Method for the diagnosis of etoposide prodrug treatable cancer

Abstract

The present invention pertains to a diagnostic and therapeutic method for assessing whether a patient is susceptible to the treatment of an ester-prodrug. The methods of the invention include the analysis of carboxyelesterase 2 (CES2)-expression in tumor samples as a predictive value for the assessment of treatment success with an ester-prodrug of a chemotherapeutic agent. Alternatively, the invention provides methods involving the analysis of the urinary ratio of the prodrug and the active therapeutic as another predictive value for assessing treatment susceptibility.

Claims

1. A method of stratifying and treating a patient suffering from biliary tract cancer (BTC), the method comprising the steps of a) providing a BTC tumor sample of the patient, b) determining the expression of carboxylesterase 2 (CES2) in the BTC tumor sample, wherein a lack of expression of CES2 in the BTC tumor sample indicates the risk of a patient that a treatment with an ester prodrug of a chemotherapeutic agent will not lead to a treatment success, and wherein the expression of CES2 in the BTC tumor sample indicates that a treatment with an ester prodrug of a chemotherapeutic agent will lead to a treatment success, and c) (i) treating the patient whose BTC tumor sample is determined to express CES2 with the ester prodrug of a chemotherapeutic, or (ii) treating the patient whose BTC tumor sample lacks CES2 expression with a treatment regimen that comprises a treatment that is different from the ester prodrug of a chemotherapeutic agent.

2. A method of assessing if a patient suffering from biliary tract cancer (BTC) is a responder to treatment with an ester prodrug of a chemotherapeutic agent and treating the patient if the patient is a responder, the method comprising the steps of a) providing a BTC tumor sample of the patient, b) determining the level of CES2 expression in the BTC tumor sample, wherein the expression of CES2 in the BTC tumor sample indicates that the patient is a responder to treatment with the ester prodrug of a chemotherapeutic agent, and wherein the lack of expression of CES2 in the BTC tumor sample indicates that the patient is a non-responder to treatment with the ester prodrug of a chemotherapeutic agent, and c) (i) treating the patient with the ester prodrug of a chemotherapeutic agent if the patient is a responder, or (ii) treating the patient with a treatment regimen that comprises a treatment that is different from the ester prodrug if the patient is a non-responder.

3. The method according to claim 2, wherein the ester prodrug is an ester prodrug of etoposide.

4. The method according to claim 3, wherein the ester-prodrug of etoposide is CAP7.1.

5. The method according to claim 1, the method consisting of: a) providing a BTC tumor sample of the patient, b) determining the expression of carboxylesterase 2 (CES2) in the BTC tumor sample, wherein a lack of expression of CES2 in the BTC tumor sample indicates the risk of a patient that a treatment with an ester prodrug of a chemotherapeutic agent will not lead to a treatment success, and wherein the expression of CES2 in the BTC tumor sample indicates that a treatment with an ester prodrug of a chemotherapeutic agent will lead to a treatment success, and c) (i) treating the patient whose BTC tumor sample is determined to express CES2 with the ester prodrug of a chemotherapeutic, or (ii) treating the patient whose BTC tumor sample lacks CES2 expression with a treatment regimen that comprises a treatment that is different from the ester prodrug of a chemotherapeutic agent.

Description

(1) The present invention will now be further described in the following examples with reference to the accompanying figures and sequences, nevertheless, without being limited thereto. For the purposes of the present invention, all references as cited herein are incorporated by reference in their entireties. In the Figures:

(2) FIG. 1: CES2 Tissue Staining: A: CAP7 responder patients; B: CAP7 non-responder patients

(3) FIG. 2: Urinary Ratios of subjects Treated with CAP7.1 Groups: 1=Patients with disease control & partial response with UR<0.0416 and CES+; 2=patients with SD with UR>0.0416 and CES + or not determined 3 =Patients with PD with UR>0.0416 and CES/+ or not determined

(4) FIG. 3: Scoring of CES expression. (A) Sections of normal tissue or (B) TMA spots from graded CRC were stained for CES 2 by immunohistochemistry (brown); nuclei with hematoxylin (blue). (A) Characteristic CES-2 distribution in normal colon tissue with strong expression in epithelial cells along the crypts. (B) Score 0: all tumor cells devoid of CES-2 expression; score 5: strong CES-2 expression in about 20% of the tumor cells; score 6 medium CES-2 expression in up to 90% of tumor cells; score 7: strong CES-2 expression in up to 90% of tumor cells; score 8: strong CES-2 expression in over 90% of tumor cells. Representative images; original magnification100. Bars represent 100 m.

