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VU Research Portal

Targetable genetic signatures of immune evasion in lymphoma

Roemer, M.G.M.

2017

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Roemer, M. G. M. (2017). Targetable genetic signatures of immune evasion in lymphoma.

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Chapter

8

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8. Discussion

In this thesis, specific genetic features of select LBCL subtypes, including PCNSL, PTL, PMBL and TCRBCL, and HL, were studied, with an emphasis on targetable genetic bases of immune evasion. In this discussion, various techniques to identify genetic alterations in these different LBCL subtypes are described, and advantages and disadvantages highlighted. The results of the different studies are summarized and discussed and their clinical significance is elucidated.

8.1. Targetable genetic features of LBCLs

In chapter 21, we comprehensively characterized recurrent CNAs and associated

putative driver genes, mutations and chromosomal rearrangements in PCNSLs, PTLs and PMBLs and compared them to a previously published dataset of primary DLBCLs.1,2 The unique combinations of genetic features in different LBCL subtypes are summarized in Table 1 and discussed in the sections below. Identification of complementary genetic alterations in PCNSL and PTL required concurrent analyses of mutations, CNAs and associated driver genes and chromosomal rearrangements. Using targeted approaches, such as FISH, qPCR and Sanger sequencing, the ability to simultaneously detect multiple contributing genetic alterations is limited and prior knowledge is required.

All ABC-type Genomic instability

CDKN2Aloss 24% (43/180)* 35% (19/55)* 88% (44/50)0 71% (15/21)0 0% (0/11)0 bi-allelic 19% (8/43)* 26% (5/19)* 77% (34/44)0 73% (11/15)0 0% (0/11)0

CNAs of additional p53/cell cycle

components multiple* multiple* no0 rare0 no0

Total CNAs high high high high low

Oncogenic TLR and BCR signaling

MYD88L265P 12% (6/49)29% (45/155)78% (38/49)0 60% (33/55)0 NA0

NFKBIZgain 9% (16/180)* 20% (11/55)* 42% (21/50)0 45% (28/62)0 0% (0/11)0 NFKBIZgain and/or MYD88L265P NA NA 92% (45/49) 83% (44/53) NA

CD79BY196mut

Total 16% (8/49)† 23% (35/155)‡ 49% (22/45)0 38% (19/50)0 NA0 Concurrent with MYD88L265P 38% (3/8)43% (15/35)91% (20/22)0 89% (17/19)0 NA0 PD-1 ligand deregulation

9p24.1/PD-L1gain and/or PD-L2gain 6% (11/180)* 7% (4/55)* 54% (26/50)0 52% (33/63)0 55% (6/11)0

PD-L1 or PD-L2 translocation NA0 NA0 4% (2/50)0 6% (4/66)0 20% (25/125)§ * Chapuy & Monti et al. Cancer Cell 2012

† Lohr et al. PNAS 2012 ‡ Ngo et al. Nature 2011

DLBCL

PTL0 EBV- PCNSL PMBL0

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Advances in technologies have opened the debate about the clinical use of comprehensive sequencing-based approaches.3 However, comprehensive sequencing-based approaches come with increased costs and additional challenges including high computational demands and generation of massive amounts of data. Low tumor purity is also an issue in some of the lymphoma subtypes, particularly those with an extensive infiltrate of normal inflammatory/immune cells (as in cHL, see section 8.3.).

8.1.1. Paucity of CNAs in PMBL

In comparison to PCNSL, PTL and DLBCLs, PMBL had an overall paucity of CNAs (Table 1). The only two recurrent CN alterations were chromosome 2p16.1 and 9p24.1 gain, as previously described.4-7 In contrast to other LBCL subtypes, PMBLs are thought to arise from Activation-Induced Deaminase (AID)-expressing, medullar, thymic B-cells.8-12 AID initiates SHM and CSR by deaminating cytidines on DNA.13-15 AID-dependent mistakes in these processes contribute to the pathogenesis of B-cell lymphomas.16 Malignant B-cells in PMBL have rearranged immunoglobulin genes and a high load of SHM, indicative of exposure to the GC reaction.12,17,18 However, the CSR rate might be lower in PMBLs, possibly due to differences in accessibility of the DNA.17,19 Although more precise identification of the malignant B-cells and the mechanism of reduced CSR in PMBL are warranted, this reduced CSR rate might result in fewer double strand breaks, resulting in a lower likelihood to acquire CNAs. An additional line of evidence in support of increased genomic stability in PMBLs is the lack of alterations that induce or tolerate genomic instability, such as CNAs of

