FULL PAPER
`
`British Journal of Cancer 2016 115 442453 I doi 101038bjc2016215
`
`Keywords Nabpaclitaxel paclitaxel pancreatic
`cancer combination chemotherapy
`
`cancer gemcitabine desmoplastic stroma mouse models of human pancreatic
`
`Superior therapeutic efficacy of
`nabpaclitaxel over cremophorbased
`in locally advanced and metastatic
`paclitaxel
`models of human pancreatic cancer
`
`N V Rajeshkumar1211 Shinichi Yabuuchi2 Shweta G Pai2 Zeen Tong 3 Shihe Houll Scott Bateman5
`Daniel W Pierce6 Carla Heise6 Daniel D Von Hoff7 Anirban Maitra and Manuel HidalgokJ
`1Department of Oncology The Johns Hopkins University School of Medicine Baltimore MD 21231 USA 2Department of
`The Johns Hopkins University School of Medicine Baltimore MD 21231 USA 3Nonclinical Development Celgene
`Pathology
`Corporation Summit NJ 07901 USA 4Strategic Platforms Celgene Corporation Berkeley Heights NJ 07922 USA 5Research
`Alliance Development Celgene Corporation San Diego CA 92121 USA 6Department Translational Development Celgene
`Corporation San Francisco CA 94158 USA 7Clinical Translational Research Division Translational Genomics Research
`Institute
`The UT MD Anderson Cancer Center Houston
`TGen Phoenix AZ 85004 USA °Department
`TX 77030 USA
`of Pathology
`The UT MD Anderson Cancer Center Houston TX 77030 USA and 10Clinical
`9Department of Translational Molecular Pathology
`Research Programme Spanish National Cancer Research Centre CNIO Melchor Fernandez Almagro 3 Madrid 28029 Spain
`
`nabPTX plus gemcitabine GEM combination
`Background Albuminbound paclitaxel nabpaclitaxel
`has demonstrated
`pancreatic ductal adenocarcinoma PDAC compared with GEM
`activity and statistically
`overall survival of patients with metastatic
`as a standard of care treatment option for patients with metastatic PDAC It
`is unclear whether
`monotherapy This regimen is currently approved
`cremophorbased PTX combined with GEM provide a similar level of therapeutic
`efficacy in PDAC
`
`efficient antitumour
`
`significant
`
`Methods We comprehensively
`explored the antitumour efficacy effect on metastatic dissemination tumour stroma and survival advantage
`following GEM PTX and nabPTX as monotherapy or in combination with GEM in a locally advanced and a highly metastatic orthotopic model of
`human PDAC
`
`Results NabPTX treatment resulted in significantly
`tumour plasma ratio 198 fold robust stromal depletion antitumour
`higher paclitaxel
`efficacy 379 fold and survival benefit compared with PTX treatment PTX plus GEM treatment showed no survival gain over GEM monotherapy
`nabPTX in combination with GEM decreased primary tumour burden metastatic dissemination
`increased median
`and significantly
`However
`survival of animals compared with either agents alone These therapeutic effects were accompanied by depletion of dense fibrotic tumour stroma
`to nabPTX plus GEM in providing
`and decreased proliferation of carcinoma cells Notably nabPTX monotherapy
`was equivalent
`indicating that nabPTX could potentially stop the progression of late stage
`advantage to mice in a highly aggressive metastatic PDAC model
`pancreatic cancer
`
`survival
`
`antitumour
`
`efficacy of PTX and nabPTX vary widely and the contention that
`Conclusions Our data confirmed that
`these agents elicit similar
`therapeutic
`response was not supported The addition of PTX to GEM showed no survival advantage
`concluding that a clinical combination of PTX
`survival advantage over GEM monotherapy
`and GEM may unlikely to provide significant
`and may not be a viable alternative to the current
`standard ofcare nabPTX plus GEM regimen for the treatment of PDAC patients
`
`E mail rajeshhopkinsgmailcom or Dr M Hidalgo Email mhidalgocnioes
`Correspondence Dr NV Rajeshkumar
`Presented in part at 105th AACR Annual Meeting 2014 59 April 2014 San Diego CA USA Abstract LB94
`11Current address Human Therapeutics Division Intrexon Corporation 20358 Seneca Meadows Pkwy Germantown MD 20876 USA
`
`Revised
`
`15 June 2016 accepted 23 June 2016 published online 21 July 2016
`
`0 2016 Cancer Research UK All rights reserved 0007092016
`
`442
`
`wwwbjcancercom 1 D01101038bjc201621 5
`
`Abraxis EX2020
`Actavis LLC v Abraxis Bioscience LLC
`1PR201701101 1PR201701103 1PR201701104
`
`

`

`Nabpaclitaxel
`
`in pancreatic cancer
