Background: Adenocarcinomas or combined adeno-neuroendocrine carcinomas (MANEC) of small bowel usually have a dismal prognosis with limited systemic therapy options. This is the first description of a patient showing a germline-related BRCA1 mutated MANEC of his ileum. The tumor presented a susceptibility to a combined chemotherapy and the PARP1-inhibitor olaparib. Case presentation: A 74-year old male patient presented with a metastasized MANEC of his ileum. Due to clinical symptoms his ileum-tumor and the single brain metastasis were removed. We verified the same pathogenic (class 5) BRCA1 mutation in different tumor locations. There was no known personal history of a previous malignant tumor. Nevertheless we identified his BRCA1 mutation as germline-related. A systemic treatment was started including Gemcitabine followed by selective internal radiotherapy (SIRT) to treat liver metastases and in the further course Capecitabine but this treatment finally failed after 9 months and all liver metastases showed progression. The treatment failure was the reason to induce an individualized therapeutic approach using combined chemotherapy of carboplatin, paclitaxel and the Poly (ADP-ribose) polymerase- (PARP)-inhibitor olaparib analogous to the treatment protocol of Oza et al. All liver metastases demonstrated with significant tumor regression after 3 months and could be removed. In his most current follow up from December 2017 (25 months after his primary diagnosis) the patient is in a very good general condition without evidence for further metastases. Conclusion: We present first evidence of a therapy susceptible germline-related BRCA1 mutation in small bowel adeno-neuroendocrine carcinoma (MANEC). Our findings offer a personalized treatment option. The germline background was unexpected in a 74-year old man with no previously known tumor burden. We should be aware of the familiar background in tumors of older patients as well. Keywords: Small bowel mixed adeno-neuroendocrine carcinoma (MANEC), BRCA1 mutation, Germline-related, PARP-inhibition, Personalized treatment * Correspondence: firstname.lastname@example.org Institute of Pathology, University of Cologne, Kerpener Strasse 62, 50937 Cologne, Germany Gastrointestinal Cancer Group Cologne, Cologne, Germany Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Quaas et al. BMC Gastroenterology (2018) 18:75 Page 2 of 7 Background Parallel sequencing Small bowel carcinomas are rare and account only for Tumor areas were marked on H&E-stained tissue slides 0.5% of all cancers. The 5 year overall survival rates for by a pathologist (AQ) and DNA was extracted from cor- patients with small-bowel adenocarcinoma is 34.9%. To responding unstained 10 μm thick slides by manual best of our knowledge no valid prognostic or survival macrodissection. After proteinase K treatment, the DNA data exist to the mixed adeno-neuroendocrine carcin- was automatically purified using the Maxwell 16 FFPE oma (MANEC)-subtype of the small bowel. There is a Tissue LEV DNA Purification Kit (Promega, Madison, strong need for a more effective and personalized systemic USA) on the Maxwell 16 Instrument (Promega) follow- treatment option in carcinoma of the small bowel. Most ing the manufacturers’ protocol. The DNA concentra- recently Schrock, A et al.  described molecular alter- tion was measured using a real-time qPCR-based ations in a very large cohort of 317 small bowel carcin- method. omas. According to this study microsatellite-instability By parallel sequencing, all tumor manifestations were (MSI) was present in 7,6% and are characterized by the analyzed for BRCA1 and BRCA2 mutation and were fur- following mutations like KRAS (53,6%), APC (26,8%), ther analyzed using a panel of 12 other genes resulting CDKN2A (14,5%), SMAD4 (17,4%) and BRAF (9,1% but altogether in a total of 452 amplicons. Additionally, in only 10% of these have the typical V600E mutation) as these tumor manifestations microsatellite status was well as ERBB2 mutation in 8,2% of the analysable tumors. checked using five different mononucleotide markers: The most important pathway in DNA double strand BAT25, BAT26, NR-21, NR-22 and NR-27 . break (DNA-dbs.) repair is homologous recombination Isolated DNA (10 ng each) was amplified with the (HR). The HR pathway is dependent on different pro- Human BRCA1 and BRCA2 GeneRead DNAseq teins including BRCA1 and BRCA2 and others (e.g. Targeted Panel V2 (Qiagen, Hilden, Germany) or with a MNR complex, CtIP, MRE11, RAD51, ATM, H2AX, customized GeneRead DNAseq Alignment and annota- PALB2, RPA, RAD52)[2, 3]. tion was done using a modified version of a previously Effective DNA repair is essential to maintain DNA in- described method . Resulting BAM files were visual- tegrity – so BRCA1/2 