Immuno-transcriptomic profiling of extracranial pediatric solid malignancies.

We perform an immunogenomics analysis utilizing whole-transcriptome sequencing of 657 pediatric extracranial solid cancer samples representing 14 diagnoses, and additionally utilize transcriptomes of 131 pediatric cancer cell lines and 147 normal tissue samples for comparison. We describe patterns of infiltrating immune cells, T cell receptor (TCR) clonal expansion, and translationally relevant immune checkpoints. We find that tumor-infiltrating lymphocytes and TCR counts vary widely across cancer types and within each diagnosis, and notably are significantly predictive of survival in osteosarcoma patients. We identify potential cancer-specific immunotherapeutic targets for adoptive cell therapies including cell-surface proteins, tumor germline antigens, and lineage-specific transcription factors. Using an orthogonal immunopeptidomics approach, we find several potential immunotherapeutic targets in osteosarcoma and Ewing sarcoma and validated PRAME as a bona fide multi-pediatric cancer target. Importantly, this work provides a critical framework for immune targeting of extracranial solid tumors using parallel immuno-transcriptomic and -peptidomic approaches

Factor XIII Transglutaminase Supports the Resolution of Mucosal Damage in Experimental Colitis.

The thrombin-activated transglutaminase factor XIII (FXIII) that covalently crosslinks and stablizes provisional fibrin matrices is also thought to support endothelial and epithelial barrier function and to control inflammatory processes. Here, gene-targeted mice lacking the FXIII catalytic A subunit were employed to directly test the hypothesis that FXIII limits colonic pathologies associated with experimental colitis. Wildtype (WT) and FXIII-/- mice were found to be comparable in their initial development of mucosal damage following exposure to dextran sulfate sodium (DSS) challenge. However, unlike FXIII-sufficient mice, FXIII-deficient cohorts failed to efficiently resolve colonic inflammatory pathologies and mucosal damage following withdrawal of DSS. Consistent with prior evidence of ongoing coagulation factor activation and consumption in individuals with active colitis, plasma FXIII levels were markedly decreased in colitis-challenged WT mice. Treatment of colitis-challenged mice with recombinant human FXIII-A zymogen significantly mitigated weight loss, intestinal bleeding, and diarrhea, regardless of whether cohorts were FXIII-sufficient or were genetically devoid of FXIII. Similarly, both qualitative and quantitative microscopic analyses of colonic tissues revealed that exogenous FXIII improved the resolution of multiple colitis disease parameters in both FXIII-/- and WT mice. The most striking differences were seen in the resolution of mucosal ulceration, the most severe histopathological manifestation of DSS-induced colitis. These findings directly demonstrate that FXIII is a significant determinant of mucosal healing and clinical outcome following inflammatory colitis induced mucosal injury and provide a proof-of-principle that clinical interventions supporting FXIII activity may be a means to limit colitis pathology and improve resolution of mucosal damage

Genetic elimination of FXIII prolongs colitis symptoms.

<p>FXIII^−/− mice (◯) challenged with DSS develop significantly worse clinical signs of disease relative to WT mice (●) based on a multiparameter Disease Activity Index score (A) and weight loss (B). Note that body mass continued to decline in FXIII^−/− mice even after DSS was withdrawn, whereas body mass stabilized and improved in WT animals during this period. <i>P</i> values were generated with a 2 way ANOVA, n = 10 mice per cohort.</p

Treatment with rFXIII ameliorates colitis severity in both WT and FXIII^−/− mice.

<p>(A) Pharmacological treatment of DSS-challenged WT mice with exogenous rFXIII (◯, n = 12) significantly reduced clinical disease parameters (disease activity index, DAI) relative to vehicle treated WT mice (●, n = 11). (B) A more modest, but significant therapeutic benefit of exogenous rFXIII (◯, n = 14) was also observed in FXIII^−/− mice relative to vehicle treatment (●, n = 14). (C) Plasma levels of rFXIII measured at the end of the 14 day experiment were comparable between FXIII^−/− and WT mice. As expected, no human FXIII protein was detectable (ND; not detected) in vehicle treated mice. (D) Total plasma FXIII catalytic activity measured at the end of the 14 day study period was predictably greatest in rFXIII treated WT mice. Note that this represents activity derived from both endogenous FXIII and exogenous rFXIII). (E) Representative microscopic view of H&E-stained section of colonic tissue harvested at the end of the 14 day study from a vehicle-treated FXIII^−/− mouse. Note the large area of inflammatory edema (*) and mucosal ulceration (**). Treatment of FXIII^−/− mice with rFXIII significantly limited colonic histopathology (F), but areas of mucosal damage, including ulceration (not shown), crypt loss (triangles) and edema (*) were still common. Consistent with prior results, colons harvested from vehicle-treated WT mice (G) exhibited significantly less evidence of residual pathology relative to vehicle-treated FXIII^−/− mice (E) when collected 7 days after withdrawal of the DSS challenge. Nevertheless, small areas of ulceration (not shown) and more frequent areas of crypt spacing associated with inflammatory infiltrates (arrowhead) were still evident. More significantly, DSS-challenged WT mice treated with rFXIII (H) exhibited generally intact, near-normal appearing mucosa. (I) A detailed microscopic evaluation of tissue sections from all FXIII^−/− mice employing the multiparameter histopathological scoring system showed that exogenous rFXIII treatment (white bars) resulted in a significant decrease in ulceration and crypt loss relative to vehicle treatment (black bars). (J) Parallel analyses in WT mice revealed a statistically significant diminution in every disease parameter analysed in rFXIII treated WT mice (white bars) relative to vehicle-treated WT mice (black bars). (K) Both FXIII genotype and treatment with rFXIII were important determinants of mucosal ulceration, shown as a percentage of colon length evaluated. Note that cohorts with the least amount of mucosal ulceration also had the highest level of plasma FXIII activity (Compare panels D and K). Bars in E-H represent 100 μm. *<i>P</i> < 0.05, <i>P</i> values were generated using a 2 way Anova (A & B) or a Mann-Whitney U test.</p