`
`BRITISH JOURNAL OF CANCER
`
`cells
`
`resistance
`
`Ijichi
`
`Pancreatic ductal adenocarcinoma PDAC is well known for its
`aggressive clinical course and remains as one of the most deadly
`malignancies in the world with a 5 year mortality of 95 Hidalgo
`2010 Oettle 2014 The unusually poor prognosis of the disease is
`and metastatic nature of these
`due to the aggressively
`invasive
`tumours and
`and
`their being impervious
`to chemotherapy
`radiotherapy A constant hallmark of PDAC is the presence of a
`largely of fibroblasts
`dense desmoplastic reaction that consists
`myofibroblasts and extracellular matrix proteins including fibro
`nectin and collagens Maitra and Hruban 2008 The stromal
`is conscripted to provide protection to carcinoma
`compartment
`and
`cancer
`progression and
`contributes
`to
`therapeutic
`et al 2011 Kadaba et al 2013 Whatcott et al
`2013 Gemcitabine GEM Gemzar has been established
`as the
`standard first line chemotherapeutic drug for the treatment of
`patients with advanced PDAC Burris et al 1997 Despite
`numerous
`evidence of robust combination
`laboratory
`activity
`trials of GEM in combination with different cytotoxic or
`phase III
`molecularly targeted agents have resulted in no substantial clinical
`the use of GEM alone Van Cutsem et al 2009
`improvement over
`Colucci et al 2010 Philip et al 2010 Combinations focused to the
`have not proven clinically impactful
`tumour
`cell compartment
`perhaps reflecting the invulnerability of this highly KRASmutated
`tumour
`to signaling inhibitors This has led to an increased focus
`on targeting the desmoplastic stroma to improve clinical outcomes
`of patients with this deadly disease Beatty et al 2011 Provenzano
`et al 2012 Kadaba et al 2013
`We previously performed pilot studies in which several advanced
`PDAC patients were successfully
`on the basis of
`treated
`robust
`patient derived xenografts
`anticancer
`response in the xenografts
`PDXs derived from the resected primary tumours of those patients
`et al 2011 Preclinical
`Hidalgo et al 2011 Villarroel
`and early
`by us confirmed that albumin bound
`conducted
`clinical studies
`nabPTX Abraxane
`formulation of Paclitaxel
`cremophorfree
`exerts substantial antitumour activity in PDAC Maitra et al 2009
`Von Hoff et al 2011 Our previous studies conducted in mice with
`established PDXs subcutaneous derived from 11 individual PDAC
`patients confirmed that nabPTX treatment
`in combination with
`GEM provided a high antitumour efficacy
`compared with GEM
`alone Maitra et al 2009 Von Hoff et al 2011 This synergy was
`later confirmed by another
`group of
`researchers
`utilising a
`engineered mouse model of PDAC Frese et al 2012
`genetically
`Positive fmdings from these studies later led to a large randomised
`trial N= 861 metastatic PDAC patients showed
`phase III clinical
`that nabPTX plus GEM was superior in efficacy to GEM alone in
`and
`terms of progression free survival
`overall
`survival
`in the
`treatment of metastatic PDAC Von Hoff et al 2013 Goldstein
`et al 2015 In addition
`showed
`that one of
`our studies
`the
`mechanisms by which nabPTX works in PDAC is by eliminating
`reaction which is considered
`the extensive desmoplastic
`as a key
`hallmark of PDAC and barrier
`to drug diffusion and treatment
`efficacy Von Hoff et al 2011 Xie and Xie 2015
`Cremophor EL CreELbased Paclitaxel PTX Taxol
`is a key
`several human
`for
`the treatment of
`chemotherapy
`component
`malignancies Von Hoff 1997 Despite the clinical benefit achieved
`with solvent based taxanes
`the treatment often produce significant
`side effects Gradishar et al 2009 NabPTX is a cremophorfree and
`watersoluble albumin based formulation of PTX consisting of 130nm
`albuminpaclitaxel nanoparticles NabPTX treatment showed higher
`response rates and improved tolerability compared with solvent based
`formulations in patients with advanced metastatic breast cancer and
`non small cell lung cancer Montana et al 2011 Viudez et al 2014
`taxanes such as PTX
`Clinical experience with solvent based traditional
`in PDAC patients were disappointing Saif et al 2010
`and docetaxel
`NabPTX plus GEM combination is currently being implemen
`ted in national and international guidelines as a standard of care
`treatment option for patients with metastatic PDAC It