deficient cells show a high degree ized using the Integrative Genomics Viewer (IGV; http:// of chromosomal instability which increases the risk of www.broadinstitute.org/igv/, Cambridge; USA). A 5% malignant transformation [4, 5]. Germline mutations of cutoff for variant calls was used and results were only BRCA1/2 (gBRCA-mut) were described in carcinoma of interpreted if the coverage was > 200×. the ovary in 5–18% and in some other solid malignant tumors including breast carcinoma, pancreatic carcin- Histopathological findings oma and less frequent in prostate carcinoma. It became The primary MANEC of the ileum showed two different apparent that BRCA mutations also occur in sporadic tumor components: One gland-forming adenocarcino carcinomas and these tumors respond to Poly (ADP-ri- ma-component including PAS-positive mucins in the bose) polymerase- (PARP) inhibition. The full signifi- tumor cell cytoplasm or gland lumina as well as a solid cance of somatic BRCA mutations as a biomarker for tumor forming neuroendocrine carcinoma-component the prediction of therapeutic response to PARP inhib- showing a strong and diffuse immunohistochemical ition is not entirely clear but ovarian cancer patients positivity for synatophysin and to a lesser content chro- with a somatic BRCA mutation are equally likely to mogranin A as well (Fig. 1). The tumor infiltrated the benefit from treatment with PARP inhibitors as those subserosal tissue of the ileum and presented with vessel with a germline mutation of BRCA. Some authors sug- invasion including a single regional lymph node metasta- gest to perform somatic BRCA testing in routine clinical sis. Both, the brain and liver metastasis showed the diagnostics of all advanced tumors, which are potentially adenocarcinoma-component. amenable to PARP inhibitors [3, 6–11]. Each BRCA variant was classified according to the Case presentation established IARC classification. This classification ranges We describe the case of a 74-year old man presented to from class 1 variants (not pathogenic or of no clinical our hospital due to a liver tumor in the segments II and significance) via class 2 (likely not pathogenic or of no III as well as satellite metastatic lesions in the right liver clinical significance), class 3 (uncertain), class 4 (likely lobe. After taken a liver biopsy from the tumor the diag- pathogenic) to class 5 variants (definitively pathogenic) nosis of an adenocarcinoma was established and inter- according to different databases (compare: BRCA mutation preted as an unresectable cholangiocarcinoma, initially database: http://arup.utah.edu/database/BRCA/orClinVar- lacking the information of the primary tumor in the database:http://www.ncbi.nlm.nih.gov/clinvar/ or Universal small intestine. The patient was treated with a mutation database BRCA share: http://www.umd.be/ mono-chemotherapy of Gemcitabine ((1000 mg/qm d1– BRCA1/). 8) 12/2015–03/2016) followed by selective internal Quaas et al. BMC Gastroenterology (2018) 18:75 Page 3 of 7 Fig. 1 a Primary MANEC of the ileum (HE, 2,5×) showing the gland-forming adenocarcinoma-component (left side) as well as the solid forming neuroendocrine carcinoma component (right side); b the gland forming adenocarcinoma showing PAS-positive mucins in gland lumina (PAS, 20×); c the solid growth-pattern of the neuroendocrine carcinoma component (HE, 20×); d Immunohistochemistry using an antibody against synaptophysin – diffuse and strong positivity of all tumor cells in the neuroendocrine-carcinoma component (synaptophysin, 5×); e Immunohisto-chemistry using an antibody against chromogranin A – more patchy positivity in the neuroendocrine-carcinoma component (chromogranin A, 5×) f1 Metastasis in liver and f2 in the brain, both formed by the adenocarcinoma component (HE, 10× and 20×) radiotherapy (SIRT) analogous to the SIRCCA study metastases showed progression. The treatment failure protocol (ClinicalTrials.gov Identifier: NCT02807181). was the reason to analyze the tumor-DNA for molecular Because of anemia and poor general condition the alterations using two panels of 14 genes including BRCA administration of cisplatin was withheld. In the further 1and BRCA 2 (452 amplicons in total; compare versions course of treatment an additional tumor site in the below). We verified the same BRCA1 mutation ileum was detected and progression with cerebral metas- (c.3700_3704delGTAAA p.V1234Qfs*8) with a very high tasis occurred. The ileum tumor and one brain metasta- allele frequency up to 96% in all locations using ultra sis were removed. The ileum tumor was classified as a deep sequencing in the formalin-fixed paraffin embed- mixed adeno-neuroendocrine carcinoma (MANEC). ded material (Fig. 2). According to different databases Tumor manifestations in the liver