Neoadjuvant chemotherapy in muscle-

invasive urinary bladder cancer:

Studies on treatment response, tumor draining

lymph nodes and blood transfusion

Robert Rosenblatt

Department of Surgical and Perioperative Sciences, Urology and

Andrology

Umeå 2020

Responsible publisher under Swedish law: the Dean of the Medical Faculty

This work is protected by the Swedish Copyright Legislation (Act 1960:729)

Dissertation for PhD

ISBN: 978-91-7855-315-0 (print)

ISBN: 978-91-7855-316-7 (pdf)

ISSN: 0346-6612 New Series Number 2091 Cover photo credit: By http://www.scientificanimations.com/ - http://www.scientificanimations.com/wiki-images/, CC BY-SA 4.0, https://commons.wikimedia.org/w/index.php?curid=81897226

Electronic version available at: http://umu.diva-portal.org/

Printed by: Tryckservice, Umeå universitet

Umeå, Sweden 2020

To Béatrice, Sasha, Noah and our newly born son

The first principle is that you must not fool yourself - and you are

the easiest person to fool.

RICHARD P. FEYMAN

i

Contents

ABSTRACT ......................................................................................... iii

LIST OF PAPERS IN THE THESIS ............................................................ v

RELATED PAPERS NOT IN THE THESIS ................................................. vi

ABBREVIATIONS ............................................................................... vii

POPULÄRVETENSKAPLIG SAMMANFATTNING .................................... xi

BACKGROUND .................................................................................... 1

Urinary bladder cancer ............................................................................. 1

The lymphatic system ............................................................................. 11

Tumor immunology ................................................................................ 12

Sentinel node detection .......................................................................... 13

Adoptive immunotherapy ....................................................................... 15

Blood transfusions .................................................................................. 15

AIM OF THE THESIS........................................................................... 21

PATIENTS AND METHODS ................................................................. 22

Paper I .................................................................................................... 22

Paper II and III ........................................................................................ 23

Paper IV ................................................................................................. 25

RESULTS AND DISCUSSION ............................................................... 26

Tumor downstaging after neoadjuvant chemotherapy (Paper I) ............... 26

Sentinel node detection after neoadjuvant chemotherapy (Paper II) ........ 29

Fewer tumor draining sentinel nodes in patients with progressing muscle

invasive bladder cancer, after neoadjuvant chemotherapy and radical

cystectomy (paper III) ............................................................................. 32

Blood transfusion during neoadjuvant chemotherapy for muscle-invasive

urinary bladder cancer may have a negative impact on overall survival

(Paper IV) ............................................................................................... 34

CONCLUSION AND PERSPECTIVES ..................................................... 38

ACKNOWLEDGEMENTS ..................................................................... 40

ii

REFERENCES ..................................................................................... 43

iii

ABSTRACT

Muscle-invasive urinary bladder cancer is a deadly disease. Mortality rates

remained unchanged for decades despite radical surgery.

After several randomized trials, we today know that cisplatin based

chemotherapy given prior to cystectomy, improves survival for every tenth

patient. Markers that predict responsiveness to chemotherapy would spare

unnecessary treatment to the majority of patients. In the search for signs of

chemosensitivity, we performed a retrospective analysis of the Nordic cystectomy

trials 1 & 2: Chemo treated patients had an almost doubled increase in tumor

downstaging compared to the controls. More importantly, this group presented

with a reduced absolute risk of death of more than 30% compared to the rest of

the patients. These results were presented in paper I.

Many cancers spread through the lymphatic system. Usually, there is at least one

tumor draining lymph node, referred to as the sentinel node. If this node is free

of metastases, there is no lymphatic spread of the disease, and consequently, no

use of excavating all neighboring lymph nodes.

Sentinel node detection, is an established method in breast cancer, penile cancer

and malignant melanoma. Based on the same principles, members of our group

developed a similar detection technique in bladder cancer. Unfortunately,

sensitivity and specificity were too low to rely on this method as a diagnostic tool

for lymphatic spread. Instead, it turned out in recent years that sentinel nodes in

muscle invasive bladder cancer are valuable for translational research-lines -

mainly in tumor immunology. As for example, sentinel nodes contain tumor

specific T cells that are useful in adoptive immunotherapy.

In paper II, we set out to test whether sentinel node detection was feasible after

chemotherapy and/or tumor downstaging. In a prospective cohort of patients, we

saw no difference in detection rates between the groups. Thus, we concluded,

neither chemotherapy nor downstaging appeared to hamper the identification of

sentinel nodes.

The concept was expanded in paper III. After recruiting more patients to the

cohort mentioned above, the average numbers of sentinel nodes in different

categories of patients were compared. We saw a pattern of decreased number of

sentinel nodes in those with locally advanced tumors. It seemed that the number

of sentinel nodes had prognostic implications.

iv

In the last study, published in paper IV, we wanted to widen our knowledge on

the clinical effects of blood transfusion. Mounting data suggests that

perioperative blood products have a negative impact on long term survival after

cancer surgery. How much allogenic blood was given during the chemotherapy

prior to surgery? It turned out that one third of the bladder cancer patients

received blood, and these patients demonstrated a significantly worse overall

survival.

Neoadjuvant chemotherapy has added a new beneficial dimension to the

treatment of muscle invasive bladder cancer. In these four studies, we addressed

the effects of chemotherapy on pathoanatomical outcomes, on tumor lymphatics

and further; we are suggesting consequences of neoadjuvant chemotherapy in

conjunction with blood transfusion. It appears that the immune system is

involved in all aspects investigated above. Most likely, an improved scientific

understanding of the immune system will be crucial for future bladder cancer

treatment options.

v

LIST OF PAPERS IN THE THESIS

I. Rosenblatt R*, Sherif A*, Rintala E, Wahlqvist R, Ullen A, Nilsson S, Malmström PU, the Nordic Urothelial Cancer Group. Pathologic downstaging is a surrogate marker for efficacy and increased survival following neoadjuvant chemotherapy and radical cystectomy for muscle-invasive urothelial bladder cancer. European Urology. 2012;61(6):1229-38.

II. Rosenblatt R, Johansson M, Alamdari F, Sidiki A, Holmström B, Hansson J, Vasko J, Marits P, Gabrielsson S, Riklund K, Winqvist O, Sherif A. Sentinel node detection in muscle-invasive urothelial bladder cancer is feasible after neoadjuvant chemotherapy in all pT stages, a prospective multicenter report. World J Urol. 2017;35(6):921-7.

III. Alvaeus J, Rosenblatt R, Johansson M, Alamdari F, Jakubczyk T, Holmström B, Hemdan T, Huge Y, Aljabery F, Gabrielsson S, Riklund K, Winqvist O, Sherif A. Fewer tumour draining sentinel nodes in patients with progressing muscle invasive bladder cancer, after neoadjuvant chemotherapy and radical cystectomy. World J Urol. 2019.

IV. Rosenblatt R*, Lorentzi G*, Bahar M, Asad D, Forsman R, Johansson M,Shareef M, Alamdari F, Bergh A, Winqvist O, Sherif A. Blood transfusions during neoadjuvant chemotherapy for muscle-invasive urinary bladder cancer may have a negative impact on overall survival. Scand J Urol. 2020:1-6

*Shared first authorship

Paper I was reproduced with the kind permission from Elsevier

Paper II and III were reproduced with the kind permission from Springer Nature

Paper IV is an accepted article published by Taylor & Francis in Scandinavian Journal of

Urology on 2020 Feb, available online http://wwww.tandfonline.com/DOI. The paper was

reproduced with their kind permission.

vi

RELATED PAPERS NOT IN THE THESIS

Rosenblatt R, Jonmarker S, Lewensohn R, Egevad L, Sherif A, Kalkner

KM, Kälkner KM, Nilsson S, Valdman A, Ullén A. Current status of

prognostic immunohistochemical markers for urothelial bladder cancer.

Tumour Biol. 2008;29(5):311-22

Rosenblatt R, Valdman A, Cheng L, Lopez-Beltran A, Montironi R,

Ekman P, Egevad L. Endothelin-1 expression in prostate cancer and high

grade prostatic intraepithelial neoplasia. Anal Quant Cytol Histol.

2009;31(3):137-42.

Winerdal ME, Marits P, Winerdal M, Hasan M, Rosenblatt R, Tolf A, et

al. FOXP3 and survival in urinary bladder cancer. BJU Int.

2011;108(10):1672-8.

Shah CH, Viktorsson K, Kanter L, Sherif A, Asmundsson J, Rosenblatt

R, Lewensohn R, Ullén A. Vascular endothelial growth factor receptor 2,

but not S100A4 or S100A6, correlates with prolonged survival in

advanced urothelial carcinoma. Urol Oncol. 2014;32(8):1215-24.

vii

ABBREVIATIONS

ABC Advanced bladder cancer

ACD Anemia of chronic disease

AI Anemia of inflammation

BCG Bacillus Calmette-Guérin

CCI Charlson comorbidity index

CD40L Cluster of differentiation 40 ligand

CIS Carcinoma in situ

CMV Cytomegalovirus

CR Complete response

CT Computer tomography

CTL Cytotoxic T lymphocyte

cTNM Clinical tumor node metastasis

DC Dendritic cell

DCE MRI Dynamic contrast-enhanced magnetic resonance imaging

ddMVAC dose dense Methotrexate Vinblastine Adriamycin Cisplatin

DSS Disease specific survival

EBRT External beam radiotherapy

ECOG Eastern cooperative oncology group

E-LND Extended lymph node dissection

ER Estrogen receptor

viii

EV Extracellular vesicles

FFP Fresh frozen plasma

FNR False negative rate

FP False positive

GC Gemicitabine cisplatin

GI Gastrointestinal

GSTM1 Glutathione-S-transferase mu 1

Hb Hemoglobin

HEV High endothelial venules

HLA Human leukocyte antigen

HR Hazard ratio

IL Interleukin

INF- γ Interferon gamma

L-LND Limited lymph node dissection

LM Lymphatic mapping

LND Lymph node dissection

MIBC Muscle-invasive bladder cancer

miRNA micro ribonucleic acid

MMT Multimodality treatment

MR Magnetic resonance

MVAC Methotrexate Vinblastine Adriamycin Cisplatin

NAC Neoadjuvant chemotherapy

ix

NAT-2 N-acetyltransferase 2

NCT Nordic cystectomy trial

NMIBC Non-muscle-invasive bladder cancer

NNT Numbers needed to treat

OR Odds ratio

ORC Open radical cystectomy

OS Overall survival

PBC Packed red cell concentrate

PBT Perioperative blood transfusion

PD Progressive disease

PD-L1 Programmed death-ligand 1

PDGF Platelet-derived growth factor

PMP Platelet-derived microparticles

pTNM Pathological tumor node metastasis

RARC Robotic-assisted laparoscopic radical cystectomy

RC Radical cystectomy

RCT Randomized controlled trial

RR Relative risk

SD Stable disease

SN Sentinel node

SNd Sentinel node detection

SNdef1 Sentinel node definition 1

x

SNdef2 Sentinel node definition 2

S-LND Standard lymph node dissection

SE-LND Super-extended lymph node dissection

TGF- β Transforming growth factor beta

TNFa Tumor necrosis factor alpha

TNM Tumor node metastasis

TRALI Transfusion related acute lung injury

TRIM Transfusion related immunomodulation

TURB Transurethral resection of the bladder

VEGF Vascular endothelial growth factor

VEGFC/D Vascular endothelial growth factor C/D

UBC Urinary bladder cancer

xi

POPULÄRVETENSKAPLIG

SAMMANFATTNING

I Sverige är urinblåsecancer den fjärde vanligaste cancerformen hos män och

åttonde vanligaste hos kvinnor. Sjukdomen uppträder som regel efter 65 års

åldern. Rökning är en stark riskfaktor, vilket delvis kan förklara varför män

hittills varit mer drabbade. Det vanligaste symtomet är synligt blod i urinen. Då

är det viktigt att man utreds med kontraströntgen av urinvägarna samt

kameraundersökning av urinblåsan (cystoskopi). Den slutgiltiga diagnosen får

man efter provtagning av tumören på operation. Vävnadsbitar från tumören

skickas på mikroskopisk analys vilket avgör tumörens allvarlighetsgrad.