`is unclear
`
`whether PTX when combined with GEM provide similar therapeutic
`efficacy in PDAC Since PTX is widely available and the treatment
`cost of nabPTX is relatively higher than PTX clinicians third
`party
`interest in under
`have a substantial
`payers and regulatory agencies
`standing whether
`these drugs share a similar level of pharmacological
`activities in PDAC
`In the present study we utilised orthotopic models of human
`PDAC which were shown to better recapitulate the histologic and
`of disease than the subcutaneous
`metastatic characteristics
`line
`xenograft models Hoffman 2015 and directly compared the
`effect on tumour stroma modulation meta
`anticancer
`activity
`following GEM
`and survival
`static spreading
`to distant organs
`PTX nabPTX and combinations of GEM plus PTX or nabPTX
`To our knowledge this is the first study which used a large number
`of mice 300 with established orthotopic tumours and compre
`hensively explored the therapeutic efficacy of PTX and nabPTX
`alone and in combination with GEM in human PDAC models
`
`cell
`
`MATERIALS AND METHODS
`
`a
`
`locally
`
`Establishment of orthotopic PDX models of pancreatic cancer
`to ten weekold male athymic nunu nude mice Harlan
`Eight
`were used for
`the study Animals were maintained in a sterile
`environment and had access to autoclaved laboratory rodent diet and
`water ad libitum All animal experiments were conducted following
`and
`in accordance with the Animal Care
`and Use
`approval
`Committee guidelines of the Johns Hopkins University which is in
`the welfare and use of animals
`line with guidelines
`for
`in cancer
`et al 2010 Two human pancreatic
`research Workman
`cancer
`and Panc265
`from the `PancXenoBank
`Panc185
`xenografts
`collection of patient derived human pancreatic
`cancer xenografts
`from the resected primary tumours of patients with
`established
`PDAC were used for the present study RubioViqueira et al 2006
`We used Panc185
`tumour
`as a representative model of
`advanced primary PDAC and Panc265 tumour as a representative
`model of highly aggressive and metastatic PDAC Briefly subcuta
`neously maintained Panc185 and Panc265 tumours in mice were
`resected aseptically at the exponential growth phase and used as the
`tumours for surgical orthotopic implantation into the
`source of
`pancreas Tumours were minced with a sterile razor and cut
`into
`15=3 Mice were
`cubes of
`anaesthetised
`isoflurane
`using
`inhalation A 1 cm left
`incision was made on the
`lateral abdominal
`splenic silhouette without
`injury to underlying organs with a sterile
`microscissor The pancreas was identified and laterally externalised A
`small cut was made at the middle of pancreas and a 15mm3 tumour
`fragment dipped in Matrigel BD Biosciences Bedford MA USA
`was immediately implanted into the middle of the pancreas and
`sutured using 30 surgical sutures The pancreas was returned into the
`cavity The abdominal wall and skin were sutured in two
`peritoneal
`layers using 30 surgical sutures Kim et al 2009 Orthotopic tumour
`by transabdominal palpations
`establishment was assessed
`confirmed by an ultrasound scan Vevo 660 VisualSonics Toronto
`ON Canada
`
`initially
`
`purchased
`
`Indianapolis
`
`IN USA and
`Drugs Gemcitabine Eli Lilly
`Squibb Company
`cremophorbased
`paclitaxel BristolMyers
`Princeton NJ USA were
`from the Johns Hopkins
`Pharmacy Baltimore MD USA Nabpaclitaxel
`Hospital
`Abraxis BioScience LLC a wholly owned subsidiary of Celgene
`Corporation was supplied by Celgene Corporation Summit NJ
`USA All
`in normal
`three drugs were reconstituted separately
`saline prepared fresh daily as required and administered to tumour
`harbouring mice within 1 h of preparation
`
`Evaluation of the in vivo efficacy of drug regimens in orthotopic
`PDAC harbouring mice Mice were orthotopically
`implanted
`tumours Engrafted tumours were
`with Panc185
`and Panc265
`
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`BRITISH JOURNAL OF CANCER
`
`Nabpaclitaxel
`
`in pancreatic cancer
`
`were humanely killed by standard CO2 asphyxiation Median
`survival was estimated and compared using Kaplan Meier method
`
`Determination of the plasma and intratumour concentration of
`Panc265
`paclitaxel Mice
`established
`harbouring
`orthotopic
`200300 mm3 were either untreated or treated with
