Clinical impact of K-ras mutation analysis in EUS-guided FNA specimens from pancreatic masses

ORIGINAL ARTICLE: Clinical Endoscopy

Clinical impact of K-ras mutation analysis in EUS-guided FNA specimens from pancreatic masses Takeshi Ogura, MD,1,5 Kenji Yamao, MD,1 Akira Sawaki, MD,1 Nobumasa Mizuno, MD,1 Kazuo Hara, MD,1 Susumu Hijioka, MD,1 Yasumasa Niwa, MD,1 Masahiro Tajika, MD,1 Shinya Kondo, MD,1 Yasuhiro Shimizu, MD,2 Vikram Bhatia, MD,4 Kazuhide Higuchi, MD,5 Waki Hosoda, MD,3 Yasushi Yatabe, MD3 Nagoya, Osaka, Japan; Delhi, India

Background: EUS-guided FNA (EUS-FNA) is considered optimal for differentially diagnosing pancreatic masses. However, the sensitivity of EUS-FNA ranges from 65% to 95%, respectively, which requires improvement. Objective: To evaluate clinical impact of K-ras mutation analysis in EUS-FNA specimens from pancreatic masses. Design: Prospective registration, single-center study. Setting: Tertiary referral center. Patients: This study involved 394 consecutive patients with pancreatic masses (307 pancreatic ductal adenocarcinomas [PDACs], 47 pancreatic inflammatory lesions, and 40 other types of tumors) who underwent EUS-FNA and analysis of K-ras mutations. Intervention: EUS-FNA, Cycleave polymerase chain reaction. Main Outcome Measurements: Improvement of the diagnostic accuracy by K-ras mutation analysis; absence of K-ras mutations in non-PDAC masses. Results: K-ras mutations were detected in 266 of 307 PDAC aspirates (87%) and in 3 of 87 non-PDAC masses (3%). K-ras mutations were detected in 18 of 39 patients (46%) who remained cytohistopathologically undiagnosed. The sensitivity, specificity, positive and negative predictive values, and accuracy of cytohistopathological and K-ras mutation analyses alone were 87%, 100%, 100%, 54%, and 89%, respectively, and, when combined, were 93%, 100%, 100%, 68%, and 94%, respectively. Adding K-ras mutation analysis to standard cytohistopathological assessment increased the sensitivity and accuracy of EUS-FNA by 6% (P ⬍ .001) and 5% (P ⬍ .001), respectively. Limitations: Single-center study. Conclusions: K-ras mutation analysis may be helpful in patients with suspected PDAC yet inconclusive EUS-FNA findings. K-ras mutations were extremely rare in pancreatic inflammation and other pancreatic tumors. (Gastrointest Endosc 2012;75:769-74.)

The prognosis of pancreatic ductal adenocarcinoma (PDAC) is one of the poorest among malignant tumors. The 5-year survival rate among all patients with PDAC is

less than 3.5%.1,2 On the other hand, the prognosis of pancreatic inflammatory lesions such as chronic pancreatitis (CP), autoimmune pancreatitis (AIP), and other rare

Abbreviations: AIP, autoimmune pancreatitis; CP, chronic pancreatitis; EUS-FNA, EUS-guided FNA; NPV, negative predictive value; PanIN, pancreatic intraepithelial neoplasia; PCR, polymerase chain reaction; PDAC, pancreatic ductal adenocarcinoma; PNET, pancreatic neuroendocrine tumor.

Received June 16, 2011; Accepted November 11, 2011.

DISCLOSURE: The authors disclosed no financial relationships relevant to this publication. This study was supported by the Research Committee of Intractable Disease, Ministry of Health, Labour and Welfare of Japan. See CME section; p. 856. Copyright © 2012 by the American Society for Gastrointestinal Endoscopy 0016-5107/$36.00 doi:10.1016/j.gie.2011.11.012

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Current affiliations: Departments of Gastroenterology (1), Gastroenterological Surgery (2), and Pathology and Molecular Diagnostics (3), Aichi Cancer Center Hospital, Nagoya, Japan, Department of Hepatology (4), Institute of Liver and Biliary Sciences (ILBS), Delhi, India, 2nd Department of Internal Medicine (5), Osaka Medical College, Osaka, Japan. Poster presented at Digestive Disease Week, May 9, 2011, Chicago, Illinois. Reprint requests: Kenji Yamao, MD, Department of Gastroenterology, Aichi Cancer Center Hospital, 1-1 Kanokoden, Chikusa-ku, Nagoya 464-8681, Japan.

