Postoperative Radiation Therapy Is Indicated for "Low-Risk" Pathologic Stage I Merkel Cell Carcinoma of the Head and Neck Region but Not for Other Locations.

Purpose: The role of postoperative radiation therapy (PORT) in early stage Merkel cell carcinoma (MCC) is controversial. We analyzed the role of PORT in preventing local recurrences (LR) among patients with low-risk, pathologic stage I MCC based on the location of the primary tumors: head/neck (HN) versus non-HN sites. Methods and Materials: One hundred forty-seven patients with MCC were identified that had "low risk" disease (pathologic T1 primary tumor, negative microscopic margins, negative pathologic node status, no immunosuppression or prior systemic therapy). LR was defined as tumor recurrence within 2 cm of the primary surgical bed, and its frequency was estimated with the cumulative incidence method. Results: Seventy-nine patients received PORT (30 HN, 49 non-HN) with a median dose of 50 Gy (range, 8-64 Gy) and 68 patients were treated with surgery alone (30 HN, 38 non-HN). Overall, PORT was associated with a decreased risk of LR (5-year rate: 0% vs 9.5%; P = .004) with 6 LRs observed in the surgery alone group. Although the addition of PORT significantly reduced LR rates among patients with HN MCC (0% vs. 21%; P = .034), no LRs were observed in patients with non-HN MCC managed with surgery alone. There was no significant difference in MCC-specific survival comparing HN versus non-HN groups, with or without PORT. Conclusions: For low-risk, pathologic stage I MCC of the extremities and trunk, excellent local control rates were achieved with surgery, and PORT is not indicated. However, PORT was associated with a significant reduction in LRs among low-risk MCC of the HN.

Increased risk of recurrence and disease-specific death following delayed postoperative radiation for Merkel cell carcinoma.

BACKGROUND: Merkel cell carcinoma (MCC) is often treated with surgery and postoperative radiation therapy (PORT). The optimal time to initiate PORT (Time-to-PORT [ttPORT]) is unknown. PURPOSE: We assessed if delays in ttPORT were associated with inferior outcomes. METHODS: Competing risk regression was used to evaluate associations between ttPORT and locoregional recurrence (LRR) for patients with stage I/II MCC in a prospective registry and adjust for covariates. Distant metastasis and death were competing risks. RESULTS: The cohort included 124 patients with median ttPORT of 41 days (range: 8-125 days). Median follow-up was 55 months. 17 (14%) patients experienced a LRR, 14 (82%) of which arose outside the radiation field. LRR at 5 years was increased for ttPORT >8 weeks vs ≤ 8 weeks, 28.0% vs 9.2%, P = .006. There was an increase in the cumulative incidence of MCC-specific death with increasing ttPORT (HR = 1.14 per 1-week increase, P = .016). LIMITATIONS: The relatively low number of LRRs limited the extent of our multivariable analyses. CONCLUSIONS: Delay of PORT was associated with increased LRR, usually beyond the radiation field. This is consistent with the tendency of MCC to spread quickly via lymphatics. Initiation of PORT within 8 weeks was associated with improved locoregional control and MCC-specific survival.

The Evolving Treatment Landscape of Merkel Cell Carcinoma.

