Clonally expanded EOMES+ Tr1-like cells in primary and metastatic tumors are associated with disease progression.

Regulatory T (Treg) cells are a barrier for tumor immunity and a target for immunotherapy. Using single-cell transcriptomics, we found that CD4+ T cells infiltrating primary and metastatic colorectal cancer and non-small-cell lung cancer are highly enriched for two subsets of comparable size and suppressor function comprising forkhead box protein P3+ Treg and eomesodermin homolog (EOMES)+ type 1 regulatory T (Tr1)-like cells also expressing granzyme K and chitinase-3-like protein 2. EOMES+ Tr1-like cells, but not Treg cells, were clonally related to effector T cells and were clonally expanded in primary and metastatic tumors, which is consistent with their proliferation and differentiation in situ. Using chitinase-3-like protein 2 as a subset signature, we found that the EOMES+ Tr1-like subset correlates with disease progression but is also associated with response to programmed cell death protein 1-targeted immunotherapy. Collectively, these findings highlight the heterogeneity of Treg cells that accumulate in primary tumors and metastases and identify a new prospective target for cancer immunotherapy.

Fruquintinib versus placebo in patients with refractory metastatic colorectal cancer (FRESCO-2): an international, multicentre, randomised, double-blind, phase 3 study.

BACKGROUND: There is a paucity of effective systemic therapy options for patients with advanced, chemotherapy-refractory colorectal cancer. We aimed to evaluate the efficacy and safety of fruquintinib, a highly selective and potent oral inhibitor of vascular endothelial growth factor receptors (VEGFRs) 1, 2, and 3, in patients with heavily pretreated metastatic colorectal cancer. METHODS: We conducted an international, randomised, double-blind, placebo-controlled, phase 3 study (FRESCO-2) at 124 hospitals and cancer centres across 14 countries. We included patients aged 18 years or older (≥20 years in Japan) with histologically or cytologically documented metastatic colorectal adenocarcinoma who had received all current standard approved cytotoxic and targeted therapies and progressed on or were intolerant to trifluridine-tipiracil or regorafenib, or both. Eligible patients were randomly assigned (2:1) to receive fruquintinib (5 mg capsule) or matched placebo orally once daily on days 1-21 in 28-day cycles, plus best supportive care. Stratification factors were previous trifluridine-tipiracil or regorafenib, or both, RAS mutation status, and duration of metastatic disease. Patients, investigators, study site personnel, and sponsors, except for selected sponsor pharmacovigilance personnel, were masked to study group assignments. The primary endpoint was overall survival, defined as the time from randomisation to death from any cause. A non-binding futility analysis was done when approximately one-third of the expected overall survival events had occurred. Final analysis occurred after 480 overall survival events. This study is registered with ClinicalTrials.gov, NCT04322539, and EudraCT, 2020-000158-88, and is ongoing but not recruiting. FINDINGS: Between Aug 12, 2020, and Dec 2, 2021, 934 patients were assessed for eligibility and 691 were enrolled and randomly assigned to receive fruquintinib (n=461) or placebo (n=230). Patients had received a median of 4 lines (IQR 3-6) of previous systemic therapy for metastatic disease, and 502 (73%) of 691 patients had received more than 3 lines. Median overall survival was 7·4 months (95% CI 6·7-8·2) in the fruquintinib group versus 4·8 months (4·0-5·8) in the placebo group (hazard ratio 0·66, 95% CI 0·55-0·80; p<0·0001). Grade 3 or worse adverse events occurred in 286 (63%) of 456 patients who received fruquintinib and 116 (50%) of 230 who received placebo; the most common grade 3 or worse adverse events in the fruquintinib group included hypertension (n=62 [14%]), asthenia (n=35 [8%]), and hand-foot syndrome (n=29 [6%]). There was one treatment-related death in each group (intestinal perforation in the fruquintinib group and cardiac arrest in the placebo group). INTERPRETATION: Fruquintinib treatment resulted in a significant and clinically meaningful benefit in overall survival compared with placebo in patients with refractory metastatic colorectal cancer. These data support the use of fruquintinib as a global treatment option for patients with refractory metastatic colorectal cancer. Ongoing analysis of the quality of life data will further establish the clinical benefit of fruquintinib in this patient population. FUNDING: HUTCHMED.

