Switch in KRAS mutational status during an unusual course of disease in a patient with advanced pancreatic adenocarcinoma: implications for translational research
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Despite the introduction of novel effective treatment regimens like gemcitabine plus nab-paclitaxel and FOLFIRINOX, pancreatic ductal adenocarcinoma (PDAC) remains one of the most aggressive epithelial tumors. Among the genetic alterations frequently found in PDAC, mutations in the KRAS gene might play a prognostic role regarding overall survival and may also have the potential to predict the efficacy of anti-EGFR treatment.
We report the clinical case of a 69 year old Caucasian female that was diagnosed with histologically confirmed locally advanced PDAC with lymph node involvement in August 2010. At the time of first diagnosis, tumor tissue obtained from an open regional lymph node biopsy showed a poorly differentiated adenocarcinoma with a wild type sequence within exon 2 (codon 12/13) of the KRAS gene. The patient initially received single-agent gemcitabine and a subsequent 5-FU-based chemoradiotherapy with a sequential maintenance chemotherapy with oral capecitabine resulting in a long term disease control. Local disease progression occurred in May 2014 and the patient underwent pancreaticoduodenectomy in September 2014. A novel KRAS gene mutation (c.35G > T, p.G12 V) in exon 2 (codon 12) was detected within the surgical specimen. As of January 2016 the patient is still alive and without evidence of the underlying disease.
Specifically in the context of clinical trials and translational research in PDAC a re-assessment of molecular biomarkers, i. e. KRAS, at defined time points (e. g. relapse, disease progression, unusual clinical course) may be indicated in order to detect a potential switch in biomarker status during the course of disease.
KeywordsPancreatic ductal adenocarcinoma (PDAC) KRAS mutation Tumor heterogeneity
Anaplastic lymphoma kinase
Carbohydrate antigen 19–9
Caudal type homeo-box transcription factor 2
Epidermal growth factor receptor
Human epidermal growth factor receptor 2
Formalin fixed paraffin embedded
Folinic acid, 5-FU, irinotecan, oxaliplatin
Kirsten rat sarcoma viral oncogene homologue
Nanoparticle albumin-bound paclitaxel
Pancreatic ductal adenocarcinoma
International union against cancer
Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive epithelial tumors worldwide. In most patients it represents a deadly disease  due to an advanced stage at the time of diagnosis and the difficulties in therapeutic treatment, but also due to genetic heterogeneity . Surgical resection remains the only curative treatment option for localized PDAC. During the last decade, systemic treatment with single-agent gemcitabine has evolved as standard chemotherapy for the adjuvant and palliative treatment setting [3, 4]. Gemcitabine offers a median survival of about 5 to 7 months in patients with advanced disease and shows comparatively good tolerability ; more recently, gemcitabine-based combination regimens with the oral epidermal growth factor receptor (EGFR) inhibitor erlotinib or together with nab-paclitaxel  showed a statistically significant improvement in overall survival (OS). The development and progression of PDAC include different genetic alterations in oncogenic activation, loss of tumor-suppressor gene function and overexpression of receptor-ligand systems [7, 8]. Among these genetic alterations, mutations in the KRAS gene, which often are already present in precursor lesions, play an important role in tumor development and progression . Gain of function mutations in the KRAS gene are detected in about 70 to 90% of PDAC cases , commonly as point mutations in exon 2 (codon 12/13), most frequently as p.G12D (c.35G > A) or p.G12 V (p.35G > T). Several studies showed that constitutively activating KRAS mutations are associated with worse OS, whereas KRAS wildtype status is associated with improved OS in PDAC [7, 10, 11]. Thus, in PDAC, KRAS mutations may be regarded as prognostic biomarker. The role of KRAS mutational status as predictive biomarker regarding the use of EGFR-targeting agents like erlotinib in advanced PDAC still remains a matter of debate to date [12, 13, 14].
Here, we report the case of a PDAC patient with an unusual clinical course: the tumor of the patient harbored a wildtype KRAS gene at the time of initial PDAC diagnosis; however, upon disease progression 4 years later, a mutation within exon 2 of the KRAS gene was detectable.