CDKN2A, RB1 or TP53.20-22 These findings corroborate the distinction of PMBLs as

a separate entity of LBCL.9

8.1.2. Cell cycle deregulation in PCNSL and PTL

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8.1.3. Near-uniform TLR signaling in PCNSL and PTL

In addition to frequent CDKN2A loss, MYD88L265P mutations were detected in 60%

and 78% of EBV negative PCNSLs and PTLs, respectively (Table 1). NFKBIZ CN gain was identified as a complementary oncogenic TLR signaling mechanism. Our functional studies are consistent with published data by others in primary ABC-type DLBCLs, where knockdown of IκB-ζ induced toxicity in ABC-type DLBCL cell lines.26 Together, MYD88L265P mutations and NFKBIZ CN gain provide complementary

structural bases for NF-κB activation in ~90% of PCNSLs and PTLs. The near-uniform alterations of these TLR/NF-κB family members highlight the potential utility of targeting the TLR/NF-κB pathway in PCNSL and PTL (see section 8.1.5.).

8.1.4. Concurrent alterations of TLR and BCR signaling components

In addition to a near-uniform activation of the TLR/NF-κB signaling pathway by

MYD88 mutations and/or NFKBIZ CN gain, CD79BY196 mutations were found in 38%

and 49% of EBV negative PCNSLs and PTLs, respectively. In the PCNSL discovery cohort, the frequency of CD79BY196 mutations by whole exome sequencing (WES)

was 64%, while in our validation cohort, we found a reduced frequency of 28% with Sanger sequencing. Although the lower frequency observed in the validation cohort might be explained by sampling differences (different cohort), it is most likely due to the higher sensitivity of the WES detection technology compared to Sanger sequencing. Overall, our detected frequencies are in line with a recent study using WES in 19 PCNSLs where MYD88 and CD79B mutations are reported in 79% and 40%, respectively.27 While these alterations are also found in a subset of ABC-type DLBCLs, we find that they are more prevalent in PCNSL and PTL (Table 1). In addition, CD79B mutations often co-occurred with MYD88 mutations. In ABC-type DLBCLs, concurrent CD79B and MYD88 mutations are less frequent (Table 1).28,29 These concurrent alterations highlight the complementary roles of BCR and TLR signaling in these lymphoma subtypes.30 Synergy between BCR and TLR signaling on downstream NF-κB activity has been reported previously (Figure 1)30,31, and recent studies suggest additional crosstalk between the two pathways more proximal in the BCR pathway.32,33 Further studies are needed to explore this crosstalk in DLBCLs, PCNSLs and PTLs.

8.1.5. Targeting TLR and BCR signaling components in PCNSL and PTL

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DLBCL can be extrapolated to help guide treatment decisions in PCNSL and PTL. For instance, the BTK inhibitor, ibrutinib, was previously explored in a phase I/II clinical trial of systemic DLBCL and complete or partial responses were seen in 37% of ABC-type DLBCLs, versus 5% of GCB-type DLBCLs.37 Patients whose tumors had mutations in BCR pathway components responded more frequently to ibrutinib, although responses also occurred in ABC-type DLBCLs that lacked BCR pathway mutations.37 Eighty percent of DLBCL patients with concordant BCR and MYD88 mutations showed responses to ibrutinib, consistent with cooperation of the BCR and TLR pathways.37 This data indicates that PCNSL and PTL might be uniquely susceptible to targeted inhibition with this drug, since we reported frequent and often co-occurring CD79B and MYD88 mutations in these LBCL subtypes (Table 1). Based on these findings, a phase I trial for PCNSL has been opened recently to investigate the efficacy of ibrutinib in patients with recurrent/refractory PCNSL and secondary CNS lymphoma.38 The first results of this trial showed that CNS lymphoma patients tolerated ibrutinib well at 560 and 840 mg and a 78% overall response rate was reported.38 However, we identified a complex and distinct genetic pattern with

CARD11 mutations in cis to the high prevalence CD79B and MYD88 mutations in

PCNSL and PTL.1 CARD11 is downstream in the pathway (Figure 1), which may limit the efficacy of BTK blockade.37,39 These findings highlight the need to characterize

Figure 1. Targeting TLR/BCR signaling components. Schematic representation of TLR (on the left) and BCR signaling

(on the right) pathways, see section 1.2.1.1., along with known drugs to target these pathways.