(5) FIG. 4: CES-2 expression in CRC. Samples of tumor and normal colon tissue from patients with CRC of different grades were stained for CES-2 by immunohistochemistry. The CES-2 expression of the tumor and or the epithelial cells was scored. (A) CES-2 expression scores from resected CRC tissue of tumor grades G2 (n=10) and G3 (n=4). Scatterplot with mean+/SD. (B) CES-2 expression scores from TMA spots of CRC tissue from tumor grades G1 (n=4), G2 (n=47) and G3 (n=35). Scatterplot with mean+/SD.

(6) FIG. 5: Cell type and localization of CES 2+ cells within and around the tumor tissue in CRC. Immunohistochemistry for CES-2 expression or proteins for different immune cell types was carried out with samples of resected CRC tumor tissue from a patient with grade 2 CRC. Nuclei were stained with hematoxylin. (A) Normal tissue areas or (B, C) tumor regions were stained for CES-2 and arrows indicate the abundance of CES-2+ cells. Tumor tissue was stained for (D) CD11b+ immune cells (red), (E) MPO+ neutrophils (red), (F) CD20+ B cells (red) and CD3+ T cells (brown), (G) CD163+ macrophages (red) and CES-2+ cells (blue) or (H) CD138+ plasma cells (red) and CD68+ macrophages (brown). Representative images for n=10 patients with grade 2-3 CRC; original magnification 100. Bars represent 100 m.

(7) FIG. 6: CES-2+ plasma cells in intestinal tissues from CRC. (A, B) Normal colon tissue from CRC patients or (C E) CRC tumor tissue were stained (A, C) for CES-2 (red) and CD138 (green) by immunofluorescence or (B, D, E) CES-2 (brown) and MUM-1 (red) by immunohistochemistry. Nuclei were stained with DAPI (A, C) or hematoxylin (B, D, E) and appear blue. Representative images for n=10 patients with grade G2-3 CRC; original magnification 400. Bars represent 20 m.

(8) Responder=patients show therapeutic benefit and received at least two cycles of CAP7.1

EXAMPLES

(9) Since the prodrug CAP7.1 requires conversion to active etoposide, local and systemic CES2 expression may be a determining factor for the efficacy and safety of CAP7.1.

(10) The IHC assessment of CES2 protein expression could possibly lead to prediction of response and tolerability of CAP7.1 and could be utilized for patient stratification in post-marketing studies. Assessment of the Urinary Ratios (UR) of CAP7.1: etoposide might provide information about the metabolism of CAP7.1 in patients with BTC, which might potentially support therapeutic monitoring of the drug to optimize drug levels.

(11) The major determinants in the conversion of CAP7.1 into etoposide are the human carboxyl esterases, especially CES2. Thus, conversion of CAP7.1 to etoposide via CES2 at the tumour site may provide benefit, especially to those patients who have CES2 highly expressed in their tumour. Moreover, the systemic conversion rate may also be of importance.

(12) It is therefore proposed to measure CES2 expression in a tumour biopsy to assess expression in tumour and in peripheral mononuclear cells (PBMC) to assess systemic expression. Moreover, the urinary ratio of CAP7.1 and etoposide will provide an indication for systemic conversion of CAP7.1 to etoposide.

Example 1

CES2 Tumor Expression Correlates with CAP7.1 Treatment Success

(13) In a new study protocol, the staining of CES2 of tumour tissue samples was introduced to test the hypothesis. Therefore data from 7 patients treated with CAP7.1 and one patient as BSC control is available on CES2+ expression in BTC tumours (Table 1).