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tumors is much better. To differentiate PDAC from these inflammatory conditions is critical because treatment strategies and prognoses differ. However, despite advances in imaging techniques, differentiating PDAC from pancreatic inflammation lesions and other tumors remains challenging.3-6 EUS-guided FNA (EUS-FNA) is considered the best method for establishing a differential diagnosis of pancreatic masses. However, the overall accuracy of EUS-FNA in this setting widely varies, with sensitivity ranging from 65% to 95% and with negative predictive values (NPVs) ranging from 50% to 70%.7-10 One limitation of EUS-FNA is the small volume of acquired specimens, which can render differentiating between malignancy and benign lesions difficult.7-10 The value of techniques such as core needle biopsy,11 elastography,12 and harmonic contrast EUS13 for patients with inconclusive diagnoses has been reported, but other modalities are still required to further improve diagnostic accuracy. The rates of various genetic abnormalities, particularly K-ras mutations in PDAC, are high.14,15 We previously suggested that a combination of K-ras mutation analysis and cytohistopathological assessment of EUS-FNA aspirates increases the accuracy of a diagnosis of PDAC and allows its differentiation from CP.16 This study extends our previous findings by prospectively investigating the clinical benefit of K-ras analysis in pancreatic EUS-FNA samples from a large number of consecutive patients.

PATIENTS AND METHODS Patients The Aichi Cancer Center Hospital Institutional Review Board approved this study of K-ras mutation analysis in 394 consecutive patients who underwent EUS-FNA of pancreatic masses between March 2004 and September 2009 at the Aichi Cancer Center Hospital. All patients provided written informed consent for all procedures associated with the study.

EUS-FNA technique We performed EUS as described9,17 at 7.5-MHz frequency by using a convex linear-array echoendoscope (GF-UGT240; Olympus Optical Co Ltd, Tokyo, Japan) connected to a US device (SSD5500; Aloka, Tokyo, Japan) and a 22-gauge needle (NA-10J or NA-11J-KB; Olympus Optical Co Ltd or EchoTip-Ultra Needle; Cook Medical, Limerick, Ireland). Aspirated material was separated into 1 part each for cytopathological evaluation, cell-block preparation, and K-ras point mutation analysis. The material aspirated from all 394 patients was immediately evaluated (by using Diff Quick staining) by a cytopathologist and/or cytotechnologist for rapid diagnosis.9 Material was immediately fixed in 10% formalin in a standard specimen bottle, centrifuged, and then embedded in paraffin for cell-block analysis. Sections then were visualized by 770 GASTROINTESTINAL ENDOSCOPY Volume 75, No. 4 : 2012

Ogura et al

Take-home Message ●

●

The sensitivity of EUS-guided FNA for diagnosing pancreatic ductal adenocarcinoma (PDAC) was improved by adding K-ras mutation analysis. K-ras mutations were absent in all pancreatic inflammatory lesions and almost all tumors other than PDAC.

hematoxylin and eosin as well as by immunohistochemical staining if necessary.

Analysis of K-ras mutations Material for the genetic study was based on either the fresh specimens or the paraffin-embedded sections of the cell blocks obtained by EUS-FNA. Total RNA was extracted from the fresh specimens, and mutational analysis was performed by using reverse transcriptase polymerase chain reaction (PCR) coupled with direct sequencing methods, as described previously.18 When the direct sequencing displayed no mutational signal when the cytological diagnosis was atypical cells, suspicious, or adenocarcinoma, we further investigated the K-ras mutation by using the corresponding cell block slides by a Cycleave PCR assay.19 This is a highly sensitive assay that can detect as little as 5% of tumor cells mixed with normal tissues.

Final diagnosis The final diagnosis was based on pathological examinations of specimens obtained by surgical resection and/or EUS-FNA. If signs of malignancy were absent at the end of follow-up (disease regression or no evidence of disease progression), PDAC was ruled out. These patients were considered to have other pancreatic diseases according to their clinical course and/or cytohistopathological diagnosis obtained by EUS-FNA. The final diagnosis was a benign disorder if the clinical course was consistent with EUS-FNA findings and the patient had been followed for at least 1 year.

Statistical analysis Continuous variables are expressed as medians and ranges. Incidences and concordance between groups were compared by using the Fisher exact test or McNemar test where appropriate. All statistical analysis were performed by using StatMate IV (ATMS Co Ltd, Tokyo, Japan). A P value of ⱕ.05 was considered statistically significant.