`tumours
`
`five consecutive
`
`the
`
`diluted
`
`at
`
`ranging
`
`plasma stock
`initial
`to make
`calibration
`from 2
`
`at
`
`to
`
`PTX 134 mg kg 1 or nabPTX 223 mg kg 1 iv once daily for
`days N=9 mice per group On day 5 blood and
`2 h after the dosing of nabPTX
`tumour
`samples were collected
`or PTX Paclitaxel
`concentration was analysed by liquid chroma
`tography tandem mass spectrometry LCMSMS as described
`elsewhere Zhang et al 2013 For the calibration curve an aliquot
`solution 02 mg ml 1 in acetonitrile
`of
`stock
`paclitaxel
`into plasma to make
`was
`spiked
`4000 ng m11 which was
`then
`standard
`concentrations
`samples
`in plasma Quality control QC samples were made
`4000 ng ml
`similarlywith concentrations of low QC at 6 ng ml1 medium QC
`at 100 ng m11 and high QC at 3200 ng m11 Dilution QC at
`20 000 ng m11 was made by spiking the stock solution into blank
`plasma and then diluted 10 fold with blank plasma Aliquots
`50 µ1 of plasma and
`tumour
`including standards
`samples
`blanks QCs were transferred into a 96 well plate To each well
`solvent mixture of acetonitrile methanol 9 1
`150 pl of a
`containing 100 ng m11 of d5paclitaxel as the internal standard
`was added The plate was capped vortex mixed and centrifuged A
`150 21 aliquot of the supernatant was transferred into a clean 96
`well plate for LCMSMS analysis Chromatographic
`separation
`was carried out using a Shimadzu LC20 system equipped with an
`Agilent Pursuit XRs 3 C18 column 100 x 2 mm Mobile phases
`in water containing 01 formic
`consisted of 5 mm ammonia acetate
`acid A and 5 mm ammonia acetate
`in acetonitrol water 9 1
`acid B at
`mixture containing 01 formic
`035 ml min1 The column was eluted with a gradient of 25
`B for 09 min linearly increased to 100 B over 15 min and then
`maintained at 100 B for another
`35 min The LC elute was
`connected directly to a Sciex API4000 QTrap mass spectrometer
`Ab Sciex Framingham MA USA equipped with an electrospray
`ionisation ESI
`ion source The ratio of intratumoral
`vs plasma
`of paclitaxel was calculated
`between
`and compared
`concentrations
`nabPTX and PTX treatment groups
`
`allowed to grow over a period of 27 weeks Panc265 and Panc185
`In order to confirm orthotopic tumour growth in
`respectively
`mouse pancreas determine the weight of primary tumours and any
`metastatic dissemination baseline five animals in Panc185 and
`Panc265 were randomly selected and exploratory laparotomy was
`performed Mice were screened visually for metastatic lesions
`in
`and peritoneum
`lungs kidneys lymph nodes
`the spleen liver
`using a x 25 lens Primary tumour was excised from pancreas
`and measured using a digital caliper Spleen liver lungs
`weighed
`kidneys lymph nodes were excised Tumours and tissues were
`formalinfixed paraffin embedded
`and were used for
`sectioned
`
`allocated
`
`into
`
`consecutive
`
`the
`
`haematoxylin and eosin HE staining
`In order to determine the efficacy of treatment regimens mice
`with demonstrable primary tumours 200300 mm3 were selected
`arms 1 Control
`and
`randomly
`following
`untreated 2 GEM 100 mg kg
`g ip twice weekly for 4 weeks
`3 PTX 134 mg kg 1 iv once daily for 5 consecutive days only
`on first week 4 nabPTX 223 mg kg 1 iv once daily for 5
`days only on first week 5 PTX and GEM at
`above mentioned dose and schedule 6 nabPTX plus GEM at the
`schedule N= 810 micegroup
`above mentioned dose
`and
`Drug doses were selected based on previous studies Desai et al
`2006 Rajeshkumar et al 2011 Mice were evaluated
`for signs
`of discomfort or morbidity Tumour growth was monitored by
`periodic transabdominal palpations Animals were killed on day
`28 Upon autopsy mice were
`screened
`visually for metastatic
`lymph nodes
`and
`in the spleen liver
`lungs kidneys
`lesions
`x 25 lens After excision
`the suspected
`peritoneum using a
`in organs were fixed in 10 buffered formalin
`metastatic lesions
`stained with HE Visible
`paraffin embedded
`and
`sectioned
`macroscopic metastatic lesions verified histologically were counted
`towards metastatic incidence Primary tumours were excised from
`and measured
`the
`pancreas
`weighed
`using a digital
`caliper
`Tumour
`volume was
`formula
`using the following
`tumour volume = length x width22 Tumour pieces fixed in 10