(14) TABLE-US-00001 TABLE 1 Expression of CES2 in patients with BTC Etoposide AUC.sub.(0-) CAP7.1/Etoposide Patient CES2 staining Response g/ml hr Urinary Ratio DE-1 2++ PR NC 0.86 DE-2 1+ SD 20.35 0.01 DE-3 3+ SD 10.22 0.01 DE-4 3+ SD NC 0.01 DE-5 3+ PD 14.12 n.a. DE-6 PD NC n.a. DE-7 PD NC 0.11 NC/n.a.: Not calculated or not available

(15) CES2 stainings of tumor samples is shown in FIG. 1. FIG. 1A shows that a high CES2 expression was observed in patients responding to CAP7.1. treatment, whereas a low CES2 expression was observed in non-responder patients (FIG. 1B).

Example 2

Urinary Drug Analysis of CAP7.1 and Etoposide in the Phase I and Phase II Studies

(16) An evaluation of the Phase I and II data with respect to the conversion of CAP7.1 to etoposide and the urinary ratio of CAP7.1: etoposide (UR) in 24 h urine collection was undertaken. The urine collection data for the patients with disease control including SD (Stable disease) and PR (partial response) response to CAP7.1 and non-responders (patients with progressive disease (PD)) to CAP7.1 with respect to UR and CES2 expression were evaluated. Results are depicted in FIG. 2: UR shows that the subjects responding to CAP7.1 with high CES2 expression fall into two groups, one with a low UR and one with a higher UR.

(17) The group with the low UR had a median (range) of 0.00716 (0.0121). The 75% quartile for this group was 0.0416. Thus any responder with high CES2 expression with a UR below 0.0416 was classified as a low UR subject and any responder with a high expression of CES2 but a UR higher than 0.0146 as a high UR subject.

(18) TABLE-US-00002 TABLE 2 Subjects responding to CAP7.1 with CES2 expression Total CAP7.1 Total Eto Total Drug Recovered Groups n (mg) (mg) UR (mg) 1 10 0.56 75.88 0.01 76.15 (0.01-1.42) (50.61-165.80) (0.01-0.01) (51.10-225.50) 2 22 19.95 56.35 0.42 76.53 (0.15-91.93) (16.80-153.40) (0.01-1.70) (30.02-173.31) 3 26 14.45 89.26 0.17 101.91 (0.01-25.72) (2.26-136.30) (0.01-0.47) (3.31-160.5) All values reported are the median and ranges; n = number of urine samples Group 1 SD with CES2+ tumours and UR < 0.0416 Group 2 SD/PR with CES2 + tumours/no CES2 data and UR > 0.0416 Group 3 PD with CES2 tumours/no CES2 data and UR > 0.0416

(19) As can be seen from table 2, subjects having a low urinary ratio a very likely to be CAP7.1 responders.

Example 3

Expression of CES-2 in Tumor Cells Does Not Correlate with the Tumor Grade in Colorectal Carcinoma

(20) Recent immunohistochemically analyses indicate a broad range of CES-2 expression levels in CRC [5, 6]. To get more information about the base level expression of CES, non-inflamed colon tissue from the resection area of surgery was analyzed using immunohistochemistry. The findings showed that the expression of CES-2 in epithelial cells increased along the crypt in normal human colon with highest expression in the surface epithelium, whereas expression was completely diminished at the base of the crypt and (FIG. 3B). This suggests that CES-2 expression increases during differentiation of epithelial cells.

(21) An initial study using a small number of patient samples (n=14) from grade G2 and G3 CRC tumor samples showed no significant correlation between CES-2 expression scores and tumor grades (Mann-Whitney test; FIG. 4A). Screening 86 samples (cecum: n=8, colon: n=56, rectum: n=22) from microarrays, representing 86 patients, resulted in CES-2 expression scores of 0-8 with no significant correlation to the tumor grade (Kruskal-Wallis test; FIG. 4B). Thus, the expression levels of CES-2 within the tumor cells did not associate to the state of the tumor.