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the comprehensive genetic signature in individual patients before initiating targeted therapy.

An alternative way to target the NF-κB pathway (Figure 1, in red) is to deplete downstream targets, such as IRF4, with lenalidomide (Figure 1).40 In patients with systemic DLBCL, treatment with lenalidomide demonstrated efficacy in ABC-type DLBCLs with overall responses of 53%.40,41 Synergy of the BTK inhibitor, ibrutinib, and lenalidomide was reported in ABC-type DLBCLs, suggesting a promising treatment strategy in this and other LBCL subtypes with alterations in TLR/BCR signaling components, such as PCNSL and PTL.41 Lenalidomide has been reported to penetrate perturbed blood-brain barriers and to be active in relapsed CNS lymphoma.42 In addition, penetration of lenalidomide through the blood-testis barrier has been described43, suggesting that this drug may be a potential therapeutic option in PCNSL and PTL.

8.2. Chromosome 9p24.1 alterations in lymphoid malignancies

We identified 9p24.1/PD-L1/PD-L2 CNAs and rare chromosomal rearrangements of the PD-1 ligands in PCNSLs and PTLs in chapter 2.1 This is of particular interest given the previous identification of 9p24.1/PD-L1/PD-L2 alterations in cHL and associated evaluation and efficacy of PD-1 blockade in this disease (see section 8.3.2.).44-47 We postulated that the high response rates of PD-1 blockade in cHL were due to the genetic activation of PD-1 signaling in this disease. Therefore, we established read-outs that allowed for a robust evaluation of 9p24.1/PD-L1/PD-L2 genetic alterations and associated increased expression of the PD-1 ligands. In this thesis, we focused on the LBCL subtypes, PCNSL, PTL and PMBL (chapter 2), TCRBCL (chapter 3),

immunodeficiency-related LPDs (chapter 4) and cHL (chapter 5). A summary of our

findings is shown in Table 2.

8.2.1. Technologies to detect and evaluate PD-1 ligand deregulation

In our comprehensive genomic approach in chapter 2, we used high-density

(HD)-SNP arrays, WES and whole-transcriptome sequencing (RNA-Seq) to evaluate 9p24.1/PD-L1/PD-L2 CNAs and chromosomal rearrangements in EBV negative PCNSLs and PTLs (Table 2). By using HD-SNP arrays, we were able to delineate the exact amplicon block on chromosome 9p24.1 and by using associated gene expression identify the potential driver genes of this alteration, including PD-L1 and

PD-L2. This allowed us to later focus on the genes specifically within this amplicon

block.

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rearrangements. Since the majority of breakpoints of chromosomal rearrangements are not in the exome, we extended the WES bait set and spiked in hybrid capture probes covering 9p24.1/PD-L1/PD-L2 breakpoints. With this approach, we were able to detect exact breakpoints and rearrangement partners with base-pair resolution. We also developed orthogonal non-sequencing based approaches to analyze the integrity of the PD-1 ligand loci and assess PD-1 ligand protein expression. Specifically, we established qPCR assays to detect CNAs of PD-L1 and PD-L2 and a split apart FISH assay with probes covering PD-L1 and PD-L2 and a control centromeric probe (CEP9) to detect CNAs and chromosomal rearrangements. The main advantage of the FISH assay is that we are able to detect low-level or subclonal events. Also, in tumor samples with a low percentage of malignant cells, such as cHL (see section 8.3.1.), the FISH assay allows us to selectively analyze alterations in tumor cells. Of note, all technologies perform well in formalin-fixed paraffin embedded (FFPE) samples, an important asset to allow future clinical implementation.

