Assessing Tumor-Infiltrating Lymphocytes in Breast Cancer: A Proposal for Combining Immunohistochemistry and Gene Expression Analysis to Refine Scoring.

Scoring of tumor-infiltrating lymphocytes (TILs) in breast cancer specimens has gained increasing attention, as TILs have prognostic and predictive value in HER2+ and triple-negative breast cancer. We evaluated the intra- and interrater variability when scoring TILs by visual inspection of hematoxylin and eosin-stained tissue sections. We further addressed whether immunohistochemical staining of these sections for immune cell surface markers CD45, CD3, CD4, and CD8 and combination with nanoString nCounter® gene expression analysis could refine TIL scoring. Formalin-fixed paraffin-embedded and fresh-frozen core needle biopsies of 12 female and treatment-naive breast cancer patients were included. Scoring of TILs was performed twice by three independent pathologists with a washout period of 3 days. Increasing intra- and interrater variability was observed with higher TIL numbers. The highest reproducibility was observed on tissue sections stained for CD3 and CD8. The latter TIL scores correlated well with the TIL scores obtained through nanoString nCounter® gene expression analysis. Gene expression analysis also revealed 104 and 62 genes that are positively and negatively related to both TIL scores. In conclusion, integration of immunohistochemistry and gene expression analysis is a valuable strategy to refine TIL scoring in breast tumors.

Intratumoral administration of CD1c (BDCA-1)+ and CD141 (BDCA-3)+ myeloid dendritic cells in combination with talimogene laherparepvec in immune checkpoint blockade refractory advanced melanoma patients: a phase I clinical trial.

BACKGROUND: Intratumoral (IT) myeloid dendritic cells (myDCs) play a pivotal role in initiating antitumor immune responses and relicensing of anti-tumor cytotoxic T lymphocytes within the tumor microenvironment. Talimogene laherparepvec (T-VEC) induces immunogenic cell death, thereby providing maturation signals and enhancing the release of tumor antigens that can be captured and processed by CD1c (BDCA-1)+ / CD141 (BDCA-3)+ myDCs, in order to reinvigorate the cancer-immunity cycle. METHODS: In this phase I trial, patients with advanced melanoma who failed standard therapy were eligible for IT injections of ≥1 non-visceral metastases with T-VEC on day 1 followed by IT injection of CD1c (BDCA-1)+ myDCs +/- CD141 (BDCA-3)+ myDCs on day 2. T-VEC injections were repeated on day 21 and every 14 days thereafter. The number of IT administered CD1c (BDCA-1)+ myDCs was escalated from 0.5×106, to 1×106, to a maximum of 10×106 cells in three sequential cohorts. In cohort 4, all isolated CD1c (BDCA-1)+ / CD141 (BDCA-3)+ myDCs were used for IT injection. Primary objectives were safety and feasibility. Repetitive biopsies of treated lesions were performed. RESULTS: In total, 13 patients were enrolled (cohort 1 n=2; cohort 2 n=2; cohort 3 n=3; cohort 4 n=6). Patients received a median of 6 (range 3-8) T-VEC injections. The treatment was safe with most frequent adverse events being fatigue (n=11 (85%)), fever (n=8 (62%)), and chills/influenza-like symptoms (n=6 (46%)). Nine (69%) and four patients (31%), respectively, experienced pain or redness at the injection-site. Clinical responses were documented in injected and non-injected lesions. Two patients (cohort 3) who previously progressed on anti-PD-1 therapy (and one patient also on anti-CTLA-4 therapy) developed a durable, pathologically confirmed complete response that is ongoing at 33 and 35 months following initiation of study treatment. One additional patient treated (cohort 4) had an unconfirmed partial response as best response; two additional patients had a mixed response (with durable complete responses of some injected and non-injected lesions). On-treatment biopsies revealed a strong infiltration by inflammatory cells in regressing lesions. CONCLUSIONS: IT coinjection of autologous CD1c (BDCA-1)+ +/- CD141 (BDCA-3)+ myDCs with T-VEC is feasible, tolerable and resulted in encouraging early signs of antitumor activity in immune checkpoint inhibitor-refractory melanoma patients. TRIAL REGISTRATION NUMBER: NCT03747744.

Overcoming the Challenges of High Quality RNA Extraction from Core Needle Biopsy.

The use of gene expression profiling (GEP) in cancer management is rising, as GEP can be used for disease classification and diagnosis, tailoring treatment to underlying genetic determinants of pharmacological response, monitoring of therapy response, and prognosis. However, the reliability of GEP heavily depends on the input of RNA in sufficient quantity and quality. This highlights the need for standard procedures to ensure best practices for RNA extraction from often small tumor biopsies with variable tissue handling. We optimized an RNA extraction protocol from fresh-frozen (FF) core needle biopsies (CNB) from breast cancer patients and from formalin-fixed paraffin-embedded (FFPE) tissue when FF CNB did not yield sufficient RNA. Methods to avoid ribonucleases andto homogenize or to deparaffinize tissues and the impact of tissue composition on RNA extraction were studied. Additionally, RNA's compatibility with the nanoString nCounter® technology was studied. This technology platform enables GEP using small RNA fragments. After optimization of the protocol, RNA of high quality and sufficient quantity was obtained from FF CNB in 92% of samples. For the remaining 8% of cases, FFPE material prepared by the pathology department was used for RNA extraction. Both resulting RNA end products are compatible with the nanoString nCounter® technology.

Intracerebral administration of CTLA-4 and PD-1 immune checkpoint blocking monoclonal antibodies in patients with recurrent glioblastoma: a phase I clinical trial.