and in the brain could (UMD, ARUP, ClinVar) this BRCA1 mutation was be characterized as metastases from the adenocarcinoma classified as pathogenic (class 5). The MANEC was component (Fig. 1). microsatellite stable. The mutational hot spots of all In the further course we patient was treated with cape- other analyzed genes (KRAS, NRAS, HRAS, BRAF, citabine (500 mg 2–0-2, 5d/week) 05–09/2016). This DDR2, ERBB2, KEAP1, NFE2L2, PIK3CA, PTEN, RHOA treatment finally failed 4 months later and all liver and BRCA2) were wild type. Quaas et al. BMC Gastroenterology (2018) 18:75 Page 4 of 7 Fig. 2 BRCA deletion (BRCA1: c.3700_3704delGTAAA; p.V1234Qfs*8) as visualized by the IGV (integrative genomics viewer; www.broadinstitute.org/igv/) According to the encouraging experiences using com- Fig. 3) – and the persistent highly necrotic liver metasta- bined platin-based chemotherapy and PARP-inhibitors sis of 6 cm could be removed. In his most current follow in BRCA-mutated ovarian carcinomas the molecular re- up from December 2017 (25 months after his primary sults have been the trigger for the personalized thera- diagnosis) the patient is in a very good general condition peutic approach using combined chemotherapy of without evidence for further metastases (compare time carboplatin, paclitaxel and the PARP1-inhibitor olaparib table, Table 2). analogous to the treatment protocol of Oza et al.  following a bridging therapy with capecitabine (olaparib Discussion and conclusion 200 mg capsules twice daily, administered orally on days To the best of our knowledge this is the first description 1–10 of each 21-day cycle) plus paclitaxel (175 mg/m , of pathogenic and treatable germline-related BRCA1 administered intravenously on day 1) and carboplatin mutation in mixed adeno-neuroendocrine carcinoma (4 mg/mL per min) in total five cycles for the following (MANEC) of the small bowel. There was no known per- 4 months. sonal history of a previous malignant tumor. Neverthe- All tumor manifestations demonstrated with a signifi- less, the high allele frequency of the BRCA1 mutation in cant tumor regression in the MINT analysis and all tumor manifestations suggested a germline back- Ga-68-Dotatate PET-CT after 3 months of treatment ground. Consequently, we tested histologically (mind analysis Table 1 and Ga-68-Dotatate PET-CT tumor-free ileum mucosa and were able to detect the Quaas et al. BMC Gastroenterology (2018) 18:75 Page 5 of 7 Table 1 Response to personalized treatment (including olaparib): MINT analysisTumor showed significant regression under personalized treatment approach using olaparib, carboplatin and paclitaxel (data shown before and 3 months after initialization of personalized treatment) Fig. 3 Response to personalized treatment (including olaparib): Ga-68 Dotatate PET/CT. Upper row (May 2016) demonstrating an 8 cm tumor in the left liver lobe below physiologic liver uptake and a SRS-positive brain metastasis. Lower row: (Nov 2016): Follow-up PET/CT with tumor shrinkage to 6 cm and disappearance of the brain metastasis According to treatment protocols established in BRCA mutated ovarian carcinomas we successfully applied combined chemotherapy of carboplatin, paclitaxel and the PARP1-inhibitor olaparib. Initially, we assumed a somatic background of the BRCA1 mutation in our treated patient, because there was no known personal history of a prior malignant tumor in the 74 year-old man. The germline background of the BRCA1 mutated small bowel carcinoma (like in ovarian adenocarcinoma) is especially promising for a successful combination therapy using platinum-based chemotherapy and a PARP inhibitor (e.g. Olaparib). It has to be questioned whether the somatic pathogenic BRCA mutation itself is the main predictor of an effective PARP inhibition . Beyond BRCA1/2 somatic mutations or the promoter methylation of BRCA some other alterations are poten- tial markers of an effective PARP inhibition (like microsatellite-instable cancers with MRE11-dominant same BRCA1 mutation with an allele frequency of 48%. negative mutations, PTEN deficiency, ATM mutation This finding clearly identifies the mutation as germline and FANCF promoter methylation) . and a deletion of the wild type BRCA1 allele in the Additionally, first evidence came up that gBRCA mu- tumor. The homogeneous distribution of BRCA1 mu- tated ovarian carcinoma are sensitive to therapies using tated tumor cells in the primary tumor and in the differ- immune-checkpoint inhibition most likely due to its ent metastasis analyzed here suggests a clonal origin and higher mutational load compared to wild type ovarian car- a causative role of this second hit. Even though a cinoma . It remains to be shown whether our BRCA gBRCA1 mutation could be a marker for prostate cancer mutated