To study PD-1 ligand expression in these lymphoma subtypes, IHC was used. Earlier commercially available PD-1 ligand antibodies performed poorly in IHC, with high background and low specificity. For this reason, we obtained newly purified, monoclonal antibodies from G. Freeman (Dana-Farber Cancer Institute) and developed an assay for the IHC detection of PD-L1 and PD-L2 in collaboration with

PD-L1/PD-L2

CN gain* PD-L1 or PD-L2 rearrangements Method of detection Chapter of thesis

DLBCL 6% (11/180) NA HD-SNP array NA†

TCRLBCL 57% (16/28) 7% (2/28) FISH Chapter 3

PTL 54% (26/50) 4% (2/50) HD-SNP array, targeted sequencing and qPCR Chapter 2

EBV- PCNSL 52% (33/63) 6% (4/66) HD-SNP array, targeted sequencing and qPCR Chapter 2

PMBL 65% (15/23) 20% (25/125) HD-SNP array, qPCR and FISH Chapter 2,‡

Immunodeficiency-related LPD 43% (10/23) 9% (2/23) FISH Chapter 4

cHL 93% (100/108) 2% (2/108) FISH Chapter 5

* 9p24.1 copy gain and amplification † Monti & Chapuy et al. Cancer Cell 2012 ‡ Twa et al. Blood 2014, Chong et al. Blood 2016

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S. Rodig (Brigham and Women’s Hospital). These antibodies were utilized to explore the expression of PD-L1 and PD-L2 in the above-mentioned lymphoid malignancies.

8.2.2. PD-1 deregulation in LBCLs

DLBCL. Increased expression of the PD-1 ligands is only infrequently seen in

systemic DLBCLs.48 Using the optimized IHC L1 antibody, we showed PD-L1 expression in only 11% of the DLBCLs – not otherwise specified49 and these percentages were confirmed by others.50 The latter study showed that increased expression of PD-L1 on tumor cells was associated with poor overall survival in DLBCLs.50 The low percentages of PD-1 ligand expression were consistent with the low frequency of 9p24.1/PD-L1/PD-L2 CN gains in systemic DLBCLs, as reported previously and described in chapter 2.1,2 A subsequent study also reported a low percentage of CN gains and amplification of PD-L1 and PD-L2 in DLBCLs (15%) and infrequent chromosomal rearrangements of the PD-1 ligand loci.51 These findings highlight the differences in frequency of 9p24.1/PD-L1/PD-L2 genetic alterations and PD-1 ligand expression in DLBCL and other LBCL subtypes, of importance in considering the use of PD-1 blockade in these diseases.

TCRBCL. We previously demonstrated that the morphologic DLBCL subtype,

TCRBCL, exhibited a high prevalence of PD-L1 expression (91%) by IHC.49 We expanded on these findings by exploring PD-L1 expression and 9p24.1/PD-L1/

PD-L2 genetic alterations by FISH in a larger cohort of TCRBCLs in chapter 3.

TCRBCL is similar to cHL in its cellular microenvironment, which is characterized by scattered malignant cells in a robust, but ineffective inflammatory microenvironment. We found frequent 9p24.1/PD-L1/PD-L2 genetic alterations in TCRBCL (Table 2); however, the percentages of 9p24.1/PD-L1/PD-L2 CN gain and amplification were lower in TCRBCL than in cHL. Additional explanations for the high PD-L1 expression by IHC in TCRBCL include secretion of IFN-γ by inflammatory cells in the microenvironment52,53, since IFN-γ responsive elements were described in PD-L1 promoter elements.5 These findings indicate that in addition to genetic events, microenvironmental factors might play a role in increased PD-L1 expression in this LBCL subtype.