(22) As the TMA samples are only from a single location within the tumor, it was important to ensure that the samples scoring 0 were representative of the CES-2 expression pattern in the larger areas of the corresponding resected tumor tissues. Only half of the CRC cases revealed identical CES-2 expression scores from the TMA and from larger areas of the resected tumors tissues (not shown). As some tumors were completely devoid of the CES-2 protein, we tested for the expression of CES-1 as a potential compensatory mechanism. All tumor spots from the TMA (n=86) and all larger sections of resected tumor tissue also tested (n=14) were negative for CES-1. Additionally, NEC of the cecum, the colon and the rectum tested negative for both CES-1 and CES-2 (not shown).

(23) In CRC, CES-2 expression was also heterogeneous within single tumors. Hence, we focused on patient-matched samples (n=41) to compare the expression of CES-2 between normal colon tissue areas retrieved during surgical resection of the tumor and the cells from CRC tumors of all grades. Epithelial cells showed moderate to strong CES-2 expression ranging between score 5 and 8. The overall expression of CES-2 was significantly higher in normal epithelial cells compared with epithelial cell-derived tumor cells in CRC (p=0.0323 with Wilcoxon matched-pairs signed ranked test).

Example 4

Immune Cells in the Immediate Vicinity of the Tumor Express CES-2

(24) Examination of the lamina propria of samples taken from non-cancerous areas distant from CRC surgery showed a moderate infiltration with leukocytes, and a number of the leukocytes strongly expressed CES-2 (FIG. 5A). In some tumors, the tissue immediately surrounding the cancerous lesion exhibited only sporadic CES-2+ cells (FIG. 5B), while in other tumors the tissue was heavily infiltrated (FIG. 5C). The number of CES-2+ cells in the tissue surrounding the tumor was independent of the level of CES-2 expression in the tumor cells. Leukocytes directly infiltrating the tumor were always negative for CES-2, whereas CES-2 expression in the leukocytes located close to the tumor was consistently high (FIG. 5B, C). Immunohistochemical analysis of the tissues surrounding the tumors, looking at CD11b+ myeloid cells (FIG. 5D), MPO+neutrophils (FIG. 5E), CD3+ T cells and CD20+ B cells (FIG. 5F), CD163+ macrophages (FIG. 5G) as well as for CD68+ macrophages and CD138+ plasma cells (FIG. 5H) revealed the presence of various types of leukocytes.

Example 5

Plasma Cells in the Tumor Vicinity Contribute to Production of CES-2 in CRC

(25) Specifically addressing the plasma cell compartment, tissues were co-stained for CES-2 and for CD138, which is expressed on plasma and epithelial cells [15], and MUM1 expressed in nucleus and cytoplasm of mostly plasma cells [16], respectively. Fluorescence (FIG. 6A, C) and bright-field microscopy (FIG. 6B, D, E) confirmed plasma cells as the producers of CES-2 in the lamina propria of normal colon tissues from patients with CRC (FIG. 4A, B) and in the vicinity of the CRC (FIG. 6C, D). Although all CES-2+ cells were plasma cells, not all plasma cells expressed CES-2; plasma cells devoid of CES-2 were also present in the tissue surrounding the tumor (FIG. 6E).

(26) These observations suggested that plasma cells in the immediate surroundings of the tumor were an important source of the enzymatic activity of CES in situ.

(27) Materials and Methods

(28) Human tissue and blood samples. The following archived formalin-fixed paraffin-embedded (FFPE) samples were retrieved from the tissue bank of the CharitUniversittsmedizin, Zentrale Biomaterialbank (ZeBanC; http://biobank.charite.de/service/) (Berlin, Germany): intestinal tissue from patients with CRC including tissue microarray (TMA; Table 2), with neuroendocrine carcinomas (NEC) of the large intestine (cecum, n=2; colon, n=3; rectum, n=2), Crohn's disease (CD, n=4) or ulcerative colitis (UC, n=5); liver tissue from patients with hepatocellular (HCC, n=5) or cholangiocellular carcinoma (CCC, n=6); normal colon tissue from patients with CRC. Normal tissues were taken from resection margins with normal intestinal morphology assessed after hematoxylin/eosin (H&E) staining. Whole blood samples from healthy donors were obtained from filters from leukapheresis. The study was approved by the ethics committee of the CharitUniversittsmedizin Berlin (registration number EA1-157-13).

