Endoscopic ultrasound-guided fine-needle aspiration biopsy coupled with KRAS mutation assay to distinguish pancreatic cancer from pseudotumoral chronic pancreatitis

• Introduction
• Patients and methods
• Patients
• EUS-FNAB and sample for KRAS analysis
• KRAS mutation analysis
• Follow up after EUS-FNAB analysis
• Statistical analysis
• Results
• Discussion
• Acknowledgment
• References

Background and study aims: Differential diagnosis between pancreatic adenocarcinoma (PADC) and pseudotumoral forms of chronic pancreatitis remains difficult. Mutation of KRAS oncogene is present in 75 % to 95 % of PADC. This study aimed to evaluate whether the combined analysis of KRAS mutation with cytopathological findings from endoscopic ultrasound-guided fine-needle aspiration biopsy (EUS-FNAB) might improve discrimination between PADC and chronic pancreatitis.

Patients and methods: This prospective multicenter study included 178 patients with solid pancreatic masses (men 104, women 74; mean age 64.5 years). Cytopathological examination and KRAS mutation analysis (codon-12 and codon-13, restriction fragment length polymorphism [RFLP] and direct sequencing) were performed on EUS-FNAB material. Final diagnoses were obtained on EUS-FNAB analysis and/or a second biopsy and/or clinical follow-up and/or surgery: PADC, n = 129; chronic pancreatitis, n = 27; other pancreatic neoplasms, n = 16; and benign lesions, n = 6.

Results: KRAS status analysis was successful in all EUS-FNAB samples. Codon-12 KRAS point mutation was found in 66 % of PADC samples. No case of chronic pancreatitis displayed KRAS mutation. Sensitivity, specificity, positive and negative predictive values, and overall accuracy of cytopathology alone for diagnosis of PADC versus chronic pancreatitis were 83 %, 100 %, 100 %, 56 % and 86 %, respectively. When KRAS mutation analysis was combined with cytopathology, these values reached 88 %, 100 %, 100 %, 63 % and 90 % respectively.

Conclusion: Although the value of KRAS analysis in addition to EUS-FNAB is limited for distinguishing pancreatic mass lesions, when chronic pancreatitis presented as a pseudotumor a negative finding (wild-type KRAS), was useful in strongly suggesting a benign lesion.

Introduction

Pancreatic cancer remains one of the most deadly tumor types. The 5-year survival rate after diagnosis is less than 3.5 % [1] [2]. Difficulties persist pertaining to early diagnosis of pancreatic adenocarcinoma (PADC) and in distinguishing PADC from benign diseases such as chronic pancreatitis; the latter frequently presenting as a pseudotumor. The differential diagnosis is of critical importance, to avoid unnecessary resection of benign lesions (such as focal lesions of chronic pancreatitis or autoimmune pancreatitis) or indeed delaying treatment of PADC in a subset of patients.

The molecular mechanisms underlying pancreatic oncogenesis remain only partially understood. Several genetic alterations are well characterized in PADC such as codon-12 KRAS mutation (75 % to 95 %) and to a lesser extent p16, DPC4 and p53 gene mutations [3] [4]. Previous studies conducted by us and others on pure pancreatic juice obtained at endoscopic retrograde cholangiopancreatography (ERCP) have found that KRAS mutation was found in 60 % to 65 % of PADC [5] [6] [7]. Also, the addition of p16 and DPC4 mutation analysis of pure pancreatic juice did not improve the sensitivity and specificity of KRAS mutation analysis alone for the diagnosis of PADC or in distinguishing PADC from chronic pancreatitis [8].

Currently, despite improvement in abdominal imaging techniques (including endoscopic ultrasound (EUS), the discrimination of PADC from focal pancreatitis remains problematic [9] [10]. Endoscopic ultrasound-guided fine needle aspiration-biopsy (EUS-FNAB) is a safe and effective technique in diagnosis and staging of PADC [11] [12] [13]. However, its accuracy for the diagnosis of malignancy varies widely with a sensitivity ranging from 65 % to 95 %; its mean accuracy is 85 % with negative predictive values ranging from 50 % to 70 %. In addition, EUS-FNAB may be inconclusive in up to 20 % of cases [11] [12] [13] [14].

