Paper - Review

DOI: 10.1016/j.ccr.2010.11.014

Summary

(engraft properties & impact)
← on patient outcome of 50 pediatric ALL (acute lymphoblastic leukemia)
← which transplant into NOD/SCID mice

TTL
← Time to Leukemia
→ was determined → for each patient sample engrafted

Short TTL
→ high risk for early relapse
→ identifying → an independent prognostic factor

This high-risk phenotype
→ is reflected ← by (a gene signature) ← upon validation in (an independent patient cohort)
→ involved in (cell growth) & (apoptosis)

∴ This pathways → can directly targeted

Introduction

ALL
← Acute Lymphoblastic Leukemia
→ is the most frequent malignant disease ← in children and adolescents

(Multi-agent chemotherapy regimens) & (support cares)
→ have led → to improvement of 1⃣ remission induction 2⃣ long-term survival

Relapse
← in 10% of all patients
→ occurs at early time points
→ associated with a substantially reduced survival of 5%

Leukemia cells
→ clearance ← in response → to steroid treatment
→ has been (a prognostic marker)

Detection ← of residual leukemia cells
← after remission induction therapy
→ qualifies for HR treatment

∴ Early identification ← of patients
← with high risk of relapse
→ led to improved outcome

Leukemia cells cannot ❌ sufficiently be cultured
→ xenograft models can overcome these limitations

Results

NOD/SCID/huALL

Leukemia cells isolated
← from diagnostic samples of 50 patients
→ were transplanted onto NOD/SCID mice

Leukemia was confirmed
← by detection of 1⃣ a high percentage of leukemia cells 2⃣ infiltration of (bone marrow & spleen)
Engraftment of leukemia
→ was also monitored ← in different organ compartments

Time to Leukemia

Observed → a prominent difference
← in time elapsing from transplantation → to manifestation of leukemia

NO ❌ onset of leukemia
→was observed ← between 9 and 12 weeks ← after transplant
∴ Distinct (engraftment phenotypes) (← of different leukemia cell samples)

TTL
← Time to Leukemia
→ quantified for each leukemia sample

Threshold of 10 weeks: TTL short & TTL long

Leukemia characteristics
→ were tested → to divide the cohort
← 1⃣ ALL-BFM high-risk stratificaiton
2⃣ TEL/AML1 fusion
3⃣ prednisone-poor response
4⃣ age
5⃣ WBC at diagnosis

(Time to overt leukemia) & (Relapse-free survival)
→ compared ← between the subgroups divided

Resulting P-values ← were adjusted ← for multiplicity

21 / 50 samples
→ did NOT ❌ expand in vivo
→ to substantial cell numbers

Stability, Consistency, and Independency of the TTL Phenotype

TTL was ← in all cases assessed
← after transplantation of 10^7 of primary patient leukemia cells

TTL was assessed
→ for both recipients of each leukemia sample

Similar TTL values ← were observed
← within the replicates of 21 primary leukemia samples
← for the replicates of the relapse samples
← comparing diagnosis-relapse pairs

Analyzed → the consistency of TTL
← in subsequent recipients
(Leukemia cells ← of diagnostic patient) & (Consecutive xenograft passages)
→ were transplanted
← in parallel onto secondary & tertiary recipient
∴ TTL phenotypes
→ did NOT ❌ change
→ remained TTL-short & TTL-long ← between diagnosis and consecutive passage

Representative xenograft samples
← for TTL-long & TTL-short
→ were re-transplanted → onto primary & tertiary & 6th recipients

Re-capitulated the TTL phenotype (← of the respective leukemia)
→ remaining stable ← after up to 5 passages

TTL and Patient Outcome

6 → TTL-short
44 → TTL-long

Analyzed → relapse-free survival of the patients
← whose leukemia cells → were used for transplant

Patients
← whose transplanted cell showed a TTL-long phenotype
→ revealed a marked superior relapse-free survival

∴ TTL-short phenotypes is characteristic
← for patients ← at high risk ← for early relapse

Analyzed → whether TTL identifies patients ← who would encounter (an early relapse)
∵ the majority of (relapse patients) ← originates from the low-risk groups
∴ The analysis was focused → on the non-high risk group patients

∴ TTL is associated ← with patient outcome independent of (risk stratification)

21 leukemia samples
→ did NOT ❌ lead to leukemia ← within the observation time
∴ included into the TTL-long group

Group of patients
← whose leukemia cells upon transplant
→ did NOT ❌ lead → to leukemia ← within 20 weeks of observation

A superior relapse-free survival was observed
→ only when patient samples leading to overt leukemia