A currently 75-year-old woman was diagnosed with locally advanced PDAC at our comprehensive cancer center (CCC) in 2010. An explorative laparotomy in August 2010 showed metastatic disease spread extensively to regional lymph nodes and thus the primary tumor in the pancreatic head was not resected. By CT imaging criteria no other distant metastatic disease was evident. Lymph nodes were sampled surgically from the right gastric artery, the hepatic artery, the coeliac trunc and from the interaortocaval region; in all samples, tumor infiltration by a poorly differentiated adenocarcinoma was confirmed by histology. Immunohistochemical staining was positive for CK7, CK20 and CA 19–9 (with CDX-2 being negative). At that time point an additional analysis for KRAS mutational status and EGFR protein expression (which were conducted within a translational research project) detected a wildtype sequence of KRAS exon 2 by pyrosequencing and a moderately positive immunohistochemical staining for membranous EGFR expression in about 80% of the tumor cells.
Up to now, no prognostic or predictive tissue biomarker is available for PDAC . In contrast to other diseases like breast, lung or colorectal cancer no specific biomarker has been validated for clinical use in pancreatic cancer and several clinical and translational trials are ongoing in order to better define the molecular basis of this disease and to search specifically for predictive markers for treatment efficacy. Thus, only limited data is available on the clinical role of biomarkers in PDAC ; specifically, there are no clear recommendations at which time points biomarkers should be assessed. In CRC for example, a good correlation between biomarker results from the primary tumor and from (metachronous) CRC metastases has been reported, resulting in the acceptance of e. g. RAS status of primary tumor tissue in patients with a metachronous relapse . In contrast, in other diseases like breast cancer a switch in e. g. Her2/neu (ERBB2) status is well known resulting in the recommendation of repeated tumor biopsies at relapse or disease progression . At least to our knowledge, studies investigating this issue have not yet been performed in PDAC.
Within this manuscript we report a rather unusual clinical course of a PADC patient, with a corresponding switch in KRAS mutational status during the course of disease. Of note, we detected the new KRAS mutation upon disease progression in September 2014; furthermore, it may be important to highlight the fact that this patient did not receive previous anti-EGFR treatment (e.g. with erlotinib) before the detection of the new KRAS mutation.
Appearance of a truly new tumor KRAS mutation upon disease progression in September 2014 without previous application of agents targeting the EGFR pathway:
The reason for tumor progression could be caused by an evolved new mutation event in the KRAS gene, specifically in the light of selection pressure during previous treatment with chemotherapy and radiotherapy. In colorectal cancer, increasing evidence exists that the appearance of new KRAS mutations during treatment with agents targeting the EGFR (like cetuximab or panitumumab) may be linked to an acquired resistance to anti-EGFR therapy [17, 18]. Of note, our patient did not receive anti-EGFR treatment for example with erlotinib before the detection of the new KRAS mutation. If other treatments like cytotoxic chemotherapy (gemcitabine, fluoropyrimidines) or radiotherapy to the pancreatic primary may also induce a “selection pressure” for the development of new genetic events remains unknown.
Tumor heterogeneity with distinct results in KRAS analysis at initial diagnosis (lymph node metastasis analyzed) and at progression (primary tumor analyzed):
There is increasing evidence for intratumoral heterogeneity in different types of cancer that could be determined by multiregion sequencing . In non-small cell lung cancer it was shown that ALK rearrangements (that were previously thought to be mutually exclusive with activating EGFR and KRAS mutations) can be found together with EGFR mutations in rare cases . Moreover, it was shown that spatially separated subclones of the same tumor harbor different oncogenic drivers . If these observations are transferable to PDAC, this might explain the differences in KRAS mutational status observed in our patient reported here. However, the scarce currently available data comparing pancreatic primary tumors and corresponding metastases, showed the same KRAS mutational status in the primary tumor and each metastatic site examined, thus supporting the idea of a newly apparent KRAS mutation [22, 23].