`buffered formalin were used for histological and immunohisto
`chemical analysis
`
`calculated
`
`Treatment
`
`effect
`
`on
`
`desmoplastic
`
`stroma
`
`tumour
`
`cell
`
`proliferation and angiogenesis We performed HE staining on
`formalinfixed paraffin embedded primary tumours and tissues
`and lymph nodes harvested
`at
`the
`spleen liver lungs kidneys
`termination of experiment one day 28 Tumours and tissues were
`sectioned 511m and deparaffinised HE staining was conducted
`
`tumours from four
`
`to five
`
`the
`
`intratumour
`
`separate
`
`assessed
`
`using standard procedures Evaluable
`used
`animals were
`for
`assessing
`tumour cell proliferation
`and stromal desmoplasia
`vascularity
`tumour
`Intratumour
`were
`and
`cell proliferation
`vascularity
`using antiCD31 antibody Cat
`DIA 310 Dianova
`GmbH Hamburg Germany and Ki67 staining Cat
`7904286
`Ventana Medical Systems Inc Tucson AZ USA respectively as
`et al 2013 Massons trichrome
`previously described Yabuuchi
`Rajeshkumar et al 2015 and collagen IV staining Cat M0785
`Dako Carpinteria CA USA were used to assess
`the extent of
`stromal desmoplasia After staining sections were observed under
`a light microscope and photographed using a digital camera
`
`Evaluation
`
`orthotopic
`conducted
`
`for
`
`the
`
`survival
`
`of
`the survival
`benefit of drug regimens
`PDAC harbouring mice A separate
`study was
`of
`treatment
`advantage
`assessing
`regimens Mice with established orthotopic Panc185 and Panc265
`tumours 200300 mm3 were randomly allocated
`to six groups
`as mentioned in the
`and treated with drug doses and schedule
`study design N= 10 micegroup
`in vivo
`efficacy
`reached
`were monitored twice daily Survival
`end points were
`and recorded when mice began to appear moribund with cachexia
`abdominal distension showed signs of hunched posture hindlimb
`occurred first Mice
`laboured breathing whichever
`paralysis or
`
`in
`
`Animals
`
`a
`
`flow rate of
`
`Evaluation of the acute effect of taxanes on tumour desmoplastic
`stroma Mice harbouring established Panc185 and Panc265 ortho
`topic tumours 200300 mm3 were either untreated or treated with
`
`PTX 134 mg kg 1 or nabPTX 223 mg kg1 iv once daily for
`five consecutive days N= 5 mice per group Tumours were resected
`the last dose of PTX or nabPTX Tumours were formalin
`24 h post
`stained with Masons
`and
`sectioned
`paraffmembedded
`fixed
`Trichrome or Collagen IV Sections were evaluated under a light
`and photographed
`using a digital camera
`microscope
`
`Statistical analysis Data were analysed using GraphPad Prism 6
`Inc La Jolla CA USA Primary
`software GraphPad Software
`using Mann
`tumour
`volumes were compared and
`analysed
`Whitney Utest Survival data were estimated and compared using
`Kaplan Meier method and statistical
`significance was determined
`using Log rank Mantel Cox test Differences
`and associations
`statistically significant where P G005
`were considered
`
`GPPRIVP
`
`Characteristics
`
`of orthotopic PDAC mouse models Upon
`in mouse
`Panc185
`tumours
`implantation
`orthotopic
`pancreas
`grew as locally advanced primary tumours lacking metastatic
`Panc265
`dissemination to other organs In contrast
`tumours
`tumour growth and metastatic spreading to
`showed
`aggressive
`
`444
`
`wwwbjcancercom1D01101038bjc2016215
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`

`

`Nabpaclitaxel
`
`in pancreatic cancer
`
`A
`
`Panc265
`
`BRITISH JOURNAL OF CANCER
`
`Liver
`
`Spleen
`
`Kidney
`
`Mesenteric
`
`lymph nodes
`
`Pancreas
`
`Liver
`
`Spleen
`
`Lung
`
`71
`
`1
`
`ok
`
`WI°
`
`1111111111111111191111INIINI
`6
`
`11111111111111111111111U
`
`11111111
`
`6
`
`4
`
`7
`
`0
`
`Control
`
`GEM
`
`PTX
`
`nabPTX
`
`GEM + PTX GEM +nabPTX
`
`11p451 1
`
`11111111111111111111111111
`
`4
`
`I
`
`I
`
`I
`
`0
`
`Panc185
`
`Panc265
`
`4000
`
`>
`
`3000
`
`E 2000
`E
`
`L
`1111111111milmNMEN=
`E
`
`co
`
`ri
`
`1000
`
`0
`
`Li
`co
`
`7 c
`
`ci
`
`cL
`
`Li
`Co
`
`cci
`
`cL
`
`1500
`
`1000 I
`
`500 I
`
`E
`
`cci
`
`=c
`
`L
`
`c
`0°
`
`zco°
`
`cep
`
`cpc
`
`e>ve
`
`cR
`
`x
`
`Li
`co
`
`7 c
`
`L
`
`Li
`Co
`
`cL
`
`Control
`
`GEM
`
`PTX
`
`nabPTX
`
`GEM + PTX
`
`GEM + nabPTX
`
`Figure 1 Characteristics of orthotopically
`implanted PDAC tumours in mice and therapeutic efficacy of treatment regimens Upon implantation in
