median period of 49 months.


   PATIENTS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patients.
From December 1995 to August 2000, a total of 162 HNSCC patients underwent bone marrow aspiration. Classification and staging of the primary tumor was performed according to the tumor-node-metastasis (TNM) system of the International Union Against Cancer (Union Internationale Contre le Cancer; ref. 19 ). At our institute, the workup for oral and oropharyngeal cancer patients is as follows. After initial consultation, an examination under anesthesia and panendoscopy with biopsy are performed for histologic diagnosis, staging, and treatment planning. Generally, early-stage tumors are treated with surgery in the majority of cases, and in the remainder of cases, they are treated with primary radiotherapy. For the clinically negative neck as assessed by ultrasound-guided fine-needle aspiration cytology in case of transoral excision, a policy of observation is followed using ultrasound-guided fine-needle aspiration cytology at frequent intervals. Advanced-stage, resectable tumors are treated by surgery including radical or modified radical neck dissection encompassing levels I to V, often with free flap reconstruction. Postoperative radiotherapy of the primary site and the neck is applied in advanced T stages (>T2), when multiple lymph node metastases or extranodal spread are present, when the margins are not tumor-free, or when severe dysplasia in the margins is detected by histopathological examination. After discharge, the patients visit the outpatient clinic every 6 weeks for 1 year, every 3 months during the 2nd year, and at gradually increasing intervals until 10 years after discharge.

Just before surgery, a bone marrow sample (2–5 mL) was obtained under general anesthesia from the upper iliac crest by needle aspiration on one side and stored in heparin-treated tubes. Patients with a primary HNSCC tumor scheduled for surgery and no history of malignancy in the past were eligible. Other criteria were histologically tumor-free margins and good-quality RNA isolated from the bone marrow sample. Based on these criteria for analysis, a total of 23 patients were excluded (in 11 patients, the surgical margins were not histologically tumor-free, and in 12 cases, the RNA quality was poor). Besides these HNSCC patients, 30 noncancer control patients underwent bone marrow aspiration. These patients were scheduled for other surgical treatments in the head and neck region such as endoscopic sinus surgery for chronic sinusitis. The study was approved by the Institutional Review Board of the VU University Medical Center (Amsterdam, the Netherlands), and all patients signed written informed consent.

Preparation of Bone Marrow Samples and RNA Isolation.
Bone marrow samples were diluted 1:1 in RPMI 1640. After centrifugation at 220 x g for 10 minutes, the erythrocytes in the pellet were lysed in 5 mL of lysis buffer containing 160 mmol/L KHCO3 and 0.1 mmol/L EDTA (pH 7.4) and incubated at 4°C for 15 minutes while the solution was mixed occasionally by inverting the tube. Nucleated cells were pelleted by centrifugation at 220 x g for 10 minutes, washed with 10 mL of RPMI 1640, and centrifuged at 220 x g for 5 minutes. Cells were resuspended in 1 mL of RPMI 1640, transferred to a 1.5-mL microcentrifuge tube, and centrifuged at 12,000 x g for 1 minute. The pellet was dissolved in 1.0 mL of RNAzol-B (Campro Scientific BV, Veenendaal, the Netherlands) and mixed. After the addition of 100 µL of chloroform, the sample was mixed vigorously for 15 seconds and put on ice for 5 minutes. After centrifugation at 12,000 x g for 15 minutes at 4°C, the aqueous phase was transferred to another microcentrifuge tube, whereas the RNA was precipitated by the addition of an equal volume of isopropanol and stored until use at –20°C. Just before use, RNA was pelleted by centrifugation at 12,000 x g for 30 minutes at 4°C. The pellet was washed by vortexing in 70% EtOH, centrifuged at 12,000 x g for 5 minutes at 4°C, dissolved in 25 to 100 µL of RNase-free H2O, and incubated at 65°C for 15 minutes. The amount of RNA was calculated from the absorbance at 260 nm, and 5 µg were assayed routinely.

E48 Reverse Transcription-Polymerase Chain Reaction and Quality Assurance.
The standardized RT-PCR technique for detection of E48 RNA transcripts in bone marrow has been described previously (18) . In short, 5 µg of total RNA were reverse transcribed in 20 µL, and a PCR was performed quadruplicate on 4 x 5 µL of reverse transcription product.