OPINION STATEMENT: Merkel cell carcinoma (MCC) has a high risk of recurrence and requires unique treatment relative to other skin cancers. The patient population is generally older, with comorbidities. Multidisciplinary and personalized care is therefore paramount, based on patient preferences regarding risks and benefits. Positron emission tomography and computed tomography (PET-CT) is the most sensitive staging modality and reveals clinically occult disease in ~ 16% of patients. Discovery of occult disease spread markedly alters management. Newly diagnosed, localized disease is often managed with sentinel lymph node biopsy (SLNB), local excision, primary wound closure, and post-operative radiation therapy (PORT). In contrast, metastatic disease is usually treated systemically with an immune checkpoint inhibitor (ICI). However, one or more of these approaches may not be indicated. Criteria for such exceptions and alternative approaches will be discussed. Because MCC recurs in 40% of patients and early detection/treatment of advanced disease is advantageous, close surveillance is recommended. Given that over 90% of initial recurrences arise within 3 years, surveillance frequency can be rapidly decreased after this high-risk period. Patient-specific assessment of risk is important because recurrence risk varies widely (15 to > 80%: Merkelcell.org/recur) depending on baseline patient characteristics and time since treatment. Blood-based surveillance tests are now available (Merkel cell polyomavirus (MCPyV) antibodies and circulating tumor DNA (ctDNA)) with excellent sensitivity that can spare patients from contrast dye, radioactivity, and travel to a cancer imaging facility. If recurrent disease is locoregional, management with surgery and/or RT is typically indicated. ICIs are now the first line for systemic/advanced MCC, with objective response rates (ORRs) exceeding 50%. Cytotoxic chemotherapy is sometimes used for debulking disease or in patients who cannot tolerate ICI. ICI-refractory disease is the major problem faced by this field. Fortunately, numerous promising therapies are on the horizon to address this clinical need.

Neoadjuvant Therapy Induces a Potent Immune Response to Sarcoma, Dominated by Myeloid and B Cells.

PURPOSE: To characterize changes in the soft-tissue sarcoma (STS) tumor immune microenvironment induced by standard neoadjuvant therapy with the goal of informing neoadjuvant immunotherapy trial design. EXPERIMENTAL DESIGN: Paired pre- and postneoadjuvant therapy specimens were retrospectively identified for 32 patients with STSs and analyzed by three modalities: multiplexed IHC, NanoString, and RNA sequencing with ImmunoPrism analysis. RESULTS: All 32 patients, representing a variety of STS histologic subtypes, received neoadjuvant radiotherapy and 21 (66%) received chemotherapy prior to radiotherapy. The most prevalent immune cells in the tumor before neoadjuvant therapy were myeloid cells (45% of all immune cells) and B cells (37%), with T (13%) and natural killer (NK) cells (5%) also present. Neoadjuvant therapy significantly increased the total immune cells infiltrating the tumors across all histologic subtypes for patients receiving neoadjuvant radiotherapy with or without chemotherapy. An increase in the percentage of monocytes and macrophages, particularly M2 macrophages, B cells, and CD4+ T cells was observed postneoadjuvant therapy. Upregulation of genes and cytokines associated with antigen presentation was also observed, and a favorable pathologic response (≥90% necrosis postneoadjuvant therapy) was associated with an increase in monocytic infiltrate. Upregulation of the T-cell checkpoint TIM3 and downregulation of OX40 were observed posttreatment. CONCLUSIONS: Standard neoadjuvant therapy induces both immunostimulatory and immunosuppressive effects within a complex sarcoma microenvironment dominated by myeloid and B cells. This work informs ongoing efforts to incorporate immune checkpoint inhibitors and novel immunotherapies into the neoadjuvant setting for STSs.

Intersection of Two Checkpoints: Could Inhibiting the DNA Damage Response Checkpoint Rescue Immune Checkpoint-Refractory Cancer?

Metastatic cancers resistant to immunotherapy require novel management strategies. DNA damage response (DDR) proteins, including ATR (ataxia telangiectasia and Rad3-related), ATM (ataxia telangiectasia mutated) and DNA-PK (DNA-dependent protein kinase), have been promising therapeutic targets for decades. Specific, potent DDR inhibitors (DDRi) recently entered clinical trials. Surprisingly, preclinical studies have now indicated that DDRi may stimulate anti-tumor immunity to augment immunotherapy. The mechanisms governing how DDRi could promote anti-tumor immunity are not well understood; however, early evidence suggests that they can potentiate immunogenic cell death to recruit and activate antigen-presenting cells to prime an adaptive immune response. Merkel cell carcinoma (MCC) is well suited to test these concepts. It is inherently immunogenic as ~50% of patients with advanced MCC persistently benefit from immunotherapy, making MCC one of the most responsive solid tumors. As is typical of neuroendocrine cancers, dysfunction of p53 and Rb with upregulation of Myc leads to the very rapid growth of MCC. This suggests high replication stress and susceptibility to DDRi and DNA-damaging agents. Indeed, MCC tumors are particularly radiosensitive. Given its inherent immunogenicity, cell cycle checkpoint deficiencies and sensitivity to DNA damage, MCC may be ideal for testing whether targeting the intersection of the DDR checkpoint and the immune checkpoint could help patients with immunotherapy-refractory cancers.