Technical aspects of the discrepant KRAS sequencing results (see Fig. 2):Potentially, the initial KRAS wildtype status detected in 2010 could be the effect of a false negative sequencing result. Both KRAS analyses in the tumor tissue of the patient reported here were performed in the same specialized and certified laboratory for molecular pathology. For both analyses, formalin fixed paraffin embedded (FFPE) tumor tissue was microdissected under visual control using a microscope to reduce contamination by adjacent normal tissue. In both situations, sufficient tumor tissue was available: In 2010 a subtotally infiltrated lymph node metastasis, 22 mm in diameter, containing insignificant residual lymphatic tissue and in 2014 whole tumor resection tissue was used for analysis. Moreover, the pyrosequencing assay employed here is highly sensitive and requires only 10% of tumor DNA in the whole DNA extracted to reliably detect the KRAS mutational status . Thus, a false negative sequencing result is a very unlikely event to explain the discrepancy in the present case.
KRAS mutational status may change during the course of disease in PDAC. Thus, in well-defined clinical scenarios (e. g. relapse after surgery in curative intent, disease progression during/after chemotherapy, unusual clinical course) a re-assessment of the KRAS status should be discussed, specifically within the setting of controlled clinical and translational trials. As KRAS is not yet established as a clinically relevant biomarker in PDAC, future translational trials in pancreatic cancer that evaluate a broad range of novel biomarkers should, at least to our opinion, include a repeated biomarker assessment during the course of disease within their prospective study protocols. Novel promising techniques like liquid biopsy approaches may thereby help to overcome the limitations of obtaining tumor tissue safely in PDAC . As it may be difficult to obtain sufficient tumor tissue in PDAC by percutaneous- or endosonography-guided biopsy techniques, a sampling error may occur specifically in the light of tumor heterogeneity. In that context, liquid biopsy techniques may also eventually help to overcome these limitations.
We thank all the lab technicians at the Institute of Pathology for their excellent technical support.
SO is supported by grants from the Friedrich-Baur-Stiftung, Munich and the association for the promotion of research and science at the medical faculty LMU (wifomed), Munich.
Availability of data and materials
SB, SO, AR, TK and AJ did the pathological investigations and the molecular-pathological analyses of the reported case. MH, SK, DPM, VH and SB were the treating oncologists. JW was the surgeon who performed the pancreaticoduodenectomy. SB, SO, VH, AJ and SB designed the study, collected the clinical data and drafted the manuscript. All authors have read and approved the manuscript of this case report.
The authors are experienced pathologists, oncologists or surgeons involved in the multidisciplinary management of pancreatic cancer patients at the comprehensive cancer center of a large tertiary care university hospital.
The authors declare that they have no competing interests.
Consent for publication
The patient reported here was included in a single-center translational study protocol of the Ludwig-Maximilians-University of Munich, named “The Informative Patient” (Patient number 1303). Within that protocol, the patient gave written informed consent for data analysis and publication. Additionally, by signing the official consent form provided by BMC Cancer (http://resource-cms.springer.com/springer-cms/rest/v1/content/6621850/data/v1/Consent-Form-PDF), the patient gave written informed consent for publication of her data in form of this case report. Both a copy of the original informed consent for study participation, data analysis and publication (in German language only) as well as a copy of the original BMC Cancer consent to publish form are available for review through the editors of this journal.
Ethics approval and consent to participate
The present translational study protocol was approved by the local ethics committee of the Ludwig-Maximilians-University of Munich (approval number 284–10).
- 1.Siegel RL, Miller KD, Jemal A. Cancer statistics, 2016. CA: A cancer journal for clinicians; 2015.Google Scholar
- 6.Goldstein, D., et al., nab-Paclitaxel plus gemcitabine for metastatic pancreatic cancer: long-term survival from a phase III trial. Journal of the National Cancer Institute, 2015. 107(2): p. dju413.Google Scholar
- 15.Allegra, C.J., et al., Extended RAS gene mutation testing in metastatic colorectal carcinoma to predict response to anti–epidermal growth factor receptor monoclonal antibody therapy: American Society of Clinical Oncology provisional clinical opinion update 2015. Journal of clinical Oncology, 2015: p. JCO 2015.63. 9674.Google Scholar
- 21.Cai, W., et al., Intratumoral heterogeneity of ALK-rearranged and ALK/EGFR coaltered lung adenocarcinoma. Journal of Clinical Oncology, 2015: p. JCO. 2014.58. 8293.Google Scholar
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