`tumour showed more aggressive tumour growth and metastatic dissemination in mouse organs A Photographs
`mouse pancreas untreated Panc265
`of resected liver spleen and mesenteric lymph nodes from an untreated Panc265 tumour harbouring mouse showing prominent metastatic lesions in
`towards metastatic lesions Representative photomicrographs HE x 40 showing primary tumour growth in
`organs upper panel Yellow arrows point
`towards metastatic deposits in organs B Gross morphology
`mouse pancreas metastatic dissemination in liver spleen and lungs Black arrows point
`representative of the resected primary tumours at the termination of efficacy experiment D28 Primary tumour volumes in different
`treatment
`regimens
`are shown in C Data represent mean primary tumour volumes ± sem N= 910 mice per group in Panc185
`and 810 mice per group in Panc265
`Baseline mean tumour volumes N= 5 are shown on the left side of graphs Statistical comparison of primary tumour volumes and Pvalues are shown in
`Supplementary Table S3 D Representative photomicrographs HE x 20 of primary tumour sections Histological evaluation of Panc185
`and
`tumours showed moderately differentiated and poorly differentiated pancreatic ductal adenocarcinoma
`Panc265
`towards desmoplastic stroma Red arrows point
`infiltrate Green arrows point
`towards areas of neutrophil
`towards normal pancreatic acinar cells
`
`Blue arrows point
`
`respectively
`
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`BRITISH JOURNAL OF CANCER
`
`Nabpaclitaxel
`
`in pancreatic cancer
`
`Table 1 Treatment
`effect of regimens
`on metastatic
`to organs and peritoneum
`dissemination
`the Panc265
`in
`PDAC model
`
`Group
`
`Vehicle
`
`GEM
`
`PTX
`
`nabPTX
`
`GEM + PTX
`
`GEM + nabPTX
`
`Spleen
`
`Liver
`
`Lungs
`410110
`1010
`910110
`
`010
`
`1010
`
`510
`
`510
`
`38
`
`010
`
`010
`
`010
`
`08
`
`010
`
`010
`
`010
`
`08
`
`Kidneys
`
`LN Peritoneum
`
`510
`
`010
`
`1010
`
`610
`
`010
`310
`010110
`010110
`08
`08
`
`810
`
`310
`
`110
`
`010
`
`010
`
`08
`
`GEM =gemcitabine
`Abbreviations
`PTX= paclitaxel N=810 micegroup
`
`LN = lymph
`
`nodes
`
`nabPTX= nabpaclitaxel
`
`and
`
`liver
`
`spleen lungs
`
`kidneys
`
`lymph nodes
`peritoneum
`Figure IA and B and Table 1 Cachexia was evident
`in control
`animals harbouring Panc265 tumours and their abdomens were
`enlarged due to tumour burden and ascites There was a 1147 fold
`increase in primary tumour volumes of control animals compared
`tumour volumes
`with baseline
`indicating the highly aggressive
`tumours Figure IC and Supplementary
`of Panc265
`phenotype
`Table S2 Both Panc185 and Panc265 tumours harbour activating
`mutations of KRAS oncogene
`and inactivating mutations in p53
`tumour
`suppressor gene the two most
`frequently found genetic
`alterations in human PDAC Jones et al 2008 Biankin et al 2012
`Rajeshkumar et al 2015
`
`efficacy
`
`increased
`
`of
`treatment
`Determination of
`the
`therapeutic
`regimens We first determined the antitumour
`efficacy of each
`drugs alone and in combination with GEM Mean primary tumour
`volumes
`and complete response of treatment
`tumour weights
`shown
`and S2 All
`in Supplementary Tables
`groups are
`SI
`treatment
`regimens tested in both Panc185 and Panc265 tumour
`models
`showed
`reductions
`in primary
`statistically
`significant
`tumour volume compared with control animals Figure IB and
`C and Supplementary Tables 5153 The primary tumour volumes
`and PTXtreated
`animals
`substantially in the control
`of Panc185 P = 00007 and 00007 respectively compared with
`volumes Figure IC and
`tumour
`the baseline pretreatment
`Supplementary Tables SI and S3 GEM and nabPTX mono
`therapy were highly effective
`in reducing the primary tumour
`volumes P< 00001 compared with the
`tumour
`volumes of
`control animals Figure IC and Supplementary Tables SI and S3
`the both combination
`treatments were
`Although
`remarkably
`effective in abolishing primary tumour growth the rate of complete
`responses were higher in the GEM plus nabPTX 89 vs GEM
`plus PTX 22 treatment supporting the higher efficacy of GEM
`plus PTX regimen Supplementary Table 51
`Baseline mean tumour volume of Panc265 tumours increased
`from 273 to 3123 mm3 P = 00007 and 751 mm3 P = 0008 by
`
`control
`
`respectively
`
`the
`
`reductions
`
`statistically significant