En viktig skiljelinje är om tumören växer in i urinblåsans muskelskikt. I tre

fjärdedelar av fallen gör den inte det. Då räcker det oftast med att hyvla bort all

synlig tumör samt vid behov ge sköljningar i urinblåsan med antingen cellgifter

eller immunologiskt verkande läkemedel.

I en fjärdedel av fallen växer tumören på djupet, vilket kallas för muskelinvasiv

blåscancer. Detta är en mycket allvarlig sjukdom där botande behandling innebär

att man tar bort hela urinblåsan (cystektomi). Trots denna omfattande operation

har cancercellerna i nära hälften av fallen hunnit sprida sig, oftast via

lymfbanorna. För att minska risken för spridning kan man förbehandla med tre-

fyra kurer cellgifter (neoadjuvant kemoterapi). Drygt hälften av patienterna med

muskelinvasiv blåscancer är friska nog att klara cellgiftsbehandling.

Den här avhandlingen består av fyra studier som tittar närmare på olika aspekter

av cellgiftsbehandling vid muskelinvasiv blåscancer. Upprepade studier visar att

enbart var tionde patient har nytta av cellgifterna. För att finna en tydlig flagga

för vem som gagnas, tittade vi närmare på två nordiska studier från 90-talet. Här

hade patienterna lottats mellan standardbehandling med eller utan föregående

cellgifter. I vår studie slog vi ihop patienterna och jämförde skillnaden i

tumörutbredning innan och efter operation. Det var dubbelt så vanligt att

tumören utplånats helt om cellgifter givits. Dessutom var överlevnaden mycket

bättre. Nio av tio patienter var i liv fem år efter operationen. Hos övriga, oavsett

cellgifter eller ej, överlevde bara hälften av patienterna femårsdagen.

Vid många cancerformer sker spridning via lymfbanorna. För att slippa leta

cancer i hela lymfsystemet kan det räcka att titta i den tumördränerande

lymfknutan. Hittar man ingen cancer här så är risken låg för spridning. Vid

bröstcancer gör denna teknik att många patienter besparas onödiga ingrepp. Men

vid urinblåsecancer är tekniken inte helt tillförlitligt. Däremot innehåller

xii

tumördränerande lymfknutor vita blodkroppar som exponerats för cancern. Tar

man ut dessa och låter de föröka sig i ett laboratorium kan de fungera som en

effektiv behandling om man ger dem tillbaka till patienten. Detta kallas för

adoptiv immunterapi.

För att kunna använda denna terapi i större patientgrupper ville vi undersöka om

tekniken för att spåra den tumördränerande lymfknutan fungerade för

urinblåsecancer även efter cellgiftsbehandling. Det visade sig fungera; antalet

tumördränerande knutor var oförändrat. Samtidigt hade våra medarbetare

undersökt lymfknutorna. Aktiviteten hos de cancerbekämpande vita

blodkropparna hade ökat tack vare cellgiftsbehandlingen!

I den tredje studien tittade vi på antalet tumördränerande lymfknutor i olika

patientkategorier. Vi såg ett tydligt mönster. Tumörer motståndskraftiga mot

cellgifter hade färre dränerande lymfknutor. Sedan tidigare vet vi att tumören kan

styra nybildningen av lymfbanor och stänga av kontakten med närliggande

lymfknutor. Detta för att skydda sig. Därför tror vi att tumördränerande

lymfknutor kan vara en användbar markör för prognos och val av behandling i

framtiden.

Blodbrist är vanligt hos människor med cancer. Cellgifter och kirurgi kan förvärra

situationen vilket leder till att blodtransfusioner ofta ges. Men blod är inte

ofarligt. Mottagarens immunförsvar kan överreagera och det finns risk för

spridning av infektioner. Vad som är mindre känt är att blodtransfusion i många

fall har en dämpande effekt på det tumörbekämpande immunförsvaret. Detta kan

förklara varför patienter som fått blod i samband med canceroperation uppvisar

en försämrad prognos. Ett välfungerande immunförsvar är nämligen viktigt för

att övervinna cancern.

I den sista studien undersökte vi om effekten av cellgifter försämras hos patienter

som samtidigt erhållit blodtransfusion. Det visade sig att en tredjedel av

patienterna hade fått blod. Dessa hade elakare tumören när man undersökte

urinblåsan efter operation. Ännu värre var att patienterna hade försämrad

långtidsöverlevnad. Resultatet är tankeväckande men bör tolkas med

försiktighet. Studien baseras på 120 patienter och studiens upplägg gör det svårt

att avgöra om det är blodet eller orsaken till att blod givits som förklarar

skillnaden i resultat.

Nämnda studier belyser olika delar av cellgiftsbehandling vid muskelinvasiv

blåscancer. Vi har visat hur krympning av tumören är ett starkt uttryck för svar

på behandling. Därefter visade vi att cellgifter inte är ett hinder för att hitta

tumördränerande lymfknutor. På samma tema såg vi i den tredje studien att

antalet tumördränerande lymfknutor är lägre hos tumörer som svarar på

xiii

behandling. Avslutningsvis undersökte vi förekomsten av blodtransfusion under

pågående cellgiftsbehandling. Patienter som fått blod hade elakare tumörer och

sämre prognos.

1

BACKGROUND

Urinary bladder cancer

Definitions, epidemiology and etiology

Urinary bladder cancer (UBC) is a major global health concern. In 2018

approximately 550, 000 were diagnosed and nearly 200, 000 died of the

disease (1). It is the sixth most common cancer for men, and the 17th for

women, worldwide. Europe and North America, are high incidence regions

with 17.3 men and 4.5 women per 100.000 people (1). In Sweden, 2859

patients were diagnosed and 732 died from the disease in 2018 (1). As of 31st

of December 2016, 25 848 people were living in Sweden with cancer in the

bladder or in the upper urinary tract (2). The disease is strongly related to age,

with a mean of 73 years at diagnosis. The age standardized incidence rate for

UBC in Sweden has been increasing since the 1970 for both men and women

(3). However, in similarity with most other western countries, there has been

a stabilization of incidence and decrease in mortality since the mid 90s (4).

The main reason for this is ascribed to diminished smoking habits in the

population.

Risk factors can be divided into inherited properties and external exposures.

Thus far, there are no identified genes that strongly predispose to UBC. Yet,

studies of twins have estimated that 30% of the risk is attributed to heritable

factors (5). Likewise, relatives of UBC patients have an almost doubled risk of

the disease compared to controls, independent of smoking status (6, 7).

The genetic polymorphism of N-acetyltransferase 2 (NAT-2) and Glutathione-

S-transferase mu 1 (GSTM1) appear to alter the risk of UBC. NAT-2 is a liver

enzyme that acetylates arylamine, one of 60 established carcinogens in

tobacco smoke that is strongly associated with UBC (8). The slow functioning

genotype of NAT-2 has been associated with increased risk of UBC in both

non-smokers (OR=3.41) and heavy smokers (OR = 8.57) (8). GSTM1 is a

conjugating enzyme involved in the detoxification of various carcinogens,

where the non-functional version of GSTM1 (null allele) has been associated

with increased risk of cancer (9).

Tobacco smoking is the most important external risk factor for UBC. More

than 60 carcinogenic substances have been described, including aromatic

amines, which is believed to be the most important agent in the formation of

UBC. In a meta-analysis that pooled the result of 83 original articles, there was

2

an increased risk of incidence (RR = 3.47) and mortality (RR=1.53) in current

smokers compared to non-smokers (10).

Exposure to aromatic amines and other chemicals present in dyes, rubber,

leather and paint, have resulted in increased incidence of UBC in certain

occupations. The list of exposed occupations is long, and is topped by tobacco

workers (RR= 1.72), dye workers (RR= 1.58) and chimney sweepers (R=1.53)

(11).

To this day we do not understand why men are diagnosed with UBC three to

four times more often than women. Differences in smoking habits and

exposure to environmental carcinogens have been suggested. Yet, studies that

controlled for these factors have still observed gender differences (12).

Gender-related differences in metabolizing enzymes and bladder

microbiomes might contribute to diverse ecosystems in the bladder (12). A

lowered risk for UBC has been recorded for women in the premenopause, with

late menarche, and women supplementing with estrogens and progesterone.

At the tissue level, the expression of the androgen receptor in urothelial

carcinoma decreased with increasing tumor stage (12).

Even though the incidence of UBC is higher for men, women tend to have a

worse prognosis compared to men, even after adjusting for tumor stage (13).

Data show that the lead time from hematuria to diagnosis is significantly

longer for women (14), and this might have implications for prognosis (15).

Discrepancies in treatment might account for part of these differences (16). A

meta-analysis encompassing 27,912 patients, looking specifically at outcome

after radical cystectomy (RC), saw a significantly worse cancer-specific

survival in women (HR 1.20, p=0.0001) (17). As mentioned above, estrogens

appear to have a protective role for the emergence of UBC. Yet, the type of

estrogen receptor also matters: ER is associated with favorable tumor

outcome while ER predicts a worse prognosis (18).

3

Figure 1. Schematic image of the urinary bladder with tumors of different stages.

Credit: Cancer Research UK Original email from CRUK, CC BY-SA 4.0,

https://commons.wikimedia.org/w/index.php?curid=34334316

Symptoms, staging and classification

Gross hematuria is the cardinal symptom of urinary bladder cancer and is present

in 85% of the cases. Dysuria, urgency and increased frequency are other clinical

signs. Evaluation of hematuria includes white light cystoscopy, cytology of urine

or bladder-washing specimen, computed tomography (CT) urography, and rectal

examination of the prostate. Diagnosis is based on pathoanatomical assessment

of tumor specimens after transurethral resection of the bladder (TURB) (19).

90% of UBCs are of primary urothelial origin. Other primary carcinomas of the

bladder include squamous cell carcinomas (5%), adenocarcinomas (2%),

sarcomas, signet ring cell and small cell carcinoma. Primary urothelial

carcinomas can also present with mixed histology with the most common variant

being squamous cell carcinomas and adenocarcinomas. Other variants include,

micropapillary, nested cell variant, clear cell variant carcinomas and

plasmacytoid tumors (20).

The TNM classification defines the stages of the disease (table 1). UBC extends

from papillary neoplasms (pTa) of low malignant potential to local advanced

disease with metastatic spread (pT4bN+M+). Correct pathoanatomical diagnosis

is crucial for correct management and prognosis.