Radiation and Modulation of the Tumor Immune Microenvironment in Non-Small Cell Lung Cancer.

Immune checkpoint inhibitors are approved for a variety of indications for locally advanced and metastatic non-small cell lung cancer (NSCLC), and trials are ongoing in the early-stage setting. There is an unmet need to understand which patients may derive benefit from immunotherapies and how to harness combined modality therapies to improve overall response rates and durability. Here, we review studies from the bench-to-bedside to examine the role of radiation therapy (RT) on the tumor immune microenvironment in NSCLC with an eye toward augmenting antitumor immunity. Together, these data provide a foundation for developing future clinical trials harnessing RT to augment antitumor immunity and highlight the need for correlative translational studies to directly characterize the impact of RT on the human NSCLC tumor immune microenvironment.

Combined Intranasal Nanoemulsion and RIG-I Activating RNA Adjuvants Enhance Mucosal, Humoral, and Cellular Immunity to Influenza Virus.

Current influenza virus vaccines are focused on humoral immunity and are limited by the short duration of protection, narrow cross-strain efficacy, and suboptimal immunogenicity. Here, we combined two chemically and biologically distinct adjuvants, an oil-in-water nanoemulsion (NE) and RNA-based agonists of RIG-I, to determine whether the diverse mechanisms of these adjuvants could lead to improved immunogenicity and breadth of protection against the influenza virus. NE activates TLRs, stimulates immunogenic apoptosis, and enhances cellular antigen uptake, leading to a balanced TH1/TH2/TH17 response when administered intranasally. RIG-I agonists included RNAs derived from Sendai and influenza viral defective interfering RNAs (IVT DI, 3php, respectively) and RIG-I/TLR3 agonist, poly(I:C) (pIC), which induce IFN-Is and TH1-polarized responses. NE/RNA combined adjuvants potentially allow for costimulation of multiple innate immune receptor pathways, more closely mimicking patterns of activation occurring during natural viral infection. Mice intranasally immunized with inactivated A/Puerto Rico/8/1934 (H1N1) (PR/8) adjuvanted with NE/IVT DI or NE/3php (but not NE/pIC) showed synergistic enhancement of systemic PR/8-specific IgG with significantly greater avidity and virus neutralization activity than the individual adjuvants. Notably, NE/IVT DI induced protective neutralizing titers after a single immunization. Hemagglutinin stem-specific antibodies were also improved, allowing recognition of heterologous and heterosubtypic hemagglutinins. All NE/RNAs elicited substantial PR/8-specific sIgA. Finally, a unique cellular response with enhanced TH1/TH17 immunity was induced with the NE/RNAs. These results demonstrate that the enhanced immunogenicity of the adjuvant combinations was synergistic and not simply additive, highlighting the potential value of a combined adjuvant approach for improving the efficacy of vaccination against the influenza virus.

Postoperative, Single-Fraction Radiation Therapy in Merkel Cell Carcinoma of the Head and Neck.