(29) TABLE-US-00003 TABLE 2 Patient characteristics of CRC tumor tissue samples. Number of patients 86 Gender 42 female/44 male Age Median 67 years (range: 22-85 years) Tumor localization Cecum (n = 8), colon (n = 56), rectum (n = 22) Tumor grade* G1 (n = 4), G2 (n = 47), G3 (n = 35) *Definition according to the WHO Classification of Tumours of the Digestive System[12]

(30) Histopathology. Thin sections of archived patient samples (1-2 m) were either stained with H&E or subjected to heat-induced epitope retrieval prior to incubation with antibodies specific for CES-1 (clone EP1376Y; Biozol Diagnostica, Eching, Germany) or CES-2 (#ab64867, polyclonal rabbit; Abcam; Cambridge, United Kingdom). These were visualized using the EnVision+HRP System (#K4011; Dako, Glostrup, Denmark), with diaminobenzidine (DAB; Dako) as chromogen. The nuclei were counterstained with hematoxylin and slides cover-slipped with glycerol gelatin (both Merck, Darmstadt, Germany). The Axiolmager Z1 microscope (Carl Zeiss MicroImaging, Jena, Germany) was used for image acquisition. All evaluations were performed in a blinded manner.

(31) To evaluate CES expression in tissue samples, the expression levels and the percentages of CES-expressing tumor cells were added to create an overall score from 2 to 8 as follows: Expression level1, low expression; 2, medium expression; 3, strong expression. Percentage1, <10%; 2, 10 30%; 3, 31 60%; 4, 61 90%; 5, >90% (FIG. 3).

(32) For detection of CES-2+ leukocytes, sections were subjected to a heat-induced epitope retrieval step prior to blocking of endogenous alkaline phosphatase (AP), using the Dual Endogenous Enzyme-Blocking Reagent (Dako). After rinsing, sections were incubated with anti-CES-2 antibodies, followed by biotinylated goat anti-rabbit antibodies (Dianova, Hamburg, Germany) and AP-labelled streptavidin (Dako). AP was visualized with the VECTOR Blue substrate kit (#SK-5300; Vector Laboratories, Burlingame, USA). Proteins were then inactivated by pressure cooking and the sections were incubated with antibodies specific for CD3 (clone M-20; Santa Cruz, San Diego, USA), CD11b (clone EP1345Y; Abcam), CD20 (clone L26; Dako), CD68 (clone PG Ml; Dako), CD138 (clone MI15; Dako), CD163 (clone 10D6; Leica Biosystems, Nussloch, Germany) or MPO (#A0389, polyclonal rabbit; Dako) followed by biotinylated secondary antibodies (anti-goat, anti-mouse or anti-rabbit; Life Technologies, Carlsbad, USA) and AP-labelled streptavidin (Dako). AP was visualized with the chromogen FastRed (Dako). The nuclei were counterstained, the slides cover-slipped as described above, and the images acquired using the Axiolmager Z1 microscope. As FastRed and Vector Blue are both fluorescent, coexpression was also detected using fluorescence microscopy (emission peaks: Red/560 nm, Vector Blue/680 nm). For immunohistochemical detection of CES-2 expressing plasma cells, sections were subjected to heat-induced epitope retrieval step prior to blocking of endogenous AP employing Dual Endogenous Enzyme-Blocking Reagent (Dako). This was followed by incubation with anti-MUM1 (clone MUM1p, Dako) followed by the LSAB+, Dako REAL Detection System (#K5005, Dako). After color development, the proteins were inactivated by pressure cooking and endogenous peroxidase was blocked by Peroxidase-blocking solution (Dako). Sections were incubated with anti-CES-2 followed by the EnVision+ HRP System and DAB. Nuclei were counterstained with hematoxylin and slides cover-slipped with glycerol gelatin. Additionally, immunohistochemistry andfluorescence were combined for costaining of CES-2 and CD138. Sections were subjected to a heat-induced epitope retrieval step prior to blocking of endogenous AP employing Dual Endogenous Enzyme-Blocking Reagent (Dako). After rinsing, sections were incubated with anti-CES-2 followed by biotinylated goat anti-rabbit antibodies (Dianova) and AP-labelled streptavidin (Dako). AP was visualized with LSAB+, Dako REAL Detection System. Proteins were then inactivated by pressure cooking and the sections were incubated with anti-CD138 (clone MI15; Dako) followed by biotinylated anti-mouse secondary antibody and Alexa488-labelled streptavidin (Invitrogen). Nuclei were counterstained with DAPI (Sigma-Aldrich, St. Louis, USA) and sections cover-slipped with Fluoromount G (BIOZOL, Eching, Germany). Negative controls were carried out as above, omitting the primary antibodies.