BACKGROUND: Patients with recurrent glioblastoma (rGB) have a poor prognosis with a median overall survival (OS) of 30-39 weeks in prospective clinical trials. Intravenous administration of programmed cell death protein 1 and cytotoxic T-lymphocyte-associated antigen 4 inhibitors has low activity in patients with rGB. In this phase I clinical trial, intracerebral (IC) administration of ipilimumab (IPI) and nivolumab (NIVO) in combination with intravenous administration of NIVO was investigated. METHODS: Within 24 hours following the intravenous administration of a fixed dose (10 mg) of NIVO, patients underwent a maximal safe resection, followed by injection of IPI (10 mg; cohort-1), or IPI (5 mg) plus NIVO (10 mg; cohort-2) in the brain tissue lining the resection cavity. Intravenous administration of NIVO (10 mg) was repeated every 2 weeks (max. five administrations). Next generation sequencing and RNA gene expression profiling was performed on resected tumor tissue. RESULTS: Twenty-seven patients were enrolled (cohort-1: n=3; cohort-2: n=24). All patients underwent maximal safe resection and planned IC administrations and preoperative NIVO. Thirteen patients (cohort-1: n=3; cohort-2: n=10) received all five postoperative intravenous doses of NIVO. In cohort-2, 14 patients received a median of 3 (range 1-4) intravenous doses. Subacute postoperative neurological deterioration (n=2) was reversible on steroid treatment; no other central nervous system toxicity was observed. Immune-related adverse events were infrequent and mild. GB recurrence was diagnosed in 26 patients (median progression-free survival (PFS) is 11.7 weeks (range 2-152)); 21 patients have died due to progression. Median OS is 38 weeks (95% CI: 27 to 49) with a 6-month, 1-year, and 2-year OS-rate of, respectively, 74.1% (95% CI: 57 to 90), 40.7% (95% CI: 22 to 59), and 27% (95% CI: 9 to 44). OS compares favorable against a historical cohort (descriptive Log-Rank p>0.003). No significant difference was found with respect to PFS (descriptive Log-Rank test p>0.05). A higher tumor mRNA expression level of B7-H3 was associated with a significantly worse survival (multivariate Cox logistic regression, p>0.029). CONCLUSION: IC administration of NIVO and IPI following maximal safe resection of rGB was feasible, safe, and associated with encouraging OS. TRIAL REGISTRATION: NCT03233152.

Intratumoral Combinatorial Administration of CD1c (BDCA-1)+ Myeloid Dendritic Cells Plus Ipilimumab and Avelumab in Combination with Intravenous Low-Dose Nivolumab in Patients with Advanced Solid Tumors: A Phase IB Clinical Trial.

Intratumoral (IT) myeloid dendritic cells (myDCs) play a pivotal role in re-licensing antitumor cytotoxic T lymphocytes. IT injection of the IgG1 monoclonal antibodies ipilimumab and avelumab may induce antibody-dependent cellular cytotoxicity, thereby enhancing the release of tumor antigens that can be captured and processed by CD1c (BDCA-1)+ myDCs. Patients with advanced solid tumors after standard care were eligible for IT injections of ≥1 lesion with ipilimumab (10 mg) and avelumab (40 mg) and intravenous (IV) nivolumab (10 mg) on day 1, followed by IT injection of autologous CD1c (BDCA-1)+ myDCs on day 2. IT/IV administration of ipilimumab, avelumab, and nivolumab was repeated bi-weekly. Primary objectives were safety and feasibility. Nine patients were treated with a median of 21 × 106 CD1c (BDCA-1)+ myDCs, and a median of 4 IT/IV administrations of ipilimumab, avelumab, and nivolumab. The treatment was safe with mainly injection-site reactions, but also immune-related pneumonitis (n = 2), colitis (n = 1), and bullous pemphigoid (n = 1). The best response was a durable partial response in a patient with stage IV melanoma who previously progressed on checkpoint inhibitors. Our combinatorial therapeutic approach, including IT injection of CD1c (BDCA-1)+ myDCs, is feasible and safe, and it resulted in encouraging signs of antitumor activity in patients with advanced solid tumors.

An atypical sarcoid-like reaction during anti-protein death 1 treatment in a patient with metastatic melanoma.

We report a case of anti-protein death 1-induced sarcoid-like reaction in a 63-year-old Caucasian male who was diagnosed with stage IV-M1a melanoma. He was initially treated with pembrolizumab monotherapy (Q3W) and had a complete response after 14 cycles. However, relapse was suspected 3 months later with appearance of hilar, mediastinal and hepatic hilar lymph nodes as well as a skin lesion. Biopsy of both the hilar lymph nodes and the skin lesion demonstrated sarcomatoid granulomatosis. Pembrolizumab was discontinued temporarily. While on F-FDG-PET/CT, all sarcoid-like lesions regressed in size and activity, a new hypermetabolic solitary skeletal lesion was detected in a lumbar vertebra, suspicious for metastasis. However, since the patient was asymptomatic, a watchful-waiting attitude was taken. During this period, a spontaneous and complete resolution of the metabolic activity was observed of the skeletal lesion. Until today, the patient remains in complete remission. Current case presents an atypical presentation and evolution of anti-PD-1-induced sarcoid-like reaction, illustrating the difficulty of differentiating it from disease progression. Before considering (re-)initiation of anti-melanoma therapy, a tissue biopsy of one of the suspected lesions may be performed to confirm diagnosis. Physicians treating patients with ICI should be aware of this difficulty and critically assess the nature of lesions suspect of progression in patients responding to ICI and presenting with a sarcoid-like reaction.