4

Table 1. Classification of urinary bladder cancer. Adapted from the Guidelines on Muscle-

invasive and Metastatic Bladder Cancer, European Association of Urology (EAU) (19).

T - Primary Tumor

Tx Primary tumor cannot be assessed

T0 No evidence of primary tumor

Ta Non-invasive papillary carcinoma

Tis Carcinoma in situ: "flat tumor"

T1 Tumor invades subepithelial connective tissue

T2 Tumor invades muscle

T2a Tumor invades superficial muscle (inner half)

T2b Tumor invades deep muscle (outer half)

T3 Tumor invades perivesical tissue

T3a microscopically

T3b macroscopically (extravesical mass)

T4 Tumor invades any of the following: prostate stroma,

seminal vesicles, uterus, vagina, pelvic wall, abdominal wall

T4a Tumor invades prostate stroma,

seminal vesicles, uterus, or vagina

T4b Tumor invades pelvic wall or abdominal wall

N - Regional Lymh Nodes

Nx Regional lymph nodes cannot be assessed

N0 No regional lymph node metastasis

N1 Metastasis in a single lymph node in the true pelvis

(hypogastric, obturator, external iliac, or presacral)

N2 Metastasis in multiple lymph nodes in the true pelvis

(hypogastric, obturator, external iliac, or presacral)

N3 Metastasis in common iliac lymph node(s)

M - Distant Metastasis

M0 No distant metastasis

M1a Non-regional lymph nodes

M1b Other distant metastasis

5

At presentation, approximately 75% of UBCs are non-muscle-invasive bladder

cancer (NMIBC). This means that the tumors have not invaded the detrusor

muscle in the bladder wall. These lesions are further categorized into low,

intermediate and high risk of recurrence and progression and are typically

managed by TURB with or without adjuvant intravesical instillations. The high

risk for recurrence and progression demands frequent cystoscopies and repeated

TURBs (21).

The remaining 25% of UBC represent muscle-invasive urinary bladder cancer

(MIBC). For MIBC, where metastatic spread is not suspected, the mainstay of

treatment is radical cystectomy (RC) and regional lymph node dissection (LND).

An overview of the different treatment options is summarized in (table 2).

Table 2. Summary of urinary bladder cancer treatment. Adapted from the Guidelines on

Muscle-invasive and Metastatic Bladder Cancer, European Association of Urology (EAU) (19).

Non-muscle-invasive bladder cancer

Low-risk Primary, solitary, TaG1, <3cm, no CIS

TURB + immediate instillation of intravesical chemotherapy

Intermediate risk All tumors not defined as low or high-risk

TURB + repeated instillations of intravescial chemotherapy/BCG

High-risk T1/G3/CIS or multiple, recurrent and >3cm

TURB + intravescial BCG 1-3 years or RC

Muscle-invasive bladder cancer

Cisplatin-fit T2-T4aN0M0

NAC+RC High risk patient NAC naïve pT3/4 and/or pN+

RC + adjuvant (GC/MVAC)

Unfit/unwilling RC EBRT/MMT

Metastatic bladder cancer

Cisplatin-fit GC, MVAC Cisplatin unfit (PD-L1 +) Pembrolizumab/Atezolizumab Cisplatin unfit (PD-L1-) Carboplatin

Second line Pembrolizumab

6

Staging and treatment of MIBC

A correct staging is pivotal for management of MIBC. The CT urography

performed in the initial investigation, accompanied with a CT of the thorax, is

used for assessing: local extension of the tumor, suspected lymph node

metastases, spread of tumor to upper urothelial tract and other organs (22).

Magnetic resonance (MR) is better at soft tissue resolution, which is valuable for

distinguishing tumors from surrounding tissue (23). However, at this point no

imaging modality can differentiate T2 from T3a, whereas identification of T3b or

higher is feasible (24). Lymph node metastases should be suspected when a

lymph node is > 8 mm in the pelvis, and >10 mm in the abdomen (19).

Comorbidity assessment

Mortality after RC increases with chronological age: 2.3% in 70-79 year of age

compared to 4.3% in 80 years and older (25). However, biological age is a more

important prognostic factor. Sarcopenia prior to abdominal surgery, and low

preoperative serum albumin levels, are some factors that are important for

overall survival after RC (26). The Charlson Comorbidity Index (CCI) is a well-

studied tool for evaluating comorbidity in MIBC. Together with instruments

developed for assessing activity level, e.g. the Eastern Cooperative Oncology

Group (ECOG), CCI can aid in determining the appropriate treatment for a given

patient (19).

Radical cystectomy

The gold standard therapy for localized MIBC (pT2-T4aN0M0) is radical

cystectomy (RC) with bilateral pelvic lymph node dissection (LND). Some other

main indications for RC are NMIBC with very high risk, BCG resistant NMIBC,

and MIBC N1 disease (19).

The standard RC implies removal of the urinary bladder and the distal ureters,

including the excision of the entire urethra, the uterus, the cervix, the ovaries, and

the anterior vagina in women, and the removal of the prostate and the seminal

vesicles in men. In men with organ-confined disease that are motivated to

preserve their sexual ability, four organ sparing techniques are described:

Preserving the neurovascular bundle, +/-, seminal vesicles and vas deferens, +/-

parts or the whole prostate (27). In women, pelvic organ-preserving techniques

maintain the neurovascular bundle, vagina, uterus and the ovaries. In

premenopausal women, preserving the ovaries will keep the hormonal

homeostasis, whereas preservation of the uterus and vagina reduce pelvic floor

disorders. Although data on functional and oncological results are limited, the

techniques can be considered in selected cases with organ-confined disease (27).

7

Controversy remains on the importance of timing of surgery for optimal oncologic

results. Some have reported worse results after delaying definitive treatment with

>3 months (28) whereas others have shown no difference (29).

Lymph node dissection (LND)

The rationale for LND is claimed to be of both prognostic and therapeutic value.

Among patients with clinical T2-4N0M0 tumors, approximately 25% have lymph

node metastases detected in removed lymph nodes post-RC (30, 31). Patients

with positive lymph nodes (pN+) are associated with a significant risk of disease

recurrence compared to pN0 (32). Nevertheless, patients without demonstrable

lymph node metastases, still suffer from recurrences and death due to metastases.

The lymphatics of the urinary bladder flows primarily to nodes in the internal

iliac, external iliac, obturator and presacral region. In a large consecutive series

of 200 patients, Abol-Enein et al observed that lymph node metastases outside

the pelvis always preceded metastases in obturator or iliac nodes first (30). Yet,

evidence of “skip lesions” i.e. metastases occurring solely in extra-pelvic regions,

have been reported (33).

Retrospective data have pointed to an oncological benefit in favour of LND

compared to no-LND (34). More controversy concerns the extent of LND which

can be classified into four templates: Limited LND (L-LND) which is restricted to

the obturator fossa (external iliac vessels laterally, iliac bifurcation proximally,

genitofemoral nerve medially). Standard LND (S-LND), includes L-LND plus the

medial internal iliac vessel medially, and the inguinal ligament distally. In

extended LND (E-END) presacral nodes up to the aorta bifurcation are added.

Super-extended LND (SE-LND) continues proximally to the root of inferior

mesenteric artery (19).

In a systematic review including 14 studies comparing E-LND with L-LND/S-

LND there was no compelling evidence in favor of E-LND (34). More importantly,

the first randomized trial comparing 198 patients with E-LND to 203 patients

with L-LND, failed to show a significant oncological advantage in the E-LND

group. Rather, the former group presented with increased need of lymphocele

drainage within 90 days of surgery, compared to the latter (35).

Retrospective comparisons of different LND templates will encounter selection

bias (36). A patient undergoing cystectomy without LND has an unknown pN

status. The more extended LND a patient is submitted to, the more defined the

pN status will be. Therefore, retrospective comparisons of different LND

templates will compare apples with pears. For instance, if a patient with pN0 after

no or limited LND is compared to a patient with pN0 after more extended LND,

8

the first patient might have been categorized as a pN+ if a more extensive

template was applied.

Robotic vs open radical cystectomy

In 2019, a Cochrane review based on five RCTs, compared open radical

cystectomy (ORC) with robotic-assisted laparoscopic radical cystectomy (RARC)

(37). With 270 and 271 patients in each arm respectively, there was no significant

difference in time-to-recurrence or major complications (Clavien 3-5). There

were fewer blood transfusions and somewhat shorter hospital stay for the RARC

arm. However, cost-effectiveness was never addressed. Similar results were

shown in a systematic review, which also demonstrated longer operative time, but

shorter hospital stay and decreased blood loss in RARC patients (38).

Urinary diversion

After the bladder is removed, the ureters can be rerouted in three principal ways:

the abdominal wall, the urethra, or the rectosigmoid. Various parts of the

gastrointestinal tract can be used as conduit or continent pouch. The most

common diversions are the ileal orthotopic neobladder and the ileal conduit. Age,

comorbidity and cognitive functions are important factors when deciding on the

type of diversion. The orthotopic neobladder is anastomosed to the urethra.

Voiding depends on abdominal pressure, intestinal movement and sphincter

opening. This alternative is generally not recommended to patients above 80

years, and it is contraindicated to patients with psychiatric illness, tumor growth

in the urethra, restricted life probability, and renal failure. Common

complications include both day- and nighttime incontinence and

ureterointestinal stenosis. With an ileal conduit, 50% of the patients experience

early complications, e.g. urinary tract infections, ureteroileal leakage and stenosis

whereas stomal complications occur in the long-term. There are no RCTs that

compare the conduit to the neobladder, but the oncological outcome is similar

(27).

Mortality

Perioperative mortality after RC at 30 and 90 days ranges between about 1.9-3.2%

and 5.7-8% with significant increased rates for low volume hospitals (39).

Oncological outcome is strongly correlated to pT stage and pN status. In one of

the larger cohorts involving 1054 patients of urothelial carcinoma, the recurrence

free survival at 5 years was 68% for the whole cohort, 92% for pT0 bladders, 89%

for pT2N0 and 78% for pT3a, 62% for pT3b, and 50% for pT4. 24% had pN+ and

these patients had a five-year disease specific survival of 35% (40). Other

prognostic factors include margin status, lymphovascular invasion, hospital

volume and age (41). Molecular subtypes are also important: luminal papillary,

luminal unstable, luminal unspecified, stroma-rich, basal/squamous and

9

neuroendocrine-like are definitions that might be used for prognosis and choice

of treatment in the future (42).

Chemotherapy

The introduction of chemotherapy as a treatment of cancer was inspired by the

use of antibiotics to treat infections in the 1930s. However, in contrast to bacteria,

cancer cells are similar to normal cells, which makes them difficult to target

without damaging healthy tissue.

Moreover, tumor cells are genomic unstable, which give rise to subclones with

varying degrees of susceptibility to drugs. It was not until chemotherapies of

different mechanisms were combined, that cancer patients started to be cured by

chemotherapy.

In MIBC, 50% of patients progress after radical cystectomy. Thus, surgery needs

to be accompanied by other treatments for survival to improve. In the mid 70s, it

was shown that advanced UBC responded to cisplatin (43) followed by

methotrexate in the early 80s (44) and then in combinations with vinblastine (45)

and doxorubicin (46). Yet, a long going debate followed, concerning the timing of

the therapy. Should the systemic treatment be given before (neoadjuvant) or after

(adjuvant) surgery?