PCNSL/PTL. We discovered frequent 9p24.1/PD-L1/PD-L2 CNAs and infrequent

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or positioning of a foreign 5’ regulatory element proximal to the start codon of

PD-L1 or PD-L2.1 Our findings are in line with subsequent studies that showed both

the presence of multiple translocation partners and the combination of promoter replacements and introduction of enhancer elements.54-57 A recent study reported infrequent structural variants, including deletions, tandem duplications, inversions and rearrangements within the 3’ region of the PD-L1 locus with elevated PD-L1 expression in different tumor types, including a small subset of DLBCLs.58 Of note, detection of increased expression caused by structural variants in the 3’ region of

PD-L1 may be limited with antibodies directed against the C-terminal region of the

PD-L1 protein58, providing a rare genetic reason for false negative results using IHC. Thus, to detect increased expression caused by these structural variants, it will be important to use antibodies directed against the N-terminal region of PD-L1.

We also demonstrated that EBV infection is an alternative mechanism for PD-1 ligand upregulation in EBV positive PCNSLs in chapter 21, as previously described.5,49,59 Since the promoter region of PD-L1 contains ISRE/IRF1 elements and STAT binding sites, increased JAK-STAT signaling induced by viral infection increases PD-L1 expression.60 In addition, viral infection increases components of the AP-1 pathway that can bind to enhancer elements of PD-L1 and thereby increase PD-L1 expression.59,61

PMBL. Frequent CN gains of 9p24.1/PD-L1/PD-L2 in PMBL, leading to overexpression

of the PD-1 ligands, have been reported by our group and others.1,4-7,62 Genomic rearrangements involving the PD-1 ligands have also been described in PMBL56,63, highlighting the importance of this immune evasion strategy in the pathogenesis of PMBL. We showed CN gains of 9p24.1/PD-L2 and increased PD-L2 expression in 72% of the PMBL patients. This is in marked contrast to DLBCL, where only 3% of the cases showed positivity.62 This feature can be used as a distinguishing feature for PMBL, compared to DLBCLs, and suggests that PMBLs might be susceptible to targeted PD-1 inhibition.

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8.2.3. PD-1 blockade in LBCLs

Since infrequent chromosomal rearrangements of PD-L2 have been found (see section 8.2.2.), we suggest that targeting the PD-1 receptor rather than PD-L1 may be preferred. This was exemplified in a recent NHL clinical trial in which a patient with mycosis fungoides achieved a partial response to Nivolumab (PD-1 blockade) therapy. The patients’ tumor had a 9p24.1 rearrangement, leading to selective increased expression of PD-L2, as determined by a combined approach using FISH and IHC.64 Targeting PD-L1 would have been ineffective in this patient.

PMBL. In PMBL, a phase 1 study of PD-1 blockade in patients with relapsed/refractory

PMBL showed an overall response rate of 40% with durable remissions.65 These encouraging findings resulted in the development of a recently opened national/ international confirmatory phase II trial (www.clinicaltrials.gov, NCT02576990).

PCNSL/PTL. Our aforementioned molecular findings in PCNSL and PTL in chapter 21 formed the basis for the treatment of a small patient cohort (5 patients) with recurrent/refractory CNS lymphoma with PD-1 blockade.66 In this small patient cohort, four patients had multiply recurrent disease (3 patients with PCNSL and 1 patient with PTL and CNS relapse) and 1 patient had primary refractory PCNSL. All patients had been treated with standard of care regimens and had no other available options. These patients were treated with Nivolumab (PD-1 blockade) under informed consent. Strikingly, all patients had objective radiographic responses on PD-1 blockade, including 4 complete responses and 1 partial response. The PFS is ~15 months and all patients are still alive at date of writing this discussion.66 Based on our preclinical molecular findings and the promising clinical results in this small patient cohort, a multi-institutional phase 2 open-label, single-arm trial of PD-1 blockade in recurrent and refractory PCNSL and PTL patients is currently recruiting (www.clinicaltrials.gov, NCT02857426).

An attractive targeted treatment regimen for PCNSL and PTL is combining PD-1 blockade with a BTK inhibitor, e.g. ibrutinib (see section 8.1.5.). Patients with 9p24.1/

PD-L1/PD-L2 CNAs and chromosomal rearrangements often have concurrent MYD88/CD79B mutations. This is of interest, because the BTK inhibitor, ibrutinib,

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PD-L2 and MYD88/CD79B genetic alterations. Therefore, a combination of these

two drugs might have clinical benefit in these patients and should be considered after evaluating the single agent activity of both agents.