Plasma levels of FXIII are a sensitive biomarker of colitis in mice.

<p>(A) Plasma FXIII activity in unchallenged WT mice and DSS-challenged WT and FXIII^−/− mice. Plasma samples were collected for analysis 7 days following withdrawal from an initial 7 day DSS-challenge. Note that FXIII activity was significantly lower in DSS-challenged WT mice than unchallenged controls, even well into the colitis resolution phase (* <i>P</i> < 0.0001, Mann Whitney U test). Predictably, no FXIII activity was detectable in plasma prepared from FXIII^−/− mice. Data were normalized to the unchallenged WT cohort. (B) Plasma FXIII activity in WT mice 7 days after withdrawal of DSS inversely correlated with residual colitis activity as assessed by the histopathology score (R² = 0.63, P < 0.05).</p

Lack of FXIII results in a failure to resolve colonic damage after DSS exposure.

<p>(A) Colon lengths observed in cohorts of WT and FXIII^−/− mice 7 days after withdrawal of DSS (n = 10 per cohort). Note that colons were significantly shorter in FXIII^−/− mice (consistent with more severe lingering disease) in comparison to WT mice. (B) Representative H&E-stained section of colon tissue harvested from a WT mouse 7 days after DSS withdrawal demonstrated substantial resolution of DSS-induced colitis in this time frame. Note the generally intact mucosa with mild inflammation, and occasional crypt loss and crypt spacing (triangle). (C) In contrast, colons harvested from FXIII^−/− mice typically had large areas of ulceration (arrowheads) and inflammatory submucosal edema (*). (D-E) Representative immunohistochemical analyses of fibrin(ogen) deposition (detected by brown staining) within colon tissue sections harvest from WT (D) and FXIII^−/− mice (E) 7 days after withdrawal of the DSS challenge. Note that interstitial fibrin(ogen) was minimal in the well-resolved colonic mucosa of WT mice; indeed, the trace fibrin(ogen) detected was generally not appreciably different from that observed in unchallenged WT mice (F). In contrast, interstitial fibrin(ogen) deposits were readily observed in the colons of FXIII^−/− mice a full week after withdrawal of DSS, and were particularly intense in the numerous areas of lingering severe mucosal damage. Size bars represent 50 μm. (G-I) Colons harvested from FXIII^−/− mice (white bars) had more severe microscopic features of disease relative to WT mice (black bars) in the resolution phase 7 days after withdrawal of DSS challenge based on comparative analyses of histopathology scores evaluating key individual (G) and combined (H) disease scores (n = 10 per cohort). (I) Shown are results of direct microscopic measurement of mucosal ulceration as a percentage of colon length (n = 10 per cohort). * <i>P</i> < 0.01, all <i>P</i> values were generated with a Mann-Whitney U test.</p

FXIII-deficiency does not alter initial colonic mucosal damage following 7 days of continuous DSS exposure.

<p>(A) Colons harvested from FXIII^−/− and WT mice immediately after 7 days of DSS challenge (n = 10 per cohort) were significantly shorter than colons harvested from unchallenged mice (n = 4 per cohort), consistent with severe colitis (<i>P</i> < 0.01 for each comparison). However, colon lengths were similar in animals of both genotypes following 7 days of DSS exposure indicating comparable levels of initial disease. H&E stained sections of colonic tissue harvested from both WT (B) and FXIII^−/− mice (C) had similar evidence of significant mucosal ulceration (arrowheads) and inflammatory edema (*). (D) An unchallenged colon cut in the same plane is shown for comparison. Size bars represent 50 μm. These observations were confirmed using a semi-quantitative multiparameter histopathological scoring system. Shown are the results for each individual parameter (E) and the summation of each parameter yielding a total disease score (F). Black bars and white bars represent WT and FXIII^−/− cohorts (n = 10 per cohort), respectively. <i>P</i> = N.S for each comparison, Mann-Whitney U test. (G) Shown are the levels of inflammatory cytokines measured in colonic homogenates harvested from WT (n = 12) and FXIII^−/− mice (n = 11) immediately following 7 days of DSS challenge as well as levels in colons harvested from unchallenged mice (n = 5). Note that levels of each cytokine measured were significantly elevated in DSS-challenged colons relative to those from unchallenged mice, but FXIII genotype had no significant effect on local cytokine levels.</p

The use of neoadjuvant larotrectinib in the management of children with locally advanced TRK fusion sarcomas.