Neoadjuvant chemotherapy (NAC)

Studies on breast cancer indicate that both neoadjuvant and adjuvant

administration of chemotherapy are effective in eradicating micrometastases

(47). However, in MIBC, the situation is different: Patients are older, have more

comorbidities, and will undergo cystectomy; an operation associated with more

complications and deconditioning than surgical treatment of breast cancer. Thus,

the arguments for neoadjuvant treatment are multiple: 1) The patient is in better

shape to tolerate systemic therapy. 2) Gross anatomy is unobstructed, with intact

blood vessels that can deliver the cytotoxic agents to the target organ. 3) The

chemotherapy is given as early as possible, before the tumor burden is too high.

4) Chemotherapy allows debulking effects on the tumor, which facilitates surgery,

and increased levels of pT0, pN0 and negative margins. 5) Chemotherapy does

not appear to affect surgical morbidity nor the eligibility for RC (48).

One of the early initiatives to investigate the role of NAC in UBC came from

Scandinavia. In 1992, the first Nordic Cystectomy Trial (NCS1) demonstrated a

significant survival advantage in patients with cT1-T4aNxM0 that were

randomized to two cycles of cisplatin + doxorubicin prior to standard care

(radiotherapy and RC) (49). In the follow-up study the Nordic Cystectomy Trial

2 (NCS2), patients with only MIBC were included and the chemotherapy regimen

was intensified (50). However, no distinct survival benefit was recorded in the

10

experimental arm until the two studies were combined in a joint analysis. Now an

absolute risk reduction was seen in favour of the experimental arm (p=0.045)

(48).

Today there is solid evidence for the benefit of NAC. The Advanced Bladder

Cancer (ABC) Meta-analysis Collaboration, encompassing individual patient data

of 3005 patients from eleven RCTs, (including the NCS1&2), showed a significant

benefit in overall survival for platinum-based combination chemotherapy

(HR=0.86, p=0.03) (51). The most recent updated meta-analysis of the ABC,

including four additional RCTs and 427 new patients showed an absolute risk

reduction of eight percent at five years (NNT=12,5) when gemcitabine cisplatin

(GC) or methotrexate, vinblastine, adriamycin and cisplatin (MVAC)

chemotherapy were considered (52).

Adjuvant chemotherapy

The arguments for adjuvant chemotherapy is that that patients with low risk of

metastatic disease are sorted out, and that curative surgery is performed without

delay. With this approach, patients with pT3-4 and/or pN+ disease with high risk

for relapse, would be candidates for postoperative treatment.

Yet, repeated trials have failed to demonstrate any benefit from this strategy.

Patients are often in weakened condition after cystectomy, with deteriorating

renal function that makes them unfit for chemotherapy. Four randomized trials

recruited less than 50% of their planned enrolments. In the largest of them,

EORTC 30994, 284 patients were randomized between early or deferred

chemotherapy (GC, MVAC or ddMVAC). The median overall survival was 6.74

years in the early compared to 4.60 years in the deferred (p= 0.13) (53). In an

updated meta-analysis of nine trials including 945 patients, a hazard ratio of 0.77

was seen in overall survival (p=0.049). The benefit was accentuated in disease

free survival for patients with lymph node positive disease (p=0.010) (54).

In contrast to NAC, trials on adjuvant chemotherapy have only examined tumors

that are locally advanced and/or pN+. This approach deprives patients, with a

potentially high load of undetected micrometastases (≥cT3pN0), from systemic

treatment. Subgroup analysis of cT3 tumors has shown greatest benefit from NAC

in this group, both in terms of tumor downstaging as well as in significantly

improved overall survival. Arguably, this is due to treatment of undiagnosed

micrometastatic disease (48).

11

The lymphatic system

The lymphatic vessels

Fluid is constantly formed in the interstitial space between cells. To maintain

tissue homeostasis, small vessels, lymph capillaries or lymphatics, drain

vascularized tissue from extracellular fluid. The absorbed fluid, lymph, travels

through a network of merging vessels that connects to a lymph node. In turn, one

lymph node is connected to a series of other lymph nodes. Accumulating lymph

eventually empty into the thoracic duct that returns to the blood circulation

through vena cava superior (55).

The lymph contains debris secreted by cells, but also antigens from invading

microbes and cells from the immune system e.g. memory T cells, Dendritic cells

(DC) and macrophages. They use the lymphatics to reach the lymph nodes from

the peripheral tissue (55).

The lymph node

The lymphatics enter the lymph node through afferent vessels that penetrate the

surrounding fibrous capsule. Here, in the subcapsular sinus, macrophages reside

for the purpose of recognizing and removing foreign organisms. Underneath lies

the outer cortex where clusters of B lymphocytes form follicles surrounded by

paracortex, which mainly constitutes T lymphocytes. In the center of the follicle

lies follicular DCs. After antigenic stimulation, the B cells start proliferating. Now

the center of the follicle stains lighter in the microscope, and is called a germinal

center, and the structure as a whole has become a secondary follicle (55).

The DC travels to the lymph node from the peripheral tissue upon activation by

antigens. Here, the ingested antigen is presented to T and B lymphocytes. The

lymphocytes access the lymph node through cortical blood vessels named high

endothelial venules (HEV). They are directed towards specific sites in the lymph

node by different chemokines expressed by stromal cells. The antigen receptors

on the lymphocytes are unique for a particular antigen. The binding of the antigen

to the correct receptor can activate the lymphocytes in the presence of the right

co-stimulatory signal and create an antigen specific immune response. This

makes the DC and the lymph node the crucial link between the innate and the

adaptive immunity (55).

Neolymphangiogenesis

A growing tumor needs oxygen. The hypoxic environment makes the tumor cells

secrete angiogenic factors that stimulate the formation of new blood vessels. As

blood plasma filters through the aberrant walls of the newly formed vessels, fluid

12

is accumulating in the interstitial space of the tumor. The increased pressure

partly drives the formation of new lymph vessels (56). In addition, growth factors,

such as vascular endothelial growth factor C/D (VEGFC/D), secreted by tumor

cells, follow the interstitial flow and lead to the proliferation of lymphatic

endothelial cells that lead to new lymph capillaries (57). The increase in lymphatic

vessel density facilitates the transport of migrating cancer cells. In fact, lymphatic

density and expression of VEGFC/D have been associated with tumor

progression (58).

Tumor immunology

The interplay between the immune system and cancer has caught the attention of

scientists since the late 1800s. In 1957 the hypothesis of “immunosurveillance”

was formulated by Burnet and Thomas (59). It stated that neoplastic alteration of

a cell will be detected by the immune system as foreign, and thus be removed

before they grow into tumors. A key player is the cytotoxic T lymphocyte (CTL)

which eradicate cells that express foreign antigens on their major

histocompatibility complex class I. However, when the immune system is

attenuated, as for organ transplanted or HIV/AIDS patients, a higher incidence

of certain types of cancer can appear (60).

In an immunocompetent state, the microenvironment of the tumor is crowded

with immune cells. The quantity of T lymphocytes and macrophages infiltrating

the tumor correlates to prognosis in several cancers (61, 62). For the neoplastic

cells to evade the immune system, they need to modify the expression of proteins

on the cell surface or downregulate the antitumoral response by secretion of

inhibiting cytokines (63).

In the tumor draining lymph node, naïve lymphocytes and macrophages are

activated to mount a tumor specific immune response (64). These lymphocytes

have been utilized for adoptive cell immunotherapy in UBC (65) and in colon

cancer (66). However, there are also many examples of how the tumor establishes

a tumor tolerant environment in the draining node (67). Immune suppression is

achieved by tumor-derived cytokines (68) that augment the number of regulatory

T cells while making cytotoxic T cells non-functional (56). Also, increased

interstitial flow can induce TGF-β that downregulates DCs in the lymph node (56)

and tumor-secreted exosomes that induce tolerance in draining lymph nodes

(69). In malignant melanoma, it has been shown that nodes closest to the primary

tumor were more immunosuppressed, whereas those furthest away lacked

immunological reactivity compared to intermediate nodes (64). This tumor

induced immunomodulation is perhaps a prerequisite for the tumor to colonize

“hostile” lymph nodes.

13

Sentinel node detection

The first draining lymph node of a tumor is unique, and often referred to as the

sentinel node (SN). Since many cancers spread through the lymphatic system, the

identification of the SN can be valuable for both diagnostic and therapeutic

reasons.

A lymphatic mapping (LM) can be performed by one or a combination of

techniques. A radioactive tracer is injected peritumorally so that a signal can

either be detected by a preoperative lymphoscintigraphy and/or by utilizing an

intraoperative handheld Geiger meter (Figure 2)(70). If blue dye is injected

around the tumor, blue lymphatics can be followed to a blue colored SN. The SN

is then excised and sent for pathological assessment (sentinel node biopsy).

Figure 2. Schematic illustration of intraoperative sentinel node detection by a handheld

Geiger meter. Credit: Malin Winerdal.

In malignant melanoma, breast cancer and penile cancer, the sentinel nodes

biopsy is a well-established diagnostic tool for detecting lymphatic metastases

(71, 72). If the SN is free of tumor cells, there is no need to proceed with regional

LND as the SN is said to reflect the status of the whole region of nodes. Thus, the

patient is spared from avoidable surgery and its associated comorbidities.

14

The implementation of SN biopsies in the above-mentioned cancers have

inspired others to investigate its role in other types of cancers. In UBC, the

pioneering work of Sherif et al, detected SNs in 11 of 13 patients (73). Metastases

were found in the SNs of four patients and not in any non-SNs. In another cohort

of 14 patients, two patients were without detectable SN, whereas all seven

patients that were pN+ had metastases in SNs and three in non SNs (74).

To improve detection rates, attempts to fuse the lymphoscintigraphy with single-

photon emission CT and conventional CT visualized 21 SNs in five out of six

patients (70).

In 2006, Liedberg and colleagues performed peritumoral injections with

radioactive tracer and blue dye in 75 patients (75). The first 30 patients

underwent preoperative lymphoscintigraphy, but the method was later

abandoned due to low sensitivity. Only two SNs were identified by blue dye, which

is remarkably low compared to the experiences in breast cancer. 19% of the lymph

node metastases were found in non-SN. The authors proposed that

macrometastases might have obstructed the flow to the true sentinel nodes.

Instead, the lymphatic flow was redirected, and detected as a false negative SN

(75).

Aljabery et al. injected intraoperative radiotracer and blue dye on 103 patients

with MIBC. 80% of the patients had SN detected with a sensitivity and specificity

for finding metastases of 67% and 90% respectively (76).

In a meta-analysis of eight studies (n=336) including the above-mentioned ones,

the pooled rate of detection and sensitivity were 91% and 89% respectively.

Interestingly, subgroup analysis showed reduced sensitivity rates for patients

with many pT3-pT4 tumors. This phenomenon is in line with SN detection in

other forms of cancer (77).