PURPOSE: Conventionally fractionated, postoperative radiation therapy (cPORT; 50 Gy in 25 fractions) is considered for patients with Merkel cell carcinoma (MCC) to improve locoregional control. However, cPORT is associated with acute toxicity, especially in the head and neck (H&N) region, and requires daily treatments over several weeks. We previously reported high rates of durable local control with minimal toxicity using 8-Gy single-fraction radiation therapy (SFRT) in the metastatic setting. We report early results on a cohort of patients with localized H&N MCC who received postoperative SFRT if a cPORT regimen was not feasible. METHODS AND MATERIALS: Twelve patients with localized MCC of the H&N (clinical/pathologic stages I-II) and no prior radiation therapy to the region were identified from an institutional review board-approved prospective registry who underwent surgical resection followed by postoperative SFRT. Time to event was calculated starting from the date of resection before SFRT. The cumulative incidence of in-field locoregional recurrences and out-of-field recurrences was estimated with death as a competing risk. RESULTS: Twelve patients with H&N MCC were identified with clinical/pathologic stages I-II H&N MCC. Median age at diagnosis was 81 years (range, 58-96 years); 25% had immunosuppression. At a median follow-up of 19 months (range, 8-34), there were no in-field locoregional recurrences. A single out-of-field regional recurrence was observed, which was successfully salvaged. There were no MCC-specific deaths. No radiation-associated toxicities greater than grade 1 (Common Terminology Criteria for Adverse Events v5) were observed. CONCLUSIONS: Preliminary data suggest that SFRT could offer a potential alternative to cPORT to treat the primary site for localized H&N MCC, particularly in elderly or frail patients, with promising in-field local control and minimal toxicity. Further data with validation in larger cohorts are needed to confirm the sustained safety and efficacy of postoperative SFRT.

Oncolytic Newcastle disease virus expressing a checkpoint inhibitor as a radioenhancing agent for murine melanoma.

BACKGROUND: Monoclonal antibodies (mAbs) targeting negative regulators, or checkpoint molecules (e.g. PD1/PD-L1 & CTLA4), of anti-tumoural T cells have demonstrated clinical efficacy in treating several neoplastic diseases. While many patients enjoy remarkable responses to checkpoint inhibitors, a majority show adverse effects. Understanding how checkpoint inhibitors may augment established chemotherapy or radiotherapy regimens or other immunotherapies like oncolytic viruses may lead to better clinical outcomes measured by improved efficacy with reduced toxicity. Here, we assess how Newcastle disease virus (NDV), an oncolytic virus in clinical testing, may interact with radiotherapy to enhance checkpoint inhibitor blockade. METHODS: An immunocompetent B16-F10 murine melanoma model, generally considered to be a poorly immunogenic or "cold" tumour, was utilised to query whether combining localised radiotherapy with NDV may be more effective than either therapy alone in controlling tumours in mice treated with anti-PD1 or anti-CTLA4 monoclonal antibodies. We also investigated whether localised administration of a checkpoint inhibitor through an intratumoural injection of NDV that expresses anti-CTLA4 single-chain variable fragment (scFv) is comparable to systemic administration of anti-CTLA4 when combined with radiation in mediating its anti-tumour efficacy. Response rates were characterised by measuring tumour size over time, observation of complete tumour regression, and overall survival. FINDINGS: Our results show that combining NDV plus radiotherapy with checkpoint inhibitors (PD1 or CTLA4 targeted mAbs) results in significantly better complete tumour regression rates with an abscopal effect in a murine model of melanoma than either single therapy combined with checkpoint inhibitors. Finally, we also show that localised administration of a recombinant NDV expressing anti-CTLA4 plus radiation is comparable to systemic anti-CTLA4 plus radiation in mediating its anti-tumour effect as assayed by survival benefit. INTERPRETATION: Our results show that oncolytic NDV plus radiotherapy work together with checkpoint inhibitors to enhance tumour clearance of murine melanoma. NDV is an effective radiotherapy dose-sparing and immunotherapeutic agent capable of transgenic, in vivo expression of an anti-CTLA4 targeted scFv antibody with the potential to spare systemic exposure. FUNDING: The National Institutes of Health grant HHSN272201400008C supported the work. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

2D kV orthogonal imaging with fiducial markers is more precise for daily image guided alignments than soft-tissue cone beam computed tomography for prostate radiation therapy.