8.2.4 PD-1 ligand deregulation in immunodeficiency-related LPDs

EBV infection and increased PD-L1 expression was frequently seen in immunodeficiency-related LPDs.49 In chapter 468, we focused on NHLs and cHLs associated with immunodeficiency and explored PD-L1/PD-L2 expression and 9p24.1/PD-L1/PD-L2 genetic alterations across this spectrum. PD-L1 expression and

PD-L1/PD-L2 genetic alterations were seen across all immunodeficiency settings and

in both EBV positive and EBV negative cases. This study suggested the presence of a shared pathogenic mechanism involving PD-1 ligand deregulation across the spectrum of B-cell LPDs. For this reason, we proposed a more “unifying approach” for classification of these immunodeficiency-related lymphomas. However, given the relatively small and heterogeneous cohort, the exact role of EBV infection on increased PD-L1 expression in B-cell LPDs is still unknown. These findings should be confirmed in larger cohorts and the intensity of PD-L1 expression in EBV negative versus EBV positive cases should be explored.

8.3. Immune evasion in Hodgkin lymphoma 8.3.1. 9p24.1/PD-L1/PD-L2 alterations in cHL

Because of the unique cellular composition of cHL, with only the minority of the tumor mass being the malignant cells, it is challenging to study genetic alterations in this disease using sequencing approaches. For this reason, we used the

PD-L1/PD-L2 FISH assay to more specifically define the incidence and type of 9p24.1/PD-L1/ PD-L2 genetic alterations and explored PD-L1 and PD-L2 expression in a uniformly

treated primary cHL cohort with long-term follow-up in chapter 5.69 Of interest, we showed that PD-L1/PD-L2 alterations are a near-universal feature of cHL, occurring in 97% of diagnostic biopsy specimens and ranging from low-level polysomy to relative copy gain and amplification. We also showed that PFS was significantly shorter for cHL patients with 9p24.1 amplification, who were also more likely to have advanced stage disease. In previous array comparative genomic hybridization or qPCR studies, enrichment for HRS cells had to be done by laser capture microdissection.5,70,71 With these approaches, 9p24.1/PD-L1/PD-L2 genetic alterations were seen in only ~40-50% of the patients.5,70,71 However, when performing laser capture microdissection, surrounding normal tissue is taken along. This might cause an underestimation of CNAs. In addition, our study in chapter 5 shows that 9p24.1/PD-L1/PD-L2 genetic

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cells in all cases. By pooling malignant cells for these approaches, the frequency of 9p24.1/PD-L1/PD-L2 genetic alterations may be underestimated. For this reason, we believe that the 9p24.1/PD-L1/PD-L2 FISH assay is a good strategy to identify tumor types with genetic bases for deregulation of the PD-1 pathway that are less approachable to other high throughput technologies due to their low tumor purity.

8.3.2. PD-1 blockade in cHL

Response rates of PD-1 blockade in pilot studies and subsequent registration trials in relapsed/refractory cHL range from 65-87% (Table 3). Successes of phase I studies of PD-1 blockade in cHL44,45 led to breakthrough therapy designation for relapsed/refractory cHL by the US Food and Drug Administration (FDA) on May 12, 2014. This is an expedited development program of the FDA indicating that there is a substantial improvement over standard therapy. This was followed by registration trials46,47 (Table 3) and accelerated approval of PD-1 blockade for relapsed/refractory cHL on May 17, 2016. In the European Union, PD-1 blockade for relapsed/refractory cHL was approved on November 22, 2016 by the European Medicines Agency. The multi-center, multi-cohort phase II registration trial of Nivolumab in patients with relapsed/refractory cHL consisted of 3 treatment arms. All patients received prior high-dose conditioning chemotherapy followed by ASCT as a part of their salvage therapy. Patients in cohort A were BV-naïve, patients in cohort B received BV after their ASCT and patients in cohort C received BV either before or after ASCT. The results of PD-1 blockade in cohort B of this phase II trial were recently reported47 and in chapter 672, we added a previously unreported series of patients from cohort C and evaluated the full combined series