`
`28 in the
`and PTXtreated
`day
`groups
`Figure IC and Supplementary Tables S2 and S3 Treatment of
`combination
`regimens resulted in statistically
`significant
`in tumour size but neither
`combination
`induced
`complete regressions Supplementary Tables S2 and S3 GEM pus
`nabPTX treatment produced
`reduction in
`tumour volumes compared with GEM treatment P = 00004
`In both Panc185 and Panc265 tumour models nabPTX mono
`therapy resulted in primary tumour regression compared with
`baseline tumours Figure IC NabPTX treatment was remarkably
`in blocking primary tumour progression compared with
`effective
`3944
`3645 fold P < 00001
`PTX monotherapy
`and
`and
`P = 00001 respectively Figure IC and Supplementary Tables
`5153 In both tumour models one mouse
`each treated with
`nabPTX achieved
`a complete response whereas
`
`observed with PTX treatment Supplementary
`responses were
`Tables SI and S2 Results of exploratory laparotomies conducted
`in mice before
`randomisation baseline confirmed the tumour
`growth confined only in pancreas
`
`evaluation
`
`of Panc185
`
`and
`
`pancreatic
`
`respectively
`
`Tumour histology Histological
`Panc265 tumours revealed moderately differentiated and poorly
`ductal adenocarcinoma
`differentiated
`Figure ID Tumours of control mice showed cribriform pattern
`cells admixed with necrotic debris moderate to
`of neoplastic
`severe nuclear atypia nuclear overcrowding
`aberrant mitoses and
`abundant desmoplastic stroma Figure ID While there was a
`reduction in neoplastic
`cells desmoplastic stroma persisted in the
`tumours of animals treated with GEM or PTX Depletion of
`in the tumours of animals
`desmoplastic stroma was clearly evident
`and in combination with GEM
`treated with nabPTX alone
`Tumours harvested from animals treated with nabPTX alone and
`in combination with GEM displayed mild to moderate neutrophil
`in tumour microenvironment
`compared with control
`regimens NabPTX alone and in
`tumours and other
`treatment
`combination with GEM treatment culminated in a sharp reduction
`of tumour cellularity and progressive
`loss of desmoplastic stroma
`Islands of mouse pancreatic acinar cells were frequently noticed in
`the Panc185
`and Panc265
`tumours harvested
`from the animals
`administered with GEM plus nabPTX Figure ID indicating the
`therapy effectiveness of this regimen in destroying tumour cells
`and desmoplasia HE staining performed on the organs spleen
`
`infiltration
`
`lymph nodes of mice killed prior
`kidneys
`lungs
`to
`liver
`randomisation baseline did not show any metastatic infiltration
`data not shown
`
`Effect of drug treatments on metastatic dissemination to organs
`tumours are highly metastatic when
`and peritoneum Panc265
`implanted orthotopically into mouse pancreas
`showing metastatic
`dissemination in spleen liver
`lungs kidneys lymph nodes
`and
`ranging from 10 to 100 of animals
`peritoneum with frequencies
`Figure IA and Table 1 Treatment effects of agents alone and in
`combination with GEM on metastatic dissemination to distant
`organs and peritoneum are summarised in Table 1 All animals
`untreated or treated with PTX developed metastasis in at
`least one
`organ Table 1 GEM treatment was ineffective
`in controlling
`as evidenced
`metastatic spreading
`by metastatic lesions
`in the
`spleen of nine out of ten 90 mice Table 1 However nabPTX
`the metastatic spreading to spleen in 5 out of 10 mice
`50 Combined treatment of GEM and nabPTX decreased
`metastatic tumour burden compared with either agent used alone
`Table 1
`
`prevented
`
`on carcinoma
`Impact of drug treatments
`cell proliferation
`stroma We
`tumour
`tumour
`and desmoplastic
`vascularity
`determined the treatment effects of agents alone and in combina
`tion with GEM on tumour cell proliferation
`and desmoplastic
`stroma Previous
`studies conducted by us revealed the presence of
`and meta
`grown PDXs
`desmoplastic stroma in subcutaneously
`static lesions of human PDAC Rajeshkumar et al 2015 Whatcott
`Tumours
`et al 2015
`harvested
`from untreated
`animals
`of Panc265 PDX showed
`higher number of carcinoma cells
`expressing Ki67 compared with tumours in Panc185
`indicating
`the aggressiveness of Panc265 tumours Figure 2A The GEM plus
`nabPTX combination effectively
`tumour
`suppressed
`cell prolif
`Figure 2A