Amplimers were loaded on a 2% agarose gel, blotted after electrophoretic separation, and hybridized with [{alpha}-32P]dCTP-labeled E48 cDNA as a probe. A serial dilution of RNA from the E48-expressing cell line UM-SCC-22A (50, 15, 5, 1.5, and 0.5 pg) was run in parallel in each experiment for calibration, as described previously (20) . All primers, probes, and buffers were prepared in a laboratory that is isolated from sample preparation and PCR product analysis. RNA isolation, cDNA synthesis, and preparation of PCR reactions were performed in a pre-PCR laboratory. To prevent amplimer carryover contamination, all materials and reagents were transported in a one-way direction from the pre-PCR laboratories to the post-PCR laboratory. Sample-to-sample carryover contamination was further avoided by using different pipette sets and filter tips (Greiner Bio-One, Kremsmünster, Austria). Preparations without RNA template were used as negative RT-PCR control. In addition, a blotting control was loaded on the agarose gels consisting of dilutions of EcoRI-cut plasmid DNA containing E48 cDNA (21) . Autoradiography was standardized using the blotting control. An assay was considered reliable when at least one of four signals was seen at 0.5 pg of UM-SCC-22A RNA. A sample was considered positive when at least one of four PCR reactions was positive. RT-PCR amplification of ß2-microglobulin transcripts was used as control for the quality of the RNA. These amplimers were loaded on a 2% agarose gel and stained with ethidium bromide. Examples are depicted in Fig. 1Citation . In addition, RNA (5 µg) isolated from bone marrow aspirates of 30 noncancer controls was analyzed as described.



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  		Fig. 1. E48 RT-PCR of bone marrow samples of HNSCC patients. Top panel, a serial dilution (50, 15, 5, 1.5, and 0.5 pg) of RNA from the E48-expressing cell line UM-SCC-22A was run in parallel in each experiment as a positive control. Bottom panel, preparations without RNA template were used as a negative RT-PCR control. In addition, a blotting control was loaded on agarose gels consisting of dilutions of EcoRI-cut plasmid DNA containing the E48 cDNA to standardize autoradiography. RT-PCR amplification of ß2-microglobulin transcripts was used as a control for the quality of the RNA. Four representative examples of bone marrow aspirates of HNSCC patients are depicted; samples P1 and P3 were scored as positive for micrometastatic cells in bone marrow.

 
Statistical Analysis.
Differences in frequency distributions of clinical characteristics were analyzed by either Student’s t test or the {chi}2 test (for two-by-two tables, Fisher’s exact test). The primary statistical end point of the prognostic study was the number of patients remaining free of recurrent disease and distant metastases using Kaplan-Meier life-table analyses and log-rank tests (Mantel-Cox). Time to recurrence or death was measured from the date of primary surgery. Patients who developed a second primary tumor were censored for all outcomes at the incidence date of the second tumor because development of recurrent disease in such a case could not be related to the primary tumor alone. Delayed lymph node metastases that developed in an untreated neck during follow-up were not regarded as recurrence. Statistical analyses were performed with the statistical software package BMPD (22) . Relative risk (RR) factors and 95% confidence intervals (CI) were calculated by Cox proportional hazards model using SPSS software. Data summaries and graphical presentations were also obtained using SPSS software (23) . P values of <0.05 were considered significant.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The RT-PCR assay detected E48 transcripts in bone marrow aspirates of 56 of 139 (40%) patients. Analysis of bone marrow aspirates of 30 noncancer controls demonstrated a negative E48 RT-PCR assay in all cases, whereas the RNA quality was good (P < 0.0001). Odds ratio and 95% CIs could not be calculated because there are no positive E48 RT-PCR assays in the noncancer control group. No significant differences in frequency distributions between the clinical characteristics of patients were seen for the presence or absence of a positive E48 RT-PCR result (Table 2)Citation . Of all patients, 37% presented with stage I or II disease, whereas 63% presented with stage III or IV disease. The primary tumors were staged pT1 (16%), pT2 (33%), pT3 (34%), and pT4 (17%). In 60 of 139 (43%) patients, lymph node metastases were detected by histopathological examination of the neck dissection specimen. Extranodal spread in one or more lymph node metastases was noticed in 46 of 60 patients (77%) with lymph node metastases.


