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Ann Thorac Surg 2007;84:1825-1829. doi:10.1016/j.athoracsur.2007.07.016
© 2007 The Society of Thoracic Surgeons
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Original Articles: General Thoracic

Effect of Formalin Fixation on Tumor Size Determination in Stage I Non-Small Cell Lung Cancer

Po-Kuei Hsu, MD*, Hsu-Chih Huang, MD*, Chih-Cheng Hsieh, MD*, Han-Shui Hsu, MD, Yu-Chung Wu, MD, Min-Hsiung Huang, MD, Wen-Hu Hsu, MD

Division of Thoracic Surgery, Department of Surgery, Taipei-Veterans General Hospital National Yang-Ming University, School of Medicine, Taipei, Taiwan

Accepted for publication July 6, 2007.

* Address correspondence to Dr Hsieh, Division of Thoracic Surgery, Department of Surgery, Taipei-Veterans General Hospital, No. 201, Sec. 2, Shih-Pai Road, Taipei, Taiwan (Email: cchsieh2{at}vghtpe.gov.tw).


   Abstract
Top
 Abstract
Introduction
 Patients and Methods
 Results
 Comment
 Footnotes
 References
 
Background: Tumor size is an important prognostic factor in non-small cell lung cancer (NSCLC), but the American Joint Committee on Cancer staging system does not mandate a specific measurement method. Moreover, measuring fresh specimens and formalin-fixed specimens may yield disparate results. Our goal was to evaluate this disparity for stage I NSCLC.

Methods: We enrolled 401 patients with stage I NSCLC who underwent surgical interventions and follow-up in our hospital between 1993 and 2002. Tumors invading visceral pleura, involving the main bronchus, or associated with atelectasis or obstructive pneumonitis were excluded. Tumor size was measured immediately after resection by surgeons and after formalin fixation by pathologists. Patients were assigned to one of three groups. Group 1 included 201 patients with tumors of 3 cm or less as indicated by both operation notes and pathology reports. Group 2 included 160 patients with tumors larger than 3 cm by both records. Group 3 included 40 patients with tumors larger than 3 cm according to operation notes but 3 cm or less according to pathology reports. Survival rates were compared.

Results: Mean follow-up was 58 months. Five-year survival was 70.1% in group 1, 49.1% in group 2, and 51.1% in group 3. As expected, there was a significant survival difference between groups 1 and 2 (p < 0.001); however, there was also a difference between groups 1 and 3 (p = 0.006).

Conclusions: Formalin fixation may cause tumor shrinkage and migration from T2 to T1. For accurate tumor staging, size measurements should be performed immediately after resection instead of after formalin fixation. TNM staging should specify how to measure tumor size and the specimen status to be measured.


   Introduction
Top
 Abstract
 Introduction
Patients and Methods
 Results
 Comment
 Footnotes
 References
 
According to the American Joint Committee on Cancer (AJCC) staging system, the pathologic classification of a carcinoma is determined by the evidence acquired before treatment and modified by additional information obtained through surgery, particularly specimen examination by a pathologist [1]. The criteria for defining the extent of a tumor (T status) vary among different tumors. In gastrointestinal tract malignancies, T status is determined by depth of invasion. In tumors arising from the larynx, nasal cavity, and paranasal sinuses, the determinant is the behavior of the neoplasm (peripheral structure invasion or not). For lung cancer, breast cancer, and musculoskeletal neoplasms, tumor size is an important factor in T status determination.

In non-small cell lung cancer (NSCLC), a tumor 3 cm or smaller is staged as T1, whereas a tumor exceeding 3 cm is staged as T2 [2]. The pathologic staging is important for making an accurate prognosis and determining the need for adjuvant treatment of lung cancer patients. The results of recent studies suggest that adjuvant chemotherapy would reduce recurrences and prolong overall survival of patients with surgically resected stage Ib-III NSCLC, but not of stage Ia patients [3–7]. These practices highlight the need for accurately distinguishing T2 from T1 lung cancers; therefore, precise measurement of a primary tumor is crucial, particularly those with dimensions that are about 3.0 cm [8].