PURPOSE: The hypothesis is that 2-dimensional kV orthogonal imaging with fiducial markers (kV-FM) and soft-tissue cone beam computed tomography (ST-CBCT) are equally reproducible for daily positional alignments for image guided (IG) intensity modulated radiation therapy (IMRT) for prostate cancer. METHODS AND MATERIALS: Ten patients undergoing definitive treatment for prostate cancer with IG-IMRT were imaged daily with kV-FM and ST-CBCT. For each acquired kV and CBCT image, offline alignments to the digitally reconstructed radiograph or planning CT, respectively, were made twice by the same physician to assess intraobserver test-retest reproducibility. The 256 kV and 142 CBCT images were analyzed, and the test-retest analysis was performed again on a subset of images by another physician to verify the results. RESULTS: The results demonstrated that kV-FM had better intraobserver test-retest reproducibility in the anterior-posterior (AP; 95% confidence interval [CI] Pearson correlation coefficient [r], 0.987-0.991), left-right (LR; 95% CI r, 0.955-0.969), and superior-inferior (SI; 95% CI r, 0.971-0.980) directions for daily IG alignments compared with ST-CBCT (AP: 95% CI r, 0.804-0.877; LR: 95% CI r, 0.877-0.924; SI: 95% CI r, 0.791-0.869). Errors associated with intraobserver test-retest reproducibility were submillimeter with kV-FM (AP: 0.4 ± 0.7 mm; RL: 0.4 ± 1.0 mm; SI: 0.5 ± 0.7 mm) compared with ST-CBCT (AP: 2.1 ± 2.2 mm; LR: 1.3 ± 1.4 mm; SI: 1.2 ± 1.8 mm). The mean shift differences between kV-FM and ST-CBCT were 0.3 ± 3.8 mm for AP, -1.1 ± 8.5 mm for LR, and -2.0 ± 3.7 mm for SI. Dose-volume histograms were generated and showed that test-retest variability associated with ST-CBCT IG-alignments resulted in significantly increased dose to normal structures and a reduced planning target volume dose in many patients. CONCLUSIONS: The kV-FM-based daily IG alignment for IMRT of prostate cancer is more precise than ST-CBCT, as assessed by a physician's ability to reproducibly align images. Given the magnitude of the error introduced by inconsistency in making ST-CBCT alignments, these data support a role for daily kV imaging of FM to enhance the precision of external beam dose delivery to the prostate.

Ataxia telangiectasia-mutated protein and DNA-dependent protein kinase have complementary V(D)J recombination functions.

Antigen receptor variable region exons are assembled during lymphocyte development from variable (V), diversity (D), and joining (J) gene segments. Each germ-line gene segment is flanked by recombination signal sequences (RSs). Recombination-activating gene endonuclease initiates V(D)J recombination by cleaving a pair of gene segments at their junction with flanking RSs to generate covalently sealed (hairpinned) coding ends (CEs) and blunt 5'-phosphorylated RS ends (SEs). Subsequently, nonhomologous end joining (NHEJ) opens, processes, and fuses CEs to form coding joins (CJs) and precisely joins SEs to form signal joins (SJs). DNA-dependent protein kinase catalytic subunit (DNA-PKcs) activates Artemis endonuclease to open and process hairpinned CEs before their fusion into CJs by other NHEJ factors. Although DNA-PKcs is absolutely required for CJs, SJs are formed to variable degrees and with variable fidelity in different DNA-PKcs-deficient cell types. Thus, other factors may compensate for DNA-PKcs function in SJ formation. DNA-PKcs and the ataxia telangiectasia-mutated (ATM) kinase are members of the same family, and they share common substrates in the DNA damage response. Although ATM deficiency compromises chromosomal V(D)J CJ formation, it has no reported role in SJ formation in normal cells. Here, we report that DNA-PKcs and ATM have redundant functions in SJ formation. Thus, combined DNA-PKcs and ATM deficiency during V(D)J recombination leads to accumulation of unjoined SEs and lack of SJ fidelity. Moreover, treatment of DNA-PKcs- or ATM-deficient cells, respectively, with specific kinase inhibitors for ATM or DNA-PKcs recapitulates SJ defects, indicating that the overlapping V(D)J recombination functions of ATM and DNA-PKcs are mediated through their kinase activities.

ATM damage response and XLF repair factor are functionally redundant in joining DNA breaks.