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  		Table 2 Clinical characteristics of 139 HNSCC patients, with presence or absence of occult micrometastatic cells as assessed by E48 RT-PCR on bone marrow aspirates

 
The median follow-up of patients was 49 months, ranging from 5 to 87 months. An overview of the events during follow-up is given in Table 3Citation . Recurrent disease occurred in 24 of 139 patients, of whom 8 had a local recurrence, 3 developed regional recurrent disease, and 13 presented with distant metastases during follow-up. A second primary tumor was diagnosed in 14 patients. In 10 patients who presented at initial diagnosis with a clinically negative neck and did not undergo elective neck dissection, lymph node metastases in the neck occurred during follow-up, and they were treated with a delayed neck dissection. None of these patients developed recurrent disease during further follow-up.


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  		Table 3 Events during follow-up and type of recurrent disease for HNSCC patients with or without micrometastatic cells in bone marrow as assessed by E48 RT-PCR at initial work-up

 
The presence of micrometastatic cells in bone marrow, as assessed by a positive E48 RT-PCR result, had no significant influence on disease-free survival (Fig. 2)Citation or distant metastasis-free survival (P = 0.1460 and P = 0.2912, respectively). Standard risk factors such as (a) the presence of multiple lymph node metastases and (b) extranodal spread, showed a significantly higher risk for development of recurrent disease (P < 0.0001; RR, 4.9; 95% CI, 2.2–17.1) and (P = 0.005; RR, 3.7; 95% CI, 1.4–9.7), respectively. Based on the number of lymph node metastases, we were able to define a good prognosis group (≤1 lymph node metastasis) and a poor prognosis group (≥2 lymph node metastases; Fig. 3Citation ). For both groups, disease-free survival was not significantly associated with the presence of E48 transcripts in the bone marrow, although a near significant trend is observed in the poor prognosis group (P = 0.7601 for patients with ≤1 lymph node metastasis and P = 0.0908 for patients with ≥2 lymph node metastases, respectively). For patients with ≤1 lymph node metastasis, no significant relation between a positive E48 RT-PCR result and distant metastasis-free survival was found (P = 0.4215), but for patients with ≥2 lymph node metastases, a positive E48 RT-PCR result was associated with a poor distant metastasis-free survival (P = 0.0210). It should be noted, however, that the number of events in this group was rather low. Kaplan-Meier life-table analyses are depicted in Fig. 4Citation . RR and 95% CI could not be calculated because there were no events in the E48 RT-PCR–negative group.



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  		Fig. 2. Kaplan-Meier life-table analysis of the disease-free survival of HNSCC patients, according to the presence or absence of micrometastatic cells in bone marrow as assessed by E48 RT-PCR at initial work-up (P = 0.1460).

 


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  		Fig. 3. Kaplan-Meier life-table analysis of the disease-free survival of HNSCC patients, related to the number of lymph node metastases (LNM) as assessed by histopathological analysis of the neck dissection specimen. The disease-free survival of patients with ≤1 lymph node metastasis versus patients with ≥2 lymph node metastases is significantly different (P < 0.0001).

 


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  		Fig. 4. Kaplan-Meier life-table analysis of the disease-free (A) and distant metastasis-free (B) survival of HNSCC patients, related to the presence of micrometastatic cells in bone marrow as assessed by E48 RT-PCR at initial work-up and to the number of lymph node metastases (LNM) as assessed by histopathological analysis of the neck dissection specimen. A. Significant difference in disease-free survival was not observed for patients with ≤1 lymph node metastasis or for patients with ≥2 lymph node metastases, although a near significant trend was observed for the latter group P = 0.0908. B. The presence of micrometastatic cells in bone marrow of patients with ≥2 lymph node metastases correlated with a poor distant metastasis-free survival (P = 0.0210). No significant difference in distant metastasis-free survival was observed for patients with ≤1 lymph node metastasis (P = 0.4215). DM, distant metastasis.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The E48 antigen is selectively expressed on normal squamous and transitional epithelia and their malignant counterparts (21 , 24 , 25) . Bone marrow aspirates of 30 noncancer controls were all negative in the E48 RT-PCR assay, which confirmed previous results (18) . Therefore, the finding of E48 RNA transcripts in bone marrow of head and neck cancer patients seems a strong indicator for the presence of micrometastatic cells, and a positive E48 RT-PCR result in bone marrow was indeed associated with a poor distant metastasis-free survival for patients with multiple lymph node metastases at the time of primary surgery. Patients with multiple lymph node metastases are known to be at risk for development of recurrent disease, particularly distant metastases, because the incidence of distant metastases can be as high as 50% (6) . The observations of our study indicate that in these patients, based on E48 RT-PCR findings, selection of a high-risk subgroup is feasible. Analysis of a larger group of patients or pooled analysis of other HNSCC studies is needed to substantiate these findings. The E48 RT-PCR assay could be a useful tool to identify patients who might benefit from experimental adjuvant systemic treatment.