Disparities, however, have been reported for tumor sizing of breast, prostate, and gastrointestinal tract specimens; that is, differences in measurements (1) immediately after resection by surgeons and (2) after formalin fixation by pathologists [9–12]. Despite the potential for size discrepancies between fresh and fixed-tissue states, there is no official agreement or recommendation on which state should be used for final staging, even in the lung cancer staging system [1, 2]. It is also possible that the composition and type of tissue affected may influence the overall amount of tumor shrinkage. Thus, studies addressing formalin fixation-induced changes in size should be organ-specific [10]. In this study, we used survival analysis to investigate the impact of formalin fixation-caused tissue shrinkage on tumor size and stage determination in stage I NSCLC.


   Patients and Methods
Top
 Abstract
 Introduction
 Patients and Methods
Results
 Comment
 Footnotes
 References
 
Subjects
Between 1993 and 2002, we enrolled 615 patients presenting at Taipei-Veterans General Hospital with resected stage I (T1 N0 M0 or T2 N0 M0) NSCLC. We recorded all clinical data, including age, gender, tumor size, location, histology, and treatment. We required that these stage I patients have no evidence of metastasis by radionuclide bone scan, brain scan, and ultrasound scanning of the abdomen.

All patients underwent surgical resection and radical ipsilateral mediastinal lymph node dissection with curative intent. On pathology, the dissected lymph nodes were all free of tumor. Postoperative follow-up was done by thoracic surgeons on regular schedules, and no adjuvant chemotherapy was given until tumor recurrence or metastasis. This study protocol was approved by the Institutional Review Board approved of Taipei-Veterans General Hospital.

Tumor Size Measurement
Our standard practice is that fresh specimens are routinely dissected by surgeons immediately after resection. The tumor is palpated and cut open by surgeons. The largest cross-sectional diameter of each specimen was measured with a handheld ruler and recorded in the operation notes. Then the specimens were preserved in 10% neutral buffered formalin and the container was sent to the pathology department. In our hospital, the pathologists do not perform their dissections in the operation room. Instead, they obtain the specimens after overnight fixation and then do measurements. In specimens without a well-defined margin, microscopic findings were correlated to avoid including areas of pneumonia or fibrosis adjacent to the tumor. The size measured by the pathologist was recorded in the pathology reports. The tumor size as recorded in the operation notes and in the pathology report of each patient was retrospectively recorded. For each patient, the surgical method in the operation notes and the cell type in the pathology report were collected.

Grouping
To specifically investigate the effect of formalin-induced tumor size change on accurate stage determination, patients with tumors that invaded the visceral pleura, involved the main bronchus, or were associated with atelectasis or obstructive pneumonitis (nonsize T2 status) were excluded. The patients were categorized into one of three groups. Group 1 included 201 patients with tumors 3 cm or smaller in the greatest dimension by both operation notes and pathology reports. Group 2 included 160 patients with tumors that exceeded 3 cm by both operation notes and pathology reports. Group 3 included 40 patients with tumors that exceeded 3 cm by the operation notes but were sized smaller than 3 cm by the pathology reports.

Statistical Analysis
Overall survival was calculated from the date of operation to the date of last follow-up. Continuous variables were expressed as mean ± standard deviation. Survival curves were estimated by the Kaplan-Meier method and compared by log-rank test. A value of p < 0.05 was deemed a statistically significant difference. Calculations were done with SPSS 13.0 software (SPSS Inc, Chicago, IL).


   Results
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 Abstract
 Introduction
 Patients and Methods
 Results
Comment
 Footnotes
 References
 
We excluded 214 of the 615 patients, leaving 401 who were eligible, were enrolled, and were used for statistical analysis. The reasons for exclusion included surgical mortality in 10 patients, visceral pleural invasion in 192, and main bronchus involvement in 14. The 401 patients had a mean age of 66.2 years (range, 19 to 90 years); 289 (72.1%) were men, and 112 (27.9%) were women.