TTL & (remission duration)
→ have a significant correlation

Prognostic Impact of TTL

Different prognostic factors → have been implemented
← in different treatment protocols
→ to stratify patients ← based on individual risk

Compared → (the prognostic impact) (← of TTL)
→ to established prognostic factors

Patients ← with TTL-short
→ exhibited a 45-fold increased risk → for relapse
→ No ❌ significant elevated risk ← for patients with PPR

TTL-short → defined by the 10-week cutoff
→ was the most significant prognostic factor
→ in comparison to additional thresholds → to determine TTL

Genetic alternations
→ are common ← in pediatric ALL
→ might be associated ← with (favorable & unfavorable) outcome
∴ Genetic alternations → are used for treatment stratification

All 19 patients
← who are positive for TEL/AML1
→ showed TTL-long

This patients
→ was high-risk stratified
∵ PPR and showed TTL-long

TTL is Confirmed in an Independent Leukemia Subset

Independent subset (← of cell bank BCP-ALL samples)
→ to evaluate (the significance of the TTL)

TTL → was significantly associated
← with the time ← (from diagnosis to relapse)

Gene Expression Analysis of Xenograft Leukemia

Gene expression profiles → were analyzed
→ using a human whole-genome array approach
→ to gain insight into (molecular mechanisms) (← which responsible for distinct in vivo leukemia proliferation)

ALL cells isolated
← from leukemia-bearing recipients
→ were investigated

Cytogenetic abnormalities
← 1⃣ leading to fusion genes 2⃣ involving transcription factors
→ are recognized ← by specific (transcription profiles)
→ might overcast other differences ← in gene regulation

∴ The analysis
→ has been focused ← on xenografts
← which derived from patients ← without gene fusions

The expression profiles → were analyzed
← employing a model-free shrinkage estimate of the variance
← across genes ranking the data set

2 genes ← which involved in regulation of mammalian target of rapamycin signaling
→ were identified
→ 1⃣ DDIT4L 2⃣ RHEB

2 genes coding
← for molecules involved in regulation apoptosis
→ were identified
→ 1⃣ PDE4A 2⃣ DAPK1

Differential Regulation of Identified Genes in Xenograft ALL

Differential regulation of transcripts
→ identified ← in gene array analysis
← by quantitative RT-PCR

Application of the Xenograft Signature on an Independent Patient Cohort

TTL signature was applied
→ onto profiles of (an independent cohort of pediatric patients) ← with BCP-ALL
→ to corroborate → the relevance of (the specific expression profile) ← identified in xenograft leukemia samples

This independent set ←of patient profiles
→ was analyzed
← with respect to the best distinction into subgroups

A Classifier Based on the TTL Signature Identifies Patients with Early Relapse

(TTL-short & Corresponding expression signature)
→ are strongly associated ← with early-relapse leukemia

A classifier → could be obtained
→ to identify patients ← with early relapse

The set → was refined
→ to further improve the robustness

Cell Death is Predominantly Induced in TTL-short but Not TTL-long Leukemia Cells

Inhibition of (mTOR & PDE4A)
→ might successfully target TTL-short/early-relapse leukemia

Xenograft ALL samples → were treated
Cell death → was analyzed

Data ← on primary pediatric ALL
→ is showing (an effect of both inhibitors & predominantly) ← on TTL-short leukemias samples

Discussion

Identified →
1⃣ in vivo proliferation ←of de novo BCP-ALL cells ← which transplanted into NOD?SCID mice
2⃣ predictive value → for patients outcome

TTL-short is found in
← patients with poor prognosis

A specific gene signature
← which identifying patients with early relapse
← which pointing to pathways regulating (cell proliferation & apoptosis)

Leukemia cells
← which engrafted in out model of NOD/SCID/huALL
→ retained the (immuno-phenotypic & clinical characteristic) (← of the primary leukemia)

A most significant correlation
← of 1⃣ in vivo leukemia growth 2⃣ overall patient outcome

1⃣ pretreatment characteristic 2⃣ initial treatment response
→ were NOT ❌ significantly associated ← with increased relapse risk

Patients
← with TTL-short
→ are NOT ❌ characterized ← by poor treatment response

Engraftment properfies
→ are indeed intrinsic → to the leukemia cell itself
→ are retained independently of the cell number transplanted

Identified → a specific TTL-gene profiles
← with 1⃣ robust clustering 2⃣ inferior relapse-free survival of the TTL-short signature group

Activated pathways ← associated with poor outcome
→ indicating additional therapeutic targets

Leukemias ← which showing down-regulated DDIT4L
→ might lack repression of mTOR

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