(33) Human cell lines. Human cell lines were from the American Type Culture Collection (ATCC; Bethesda, USA) or the German Collection of Microorganisms and Cell Cultures (DSMZ; Braunschweig, Germany). Burkitt's lymphoma-derived Raji cells (ATCC CCL86) and T cell leukemia-derived Jurkat cells (DSMZ ACC282) were used as models for lymphoblastoid; diffuse histiocytic lymphoma-derived U937 cells (ATCC CRL1593) and chronic myelogenous leukemia-derived K562 cells (ATCC CCL243) for myeloid; colorectal adenocarcinoma-derived HT-29 cells (ATCC HTB38) and embryonic kidney-derived HEK293 cells (ATCC CRL1573) for epithelial cells. All cell lines were maintained in standard culture medium consisting of RPMI1640 (Gibco; Life Technologies, Darmstadt, Germany) supplemented with 10% fetal bovine serum (Linaris, Bettingen, Germany), 2 mmol/L L-glutamine and 1 mmol/L sodium pyruvate (both from Biochrom, Berlin, Germany). For passaging twice weekly, cells were detached with trypsin (0.25%)/ethylenediaminetetraacetic acid (1 mmol/L; Life Technologies). Cells were routinely tested using a Mycoplasma-specific polymerase chain reaction (VenorGEM; Biochrom) and positive cells were discarded. Detached cells from standard culture (2108) were fixed with formaldehyde and collected in paraffin blocks as described earlier [14].

(34) Peripheral blood mononuclear cells (PBMC). PBMC were prepared using Ficoll density gradient centrifugation (p=1.078 g/mL; GE Healthcare, Frankfurt, Germany) according to the manufacturer's instructions. For immunocytochemistry, 2105 freshly prepared PBMC were spun to slides using a Shandon cytospin centrifuge (Thermo Fisher Scientific, Waltham, USA).

(35) Quantitative real-time polymerase chain reaction (PCR). If not noted otherwise, reagents were purchased from Life Technologies. Total RNA (1 g) from PBMC and cell lines (5 10106) prepared by RNApure (Peqlab, Erlangen, Germany) was subjected to reverse transcription using the High Capacity cDNA Reverse Transcription Kit. The mRNA expression was assessed by TaqMan PCR using the following Gene Expression Assays: CES1 (#Hs00275607_m1), CES2 (#Hs01077945_m1) and glyceraldehyde 3-phosphate dehydro-genase (GAPDH; #Hs99999905_m1) and the TaqMan Universal PCR Master Mix in a StepOne Plus device (all from Applied Biosystems, Foster City, USA). For quantification, reference sequences for each of the transcripts were cloned into pCR2.1 TOPO (Invitrogen, Groningen, The Netherlands) and used in standard titration curves from 102 to 109 copies. Expression of CES-1 and CES-2 was quantified in relation to GAPDH.

(36) Statistics. The software GraphPad Prism 6 (Prism Software, San Diego, USA) was used for statistics tests. P values0.05 were considered significant.

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