Squamous cell carcinoma accounted for 33.5% of patients and adenocarcinoma for 44.6%. Lobectomy was performed in 310 patients (77.3%). Bilobectomy or pneumonectomy was performed in 50 patients (12.4%) owing to tumor location or severe pleural adhesion. Poor pulmonary reserve resulted in sublobar resection (wedge resection or segmentectomy) in 41 patients (10.2%). The mean follow-up period was 58 months. The overall 5-year survival rate was 59.7%.

There were 201 patients in group 1, 160 in group 2, and 40 in group 3. Patient demographics are summarized in Table 1. A factor that distributed unevenly was tumor histology. Group 1 had more patients with adenocarcinoma and bronchoalveolar carcinoma, and the other two groups had more patients with squamous cell carcinoma. The results of tumor measurements before and after formalin fixation are listed in Table 2. Although not all lung tumors showed shrinkage after formalin fixation and the shrinkage rate was also not constant, group 3 represented a group of tumors that showed significant shrinkage after fixation. This difference in size measurement even caused stage migration from T2 to T1.


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  		Table 1 Patient Demographics
 

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  		Table 2 Tumor Size Measured Before and After Formalin Fixation a
 
The overall 5-year survival rates were 70.1% for group 1, 49.1% for group 2, and 51.1% for group 3. The survival curves for each group are shown in Figure 1. The difference in survival between groups 1 and 2 was significant (p < 0.001). The survival difference between groups 1 and 3 was also statistically significant (p = 0.006). The difference between groups 2 and 3 was not significant (p = 0.710); indeed, the survival curve of group 3 was almost identical to the survival curve of group 2.


Figure 1
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  		Fig 1. Overall survival curves for patients with stage I non-small cell lung cancer (group 1, dotted line; group 2, thin solid line; group 3, thick solid line). Survival curves were plotted using Kaplan-Meier methods. Statistically significant differences in survival between groups were analyzed by the log-rank test.

 

   Comment
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Comment
Footnotes
 References
 
The effects of formalin fixation on tissue specimens include two phases. During the first phase, the fixative penetrates the tissue by diffusion and accumulates to a sufficient concentration for the next phase to take place. In the second phase, formalin exhibits a gelling action and chemically binds to protein amino groups, eventually leading to extensive cross-links between proteins or between proteins and nucleic acids [13, 14]. Although formalin is used to stabilize and preserve tissue ultrastructure, it occasionally causes some histologic changes, including distortion, vacuolization, and cell shrinkage [13]. These reactions not only affect tissue immumohistochemical reactivity but also possibly cause gross changes in specimen shape and size. Many studies have shown that formalin fixation will cause a change in tissue size, which accounts for the tumor size discrepancies between surgeons and pathologists [9–12, 15–18].

The length of a gastrointestinal tract specimen changes significantly after formalin fixation. Siu and colleagues [12] reported that the overall shrinkage for the whole esophagus from in situ to fixed specimen was 50%, and 20% of the shrinkage was attributed to the effect of formalin fixation.

Goldstein and colleagues [9] reported that colorectal specimens shrank 57% of their in vivo length after fixation and 30% of the shrinkage was due to formalin fixation. The effect of formalin fixation on colon polyps is controversial. Schoen and colleagues [15] reported that polyps neither consistently shrank nor enlarged while in formalin. However, in the study by Gopalswamy and colleagues [16], the size of the polyps after fixation was 12% to 18% smaller than the size measured soon after retrieval.

In cutaneous malignancy, the thickness of invasion is important in prognosis prediction. Salmhofer and colleagues [17] reported that formalin fixation may induce tissue retraction and shrinkage, which affects thickness measurements.