RESULTS Patient’s characteristics Tables 1 and 2 show patient’s characteristics. Among 394 patients, 307 had PDAC (185 male, 122 female; mean age, 64.7 years; mean size of mass, 31.3 mm), 47 had www.giejournal.org

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K-ras mutation analysis in EUS-FNA specimens from pancreatic masses

TABLE 1. Patient characteristics (N ⴝ 394) PDAC

Pancreatic inflammatory lesions

Other tumors

No. of patients (M/F)

307 (185/122)

47 (38/9)

40 (20/20)

Mean age, y (range)

64.7 (35-84)

64.2 (41-84)

53.9 (23-81)

2.3 (1-4)

2.3 (1-4)

2.3 (1-4)

31.3 (7.0-50.0)

20.4 (10.0-58.5)

25.6 (8.0-90.0)

Mean no. of needle passes (range) Mean size of mass, mm (range)

PDAC, Pancreatic ductal adenocarcinoma; M, male; F, female.

TABLE 2. Final diagnosis (N ⴝ 394)

TABLE 3. Detection of K-ras mutations No. of patients

PDAC

K-ras mutations, no. (%) Positive Negative P value

307 PDAC (n ⫽ 307)

Pancreatic inflammatory lesions Focal chronic pancreatitis

24

AIP

23

Other tumors

Pancreatic inflammatory lesions and other tumors (n ⫽ 87)

267 (87)

40 (13)*

3 (3)†

84 (97)

⬍.001

PDAC, Pancreatic ductal adenocarcinoma. *Including patient in whom K-ras mutation was not possible. †Comprising 1 each of poorly differentiated pancreatic neuroendocrine tumor and metastasis from the stomach and ovary.

PNET

20

Metastatic tumor*

8

Acinar cell carcinoma

3

Malignant lymphoma

3

Detection of K-ras mutations

Solid pseudopapillary tumor

2

Serous cystic tumor, lymphoepithelial cyst

1

Table 3 shows the results of K-ras analysis of the 394 patients. K-ras analysis was successfully performed in 99.7% of patients (393/394). The aspirate of 1 patient did not contain any cell components in PDAC. K-ras mutations were detected in 266 of 307 aspirates from PDAC (87%) and in only 3 of 87 non-PDAC masses (3%), including 1 poorly differentiated PNET and 2 metastatic tumors (1 each in the stomach and ovary). K-ras mutations were significantly more frequent in PDAC (P ⬍ .001). Among 307 patients with PDAC, 68 were found to be negative for the K-ras mutation by direct sequencing, and 27 were found positive for the K-ras mutation by Cycleave PCR. As a result, 78% of PDAC patients were positive for the K-ras mutation by direct sequencing alone, but this improved to 87% with the additional use of Cycleave PCR. The 3 patients with conditions other than PDAC who were positive for the K-ras mutation were also positive by direct sequencing.

PDAC, Pancreatic ductal adenocarcinoma; AIP, autoimmune pancreatitis; PNET, pancreatic neuroendocrine tumor. *Metastatic tumors 8 (2 from lung, 2 from the stomach, 1 from the ovary, 1 from the esophagus, 1 from the breast, 1 from the kidney).

pancreatic inflammatory lesions (38 male; mean age, 64.2 years; mean size of mass, 20.4 mm), and 40 had other tumors (20 male; mean age, 53.9 years; mean size of mass, 25.6 mm). The mean number of needle passes in all patients was 2.3. Pancreatic inflammatory lesions constituted AIP in 24 patients and focal CP in 23 patients. The miscellaneous tumors included pancreatic neuroendocrine tumors ([PNETs] n ⫽ 20), metastatic tumors (n ⫽ 8), acinar cell carcinomas (n ⫽ 3), malignant lymphomas (n ⫽ 3), solid pseudopapillary tumors (n ⫽ 3), serous cystic tumors (n ⫽ 2), and a lymphoepithelial cyst (n ⫽ 1). The primary sites for metastasis to the pancreas were lung (n ⫽ 2), stomach (n ⫽ 2), and ovary, esophagus, breast, and kidney (n ⫽ 1 each). The average follow-up period was 341 ⫾ 313 days for PDAC, 1090 ⫾ 493 days for pancreatic inflammatory lesions, and 672 ⫾ 371 days for other tumors. www.giejournal.org

Diagnostic flow of PDAC Figure 1 shows the diagnostic flow of 307 patients with a final diagnosis of PDAC. Cytological evaluation alone diagnosed PDAC in 255 of them, malignancy was suspected in 31, and 21 were negative for malignancy. In addition, histological assessment diagnosed malignancy in 13 of the total of 52 patients with inconclusive or negative cytological results. Of the remaining 39 patients who reVolume 75, No. 4 : 2012 GASTROINTESTINAL ENDOSCOPY 771

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Figure 1. Diagnostic flow of pancreatic ductal adenocarcinoma. Of the remaining 39 patients who remained undiagnosed by cytological and histological assessment, K-ras mutations were detected in 18 (46%) of them. *Including the patient in whom the K-ras mutation was not possible. **Malignancy was diagnosed by ERCP, surgery, and aspiration from metastasis foci.