15

Adoptive immunotherapy

The sentinel node biopsy appears incomplete as a diagnostic tool for identifying

lymph node metastases in UBC. Instead, the technique is promising for

harvesting tumor-reactive lymphocytes for adoptive immunotherapy (78). In a

pilot study on 14 patients with UBC, the lymphocytic activity increased in SNs

after stimulation with autologous tumor extract (74). In a follow-up study, 12

patients with metastatic UBC had the lymph nodes that drained their metastatic

nodes, the so-called metinel nodes, extirpated. In vitro, T-cells from the metinel

nodes were cultured and expanded by autologous tumor extract and the

mitogenic signal IL-2 (79). After reinfusion of nine patients with expanded T-

cells, two patients showed objective radiological response with an overall survival

of 35 months and 11 months respectively (65). Adding NAC has demonstrated a

dose-dependent, immunostimulatory effect of T cells in SNs (80), a plausible way

to improve the therapy further.

Blood transfusions

Early attempts to transfuse blood originates from the 1600s. The first successful

transfusion was in 1838 by Dr James Blundel, yet the treatments were risky and

often led to death of the patient. The discovery of blood groups by Karl

Landsteiner led to safer transfusions and a Nobel Prize in the 1930s. With

anticoagulants and refrigeration, the blood could be stored for transfusion at a

later time, which laid the basis for blood banks. In the 1940s, blood fractionation

allowed separation of blood into three main compartments: plasma (55%), buffy

coat consisting of leukocytes and platelets (<1%) and erythrocytes (45%)(81).

Today a unit of donated blood is converted into different blood products. Packed

red blood cells (PBC) can be stored up to 35 days and are used for increasing the

oxygen-carrying capacity of the receiver (Figure 3). Fresh frozen plasma (FFP)

contains plasma proteins including coagulation factors, protein C and S,

fibrinogen and antithrombin. It is used for treating coagulations deficiencies and

for reversal of anticoagulant medication. Platelet transfusion is indicated for

thrombocytopenia after hemorrhage or as a prophylaxis in patients with <10

000/μL (82).

16

Figure 3 Image of bag with packed red cell concentrate (PBC). Credit: ICSident at German WikiWikipedia, CC BY-SA 3.0, https://commons.wikimedia.org/w/index.php?curid=29492562

Blood group systems

The transfusion of donated blood is termed allogeneic blood transfusion. Foreign

blood contains many elements that can be antigenic in the receiver such as

carbohydrate and protein structures on the surface of erythrocytes, thrombocytes

and leukocytes as well as plasma proteins themselves. Antibodies with affinity to

these antigens are called alloantibodies. Matching of blood is important in order

to prevent alloantibodies from attacking the allogeneic blood. Today, there are 36

blood group systems that categorize more than 350 antigens (82).

The first discovered and most important blood group system, AB0, classifies the

erythrocytes on the type of carbohydrate structure that is attached to their cell

surface. An individual with type A will carry anti-B antibodies and vice versa,

whereas an AB individual will carry neither anti-A nor anti-B antibodies. In type

0 individuals, there are neither A nor B structures on their cells, instead both anti-

A and anti B antibodies are present in their blood. In case a type A person would

receive type B blood, the anti-B antibodies in that individual would attack all

foreign erythrocytes carrying the B antigen and vice versa. Similarly, a type 0

could neither receive blood from A, B or AB since that individual carries both anti-

A and anti-B antibodies. Other blood group systems include Rh, Lewis, Kell,

Duffy, Kidd, I and MNS Ralston, Stuart and Penman (83).

The pretransfusion testing consists of determining the AB0 and Rh phenotype by

applying antibodies against A, B and D antigens. The alloantibody screen detects

antibodies against other erythrocyte antigens.

17

Adverse reactions

Despite pretransfusion testing, adverse reactions do occur after allogeneic blood

transfusion. Lethal outcome is rare and accounts for 0,3 cases per 100 000

transfusions. Mild reactions like fever and urticaria are seen in 3%.

Immune-mediated reactions are of different kinds. Acute hemolytic reactions

present with hypotension, tachypnea, tachycardia and fever. The condition

occurs when preformed antibodies destruct donor erythrocytes. A less dramatic

presentation is the delayed hemolytic reaction which arises when allogenic blood

induces the production of alloantibodies detectable 1-2 weeks post transfusion.

Febrile nonhemolytic reactions can appear when antibodies are directed against

donor leukocytes and HLA antigens. The most feared complication is Graft-

versus-host disease. Here, donor T lymphocytes recognize host HLA antigens as

alien and attack the host. Another, mostly fatal, complication is transfusion-

related acute lung injury (TRALI). Donor antibodies aggregate with host

leukocytes in the blood vessels of the lungs leading to increased capillary

permeability and pulmonary edema (82).

Infectious complications are rare. Blood donations are tested for HIV, hepatitis B

and C. Cytomegalovirus can be transmitted through infected donor leukocytes.

Receivers at risk such as immunosuppressed patients and neonates should

receive leukocyte-depleted blood products (83).

Anemia in the cancer patient

In 2005, the European Cancer Anaemia Survey (ECAS) enrolled more than 15

000 patients with solid and hematological malignancies of various stages and

treatments. The Patients were followed for up to 6 months. The prevalence of

anemia at enrollment was 39.3% (Hb < 12.0 g/dL) and 67.0% of the patients were

anemic at least once during the survey. Anemia was often seen after

chemotherapy, and increased with the number of cycles given (84).

In a study of MIBC, 53.3% of patients were anemic prior to RC, and NAC was an

independent predictor for preoperative anemia (85).

18

There can be many causes of anemia in cancer patients of which some are listed

below (86):

1. Anemia of chronic disease/inflammation ACD/AI.

2. Tumor bleeding (eg GI bleeding, hematuria).

3. Malnutrition (deficiency in iron, folate, B12)

4. Renal failure with defective erythropoietin production.

5. Bone marrow toxicity from chemo/radiotherapy

6. Bone marrow metastases

7. Autoimmune hemolytic anemia

In solid tumors, the most common etiology of anemia is that of chronic

disease/inflammation. In similarity with other chronic inflammatory conditions,

cancer can cause an increased activation of macrophages and T cells. These

immunocytes secrete cytokines like TNFα, IFN-γ and IL-1 that suppress the

erythropoiesis in the bone marrow and interfere with iron utilization. In addition,

the cytokines induce hepcidin production in the liver. This polypeptide hormone

blocks ferroportin in the enterocytes, so that iron is retained in the enterocytes

establishing a state of sequestrated anemia. Thus, it might be challenging to

differentiate ACD/AI from iron deficiency. High doses of intravenous iron have

been proposed as means to evade the hepcidin barricade (86).

Regardless of the etiology, the high prevalence of anemia in cancer patients make

them predisposed to receive allogeneic blood transfusion during the course of the

disease (87). Apart from the above described adverse reactions, there is evidence

that blood transfusions can affect long term oncological outcome; an effect known

as transfusion related immunomodulation (TRIM).

Transfusion related immunomodulation

In 1973 Opelz et al observed improved renal-allograft survival in patients that

received allogeneic blood (88). This beneficial immunomodulatory effect, later

referred to as transfusion related immunomodulation (TRIM) became utilized

by renal transplant units to increase graft tolerance. However, in 1981, concerns

were raised whether the immunosuppressive effect of blood could lead to tumor

progression in cancer patients (89). In the following years, many observational

studies saw an association between blood transfusions and increased cancer

recurrence, and also increased risk for postoperative bacterial infections (90). In

19

addition, a number of RCTs revealed increased mortality in patients receiving

leucocyte containing vs leukocyte depleted blood products in cardiac surgery (91).

Hence, donor leukocytes were suggested as an important mechanism in TRIM,

partly through the release of cytokines and inflammatory lipids (92).

In the late 1990s, blood products were leukocyte reduced as a routine in many

western countries. Febrile transfusion reactions and CMV transmissions

decreased. In vitro studies observed reduction in the proinflammatory cytokine

IL-1β, compared to non-leukocyte reduced blood (93), whereas levels of IL-6, IL-

10 and TNFα were repeatedly high (93, 94). Yet, both leukoreduced and non-

leukoreduced blood increase the level of T regulatory cells (95).

Prolonged blood storage has been linked to tumor growth in animal models.

Release of degraded lipid membrane-derived factors and raised tissue hypoxia

are plausible tumor promoting factors (96). In addition, storage of blood leads to

the deterioration and apoptosis of blood cells. Leukocytes and platelets release

the immunosuppressive TGF-β. Apoptotic cells present phosphatidylserine on

their surface, which attracts phagocytic cells like macrophages, that in turn

triggers release of TGF-β (97).

Moreover, the disruption of erythrocytes leads to the release of hemoglobin and

non-heme-bound iron. The cell-free hemoglobin binds nitric oxide, resulting in

vasoconstriction, hypertension, and inflammation. These factors have been

attributed to the increase in multi-organ failure and mortality seen after massive

transfusions (92).

Lastly, microparticles can have immunomodulatory effects. These extracellular

vesicles (EV) contain lipids, proteins and miRNA. They are constantly discarded

from all cells, a process that is exacerbated by the handling of blood ex vivo. The

effect of EVs on the immune system depends on many factors: cell origin of EV,

different methods of manipulation of blood products, storage time etc. A myriad

of effects on the immune system has been observed including neutrophil priming,

transfer of miRNA, expression of CD40L and the stimulation of T cells (98).

TRIM and clinical outcome

In 1981, Gantt et al raised the question whether blood transfusion could have a

negative impact on clinical outcome in cancer patients (89). Since then, a number

of studies, predominately of retrospective design, have addressed this specific

issue. Today, there are meta-analyses encompassing 5000-25,000 patients who

have investigated the impact of perioperative blood transfusion for patients

undergoing surgery for colorectal cancer (99), lung cancer (100), urinary bladder

cancer (101), and prostate cancer (102). They all show an increased risk of

mortality associated with allogeneic blood transfusion.

20

Whether autologous blood is safer is not completely clear. Intraoperative blood

salvage or cell salvage, is a medical procedure that allows reinfusion of blood

during surgery. It was speculated that this form of autotransfusion could

reintroduce malignant cells from the surgical removal of the primary tumor (103).

In 1993, 475 patients were randomized between autologous and allogeneic blood

transfusions in patients undergoing surgery for colorectal cancer. The risk of

recurrence increased after transfusions of both autologous and allogeneic blood

(104). In contrast, data from surgery of hepatocarcinoma concluded that

autologous blood transfusion does not increase recurrence of disease (105).

21

AIM OF THE THESIS

The aim of this thesis was to elucidate different clinical aspects of neoadjuvant

chemotherapy (NAC) on muscle-invasive bladder cancer (MIBC).

Paper I. 1. To clarify the role of tumor downstaging as a surrogate marker for

survival after NAC in MIBC.

Paper II. 2 To assess the feasibility of sentinel node detection in MIBC after NAC

and/or after completely downstaged (pT0) tumors post-NAC.

Paper III. 3. To assess the number of sentinel nodes in radical cystectomy for

MIBC as a prognostic marker in NAC-treated patients.

Paper IV. 4. To evaluate the extent of and the effects of blood transfusions

during NAC on pathoanatomical outcome and overall survival in patients with

MIBC undergoing radical cystectomy (RC) with curative intention (cT2-

T4aN0M0).

22

PATIENTS AND METHODS

Paper I

Cohort NCS1

In 1985-1989 the Nordic Cooperative Bladder Cancer Study Group included 325

patients from Sweden, Norway and Finland, who were randomized between +/-

NAC and standard care (low dose irradiation and RC) or standard care alone.