Study Response rate (# pts) DOR or PFS

Nivolumab pilot* 87% (20/23) PFS (24 wks) 86%

Nivolumab registration trial

Cohort B† 66% (53/80) PFS (26 wks) 77%

Pembrolizumab pilot‡ 65% (20/31) PFS (24 wks) 69%

Pembrolizumab registration trial§

Cohort 1 (ASCT/BV) 70% (21/30) NA

Cohort 2 (ASCT ineligible) 80% (24/30) NA

* Ansell et al. NEJM 2015 † Younes et al. Lancet Oncol 2016 ‡ Armand et al. JCO 2016

Table 3. Clinical evaluation of PD-1 blockade in relapsed/refractory cHL

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for associations between 9p24.1/PD-L1/PD-L2 genetic alterations and PD-L1 expression and both BOR and PFS. Consistent with results of the pilot study and cohort B44,47, we demonstrated that all evaluable patients in this study had genetic alterations of 9p24.1/L1/L2 and CN-dependent increased expression of PD-L1 in HRS cells. Of note, in this study we linked high-level alterations of

9p24.1/PD-L1/PD-L2 and increased PD-L1 expression to a more favourable outcome to targeted

PD-1 blockade. This is in contrast with responses to standard therapy, where high-level alterations of 9p24.1/PD-L1/PD-L2 are linked to an inferior outcome.69

Based on the promising clinical results of PD-1 blockade in relapsed/refractory cHL44-47 and near-uniform presence of 9p24.1/PD-L1/PD-L2 genetic alterations in newly diagnosed cHL (chapter 5)69, a clinical trial of PD-1 blockade in patients with newly diagnosed advanced stage cHL was developed (www.clinicaltrials. gov, NCT02181738, cohort D). In addition, these data prompted multiple groups to evaluate the best timing of PD-1 blockade in cHL therapy. Anticipating that the promising results from the phase I/II studies hold, this might lead to a paradigm shift in the treatment of cHL.

8.3.3. Potential biomarkers for response to PD-1 blockade

PD-1 blockade produces clinical responses in the majority of cHL patients, but the exact mechanism by which this occurs has not been fully characterized and biomarkers that are predictive for response and resistance are needed. The predictive value of 9p24.1/PD-L1/PD-L2 genetic alterations to PD-1 blockade in cHL suggests the development of the PD-L1/PD-L2 FISH assay as a Clinical Laboratory Improvement Amendments (CLIA)-approved test. In addition, IHC can be used to determine expression of PD-1 ligands. Given the clonality and heterogeneity of 9p24.1/PD-L1/

PD-L2 genetic alterations, we believe that the modified H-score, which reflects both

the intensity of PD-L1 expression and the percentage of positive malignant cells is the best approach. So far, different studies have used different antibodies and various thresholds to score positivity or negativity of the PD-1 ligands49,50, resulting in slight differences in percentages and interpretation. After carefully exploring different antibodies for the PD-1 ligands, we saw that PD-1 ligand expression might be present on some malignant cells, but not on others, likely due to subclonal 9p24.1/

PD-L1/PD-L2 genetic alterations. In addition, applying a double stain of PD-L1 with

PAX5, a B-cell lineage marker, is necessary to distinguish PD-1 ligand expression on malignant and non-malignant cells.

8.3.4. Additional immune evasion strategies

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effective immune response by downregulating expression of MHC class I and/or MHC class II on the tumor cell surface. In a recent study on flow-sorted HRS cells,

B2M alterations were described in 7/10 (70%) of the cHL patients.73 Also, inactivating

alterations of the MHC class II transactivator CIITA have been described.56 In

chapter 774, we explored the expression and prognostic significance of antigen

presentation components in cHL. We showed that 79% of the cHL patients had decreased/absent cell surface expression of β2M/MHC class I, and 70% exhibited decreased/absent expression of MHC class II. Patients with decreased/absent β2M/ MHC class I expression on HRS cells had shorter PFS, independent of PD-L1/PD-L2 amplification and advanced clinical stage. These findings highlight the importance of MHC class I-mediated antigen presentation by HRS cells to CD8+ T-cells for

responses to standard induction therapy. While our finding is consistent with the favorable outcome of patients with expression of MHC class I to standard therapy in other tumor types75-78, a single center retrospective study in cHL reported that absence of β2M expression was associated with a lower stage of disease, younger age at diagnosis, and better overall survival and PFS.73 Contrary to this report, patient samples in our study had a representative distribution of morphological subtypes, a balanced representation of clinical risk-groups and were collected in the context of a clinical trial with pre-specified, long-term clinical follow-up.

