Purpose: To report the 4-year outcomes of a consecutive series of anal cancer patients treated with concurrent chemo-radiation delivered with intensity-modulated radiotherapy (IMRT), employing a simultaneous integrated boost (SIB) approach. Methods: A consecutive series of 54 patients was enrolled between 2007 and 2013. Treatment schedule consisted of 50.4 Gy/28 fractions (1.8 Gy daily) to the gross tumor volume, while the elective nodal volumes were prescribed 42 Gy/28 fractions (1.5 Gy/daily) for patients having a cT2N0 disease. Patients with cT3-T4/N0-N3 tumors were prescribed 54 (T3) or 60 (T4) Gy/30 fractions (1.8–2 Gy daily) to the gross tumor volume; gross nodal volumes were prescribed 50.4 Gy/30 fr (1.68 Gy daily) if sized ≤ 3 cm or 54 Gy/30 fr (1.8 Gy daily) if > 3 cm; elective nodal regions were given 45 Gy/30 fractions (1.5 Gy daily). Chemotherapy was administered concurrently according to the Nigro's regimen. Primary endpoint was colostomy-free survival (CFS). Secondary endpoints were local control (LC), disease-free survival (DFS), cancer-specific survival (CSS), overall survival (OS), and toxicity profile. Results: Median follow up was 32.6 months (range 12–84). The actuarial probability of being alive at 4 years without a colostomy (CFS) was 68.9% (95% CI: 50.3%–84.7%). Actuarial 4-year OS, CSS, DFS, and LC were 77.7% (95% CI: 60.7–88.1%), 81.5% (95% CI: 64%–91%), 65.5% (95% CI: 47.7%–78.5%), and 84.6% (95% CI: 71.6%–92%). Actuarial 4-year metastasis-free survival was 74.4% (95% CI: 55.5%–86.2%). Maximum detected acute toxicities were as follows: dermatologic –G3: 13%; GI-G3: 8%; GU-G3: 2%; anemia-G3: 2%; neutropenia-G3:11%; G4: 2%; thrombocytopenia- G3:2%. Four-year G2 chronic toxicity rates were 2.5% (95% CI: 3.6–16.4) for GU, 14.4% (95% CI: 7.1–28) for GI, 3.9% (95% CI: 1%–14.5%) for skin, and 4.2% (95% CI: 1.1–15.9) for genitalia. Conclusions: Our study shows the feasibility of IMRT in the combined modality treatment of anal cancer, with comparable results to the literature with respect to LC, sphincter preservation and survival. Acute toxicity is lower if compared to series employing standard techniques. Our results support the use of IMRT on a routine basis for the treatment of anal cancer.
<div>AbstractPurpose:<p>Current glioma diagnostic guidelines call for molecular profiling to stratify patients into prognostic and treatment subgroups. In case the tumor tissue is inaccessible, cerebrospinal fluid (CSF) has been proposed as a reliable tumor DNA source for liquid biopsy. We prospectively investigated the use of CSF for molecular characterization of newly diagnosed gliomas.</p>Experimental design:<p>We recruited two cohorts of newly diagnosed patients with glioma, one (<i>n =</i> 45) providing CSF collected in proximity of the tumor, the other (<i>n =</i> 39) CSF collected by lumbar puncture (LP). Both cohorts provided tumor tissues by surgery concomitant with CSF sampling. DNA samples retrieved from CSF and matched tumors were systematically characterized and compared by comprehensive (NGS, next-generation sequencing) or targeted (ddPCR, droplet digital PCR) methodologies. Conventional and molecular diagnosis outcomes were compared.</p>Results:<p>We report that tumor DNA is abundant in CSF close to the tumor, but scanty and mostly below NGS sensitivity threshold in CSF from LP. Indeed, tumor DNA is mostly released by cells invading liquoral spaces, generating a gradient that attenuates by departing from the tumor. Nevertheless, in >60% of LP CSF samples, tumor DNA is sufficient to assess a selected panel of genetic alterations (<i><u>I</u>DH</i> and <i><u>T</u>ERT</i> promoter mutations, <i><u>E</u>GFR</i> amplification, <i><u>C</u>DKN2A/B</i> deletion: ITEC protocol) and <i>MGMT</i> methylation that, combined with imaging, enable tissue-agnostic identification of main glioma molecular subtypes.</p>Conclusions:<p>This study shows potentialities and limitations of CSF liquid biopsy in achieving molecular characterization of gliomas at first clinical presentation and proposes a protocol to maximize diagnostic information retrievable from CSF DNA.</p></div>