Patients with urothelial, squamous cell, and undifferentiated carcinoma of the

bladder, T1G3NxM0 and T2-T4aNxM0 were included. Staging included

cystoscopy, bimanual palpation, iv pyelography, and chest radiography. CT was

not obligatory. All patients received 4 Gy daily for five consecutive days targeting

the bladder, and lymph nodes in the iliac and obturator region. The

chemotherapy consisted of 2 cycles of 70mg/m2 cisplatin and 30mg/m2

doxorubicin. 311 patients were eligible of which 151 in the experimental arm and

160 in the control. 21 and 26 patients respectively were not cystectomized. In the

experimental arm, ten patients received no chemotherapy, whereas eight patients

received one course. 58 patients had T1 disease. At cystectomy, only enlarged

lymph nodes in the iliac and obturator regions were removed

Cohort NCS2

317 patients with T2-T4NXM0 were recruited between 1991-1997 from Sweden,

Norway and Finland. Squamous and adenocarcinoma of the bladder were

excluded. Staging encompassed TURB, bimanual palpation, and iv pyelography.

No preoperative radiation was used. The chemotherapy regimen comprised three

cycles of cisplatin 100mg/m2 and methotrexate 250mg/m2 given with a three-

week interval. 309 patients were eligible, 155 in the experimental arm and 154 in

the control. 14 patients received no chemotherapy, nine received one course cycle,

and 14 received two cycles. 23 patients were not cystectomized compared to 15 in

the control. Cystectomy included dissection of obturator and iliac nodes.

Method and statistics

The aim of the analysis was to compare clinical data and pathologic outcome in

patients with urinary bladder cancer cT2-T4aN0M0 from NCS1&2. Data was

retrieved from the Regional Oncologic Centre in Uppsala/Örebo (Regionalt

Onkologiskt Centrum Uppsala/Örebro region).

23

From the above described 311 eligible patients in NCS1, all 53 patients with T1

tumors, 5 patients with missing cT-stage, and 50 patients with missing pNx and

pTx were excluded, resulting in 203 patients for further analysis. From the 309

eligible patients in the NCS 2, one patient with cTx, and 62 patients with pNx/pTx

were excluded, resulting in 246 patients. A total of 449 patients remained for a

joint analysis. The experimental arms of NCS1&2 were grouped together (n=225)

and so were the control arms (n= 224).

The primary endpoint was the rate of pathologic downstaging, i.e. a pathologic

TNM lower than the clinical TNM. The secondary endpoint was overall survival,

measured from date of randomization to death or last follow-up. SPSS 19 was

used for statistical analyses. Chi-squared tests compared categorical data.

Differences in survival were plotted in Kaplan-Meier curves, and tested by log-

rank. A Cox-regression model was used to assess factors of importance for overall

survival. P≤ 0.05 was deemed significant.

Paper II and III

Patients

In 2013, a prospective non-randomized, multicenter trial was initiated at the

Urological Department, University Hospital of Umeå (Norrlands

universitetssjukhus) and the Department of Surgical and Perioperative

Sciences, Division of Urology and Andrology, Umeå University in cooperation

with the Department of Medicine, Unit of Translational Immunology,

Karolinska Institutet, Stockholm. Patients with suspected muscle-invasive

urinary bladder cancer were recruited from up to 10 different urological

centers in Sweden. Blood, urine, and specimens from macroscopically healthy

bladder and bladder tumors were collected at the time of the first TURB.

Patients were excluded if histopathology showed non-muscle invasive

urothelial bladder cancer, primary bladder cancer of non-urothelial origin,

benign tissue, patients unfit for radical cystectomy, logistical problems,

incurable cancer, robot-assisted cystectomy or prior BCG treatment. Based on

clinical routines at the recruiting center, patients were either selected for

neoadjuvant chemotherapy plus radical cystectomy, or radical cystectomy

alone.

Method and statistics

Sentinel node detection was performed on the day of cystectomy prior to

surgery. 1 ml of 80 MBq Technetium was deposited intravesically by

cystoscopy and a Williams injection needle (Cook Medical®), at four positions

around the primary tumor or around the resection scar in case of visual pT0.

24

The patient then proceeded for cystectomy in the same surgical session. After

the bladder specimen was removed, the lymph node dissection was

performed. All dissected lymph nodes were registered and probed by a Geiger

meter. Radioactivity was quantified in counts per minute (CPM).

Two definitions of SN were used: SN definition 1, (SNdef1): a lymph node of

10 CPM. SN definition 2 (SNdef2), the lymph node with the highest CPM (the

hottest) and with CPM 10% of the hottest node in each patient.

Paper II

For the analysis presented in paper II, a total of 99 patients were recruited

between May 2013 and December 2015 from six different centers. 34 were

excluded due any of the above-mentioned reasons of which 18 was due to

NIMBC. 65 patients remained for cystectomy and SNd of which 47 patients

were fit for NAC.

Paper III

The cohort of paper II was expanded. 230 patients with suspected MIBC were

recruited between May 2013 and December 2018 from 10 different centers.

114 were excluded of which the majority (n=56) was due to NIMBC. 116

patients were enrolled for RC and SND of which 83 patients were fit for NAC.

Only SNdef1 was used in the analysis.

Statistics

SPSS 23 was used for statistics in paper II and SPSS 25/26 for paper III. The

mean and median rates of true SN and false positive SN were compared using one

way ANOVA. Categorical variables were tested with chi-squared tests. A general

linear model (ANCOVA) was used for testing clinical factors (independent

variables) of value for SN outcome (dependent variable). In paper II, independent

variables were first tested univariately. Variables with a p-value <0.05 were then

tested together in a multivariate general linear mode. In paper III four clinical

variables were tested directly in a multivariate analysis (ANCOVA).

25

Paper IV

Patients, methods and statistics

All patients undergoing radical cystectomy for UBC in 2008-2014 were retrieved

from four urological centers in Sweden: the University Hospital of Umeå,

Sundsvall-Härnösand County Hospital, Gävle County Hospital and Västmanland

County Hospital (n= 372). Patients with muscle-invasive urothelial urinary

bladder cancer (cT2-T4aN0M0) that underwent NAC and RC (n= 120) were

included for further analysis. Medical records, including charts of red cell

concentrates administered from time of diagnosis until discharge from the

hospital after the cystectomy were collected. The patients were divided into two

groups: blood yes that received blood transfusions during NAC and blood no that

were blood naïve during NAC. Clinicopathological factors were tested using t-test

and Chi-square, whereas survival rates between the groups were compared using

Kaplan-Meier and Log Rank test. IBM SPSS statistics version 24 and 26 were

used.

26

RESULTS AND DISCUSSION

Tumor downstaging after neoadjuvant chemotherapy (Paper

I)

From the original 620 patients in NCS 1 & 2, 449 patients were eligible for the

retrospective analysis. The number of patients remained balanced between the

groups with 225 patients in the experimental arm, and 224 patients in the control.

The aim of the analysis was to compare the rate of tumor downstaging between

the arms, and to see if these findings were associated with survival benefits.

The notion that preoperative chemotherapy could impact pathologic outcome in

MIBC is not new. In 1988, a phase I trial on neoadjuvant chemotherapy on T2-4

tumors, showed that 33% of the cystectomized patients downstaged to pT0 (106).

Since then, numerous trials have presented pathological response rates between

14-38% after cisplatin based neoadjuvant chemotherapy (107). One of the more

recent and largest of these trials, the Intergroup 0080, randomized 307 eligible

patients between neoadjuvant MVAC+ RC versus RC alone. 38% of the patients

downstaged to pT0 in the NAC arm and 15% in the control (108). In a multicenter,

retrospective, real-world assessment of pathologic response rates, 22.7% were

pT0N0 after at least three cycles of NAC (109). In the same study, there were no

difference in downstaging rates between MVAC and GC.

Figure 4. Percentage of complete downstaging tumors (pT0N0) stratified by treatment arm and

clinical stage

27

In the present analysis, the rate of patients with completely downstaged tumors

(pT0N0) was almost doubled (22.7%) in the arm that received preoperative

chemotherapy compared to the controls (12.5%) (p=0.006). In patients with cT3

tumors, the difference was almost three-fold (Figure 4). In patients that

downstaged to a non-invasive state or less (pT0/pTis/pTaN0), the same pattern

emerged (not shown).

In survival analysis, patients were stratified by treatment arms and downstaging

group: 88.2% of the patients with complete downstaging tumors after NAC were

alive after 5 years. This translated into an absolute risk reduction of 31.1%

compared to downstaging tumors in the control arm (p=0.001). Furthermore, the

latter group retained no survival benefits compared to patients with non-

downstaging tumors in the controls (p= 0.550) nor in the experimental arm

(p=0.612) (Figure 5).

Figure 5. Kaplan-Meier curve plotting overall survival stratified by treatment arms and

pathoanatomical outcome. Downstaging tumor after NAC (blue). Downstaging tumor without NAC

(green). Non-downstaging tumor after NAC (yellow). Non-downstaging tumor without NAC (purple

28

In the multivariate analysis, the combination of downstaging and NAC was an

independent positive prognostic factor when downstaging was defined as pT0N0

(HR= 0.26), <pT1 (HR= 0.43) and organ confined disease (HR = 0.42). In the

original paper of 2012 we postulated that the increased downstaging of the

primary tumor after chemotherapy was translated into a survival benefit through

its efficacy on micrometastatic deposits. In line with this argument, we saw how

the discrepancy in downstaging and survival between the treatment arms was

most prominent for clinical T3 tumors (ARR=40.5%, p=0.023). In an autopsy

study of UBC it was clearly displayed how the frequency of metastases increased

with the increment in pT stage (110). Thus, it is very likely that locally, more

advanced tumors (i.e. cT3) would yield a higher rate of dissemination, and

thereby benefit more from systemic treatment compared to clinical T2 tumors.

Interestingly, our results deviate from Intergroup 0080 where pT0 patients in

both arms experienced an 85% survival rate at five years (108). In 2012, when our

results were published, we argued that one of the reasons for these discrepancies

could be that NCS1&2 represented a more homogenous group of patients derived

from a smaller recruitment area of 41 hospitals in three Nordic countries. In

contrast, patients in the Intergroup 0080 were enrolled over an 11-year period

and came from 126 institutions.

Moreover, there were different chemotherapeutic regimens. The Intergroup

0080 used 3 cycles of MVAC compared to two cycles of cisplatin/doxorubicin in

NCS1 and 3 cycles of cisplatin/methotrexate in NCS2. Yet, this cannot explain the

low survival rate of pT0 in the control arm of our analysis.

Plausibly, differences in preoperative staging could have a role here. In the

NCS1&2, patients were staged T1-T4aNxM0 preoperatively. CT of the bladder was

optional. In NCS1 only enlarged lymph nodes in the iliac and obturator regions

were removed, whereas dissection of obturator and iliac nodes were performed

in NCS2. In contrast, patients in the Intergroup 0080 were staged T2-4aN0M0.

Yet, how the preoperative staging was accomplished, was not accounted for in the

article, and the LND included bilateral pelvic lymph nodes (108).

This leaves us with the possibility that patients from the NCS cohort might

comprise unidentified lymph node metastases to a higher degree than in the

Intergroup 0080. In that case, patients with pT0 in the control group of NCS1

and to a certain degree in the NCS2 might encounter a worse survival due to

undiagnosed and untreated lymph node metastases. Conversely, undiagnosed

lymph metastases in the pT0 of the experimental arm would have benefited from

systemic treatment.