Recent studies have revealed that the KRAS mutation can be detected in cellular materials obtained in EUS-FNAB samples [15] [16] [17] [18]. KRAS mutation analysis when combined with cyto-/histological findings obtained via EUS-FNAB appeared to be highly accurate in distinguishing benign and malignant solid pancreatic lesions [15] [16] [18]. These single-center studies concluded that KRAS mutation analysis may be essential to reinforce the diagnosis of pancreatic masses assessed using EUS. Herein, we conducted a prospective multicenter study to assess whether combining EUS-FNAB with KRAS mutation analysis might effectively distinguish between PADC and a pseudotumoral form of chronic pancreatitis.

Patients and methods

Patients

A total of 178 patients (men 104, women 74; mean age 64.5 ± 11.6 years) with solid pancreatic masses underwent EUS-FNAB procedures between January 2005 and April 2007 at four French referral centers (Clichy, Marseille, Montpellier, and Toulouse). All patients were referred for EUS-FNAB based on clinical data and results of previous imaging techniques (abdominal ultrasound, EUS, computed tomography [CT] scan or magnetic resonance cholangiopancreatography [MRCP]) that suggested or raised suspicion of a pancreatic mass. Patients with pancreatic cystic lesions, those who had undergone previous chemotherapy or pancreatic surgery, and those with contraindications for EUS were excluded.

Informed consent for KRAS analysis was obtained from all patients. The protocol has been approved by the regional ethical committee (CCPPRB Midi-Pyrénées II 6 January 2005 – protocol N°03 042 02 including external audit of the data).

EUS-FNAB and sample for KRAS analysis

EUS was carried out with the patient under intravenous propofol anesthesia, as previously described in [12] [19], using curved linear array echo endoscopes, FG-36 UA Pentax (Argenteuil, France) or UCT140T Olympus (Rungis, France), connected to Hitachi or Aloka ultrasound devices, respectively. EUS-FNAB was done using the EUS N1 – 22 gauge needle (Wilson-Cook, Limerick, Ireland) [19] [20]. At each center, at least two needle passes were done until sufficient tissue material was collected.

Core biopsy samples of pancreatic tissues were transferred into either Dubosq-Brazil or Cytolyte medium [20] with the needle stylet, for further cytological and histological diagnosis. Once the core biopsies had been transferred, the stylet was removed and the cellular material remaining in the needle catheter was air-flushed with a sterile 20-ml syringe and put into a sterile 1-ml Eppendorf tube and immediately frozen at – 20 °C until DNA extraction.

KRAS mutation analysis

The EUS-FNAB samples were centrifuged for 30 minutes at 7000 r. p. m. DNA was extracted from the pellets using the QIAamp DNA micro kit (Qiagen, Les Ulis, France) and eluted in 20-µl volumes. To identify KRAS codon-12 mutations, we performed a two-step nested polymerase chain reaction (PCR) amplification, followed by restriction fragment length polymorphism (RFLP) analysis, as previously described [6] [8] [21] with minor modifications.

DNA sequencing using the BigDye Terminator v3.1 kit in an automatic ABI 3100 sequencer (Applied Biosystems, California, USA) allowed verification and identification of mutations of the first or second nucleotide of codon-12 and a possible mutation of codon-13 in case of a wild-type codon-12 [8]. DNA extracted from human pancreatic cancer cells Capan-1 and BxPC-3, respectively, was used as positive (mutated KRAS) and negative (wild-type KRAS) control. KRAS mutation assays were performed by A.S. and M.B., who were blinded to all clinical data.

Follow up after EUS-FNAB analysis

After an initial EUS-FNAB procedure coupled with a KRAS mutation analysis, patients were clinically followed up with additional imaging examinations (including a second EUS-FNAB if required), especially in the case of a lack of a definite diagnosis and/or benign lesions. Data recorded comprised the results of subsequent imaging and cytopathology (as well as KRAS mutation analysis) if performed, treatments, date and cause of death (patients with PADC were followed monthly), new clinical events, and last contact.