BackgroundThe highly selective oral tropomyosin receptor kinase (TRK) inhibitor larotrectinib has demonstrated significant activity in adult and pediatric TRK fusion cancers. In the current study, the authors describe the clinical course of children with locally advanced TRK fusion sarcoma who were treated preoperatively with larotrectinib and underwent subsequent surgical resection.MethodsA total of 24 children were treated on a pediatric phase 1 trial of larotrectinib (ClinicalTrials.gov identifier NCT02637687). Five children who had a documented TRK fusion sarcoma and underwent surgical resection were included in the current analysis. Tumor response (Response Evaluation Criteria In Solid Tumors [RECIST] version 1.1) and surgical outcomes were collected prospectively.ResultsA total of 5 patients (median age, 2 years; range, 0.4-12 years) had locally advanced infantile fibrosarcoma (3 patients) or soft-tissue sarcoma (2 patients). Four patients had disease that was refractory to standard therapy. All 5 patients achieved a partial response to larotrectinib by version 1.1 of RECIST and underwent surgical resection after a median of 6 cycles (range, 4-9 cycles) of treatment. Surgical resections were R0 (negative resection margins with no tumor at the inked resection margin) in 3 patients, R1 (microscopic residual tumor at the resection margin) in 1 patient, and R2 (macroscopic residual tumor at the resection margin) in 1 patient. Three patients achieved complete (2 patients) or near-complete (&gt;98% treatment effect; 1 patient) pathologic responses. These patients remained in follow-up and were no longer receiving larotrectinib for a minimum of 7 to 15 months postoperatively. Two patients had viable tumor at the time of surgical resection and positive resection margins and continued to receive adjuvant larotrectinib. No patients experienced postoperative complications or wound healing issues.ConclusionsChildren with locally advanced TRK fusion sarcomas may proceed to surgical resection after treatment with the selective TRK inhibitor larotrectinib, thereby sparing them the potentially significant morbidity noted with current approaches. These results support the evaluation of larotrectinib as presurgical therapy in children with newly diagnosed TRK fusion sarcomas

The use of neoadjuvant larotrectinib in the management of children with locally advanced TRK fusion sarcomas

BackgroundThe highly selective oral tropomyosin receptor kinase (TRK) inhibitor larotrectinib has demonstrated significant activity in adult and pediatric TRK fusion cancers. In the current study, the authors describe the clinical course of children with locally advanced TRK fusion sarcoma who were treated preoperatively with larotrectinib and underwent subsequent surgical resection.MethodsA total of 24 children were treated on a pediatric phase 1 trial of larotrectinib (ClinicalTrials.gov identifier NCT02637687). Five children who had a documented TRK fusion sarcoma and underwent surgical resection were included in the current analysis. Tumor response (Response Evaluation Criteria In Solid Tumors [RECIST] version 1.1) and surgical outcomes were collected prospectively.ResultsA total of 5 patients (median age, 2 years; range, 0.4-12 years) had locally advanced infantile fibrosarcoma (3 patients) or soft-tissue sarcoma (2 patients). Four patients had disease that was refractory to standard therapy. All 5 patients achieved a partial response to larotrectinib by version 1.1 of RECIST and underwent surgical resection after a median of 6 cycles (range, 4-9 cycles) of treatment. Surgical resections were R0 (negative resection margins with no tumor at the inked resection margin) in 3 patients, R1 (microscopic residual tumor at the resection margin) in 1 patient, and R2 (macroscopic residual tumor at the resection margin) in 1 patient. Three patients achieved complete (2 patients) or near-complete (&gt;98% treatment effect; 1 patient) pathologic responses. These patients remained in follow-up and were no longer receiving larotrectinib for a minimum of 7 to 15 months postoperatively. Two patients had viable tumor at the time of surgical resection and positive resection margins and continued to receive adjuvant larotrectinib. No patients experienced postoperative complications or wound healing issues.ConclusionsChildren with locally advanced TRK fusion sarcomas may proceed to surgical resection after treatment with the selective TRK inhibitor larotrectinib, thereby sparing them the potentially significant morbidity noted with current approaches. These results support the evaluation of larotrectinib as presurgical therapy in children with newly diagnosed TRK fusion sarcomas