29

In the background section of this thesis, I reasoned that the clinical gain from

LND observed in retrospective studies is foremost an effect of improved

diagnostic staging in chemonaïve patients. It is plausible that many pT0 in NCS1

consisted of undiagnosed pN+. In that case, it is conceivable that NAC

compensates for potentially undiagnosed pN+ in NCS1, resulting in a larger

survival difference between downstaging patients in the experimental arm versus

the control.

Sentinel node detection after neoadjuvant chemotherapy

(Paper II)

65 patients were eligible for the intended analysis. 47 patients received NAC prior

to sentinel node detection (SNd) and RC and 18 patients underwent SNd and RC

alone. In accordance with the aim of the study, to assess the feasibility of SNd

after NAC and/or pT0, the cohort was divided into three groups: Group 1,

downstaging patients after NAC (n=24). Group 2, NAC patients without

downstaging (n=23). Group 3, No NAC patients (n=18).

The rate of downstaging tumors among the NAC treated was 51.1% compared to

the noNAC group 11.1%. This is congruence with reports using modern regimen

of chemotherapy (108). Patients in group 3 were older, and consisted of 50%

women compared to 20.8% and 17.4% in group 1 and 2 respectively. The mean

number of harvested lymph nodes were similar between the groups with an

average of 16.4. The total number of detected SNs were 222 according to SNdef1

and 227 according to SNdef2. The average number of SNs were 3.42 and 3.49

respectively, and when the average number was compared between the groups,

there was no statistical difference between groups for neither definition (p= 0.544

and p= 0.762). Radioactive specimen above the cut-off value, where the pathology

report showed no lymphatic tissue was labeled false positive (FP). The average FP

nodes were 0.51 and 0.46 for the two definitions, and no statistical difference was

recorded between the groups (p= 0.975, p=0.976).

30

Figure 6. Schematic illustration of detected sentinel nodes according to definition 1. The nodes were

scattered in the pelvic region in a similar fashion in the patient groups 1,2 and 3. Red arrows pointing

at red nodes indicate lymph node metastases.

In the whole cohort, only 8 patients (12.3%) had metastatic lymph nodes (n=22).

The majority of lymph node metastases (n=18) were found in non-SNs,

equivalent to a false negative rate (FNR) of 82%. Three patients had metastases

in SNs (one from group 2 and two from group 3) (Figure 6). One patient (group

3), had metastases in both SN and non-SN. In the remaining five patients (group

2), all metastases were found in non-SN.

For comparison, the studies on SNd in chemonaïve UBC patients previously

accounted for, consisted of a higher rate of metastatic lymph nodes (30-43%) but

with a lower rate of false negative SNs (0-20%) (75, 76).

When sentinel node biopsy was introduced in breast cancer the false negative

rates were around 5% or below (111). The implementation of the technique in the

broader surgical community has resulted in rates between 8-11% (111). The effect

of NAC on SN biopsies in breast cancer was systematically investigated in the

SENTINA trial. Here, more than 2000 patients were included in a prospective,

multicenter study (112). In the arm of chemonaïve patients, the detection rate was

99.1% whereas patients that first underwent NAC had a detection rate of 80.1%

and a false negative rate of 14.2% (112). In one arm, patients underwent SN

biopsies twice, before and after NAC. Less than two –thirds of the SN were

detected after NAC, and the false-negative rate was 51.6%. The number of the

lymph node metastases did not impact the detection rates, which made the

authors suggest that factors like fibrosis, and blockage of lymphatic flow were the

main factors to impair SNd after NAC (112).

The SENTINA trial is contrasted by the ACOSOG trial. Here 649 patients with

lymph node metastatic breast cancer received chemotherapy prior to SN biopsies

31

and axillary LND. The primary endpoint was FNR, which was 12.6% for the whole

cohort (113).

Despite solid data pointing at impaired SNd after NAC in breast cancer, the

present study showed no statistical difference in the number of detected SN

between the groups with an average of 3.42 per patient. In multivariate analysis,

the most important factor for SN yield was the number of harvested nodes. In

contrast, the Liedberg cohort of mainly chemonaïve patients, had a mean number

of 2.4 SNs, whereas the mean number of dissected nodes were 40 per patient (75).

The combination of low number of harvested lymph nodes and usage of NAC

might explain the low rate of detected lymph node metastasis in the present

cohort compared to Liedberg and others (75, 76). Likewise, the high rate of false

negative nodes could be attributed to NAC. It is tempting to speculate that

patients with lymph node metastases after NAC represent a distilled subgroup of

patients with more advanced disease resistant to chemotherapy. Presumably,

these patients retain chemoresistant tumor cells that obstruct and redirect the

lymphatic flow.

Nevertheless, the high false negative rate of SNs after NAC in breast cancer is

more concerning than in UBC. In breast cancer, SNB is a diagnostic tool. A missed

lymph node metastasis would lead to understaging and undertreatment due to

lack of axillary node dissection in that particular patient. In UBC, however, SNB

is not sufficient to detect all lymph node metastasis even in chemo naïve patients.

Rather, we believe the technique is valuable for identifying tumor reactive

immunocytes suitable for adoptive immunotherapy.

32

Fewer tumor draining sentinel nodes in patients with

progressing muscle invasive bladder cancer, after neoadjuvant

chemotherapy and radical cystectomy (paper III)

In paper II we investigated the feasibility of performing SNd in NAC treated

patients with MIBC. In paper III, we expanded the cohort to see if the number of

SNs were associated with pathoanatomical outcome. 230 patients were recruited

retrospectively, of which 116 were eligible for analysis. 83 patients received NAC

and were stratified by pathoanatomical outcome into Complete Responders

(pT0N0M0), Stable Disease (pTisT4aN0M0) and Progressive Disease

(pTanyN+M+). 33 NAC-naive patients were designated as no-NAC, and kept for

comparison (Figure 7).

Figure 7. Flow chart of patient inclusions in paper III. Patients were stratified into different groups

based on treatment (NAC vs noNAC) and response to NAC (CR, SD and PD).

There was no statistical difference in the distribution of age, gender or amount of

NAC between the subgroups. However, there were less advanced clinically staged

tumors in the subgroup of complete responders compared to the other subgroups

(p= 0.04) preoperatively.

The proportion of complete responders (CR) was 43.4% which is in line with

others (107, 108).

The mean number of harvested lymph nodes were 17.1, 16.3 and 13.5 in CR, stable

disease (SD) and progressive disease (PD) respectively (p=0.5). Interestingly, the

33

mean number of SNs was less than half in PD (1.4) compared to the two other

groups (p=0.034). Furthermore, the detection rate of SN, i.e. a true radioactive

lymph node, was found in 91.7% of the patients in CR compared to 58.3% in PD.

The mean number of false positive nodes were similar between the groups

(p=0.36). However, in multivariate analysis the most important factor for SN

yield was the number of harvested nodes (p=0.0004).

Could the reduced number of SN in the more advanced and plausibly

chemoresistant tumors reflect a less functioning lymphatic system? On the one

hand, tumors secrete factors that stimulate lymphangiogenesis (58). In fact,

lymph vessel density has been coupled to more advanced disease (56). Yet, as

tumors progress, there is a more extensive shedding of tumor cells into the

lymphatics, ultimately leading to the establishment of macrometastatic colonies

in the regional lymph nodes (110). The sensitivity of finding metastases in SNs is

reduced in more advanced bladder tumors (77). Conceivably, this is due to

obstructed lymphatic flow as was proposed by others (75) and most probably

observed in paper II, where the majority of metastatic lymph nodes were found

in non-SNs (114).

In addition, tumor lymphatics are oftentimes dysfunctional (115). Aberrant

lymph vessels seem to shield the tumor from attacking immune cells so that it can

maintain a tumor protective microenvironment (116). Hereby, we hypothesize

that the number of SNs detected could be an indicator of tumor lymphatic

function, and in a broader sense an indirect marker of an individual anatomical

lymphatic and tumor immunological constitution of each patient.

The association between the SN number and tumor stage is intriguing but should

be interpreted with caution for several reasons:

1) The analysis was performed retrospectively, based on post-hoc defined patient

groups. 2) The cohort of 116 patients was recruited from 10 urological centers,

where the LND was defined yet never controlled for. 3) The time between

peritumoral injections of the radioactive tracer and the SN detection depend on

many factors. In theory, logistics, surgical experience, anesthetic problems,

patient body compositions and local tumor growth might have influenced

operation time and thus delayed SNd, permitting the tracer to recede. 4) A larger

primary tumor would imply a technically more difficult injection of the tracer. 5)

The registration of the radioactivity with the handheld Geiger meter comes with

a certain degree of unpredictability. The reading can be influenced by the position

of the Geiger meter in relation to the tissue etc.

Despite the abovementioned caveats, it is a noteworthy observation that

aggressive tumors are associated with fewer sentinel nodes in our material.

34

Blood transfusion during neoadjuvant chemotherapy for

muscle-invasive urinary bladder cancer may have a negative

impact on overall survival (Paper IV)

The aim of paper IV was to map the extent of blood transfusion during

neoadjuvant chemotherapy, and to see if patients that received blood were

associated with a worse pathological and clinical outcome.

The definitive cohort consisted of 120 patients with curable muscle-invasive

urothelial bladder cancer (T2-T4aN0M0) that underwent 1-4 cycles of

neoadjuvant chemotherapy and radical cystectomy. One-third of the cohort

(n=40) were given red cell concentrate (RBC) during the course of chemotherapy

and were referred to as blood yes. The remaining 80 patients were blood naïve

from diagnosis until surgery, and were referred to as blood no. Interestingly, the

majority of patients in the whole cohort (87.5%) received perioperative blood

(PBT) and there was no statistical difference in perioperative transfusion between

the groups (p=0.079).

The two groups were similar in age, comorbidity, no of NAC-cycles and cTNM,

albeit the proportion of cT4a tumors were more than doubled in Blood yes

(p=0.314). Other differences included the percentage of women that were more

than doubled in Blood yes (p=0.033) and the hemoglobin level at time of

diagnosis that was significantly lower in this group (p=0.0001).

There was a pronounced difference in pathological outcome. Patients in the Blood

yes group had proportionally half as many pT0 tumors but more than ten times

the proportion of pT4b tumors (p=0.044). Similarly, the percentage of patients

with lymph node metastases or unknown lymph node status (pNx) were

significantly higher in the Blood yes group (p=0.007).

Survival curves revealed a 19,2% absolute risk increase at five years of

observation, for patients in the Blood yes (Figure 8). In multivariate analysis,

advanced pT stage (p<0.0001) and pNx (p=0.002) were independent markers for

unfavorable prognosis.

35

Figure 8. Kaplan Meier curve of overall survival stratified by blood transfusion status during NAC.

No blood transfusions (blue) and blood transfused (red).

This is the first time that the extent and the plausible implications of blood

transfusions during neoadjuvant chemotherapy in MIBC are investigated. As

reviewed in the introduction of this thesis, the immunomodulatory effect of

allogeneic blood has been recognized since the 1970s. This phenomenon, also

known as transfusion related immunomodulation (TRIM), has been assessed

both on a molecular as well as a clinical level.

In colorectal cancer, a Cochrane review of 36 studies including seven RCTs,

pooled data revealed an OR of 1.42 for cancer recurrence after PBT. In 14 of the

studies, PBT was an independent risk factor for recurrence (117).