In contrast to previous studies73,79, we used a 3-tiered scoring system to evaluate β

2M,

MHC class I and MHC class II expression, since we noticed extensive heterogeneity of staining among HRS cells. In addition to cases that were clearly positive or negative, we observed cases with unequivocally positive but reduced membrane staining, relative to adjacent non-malignant cells, and cases with a combination of reduced and complete loss of staining in a subset of cells. These cases were categorized as “decreased”. Decreased expression might be caused by single allelic loss or heterozygous inactivating mutations in genes directly encoding these proteins or alterations in genes encoding for critical transcriptional regulators of MHC class I and MHC class II expression, such as NLRC5 and CIITA, respectively.56,80,81

8.3.5. Mechanistic implications of competing immune evasion strategies

As noted above, cHL is the among the most sensitive tumor types to PD-1 blockade. In other tumor types, clinical responses to checkpoint blockade have been associated with the presence of CD8+ T-cells within the tumor microenvironment, underscoring

the importance of MHC class I-mediated antigen presentation by malignant cells.82 However, the low percentage of cHLs in which the HRS cells retain intact MHC class I (18%) or β2M (16%) expression suggests that CD8+ cytotoxic T-cell mediated killing

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PD-1 blockade. Additional mechanisms of anti-tumor immunity that have yet to be explored in cHL include MHC class II presentation to CD4+ effector cells, activated

NK cell subsets, and antibody-mediated responses. Recent studies highlight the essential role of neoantigen-specific CD4+ cells for an effective anti-tumor immune

response83,84 and the function of CD4+ cytotoxic T-cells in the eradication of certain tumors in model systems and following checkpoint blockade85-88. Nevertheless, we also find reduced/absent expression of MHC class II on HRS cells in ~70% of examined cHLs. In PCNSL and PTL, deletions of the MHC loci and infrequent

B2M mutations have also been reported in our and other studies1,89,90. Ongoing investigations in the phase II trials of PD-1 blockade and the upfront studies in lymphoid malignancies should aim to examine the significance of impaired antigen presentation by malignant cells on the quality and durability of clinical responses to PD-1 inhibitors.

8.3.6. Importance of immune cell subsets

Analyses of peripheral blood samples of cHL patients treated with PD-1 showed an increase in number of CD4+, CD8+ and NK cells45, but the correlation with treatment response is still unknown. In mouse models, a selective proliferative burst of CXCR5+PD-1+CD8+ T-cells was seen after PD-L1 blockade, suggesting

the importance of a CD8+ T-cell subset.91 Additionally, proliferation of CD8+ T-cells has been shown in melanoma patients responding to PD-1 blockade.82 They also performed TCR sequencing to examine α- and β-chain diversity in CD4+ and CD8+

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Ideally, pre-treatment and on-treatment tumor biopsies should be obtained to look at changes in the tumor microenvironment before and during treatment. The importance of this was shown in a recent publication in melanoma patients treated with CTLA-4 and PD-1 blockade.99 Assessment of adaptive immune responses early in the course of therapy showed overlapping signatures in responders versus non-responders99, indicating the importance for collecting early on-treatment biopsies. However, on-treatment biopsies might be difficult to obtain.

8.3.7. Overall mutational load

A recent report of non–small cell lung cancer patients treated with PD-1 blockade indicated that higher nonsynonymous mutation burden in tumors was associated with improved objective response, durable clinical benefit, and PFS.100 A recent study of WES in a small cohort of 10 cHL patients suggests that there is a high mutational burden in cHL (median: 8.3 per megabase, range: 3.5-16.8 per megabase)73 that is comparable to the mutation rate found in melanomas or lung cancers and almost three times higher than that of DLBCLs.101 This might be of significance for responses to PD-1 blockade.

8.4. Conclusions

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8

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