Statistical analysis

Results are expressed as mean ± SD. Analysis was based on two-way contingency tables, sensitivity, specificity, predictive values, and accuracy, and on 95 % confidence intervals of relative frequencies, using of GraphPad InStat software.

Results

EUS-FNA was done successfully in the 178 patients included in this prospective study, without any complications. Codon-12 and codon-13 mutations of the KRAS gene were analyzed in 100 % of patient samples with no failure of amplification and sequencing. Four groups of patients were considered: those with PADC, with pseudotumoral chronic pancreatitis, with other pancreatic neoplasia, and with benign lesions without chronic pancreatitis.

The main clinical characteristics and follow-up, and results of the first FNAB together with KRAS mutation analysis, are detailed in [Tables 1] and [2]. The appearance of adenocarcinoma and pseudotumoral pancreatitis at EUS examination is illustrated in [Fig. 1]. The management algorithm of EUS-FNAB and the steps leading to a confirmed diagnosis at each stage are detailed in [Fig. 2].

Patients with pancreatic adenocarcinoma (n = 129). Cytological and/or histological analyses had established a definitive diagnosis of adenocarcinoma in 108 of the 129 cases after the first EUS-FNAB. The diagnosis was unequivocal, based on the presence of adenocarcinoma cells with positive staining for cytokeratin-7 (histological diagnosis using paraffin-embedded cores from microbiopsies can be finally achieved in 90 % of cases). A mutated codon-12 KRAS gene was detected in 79 cases.

In the remaining 21 patients, the FNAB analysis was inconclusive due to poor cellularity, absence of epithelial cells, presence of only blood clot, or presence of only normal cells. A mutated codon-12 KRAS was detected in 7 out of these 21 inconclusive cases. The final diagnosis of PADC was subsequently established by a second EUS-FNAB (n = 10), an FNA on a metastatic site at CT scan (n = 8), or during surgery (n = 3). No mutation of the KRAS gene at codon-13 was detected in codon-12 KRAS-negative samples (data not shown).

Patients with pancreatic mass lesion associated with chronic pancreatitis (n = 27). The final diagnosis of pseudotumoral chronic pancreatitis was determined from the following data: clinical context (previous symptoms or complications of chronic pancreatitis in 18 cases); imaging (17 had calcified pancreatitis); histological diagnosis obtained from one or two EUS-FNAB analyses, and a clinical follow up of at least 12 months. Of the chronic pancreatitis cases, 21 were related to alcohol abuse ([Fig. 1 c]), 2 to autoimmune pancreatitis ([Fig. 1 d]), and 4 were idiopathic.

In 21 cases, cytology and/or histology revealed features/lesions highly suggestive of chronic pancreatitis (inflammatory changes together with fibrosis) at the first EUS-FNAB. In 6 cases, EUS-FNAB was inconclusive. Chronic pancreatitis was confirmed at a second EUS-FNAB in all 6 cases. No mutation of the KRAS gene at codon-12 or codon-13 was detected in samples from chronic pancreatitis patients, both on the first and second EUS-FNAB. Patients with chronic pancreatitis were clinically reviewed every 2 months for 6 months, then every 6 months if there were no new events. A systematic CT scan was done in all patients at 6 months after initial EUS and treatment (biliary stent and/or surgical bypass if necessary). Subsequent investigations were done in 15 patients. No development of pancreatic lesions has been detected. All the 27 chronic pancreatitis patients are currently alive and well.

Patients with other pancreatic neoplasia. Definite diagnoses (including immunohistochemical findings) of endocrine tumors and pancreatic metastasis, respectively, were obtained in 12 and 4 cases; the patients with metastases had a previous history of either primary colorectal cancer (n = 2) or renal cell cancer (n = 2). Two initial FNABs were inconclusive but diagnosis was confirmed following a second EUS-FNAB. Codon-12 KRAS mutation was detected in only one case of colorectal metastasis.