In UBC, the most recent meta-analysis by Cata et al in 2016, analyzed eight

retrospective studies with more than 15 000 patients. Patients transfused within

one month before or after radical cystectomy, were associated with a 27%

reduction in overall survival (101). Similarly, large meta-analyses from colon

cancer (99), lung cancer (100), and prostate cancer (102) have shown negative

oncological outcomes associated with allogeneic blood transfusions.

36

In the current analysis, there were no statistical differences in age, clinical tumor

stage, comorbidity or amount of chemotherapy prior to surgery between the

groups. However, the fact that one third of the cohort was transfused

preoperatively may signal a more advanced tumor stage underlying these

patients, than was demonstrated clinically and could be distinguished

statistically.

The similarity in age distribution is perhaps not surprising considering the fact

that the age-limit is 75 years to receive neoadjuvant chemotherapy. More

importantly, the percentage of women was significantly higher in the Blood yes.

As discussed in the introduction of this thesis, women with UBC tend to have

worse prognosis than men. Although the exact mechanism is not understood, this

might have contributed to the difference in clinical outcome.

In the multivariate analysis of the present study, advanced pTstage (>pT2) and

pNx were independent risk factors for overall survival. The fact that pNx and not

pN+ turned out as a significant risk factor warrants alertness. In the 12 patients

where lymph node dissections were lacking, eight belonged to the blood yes

group, i.e. 20% of the participants compared to 5% in the blood no group. The

reasons were local spread (n=2), extensive fibrosis (n=5) and unknown (n=5). We

interpreted the pNx as a surrogate marker for regional spread of disease.

The core question remains, is the distinct difference in pathological outcome a

consequence of the administered blood and its proposed TRIM effects or rather

an example of confounding by skewed inclusion?

Plausbibly, the immunosuppressive effect of the allogeneic blood might have

prevented full treatment effect of NAC. Apart from the direct toxic effect of the

chemotherapy on the tumor, cell death triggers release of intracellular danger

signals that alert the immune system and elicits an antitumoral response. In this

way, an immunologically “cold” tumor, i.e. without infiltrating lymofocytes, can

become “hot” after chemotherapy, surgery or radiation (118).

Beside immunosuppression, there are other factors that could promote

tumorigenesis after blood transfusion. As accounted for previously,

microparticles (MP) are constantly shed into the bloodstream (119), and the

formation is accentuated when blood is processed ex-vivo. For instance, platelet

derived microparticles (PMP), can contain growth factors like VEGF and PDGF

that fuel blood vessel and lymph vessel formation. In experimental models the

exposure to PMP resulted in tumor growth in lung, prostate and breast cancer

(92).

37

Despite interesting mechanistic concepts on the tumor enhancing effect of

allogeneic blood, the causality, i.e. the order in which blood affects tumor

outcome, and not the reverse is only speculative. Retrospective analyses can be

confounded by many factors including nutritional and functional status of the

patient, tumor stage, tumor manipulation and resectability, method of

anesthesia, use of opioids, hyperglycemia and hyperthermia (96).

The results of this small, retrospective study with a limited follow up time should

be interpreted with caution. The detrimental effect of blood transfusion shown by

others is partly blurred in the current study by the large number of patients that

received PBT in both groups: 83% in the blood no and 95% in the NAC blood yes.

These transfusion rates are extraordinarily high compared to the average

transfusions rates (38%) in other studies (101). Still, in this small cohort we study

a relatively fit and selected group of patients who all qualified for chemotherapy

prior to surgery. The results are merely an observation, yet a rather interesting

one that we believe deserves attention.

38

CONCLUSION AND PERSPECTIVES

Localized muscle-invasive bladder cancer is a lethal condition. After decades of

randomized trials, the uro-oncologic community has appreciated the use of

neoadjuvant cisplatin based chemotherapy to improve survival after radical

cystectomy. The aim of this thesis has been to elaborate on a number of aspects

related to the use of chemotherapy in the neoadjuvant setting.

First, we observed distinct pathoanatomical differences after preoperative

chemotherapy. Tumor downstaging was far more prevalent, and appeared to be

a marker for responsiveness to chemotherapy and long-term survival. Yet, the

question lingers: how do we make use of this finding in our daily clinical practice?

Early attempts to perform post chemotherapy assessment with TURB or imaging

techniques, understaged patients in 38% of the cases (106). Post-treatment

inflammation and fibrosis made prediction of complete pathological response

difficult to discern. Thus, cystectomy has remained gold standard despite

preoperative indications of response to chemotherapy. However, imaging

techniques are becoming more sophisticated. A dynamic contrast enhanced MRI

(DCE MRI) appears promising in differentiating the post-treatment effect from

residual tumor mass (120). Moreover, complete downstaging has been proposed

as a valuable marker for accelerated drug development (121) and useful in real-

world analysis to compare GC and MVAC efficacy (109).

Next, we explored the concept of tumor draining lymph nodes. Urinary bladder

cancer metastasizes to the lymphatic system at an early point. Yet, attempts to

use sentinel node detection for identifying lymph node metastases, have revealed

inconsistencies. Instead, sentinel nodes in UBC contain reactive immunocytes

useful for adoptive immunotherapy. The immunostimulatory effect of

chemotherapy makes the combination of NAC and adoptive immunotherapy

particularly appealing. In paper II we demonstrated that the number of detected

SNs was not affected by NAC nor tumor downstage, albeit an increase in false

negative rates were observed.

Hereafter, the cohort from paper II was enlarged. Interestingly, a reduced

number of SNs was observed in patients with disease progression. The finding

left us speculating: perhaps the number of SNs could be a feasible way to

determine lymphatic function? Could an intact lymphatic system, i.e. open

communication between the primary tumor and draining lymph nodes, be a good

therapeutic marker for immunomodulating drugs, such as checkpoint inhibitors?

39

Lastly, we wanted to estimate the impact of blood transfusions during

neoadjuvant chemotherapy. Anemia is common in the cancer patient and can

have many etiologies. Despite several treatment options like iron substitution and

erythropoiesis stimulating agents, clinicians often choose allogeneic blood

transfusions to treat anemia. It seems like many medical doctors are not aware of

the accumulating data that have linked blood transfusions with unfavorable long-

term outcomes after cancer surgery.

In the fourth study of this thesis, we observed more advanced tumors and worse

overall survival in patients that underwent blood transfusions during the

neoadjuvant chemotherapy. Although one must be careful with the conclusions

from this small study, the accumulating data on this topic proclaims a more

thoughtful approach to anemia and its management in the cancer patient.

40

ACKNOWLEDGEMENTS

This thesis is the result of many hard-working people including clinicians,

biologists, statisticians, teachers and staff members from laboratories, hospitals

and academic centres. I would like to extend my gratitude to a few persons in

particular:

Amir Sherif, my principal supervisor. After we met for the first time, 12 years ago,

my life took a new direction. You introduced me to bladder cancer as a research

field and urology as a clinical profession. Your working stamina is immense. Your

energy can charge my mental batteries again and again. If not for you, this or

something alike would never have happened.

Ola Winqvist, my co-supervisor. I am jealous of your intellect. When you talk on

tumor immunology, nothing feels more important. Thank you for all the valuable

feedback I received throughout the years.

Per Marits, my co-supervisor. It has been a privilege to have your support.

Börje Ljungberg for his assistance with my registration as PhD-student at Umeå

university.

Christina Ljungberg and the staff of the Department of Surgical and Perioperative

sciences, Division of Urology and Andrology.

Per-Uno Malmström. The support from someone senior and still active in bladder

cancer research means a lot.

The Nordic Cooperative Bladder Cancer Study Group, for granting me access to

invaluable data.

Julia Alvaes, co-author on paper III. Although we never met in person, it was a

pleasure to work with you. I am grateful for your many thoughtful insights.

Gabriella Lorentzi, co-author on paper IV. Only a few times have we met in

person, yet we had many intense and fruitful moments in the ether. Thank you!

David Krantz, one of my dearest friends with whom I share so many memories.

We have pushed and pulled each other, in and out from childhood, high school,

travels, medical school and research. Thank you for seeing the world differently,

it is both nurturing and challenging.

41

Malin Winerdal, former co-worker in the KI lab with deep knowledge on

immunology. Thank you for sharing your fine illustrations.

Co-workers in the KI lab. You delivered breakthrough research and kept a good

spirit despite long working hours and noisy machines: Thank you Ali Zirakzadeh,

Ciptura Adijaya Hartana, Michael Mints and Emma Ahlén Bergman.

Marcus Thuresson, statistician at Statisticon AB who assisted us in all our papers.

Anna-Maria Eriksson, Umeå university, for your good will, help and support.

The organizers of NatiOn IV, Nationell forskarskola i klinisk och translationell

cancerforskning: Ingemar Ernberg, Dan Grandér, Svetlana Bajalica Lagercrantz,

Daria Glaessgen, Ulrika Segersten, Karin Lindberg. The research school you

organized, blended interesting courses with tickling discussions that truly

developed me as a researcher.

Collaborators in all the hospitals and academic centers who provided us with

research material, logistics and thoughtful reflections: Erkki Rintala, Rolf

Wahlqvist, Anders Ullén, Sten Nilsson, Markus Johansson, Alexander Sidiki,

Benny Holmström, Farhood Alamdari, Marwan Shareef, Johan Hansson, Janos

Vasko, Tomasz Jakubczyk, Tammer Hemdan, Firas Aljabery, Ylva Huge, Katrine

Riklund, Maryam Bahar, Danna Asad, Ramona Forsman, Anders Bergh. Thank

you for invaluable resources.

Colleagues and staff at the Department of Urology, Södersjukhuset. You endorsed

my research and taught me urology as a profession. Thank you Magnus

Wijkström, Tim Fagerström, Jesper Rosvall, Ulf Norming, Ulrika Westlund,

Elisabeth Farrelly, Karin Falkman, Hans Thorsson, Ann-Helén Sherman Plogell,

Thomas Hopfgarten, Firas Tarish, Susanne Hagedorn, Malin Nyberg Isacson,

Mats Hedlund, Stefan Anania, Emma Andersson, Camilla Malm, Per Nordlund,

Abdulghafour Halawani, Yashar Khoshkar, Ziga-Maria Johansson, Pia Simonsen

and many others.

A warm thanks to all my fantastic friends. What a luxury to have you close. You

calibrate me, ensuring me a multi-dimensional life.

My dear family. Alicja och Freddy, you have indulged me with infinite quantities

of love and support. You have laid the foundation for what is me. Paula, Gustaf,

Stella, Ruben and Elle: Thank you for your enormous hospitality. With you I

always feel at ease.

42

To my extended family: My, Per, Louise, Casper, Elisabeth, John, Gustaf, Lotta,

Maxine, Fabian, Casimir: Thank you for creating a vibrant atmosphere where I

always feel welcome, in particular at Östanå, a place where I have written large

portions of this thesis.

My children Sasha, Noah and their little brother, who came to this world as I was

writing the last finishing words of this thesis. Everyday I long for our meals, our

talks, and our time together. You make sure my priorities in life are right.

At last, Béatrice, Bisse, Bitsky: my precious love. You add so much meaning and

flavour to my life. You see things I easily miss. Thank you for the enormous

support you have given me from the very start of this thesis to the intense and

thrilling end.

43

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