TABLE 4. Cytohistopathological and K-ras mutation analysis alone and combined with EUS-FNA material in the differential diagnosis of PDAC (n ⴝ 307) and pancreatic inflammatory lesions (n ⴝ 47) Sensitivity, %

Specificity, %

PPV, %

NPV, %

Accuracy, %

K-ras mutation analysis alone

87

100

100

54

89

Cato-/histopathology alone

87

100

100

54

89

Combined cytohistopathological and K-ras mutation analysis

93

100

100

68

94

PDAC, Pancreatic ductal adenocarcinoma; PPV, positive predictive value; NPV, negative predictive value.

mained undiagnosed by cytological and histological assessment, K-ras mutations were detected in 18 (46%) of them. Finally, 3 of the 18 who were positive for the K-ras mutation were diagnosed with PDAC by a second EUSFNA. Of the remaining 15, PDAC was diagnosed by surgical resection (n ⫽ 9) and by biopsy specimens from metastatic foci (n ⫽ 6). Among the 21 who were negative for the K-ras mutation, 3 were diagnosed with PDAC by a second EUS-FNA. Of the remaining 18, PDAC was diagnosed by surgical resection (n ⫽ 3), pancreatic juice cytology (n ⫽ 2), and biopsy specimens from metastatic foci (n ⫽ 13). For the purposes of this study, a cytology result was considered negative for malignancy if identified as either atypical or suspicious for malignancy.

Differential diagnosis of PDAC and pancreatic inflammations Table 4 shows that the sensitivity, specificity, positive predictive value, NPV, and accuracy of the cytohistopathological examination alone and of the K-ras analysis alone were each 87%, 100%, 100%, 54%, and 89%, respectively, whereas these values of the 2 analyses combined were 772 GASTROINTESTINAL ENDOSCOPY Volume 75, No. 4 : 2012

93%, 100%, 100%, 68%, and 94%, respectively. The sensitivity and accuracy of EUS-FNA increased by 6% (P ⬍ .001) and 5% (P ⬍ .001), respectively, when K-ras analysis was added to standard cytohistopathological assessment.

Complications No complications were associated with EUS-FNA in any of the 394 patients.

DISCUSSION Various genetic abnormalities, such as K-ras, p53, p16, and DPC4, have been demonstrated in PDAC.14,15,20-22 K-ras mutations are frequent and are found in 75% to 90% of cases of PDAC. Several authors suggested that K-ras mutation analysis of EUS-FNA specimens is valuable.16,23-28 However, published reports have included relatively small numbers of patients with PDAC and other pancreatic tumors for comparison (Table 5). Here, we prospectively investigated the most patients in a single study to date and examined other pancreatic diseases for a differential diagnosis. K-ras analysis was informative for all of our patients except 1, whose aspirate did not contain any cellular compowww.giejournal.org

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K-ras mutation analysis in EUS-FNA specimens from pancreatic masses

TABLE 5. Results of K-ras mutation analysis of EUS-FNA specimens from pancreatic masses in recent studies % (K-ras positive/all) Study/year

No.

PDAC

CP

AIP

PNET

Tada et al23/2002

34

77 (20/26)

0 (0/8)

—

—

—

—

Pellise et al24/2003

57

73 (24/33)

0 (0/5)

—

0 (0/6)

50 (1/2)

0 (0/11)

al25/2003

66

83 (45/54)

0 (0/5)

—

0 (0/3)

—

0 (0/4)

77

74 (46/62)

0 (0/15)

—

—

—

—

21

73 (11/15)

0 (0/2)

25 (1/4)

—

—

—

74

63 (34/57)

9 (1/11)

—

0 (0/6)

—

—

Bournet et al28/2009

178

67 (86/129)

0 (0/27)

—

0 (0/10)

17 (1/6)

0 (0/6)

Total patients in studies

507

71 (266/376)

1 (1/73)

25 (1/4)

0 (0/25)

25 (2/8)

0 (0/21)

Patients in current study

394

86 (266/376)

0 (0/24)

0 (0/23)

5 (1/20)

25 (2/8)

0 (0/12)

Total

901

78 (532/682)

1 (1/97)

4 (1/27)

2 (1/45)

25 (4/16)

0 (0/33)