Patients with a benign pancreatic lesion. In 6 patients, histo-/cytopathological analysis revealed either necrotic, inflammatory, or normal pancreatic tissue (wild type KRAS in all cases). The clinical follow-up (every 2 months for 6 months, then every 6 months) and subsequent investigations at 4 months (CT scan, n = 4; CT scan and EUS, n = 2) showed regression or disappearance of the pseudotumoral appearance in the pancreas in all cases, and both pancreatic cancer and chronic pancreatitis could be ruled out. These patients were deemed to have had sequelae of inflammatory pancreatitis. All these patients had previously experienced episodes of acute pancreatitis, and they are currently alive and well.

Inconclusive first FNAB: summary. Overall, FNAB was inconclusive in 29 cases ([Fig. 2]), and among these codon-12 KRAS mutations were found in 7 cases. A second FNAB was performed in all cases: wild type KRAS was observed in 22 cases and a codon-12 KRAS mutation in 7. The final diagnoses were PADC in 21 cases (including all the 7 cases of KRAS mutation), and chronic pancreatitis and endocrine tumor in 6 and 2 cases, respectively. (The second FNAB established the final diagnosis in 18/29 inconclusive cases; the remaining 11, all PADC, were established at CT or surgery as described above.)

Diagnostic accuracy. Sensitivity, specificity, predictive values, and overall accuracies of cyto-/histopathology alone, KRAS analysis alone, and the combination of two tests are given in [Table 3] (all pancreatic neoplasias versus all benign pancreatic lesions) and in [Table 4] (PADC versus chronic pancreatitis). The diagnostic performances were not significantly different between the four centers (data not shown).

We observed that combining KRAS mutation analysis and histo-/cytopathological findings increases the accuracy in diagnosing solid pancreatic tumors. However, the gain appears minimal. In patients with a strong clinical and radiological suggestion of pancreatic adenocarcinoma and an inconclusive biopsy, a repeat FNAB is recommended, even if KRAS mutation is highly suggestive of malignancy. However, in patients with clinical and morphological data suggesting a pseudotumoral form of chronic pancreatitis, where FNAB is frequently nondiscriminatory, malignancy can be ruled out in the presence of a wild-type KRAS gene.

Discussion

In the current prospective multicenter study, we demonstrated that combining histo-/cytopathology and KRAS mutation analysis in EUS-FNAB materials may be helpful in distinguishing PADC and pancreatic mass associated with chronic pancreatitis. We also demonstrated for the first time that, despite multiple centers and operators (two or three per center performing EUS-FNAB), that KRAS mutation analysis (both codon-12 and codon-13) is technically possible in 100 % of cases (including sequencing). Despite the minute quantities of material collected for molecular analysis, KRAS mutation screening can be readily transferred into routine clinical practice. The use of a new extraction micro-kit may explain this success (the literature reports 6 % to 13 % failure rates for amplification both with tissue samples and with pure pancreatic juice) [5] [6] [7] [8]. Collection of both core biopsies for histo-/cytology (using the stylet to empty the needle contents) and cellular material for KRAS analysis (by simply flushing the biopsy needle) can be done during the same procedure. The total cost for one KRAS assay, including sequencing, is estimated at € 45 to € 49.

Several authors have suggested that the combination of histo-/cytopathology and KRAS analysis on EUS-FNAB increases the positive diagnosis of malignant pancreatic mass [16] [18]. In the present study, KRAS mutation analysis improved on the accuracy of cytopathology alone only from 86 % to 90 % when the subgroup of patients with adenocarcinoma was considered. Thus, adding KRAS mutation analysis to histo-/cytopathology does not seem to make a major contribution to the positive diagnosis of PADC. However, when the histo-/cytopathology is inconclusive or perhaps inadequate in the case of a pancreatic mass, the presence of a KRAS mutation is highly suggestive of adenocarcinoma. Several studies have been conducted in order to increase the diagnostic yield of EUS-FNAB. It has been demonstrated that on-site cytopathology interpretation has a significant clinical impact and is cost-effective [22] [23]. This could be one of the limitations of our study, as none of the four centers had an on-site pathologist during EUS-FNAB. In addition, EUS elastography and contrast-enhanced EUS improve pancreatic tissue characterization [24] [25]. However, the distinction between chronic pancreatitis and hard tumors still appears to be difficult using elastography, and EUS-FNAB remains necessary [26].