Formalin fixation also effects solid tissue measurement. Pritt and colleagues [10] observed that 4% of breast cancer specimens showed decreased size after overnight formalin fixation. As for measurements of human brains, Quester and colleagues [18] reported that formalin fixation leads to a longitudinal shrinkage of 1% to 8% in the brain stem in contrast to measurements in the cerebrum and cerebellum, which resulted in an increase in weight and size.

The amount of change in size is organ-specific owing to different tissue constitutions. Similar to the results of these other studies, our study demonstrated an effect of formalin on lung tumor size. Although not all lung tumors showed shrinkage after formalin fixation and the shrinkage rate was not constant, formalin fixation indeed caused a tumor stage shift from stage Ib to stage Ia in 40 of 401 patients (9.97%) with NSCLC.

Tumor size is one of the important components in the AJCC staging system. In lung cancer, it is not only a prognostic factor but is also a guide for treatment [19]. The results of recent reports suggest that patients with a higher stage NSCLC may benefit from postoperative adjuvant chemotherapy, even with stage Ib [3–7]. In NSCLC, a tumor size of 3 cm is the dividing line between T1 and T2. Thus, accurate tumor measurement, especially for those tumors of approximately 3 cm in size, is important in postoperative treatment strategy.

Tumor size measurements in fresh and fixed states may be associated with disparate results, however. In this circumstance, patients and doctors will be confused about stage determination. By the definition in the AJCC cancer staging manual, the pathologic classification uses evidence acquired before treatment, supplemented or modified by the additional evidence acquired during and from surgery, particularly from pathologic examination [1]. However, the pathologists do not routinely measure fresh resected specimens in the operating room in Taiwan. Instead, they obtain specimens that have undergone overnight formalin fixation. We believe that this is also the situation in many smaller institutions without a sufficient number of pathologists. Not all institutions have their pathologists handle the fresh specimens immediately after resection in the operation room. In most cases, the specimens are sent to pathology laboratories after formalin fixation and then are measured.

There is no guideline to categorize the tumor when measurements between surgeons (before fixation) and pathologists (after fixation) are different. We wondered whether formalin fixation causes tumor size change and stage shift. On the basis of survival analysis in this study, tumor staging may shift downward because of formalin fixation. Patients whose tumors shifted from T2 to T1 due to the effect of formalin fixation-induced size change had a similar survival with T2 tumors. This result implies that tumors that are approximately 3 cm should be staged according to the size measured before formalin fixation.

Our study has some limitations. First, it is retrospective. Many clinical parameters, such as tumor histology and operation methods, were distributed unevenly among groups. However, significant outcome differences among various histological types were not demonstrated in studies by Gail and colleagues [20], Christian and colleagues [21], Pelletier and colleagues [22], and Pairolero and colleagues [23]. The impact of type of resection on prognosis was also conflicting. Reports by the research groups of Gail [20], Martin-Ucar [24], Jazieh [25], and Wertzel [26] showed that the extent of resection did not influence survival in resected stage I NSCLC.

Another limitation is that the time between tumor removal and formalin fixation and the fixation duration were variable. Bias may arise because delayed fixation (>30 min) increases proteolytic tissue degradation and may affect tumor size measurement [14]. The fixation duration also has an effect. Short fixation times have an effect only at the periphery of the tissue block, whereas prolonged fixation (>24 to 48 hours) leads to excessive structural change [13, 14]. Furthermore, interobserver bias may exist during the period of the study.

In conclusion, tumor size measurement is important in NSCLC stage determination, treatment strategy, and making an accurate prognosis; however, tumor shrinkage in NSCLC may occur after formalin fixation. Thus, if precise tumor size measurements are to be obtained for prognosis and adjuvant therapy strategies, the size measurement must be performed immediately after the specimen is resected from the patient instead of after formalin fixation. We recommended that the AJCC staging system be revised so that it specifies the status of specimens to be measured in future versions.


   Footnotes
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Comment
 Footnotes
References
 
* Dr Po-Kuei Hsu and Dr Hsu-Chih Huang contributed equally to this article. Back


   References
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Comment
 Footnotes
 References
 

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