Zheng et

Takahashi et Khalid et

al16/2005

al26/2006

Maluf-Filho et

al27/2007

Metastasis

Other

PDAC, Pancreatic ductal adenocarcinoma; CP, chronic pancreatitis; AIP, autoimmune pancreatitis; PNET, pancreatic neuroendocrine tumor.

nents. We detected K-ras mutations in 87% of the patients with PDAC. These results were similar to published findings.14,15,29,30 Two findings in this study are significant. First, the sensitivity of EUS-FNA for diagnosing PDAC was significantly improved by adding K-ras mutation analysis. Among 39 patients whose EUS-FNA results were inconclusive or negative, 18 had K-ras mutations and were finally diagnosed with PDAC. These findings indicate that K-ras analysis is clinically useful when EUS-FNA specimens are sufficiently limited or inadequate as to render differentiation between malignant or benign conditions difficult. Second, K-ras mutations were absent in all pancreatic inflammatory lesions and almost all tumors other than PDAC. The sensitivity, NPV, and accuracy of EUS-FNA for differentiating PDAC from pancreatic inflammatory lesions were improved by adding K-ras mutation analysis. Thus, when pancreatic inflammatory lesions or other tumors are suspected based on the clinical course or imaging findings, the absence of K-ras mutations in EUS-FNA specimens would help to exclude PDAC. Despite the inherent limitation of being a single-center study, these findings may be deemed reliable because a large number of consecutive patients and a variety of diseases were included, and the study was prospectively registered. Table 5 shows recent studies of K-ras mutation analyses in EUS-FNA specimens. K-ras mutations were detected in 63% to 83% of PDAC, but in only 3% and 7% of pancreatic inflammatory lesions and other tumors, respectively. Our K-ras accuracy for PDAC was 89%, which is higher than that of other reports. There are 2 possible reasons why our K-ras accuracy was high compared with other studies: first, because the results of EUS-FNA themselves were good, including a high number of tumor cells and a low false-negative rate for K-ras mutations compared with other reports and, second, although direct sewww.giejournal.org

quencing or the restriction fragment length polymorphism/ PCR method was used in other studies, we also used the more sensitive Cycleave PCR system when direct sequencing was inconclusive in our study. In fact, 78% of patients were positive for the K-ras mutation by direct sequencing only, whereas these results improved to 87% with the additional use of the Cycleave PCR system. Including the results of our study, pooled analysis demonstrated that K-ras mutations were detected in 78% of cases of PDAC and in only 2% and 7% of pancreatic inflammatory lesions and other tumors, respectively. According to these results, the presence of K-ras point mutations in a pancreatic mass indicates PDAC. This study did not detect K-ras mutations in pancreatic inflammation, a finding that is consistent with previous reports indicating that this might be a useful marker with which to differentiate PDAC from CP.3-6 However, CP might be a risk factor for PDAC,31 and several reports have found associations between PDAC and CP.32,33 K-ras mutations have also been detected in AIP. Khalid et al26 detected K-ras mutations in 1 of 4 patients with AIP. However, pathological specimens from that patient included pancreatic intraepithelial neoplasia (PanIN) lesions. On the other hand, Kamisawa et al34 described K-ras mutations in 8 patients with AIP from whom pancreatic specimens were obtained at surgery (n ⫽ 6), surgical biopsy (n ⫽ 1), and autopsy (n ⫽ 1). Almost all of these pathological tissues included PanIN lesions, and the report suggested that AIP is a risk factor for PDAC. Because pathological tissue samples were obtained by EUS-FNA in the current study, relatively few cell components from PanIN lesions were present. Therefore, the differential frequency of K-ras mutations might depend on whether sampled tissues include PanIN lesions. If CP or AIP is suspected based on clinical features or imaging together with K-ras mutations in EUS-FNA Volume 75, No. 4 : 2012 GASTROINTESTINAL ENDOSCOPY 773

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aspirates, close clinical follow-up or repeat EUS-FNA would be warranted. On the other hand, we detected K-ras mutations in PNET. A proportion of PNET was positive for CK7 and CDX2. Because these 2 molecules are expressed in the pancreatic duct, it is possible that expression of these molecules represents differentiation of the tumor cells from ductal epithelium. Therefore, the K-ras mutation could be a hallmark of malignancy.35K-ras mutations have also been detected in metastasis from ovarian and gastric carcinomas, although at relatively low frequency.36,37 The possibility of a pancreatic mass being metastatic cancer should be considered when patients have previously had carcinomas, and the K-ras mutation in such tumors should be analyzed. In conclusion, K-ras mutation analysis may be helpful in patients with suspected PDAC yet inconclusive EUS-FNA findings. The frequency of K-ras mutations is low in areas of pancreatic inflammation and in other types of pancreatic tumors.

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