The current sensitivity and specificity of the E48 RT-PCR assay to predict patients at risk for distant metastases are 54% and 61%, respectively. Of the 83 patients with a negative E48 RT-PCR result, a total of 11 patients developed recurrent disease: 3 developed local recurrences; 2 developed regional recurrences, and 6 developed distant metastases. In particular, the development of distant metastases should be regarded as a false-negative result of the E48 RT-PCR assay. An explanation for false-negative findings might be heterogeneous expression of the E48 antigen. In primary HNSCC, immunohistochemical data showed homogeneous expression of the E48 antigen in 70% of primary tumors, and heterogeneous expression in an additional 19% (25) . No data are available on E48 expression in micrometastatic HNSCC cells. False-negative findings could also be explained by sampling error related to the low number of micrometastatic cells in bone marrow of HNSCC patients. This phenomenon is well known from studies with other tumor types. In breast cancer patients, it has been demonstrated that collection of four bone marrow samples instead of one increases the rate of detection of micrometastatic cells (26) . It is very likely that the number of micrometastatic cells is even lower for HNSCC than for breast cancer patients, thus requiring multiple bone marrow sampling at different sites. It seems unlikely that the analytical sensitivity of the E48 RT-PCR assay itself is responsible for false-negative results. The level of analytical sensitivity of the E48 RT-PCR has been assessed in a seeding experiment with the HNSCC cell line UM-SCC-22A and showed reproducible detection of a single tumor cell in a background of around 2 x 107 white blood cells (18 , 20) .

A low specificity of the E48 RT-PCR would make the assay less suitable for selection of patients for adjuvant therapy. In our study, positive E48 RT-PCR results were observed in 31 of 81 patients who did not develop recurrent disease despite a sufficient median duration of follow-up. An explanation might be that these positive E48 RT-PCR results indeed indicate the presence of micrometastatic cells but that they represent nonproliferating or dormant metastatic tumor cells (11) . Postoperative bone marrow sampling has been suggested for identification of persistent populations of micrometastatic cells that might be best equipped for metastasis outgrowth, but initial data do not support this (27) .

The E48 RT-PCR assay detected micrometastatic cells in bone marrow aspirates in 40% of the patients, which is in concordance with other reports using immunocytochemistry on bone marrow aspirates in HNSCC patients (15 , 16) . In these studies, an association between the presence of cytokeratin-positive cells and clinical outcome of HNSCC patients was observed. In a recent report on a smaller group of HNSCC patients by Partridge et al. (27) , detection of micrometastatic cells in bone marrow both with cytokeratin immunostaining and E48 RT-PCR was associated with a higher risk for both local and distant relapse, as well as reduced survival. The E48 RT-PCR on bone marrow aspirates in their study was positive in just 9 of 36 patients who underwent surgery for primary HNSCC, and recurrent disease occurred in 7 of these 9 patients, 4 of whom developed distant metastases. The concordance between immunocytochemistry and E48 RT-PCR was good, and each single test predicted development of distant metastases.

We conclude that presence of micrometastatic cells in bone marrow of HNSCC patients with ≥2 lymph node metastases at the time of primary surgery is associated with a poor distant metastasis-free survival. Nevertheless, the current E48 RT-PCR assay still has limitations to overcome before being of use to select HNSCC patients for future adjuvant systemic treatment because of a suboptimal sensitivity and specificity. Increasing the number and time points (postoperatively) of bone marrow aspirates might be necessary to further improve the sensitivity and specificity of the assay to be of value for selection of HNSCC patients at risk for development of recurrent disease at distant sites.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Requests for reprints: Ruud H. Brakenhoff, Department of Otolaryngology/Head and Neck Surgery, VU University Medical Center, De Boelelaan 1117, 1081 HV, Amsterdam, the Netherlands. Phone: 31-20-4443690; Fax: 31-20-4443688; E-mail: rh.brakenhoff{at}vumc.nl

Received 6/ 3/04; revised 7/27/04; accepted 8/26/04.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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Annual Meeting Education Book Meeting Abstracts Online