`PDXs
`eration in both Panc185
`and Panc265
`cell populations were
`Comparable CD31positive
`noticed in the blood vessels of untreated and drug treated tumours
`indicating that drug treatments did not alter the tumour vascularity
`Figure 2B Abundance
`of desmoplastic stroma a hallmark of
`human PDAC was evident
`in tumours harvested
`from untreated
`animals Figure 3A and B While GEM and PTX treatments were
`tumour stroma nabPTX
`
`largely
`
`ineffective
`
`in reducing
`
`endothelial
`
`no complete
`
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`

`Nabpaclitaxel
`
`in pancreatic cancer
`
`BRITISH JOURNAL OF CANCER
`
`Ki67
`
`CD31
`
`Panc185
`
`Panc265
`
`Panc185
`
`Panc265
`
`72
`
`2 L
`
`u
`
`X
`
`z
`
`PTX
`
`+
`
`GEM
`
`PTX
`
`nab
`
`+
`
`GEM
`
`1
`
`4
`
`41P
`
`ik
`
`Aft
`
`A
`
`72
`
`2L
`
`u
`
`R3
`
`PTX
`
`+
`
`GEM
`
`PTX
`
`nab
`
`+
`
`GEM
`
`on tumour cell proliferation and tumour vascularity The nabPTX plus GEM
`Figure 2 Treatment effect of agents alone and in combinations
`by reduced number of Ki67stained carcinoma cells
`combination was highly effective in controlling
`tumour cell proliferation as evidenced
`Tumours harvested from four to five mice of Panc185
`and Panc265
`on day 28 first experiment were processed and stained with Ki67 or CD31
`antibodies Ki67 a nuclear protein which is expressed in the proliferating carcinoma cells is visualised as dark brown There was no difference in
`various sized blood vessel density in tumours harvested from drug treated animals compared with tumours in the untreated animals CD31stained
`cells in blood vessels are visualised as brown stain A B Representative photomicrographs
`of Ki67 or CD31 stained tumour sections
`endothelial
`x 20
`
`and
`
`in combination with GEM facilitated
`monotherapy
`stroma Figure 3A and B These
`destruction of dense tumour
`results are in agreement with our previous preclinical and clinical
`results Maitra et al 2009 Alvarez et al 2013
`
`the
`
`effect
`
`on
`of
`the survival of mice
`Therapeutic
`regimens
`harbouring human PDAC We evaluated
`the impact of drug
`treatments on the survival of mice harbouring established PDAC
`Kaplan Meier survival curves of different
`treatment groups and
`
`the comparisons are shown in Figure 4 and Supplementary Figures
`locally advanced model Panc185 GEM
`Si and S2 In the
`monotherapy prolonged the median survival from 58 to 114 days
`Figure 4A PTX monotherapy
`also showed
`a positive albeit
`smaller effect and extended median survival to 815 days NabPTX
`compared with PTX
`showed significant advantage
`monotherapy
`by extending the median survival
`to 115
`days
`monotherapy
`P=00024
`Figure 4A and Supplementary Figure S1The
`combination of PTX and GEM showed
`a median survival of 125
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`BRITISH JOURNAL OF CANCER
`
`Nabpaclitaxel
`
`in pancreatic cancer
`
`Massons trichrome
`
`Collagen IV
`
`Panc185
`
`Panc265
`
`Panc185
`
`Panc265
`
`0
`
`2 L
`
`u
`
`z
`
`PTX
`
`+
`
`GEM
`
`PTX
`
`nab
`
`+
`
`GEM
`
`rtriVrint3A
`Ohe
`
`t6`
`
`ZeCtsli
`
`WOi4v
`
`re
`
`rgitiF12ti
`
`A
`
`0
`
`2L
`
`u
`
`z
`
`PTX
`
`+
`
`GEM
`
`z 2L
`
`u
`
`Figure 3 Treatment effect of agents alone and in combinations
`tumour stroma NabPTX monotherapy
`or combined with
`on desmoplastic
`GEM robustly depleted
`the dense tumour stroma as evidenced by less number of collagen fibres in the primary tumours We performed
`immunohistochemistry on primary tumours harvested from untreated or treated with GEM PTX nabPTX and combinations of GEM plus PTX or
`GEM plus nabPTX D28 samples first experiment The fibrotic tissue is visualised as blue Masons trichrome or brown stain Collagen IV
`A B Representative
`x 20
`of Masons trichrome and Collagen IV stained tumour sections
`photomicrographs
`
`effective
`
`days The combination of nabPTX and GEM was the most
`treatment Median survival
`in this group was 161 days
`Figure 4A and the increase was statistically significant compared
`with PTX plus GEM treatment P = 00455 Figure 4A Finally the
`plus GEM combination
`nabPTX
`treatment
`resulted in a
`in median survival compared
`improvement
`with GEM treatment P = 00039 Figure 4A and Supplementary
`Figure Si
`Mice implanted with Panc265 tumours showed a more aggressive
`disease course with a median survival of