From our results, KRAS mutation analysis may be useful to distinguish PADC from chronic pancreatitis especially in its pseudotumoral form. The multicenter design of the study allowed us to recruit 27 patients with pseudotumoral chronic pancreatitis who were followed up after initial histo-/cytopathological analysis and KRAS mutation assay. None of these patients developed a PADC, despite the relatively advanced disease associated with the high mean age (59 years) and the frequency of calcifications (85 %) found with disease of alcoholic origin. In fact, the incidence of PADC is known to increase with time during the course of chronic pancreatitis. Based on the combination of histo-/cytopathological findings (including a second biopsy in the case of negative results at first biopsy) and KRAS mutation analysis, conservative medical or surgical treatment (bypass, n = 3) was possible in all 27 of these patients, thus avoiding unnecessary pancreatic resection. With a clinical and radiological presentation of chronic pancreatitis, the presence of wild type KRAS at EUS-FNAB is therefore highly suggestive of benign lesions. As previously observed in single-center studies, in chronic pancreatitis the rate of KRAS mutations is low or zero in material obtained by EUS-guided FNAB [15] [16] [17] [18] [27] [28]. With chronic pancreatitis, the rate of KRAS mutation in tissue samples is lower than that observed in pancreatic juice; the pancreatic juice may be enriched by a cell population that is preferably shed from the epithelial cell lining including those mutated for KRAS [27] [28] [29]. The importance of KRAS mutation during the course of apparently benign chronic pancreatitis remains poorly understood. Controversial data have been published from single-center studies that included long-term follow-up of chronic pancreatitis with codon-12 KRAS mutation found in pancreatic juice. Only a low percentage of patients with KRAS mutation developed a PADC [30] [31]. Indeed, KRAS mutation is a possible event during chronic pancreatitis especially in ductal hyperplasia and in the absence of adenocarcinoma [27] [28] [29]. However, with a clinical and radiological presentation of chronic pancreatitis, the presence of mutated KRAS at EUS-FNAB may justify a second biopsy and a follow-up to rule out a PADC.

In conclusion, following a multicenter evaluation, the current study demonstrates that the combination of histo-/cytopathology and KRAS analysis on samples obtained at EUS-FNAB does not significantly increase the positive diagnosis of malignant pancreatic mass. Conversely, when confronted with a clinical and radiological presentation of pseudotumoral chronic pancreatitis both histo-/cytopathological analysis (inflammation, fibrosis) and wild-type KRAS are strongly suggestive of a benign condition.

Acknowledgment

Financial support was given by the SNFGE (Société Nationale Française de Gastroentérologie), IRMAD (Institut de Recherche des Maladies de l’Appareil digestif), INSERM and Toulouse Hospital (Centre Hospitalo-Universitaire de Toulouse).