MANEC also benefit from immune-checkpoint we did not find any indications for prostate cancer . inhibition as an additional future treatment option. Quaas et al. BMC Gastroenterology (2018) 18:75 Page 6 of 7 Table 2 Time table Authors’ contributions AQ, DW and CB were responsible for study concept and design. AQ, DW, TG, Time period Therapy MD, CB, AB, WR, NH, MS and LT were responsible for acquisition of data. AQ, 12.2015 Pat. presented with liver and brain metastases. AB, CH and MS were responsible for analyses and interpretation of data. AQ and SM were responsible for writing of the manuscript. TZ was responsible 12.2015–03.2016 Gemcitabine mono 1000 mg/qm d1–8 15 and for critical revision of the manuscript. HA, TZ, CH, PK, PP, HG, LT and SB were selective internal radiotherapy (SIRT) analogous responsible technical or material support. CH, RB and SM were responsible to the SIRCCA study protocol.Excision of the for study supervision. All authors have fully read the manuscript and have tumor in the ileum and one brain metastasis approved the accuracy and completeness of the content of all parts of the (02.2016) manuscript and assume responsibility for it. 05.2016–09.2016 Capecitabine 500 mg (2–0-2, 5d/week) Ethics approval and consent to participate 12.09.16 1. Cycle: Olaparib (200 mg capsules twice Procedures were followed as outlined in accordance with ethical standards daily, administered orally on days 1–10 of formulated in the Helsinki Declaration 1975 (and revised in 1983). Informed each 21-day cycle) plus paclitaxel (175 mg/m , consent was obtained prior to participation in this study with approval by the administered intravenously on day 1) and Ethics Committee at the University Hospital, Cologne (reference number: carboplatin (4 mg/mL per min) 13–091). This approval was necessary due to gene analysis made in this study. 04.10.2016 2. Cycle: Olaparib (200 mg capsules twice daily, administered orally on days 1–10 of Consent for publication each 21-day cycle) plus paclitaxel (175 mg/m , Written informed consent was obtained from the patient for publication of administered intravenously on day 1) and this case report and any accompanying images, and is available on request. carboplatin (4 mg/mL per min) Competing interests 31.10.2016 3. Cycle: Olaparib (100 mg capsules twice daily The authors declare that they have no competing interests. (reduced dosage), administered orally on days 1–10 of each 21-day cycle) plus paclitaxel (175 mg/m , administered intravenously Publisher’sNote on day 1) and carboplatin (4 mg/mL per min) Springer Nature remains neutral with regard to jurisdictional claims in 05.12.2016 4. Cycle: Olaparib (100 mg capsules twice daily published maps and institutional affiliations. (reduced dosage), administered orally on days 1–10 of each 21-day cycle) plus paclitaxel Author details (175 mg/m , administered intravenously Institute of Pathology, University of Cologne, Kerpener Strasse 62, 50937 on day 1) and carboplatin (4 mg/mL per min) Cologne, Germany. Department of Visceral Surgery, University of Cologne, Kerpener Strasse 62, 50937 Cologne, Germany. Department of Oncology 09.01.2017 5. Cycle: Olaparib (100 mg capsules twice daily and Hematology, University of Cologne, Kerpener Strasse 62, 50937 Cologne, (reduced dosage), administered orally on Germany. Department of Hepato- and Gastroenterology, University of days 1–10 of each 21-day cycle) plus paclitaxel Cologne, Kerpener Strasse 62, 50937 Cologne, Germany. Institute of (175 mg/m , administered intravenously Neuropathology, University of Cologne, Kerpener Strasse 62, 50937 Cologne, on day 1) and carboplatin (4 mg/mL per min) Germany. Institute of Pathology, University of Mainz, Langenbeckstraße 1, 02.2017 liver metastases could be removed 55131 Mainz, Germany. Department of Radiology, University of Cologne, Kerpener Strasse 62, 50937 Cologne, Germany. Department of 12.2017 Last CT-scan staging: still no further tumor Nuclear-Medicine, University of Cologne, Kerpener Strasse 62, 50937 Cologne, manifestations/still no metastasis Germany. Gastrointestinal Cancer Group Cologne, Cologne, Germany. Received: 21 October 2017 Accepted: 23 May 2018 Our findings are remarkable in many ways: Here we provide first evidence of a therapy susceptible BRCA1mu- References tation in a metastasized mixed adeno-neuroendocrine car- 1. Schrock AB, Devoe CE, McWilliams R, Sun J, Aparicio T, Stephens PJ, Ross JS, cinoma (MANEC) of small bowel. Wilson R, Miller VA, Ali SM, et al. Genomic profiling of small-bowel adenocarcinoma. JAMA Oncol. 2017;3(11):1546–53. The germline background of our patient was unex- 2. Lupo B, Trusolino L. 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Published: May 31, 2018
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