Classical non-homologous DNA end-joining (NHEJ) is a major mammalian DNA double-strand-break (DSB) repair pathway. Deficiencies for classical NHEJ factors, such as XRCC4, abrogate lymphocyte development, owing to a strict requirement for classical NHEJ to join V(D)J recombination DSB intermediates. The XRCC4-like factor (XLF; also called NHEJ1) is mutated in certain immunodeficient human patients and has been implicated in classical NHEJ; however, XLF-deficient mice have relatively normal lymphocyte development and their lymphocytes support normal V(D)J recombination. The ataxia telangiectasia-mutated protein (ATM) detects DSBs and activates DSB responses by phosphorylating substrates including histone H2AX. However, ATM deficiency causes only modest V(D)J recombination and lymphocyte developmental defects, and H2AX deficiency does not have a measurable impact on these processes. Here we show that XLF, ATM and H2AX all have fundamental roles in processing and joining DNA ends during V(D)J recombination, but that these roles have been masked by unanticipated functional redundancies. Thus, combined deficiency of ATM and XLF nearly blocks mouse lymphocyte development due to an inability to process and join chromosomal V(D)J recombination DSB intermediates. Combined XLF and ATM deficiency also severely impairs classical NHEJ, but not alternative end-joining, during IgH class switch recombination. Redundant ATM and XLF functions in classical NHEJ are mediated by ATM kinase activity and are not required for extra-chromosomal V(D)J recombination, indicating a role for chromatin-associated ATM substrates. Correspondingly, conditional H2AX inactivation in XLF-deficient pro-B lines leads to V(D)J recombination defects associated with marked degradation of unjoined V(D)J ends, revealing that H2AX has a role in this process.

ATM-deficient thymic lymphoma is associated with aberrant tcrd rearrangement and gene amplification.

Ataxia telangiectasia mutated (ATM) deficiency predisposes humans and mice to T lineage lymphomas with recurrent chromosome 14 translocations involving the T cell receptor alpha/delta (Tcra/d) locus. Such translocations have been thought to result from aberrant repair of DNA double-strand breaks (DSBs) during Tcra locus V(D)J recombination, and to require the Tcra enhancer (Ealpha) for Tcra rearrangement or expression of the translocated oncogene. We now show that, in addition to the known chromosome 14 translocation, ATM-deficient mouse thymic lymphomas routinely contain a centromeric fragment of chromosome 14 that spans up to the 5' boundary of the Tcra/d locus, at which position a 500-kb or larger region centromeric to Tcra/d is routinely amplified. In addition, they routinely contain a large deletion of the telomeric end of one copy of chromosome 12. In contrast to prior expectations, the recurrent translocations and amplifications involve V(D)J recombination-initiated breaks in the Tcrd locus, as opposed to the Tcra locus, and arise independently of the Ealpha. Overall, our studies reveal previously unexpected mechanisms that contribute to the oncogenic transformation of ATM-deficient T lineage cells.

Lymphocyte-specific compensation for XLF/cernunnos end-joining functions in V(D)J recombination.

Mutations in XLF/Cernunnos (XLF) cause lymphocytopenia in humans, and various studies suggest an XLF role in classical nonhomologous end joining (C-NHEJ). We now find that XLF-deficient mouse embryonic fibroblasts are ionizing radiation (IR) sensitive and severely impaired for ability to support V(D)J recombination. Yet mature lymphocyte numbers in XLF-deficient mice are only modestly decreased. Moreover, XLF-deficient pro-B lines, while IR-sensitive, perform V(D)J recombination at nearly wild-type levels. Correspondingly, XLF/p53-double-deficient mice are not markedly prone to the pro-B lymphomas that occur in previously characterized C-NHEJ/p53-deficient mice; however, like other C-NHEJ/p53-deficient mice, they still develop medulloblastomas. Despite nearly normal V(D)J recombination in developing B cells, XLF-deficient mature B cells are moderately defective for immunoglobulin heavy-chain class switch recombination. Together, our results implicate XLF as a C-NHEJ factor but also indicate that developing mouse lymphocytes harbor cell-type-specific factors/pathways that compensate for the absence of XLF function during V(D)J recombination.