<div>AbstractPurpose:<p>Current glioma diagnostic guidelines call for molecular profiling to stratify patients into prognostic and treatment subgroups. In case the tumor tissue is inaccessible, cerebrospinal fluid (CSF) has been proposed as a reliable tumor DNA source for liquid biopsy. We prospectively investigated the use of CSF for molecular characterization of newly diagnosed gliomas.</p>Experimental Design:<p>We recruited two cohorts of newly diagnosed patients with glioma, one (<i>n =</i> 45) providing CSF collected in proximity of the tumor, the other (<i>n =</i> 39) CSF collected by lumbar puncture (LP). Both cohorts provided tumor tissues by surgery concomitant with CSF sampling. DNA samples retrieved from CSF and matched tumors were systematically characterized and compared by comprehensive (NGS, next-generation sequencing) or targeted (ddPCR, droplet digital PCR) methodologies. Conventional and molecular diagnosis outcomes were compared.</p>Results:<p>We report that tumor DNA is abundant in CSF close to the tumor, but scanty and mostly below NGS sensitivity threshold in CSF from LP. Indeed, tumor DNA is mostly released by cells invading liquoral spaces, generating a gradient that attenuates by departing from the tumor. Nevertheless, in >60% of LP CSF samples, tumor DNA is sufficient to assess a selected panel of genetic alterations (<i><u>I</u>DH</i> and <i><u>T</u>ERT</i> promoter mutations, <i><u>E</u>GFR</i> amplification, <i><u>C</u>DKN2A/B</i> deletion: ITEC protocol) and <i>MGMT</i> methylation that, combined with imaging, enable tissue-agnostic identification of main glioma molecular subtypes.</p>Conclusions:<p>This study shows potentialities and limitations of CSF liquid biopsy in achieving molecular characterization of gliomas at first clinical presentation and proposes a protocol to maximize diagnostic information retrievable from CSF DNA.</p></div>
<p>GC PDXs are annotated for: CNV of HER2, EGFR, FGFR2, MET, KRAS ({greater than or equal to}8 gene copies); COSMIC Mutations/Frameshifts (Allelic frequency >0.3)</p>
<p>Statistics of multiple experimental arms from in vivo immunotherapy experiments. Unpaired two-tailed Student’s t-test of different comparisons between the following experimental conditions: IgG2, silibinin and immune checkpoint blockade (ICB) (Anti-PD1 plus Anti-CTLA4) alone or in combination with silibinin in mice intracardially injected with B16/F10-BrM cells. Unpaired two-tailed Student’s t-test of different comparisons between the following experimental conditions: IgG2, cKOGFAP-Timp1 and immune checkpoint blockade (ICB) (Anti-PD1 plus Anti-CTLA4) alone or in combination in mice intracardially injected with E0771-BrM cells. The table contains information corresponding to Figure 7C and Supplementary Figure 8L.</p>
<p>TIMP1 levels from ELISA applied to CSF from patient samples. Levels of TIMP1 in the blood or in the cerebrospinal fluid (CSF) of non-cancer patients and brain metastasis patients from different primary tumors. Immune Cluster is shown for patients in Figure 7N. The table contains information corresponding to Figure 7L, 7N and Supplementary Figure 9A,C-D.</p>
<p>GSEA of CD8+ T cells incubated with pSTAT3+ conditioned media. Gene set enrichment analysis (GSEA) of top 25 upregulated and downregulated signatures and NES of Biological Process (GOBP) pathways related to T cell function comparing CD8+ in vitro cultures incubated with the pSTAT3- (d1) or pSTAT3+ (d2) secretome. The table contains information corresponding to Figure 2B.</p>