Competing interests: None

References

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L. BuscailMD PhD 

Department of Gastroenterology
CHU Rangueil

1 avenue Jean Poulhès, TSA 50032
31059 Toulouse Cedex 9
France

Fax: +33-5-61323599

Email: Buscail.L@chu-toulouse.fr

References

• 1 Warshaw A L, Fernandez del Castillo C. Pancreatic carcinoma.  N Eng J Med. 1992;  326 455-465
  PubMedSearch in Google Scholar
• 2 Buscail L, Pagès P, Berthelémy P. et al . Role of EUS in the management of pancreatic and ampullary carcinoma: a prospective study assessing resectability and prognosis.  Gastrointest Endosc. 1999;  50 34-40
  PubMedSearch in Google Scholar
• 3 Hahn S A, Schmiegel W H. Recent discoveries in cancer genetics of exocrine pancreatic neoplasia.  Digestion. 1998;  59 493-501
  CrossrefPubMedSearch in Google Scholar
• 4 Koorstra J BM, Hustinx S R, Offerhaus G JA. et al . Pancreatic carcinogenesis.  Pancreatology. 2008;  8 110-125
  CrossrefPubMedSearch in Google Scholar
• 5 Van Laethem J L, Vertongen P, Deviere J. et al . Detection of c-Ki-ras gene codon 12 mutations from pancreatic duct brushings in the diagnosis of pancreatic tumours.  Gut. 1995;  36 781-787
  PubMedSearch in Google Scholar
• 6 Berthélemy P, Bouisson M, Escourrou J. et al . Identification of KRAS mutations in pancreatic juice in the early diagnosis of pancreatic cancer.  Ann Intern Med. 1995;  123 188-191
  PubMedSearch in Google Scholar
• 7 Tascilar M, Caspers E, Sturm P D. et al . Role of tumor markers and mutations in cells and pancreatic juice in the diagnosis of pancreatic cancer.  Ann Oncol. 1999;  10 (Suppl 4) 107-410
  CrossrefPubMedSearch in Google Scholar
• 8 Costentin L, Pagès P, Bouisson M. et al . Frequent deletions of tumor suppressor genes in pure pancreatic juice from patients with tumoral or nontumoral pancreatic diseases.  Pancreatology. 2002;  2 17-25
  PubMedSearch in Google Scholar
• 9 Varadarajulu S, Tamhane A, Eloubeidi M A. Yield of EUS-guided FNA of pancreatic masses in the presence or the absence of chronic pancreatitis.  Gastrointest Endosc. 2005;  62 728-736
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• 10 Fritscher-Ravens A, Brand L, Knöfel W T. et al . Comparison of endoscopic ultrasound-guided fine needle aspiration for focal pancreatic lesions in patients with normal parenchyma and chronic pancreatitis.  Am J Gastroenterol. 2002;  97 2768-2775
  CrossrefPubMedSearch in Google Scholar
• 11 Wiersema M J. Accuracy of endoscopic ultrasound in diagnosing and staging pancreatic carcinoma.  Pancreatology. 2001;  1 625-632
  CrossrefPubMedSearch in Google Scholar
• 12 Buscail L, Faure P, Bournet B. et al . Interventional endoscopic ultrasound in pancreatic diseases.  Pancreatology. 2006;  6 7-16
  CrossrefPubMedSearch in Google Scholar
• 13 Bhutani M S, Logroño R. Endoscopic ultrasound-guided fine-needle aspiration cytology for diagnosis above and below the diaphragm.  J Clin Ultrasound. 2005;  33 401-411
  CrossrefPubMedSearch in Google Scholar
• 14 Savides T J, Donohue M, Hunt G. et al . EUS-guided FNA diagnostic yield of malignancy in solid pancreatic masses: a benchmark for quality performance measurement.  Gastrointest Endosc. 2007;  66 277-282
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• 15 Tada M, Komatsu Y, Kawabe T. et al . Quantitative analysis of K-ras gene mutation in pancreatic tissue obtained by endoscopic ultrasonography-guided fine needle aspiration: clinical utility for diagnosis of pancreatic tumor.  Am J Gastroenterol. 2002;  97 2263-2270
  CrossrefPubMedSearch in Google Scholar
• 16 Takahashi K, Yamao K, Okubo K. et al . Differential diagnosis of pancreatic cancer and focal pancreatitis by using EUS-guided FNA.  Gastrointest Endosc. 2005;  61 76-79
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• 17 Khalid A, Nodit L, Zahid M. et al . Ultrasound fine needle aspirate DNA analysis to differentiate malignant and benign pancreatic masses.  Am J Gastroenterol. 2006;  101 2493-2500
  CrossrefPubMedSearch in Google Scholar
• 18 Maluf-Filho F, Kumar A, Gerhardt R. et al . Kras mutation analysis of fine needle aspirate under EUS guidance facilitates risk stratification of patients with pancreatic mass.  J Clin Gastroenterol. 2007;  41 906-910
  PubMedSearch in Google Scholar
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• 20 Voss M, Hammel P, Molas G. et al . Value of endoscopic ultrasound guided fine needle aspiration biopsy in the diagnosis of solid pancreatic masses.  Gut. 2000;  46 244-249
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L. BuscailMD PhD 

Department of Gastroenterology
CHU Rangueil

1 avenue Jean Poulhès, TSA 50032
31059 Toulouse Cedex 9
France

Fax: +33-5-61323599

Email: Buscail.L@chu-toulouse.fr