An optimized SpCas9 high-fidelity variant for direct protein delivery.

Electroporation of the Cas9 ribonucleoprotein (RNP) complex offers the advantage of preventing off-target cleavages and potential immune responses produced by long-term expression of the nuclease. Nevertheless, the majority of engineered high-fidelity Streptococcus pyogenes Cas9 (SpCas9) variants are less active than the wild-type enzyme and are not compatible with RNP delivery. Building on our previous studies on evoCas9, we developed a high-fidelity SpCas9 variant suitable for RNP delivery. The editing efficacy and precision of the recombinant high-fidelity Cas9 (rCas9HF), characterized by the K526D substitution, was compared with the R691A mutant (HiFi Cas9), which is currently the only available high-fidelity Cas9 that can be used as an RNP. The comparative analysis was extended to gene substitution experiments where the two high fidelities were used in combination with a DNA donor template, generating different ratios of non-homologous end joining (NHEJ) versus homology-directed repair (HDR) for precise editing. The analyses revealed a heterogeneous efficacy and precision indicating different targeting capabilities between the two variants throughout the genome. The development of rCas9HF, characterized by an editing profile diverse from the currently used HiFi Cas9 in RNP electroporation, increases the genome editing solutions for the highest precision and efficient applications.

Demystifying BRAF Mutation Status in Colorectal Liver Metastases : A Multi-institutional, Collaborative Approach to 6 Open Clinical Questions.

OBJECTIVE: To investigate the clinical implications of BRAF -mutated (mut BRAF ) colorectal liver metastases (CRLMs). BACKGROUND: The clinical implications of mut BRAF status in CRLMs are largely unknown. METHODS: Patients undergoing resection for mut BRAF CRLM were identified from prospectively maintained registries of the collaborating institutions. Overall survival (OS) and recurrence-free survival (RFS) were compared among patients with V600E versus non-V600E mutations, KRAS/BRAF comutation versus mut BRAF alone, microsatellite stability status (Microsatellite Stable (MSS) vs instable (MSI-high)), upfront resectable versus converted tumors, extrahepatic versus liver-limited disease, and intrahepatic recurrence treated with repeat hepatectomy versus nonoperative management. RESULTS: A total of 240 patients harboring BRAF -mutated tumors were included. BRAF V600E mutation was associated with shorter OS (30.6 vs 144 mo, P =0.004), but not RFS compared with non-V600E mutations. KRAS/BRAF comutation did not affect outcomes. MSS tumors were associated with shorter RFS (9.1 vs 26 mo, P <0.001) but not OS (33.5 vs 41 mo, P =0.3) compared with MSI-high tumors, whereas patients with resected converted disease had slightly worse RFS (8 vs 11 mo, P =0.01) and similar OS (30 vs 40 mo, P =0.4) compared with those with upfront resectable disease. Patients with extrahepatic disease had worse OS compared with those with liver-limited disease (8.8 vs 40 mo, P <0.001). Repeat hepatectomy after intrahepatic recurrence was associated with improved OS compared with nonoperative management (41 vs 18.7 mo, P =0.004). All results continued to hold true in the multivariable OS analysis. CONCLUSIONS: Although surgery may be futile in patients with BRAF -mutated CRLM and concurrent extrahepatic disease, resection of converted disease resulted in encouraging survival in the absence of extrahepatic spread. Importantly, second hepatectomy in select patients with recurrence was associated with improved outcomes. Finally, MSI-high status identifies a better prognostic group, with regard to RFS while patients with non-V600E mutations have excellent prognosis.

Delphi Initiative for Early-Onset Colorectal Cancer (DIRECt) International Management Guidelines.

BACKGROUND & AIMS: Patients with early-onset colorectal cancer (eoCRC) are managed according to guidelines that are not age-specific. A multidisciplinary international group (DIRECt), composed of 69 experts, was convened to develop the first evidence-based consensus recommendations for eoCRC. METHODS: After reviewing the published literature, a Delphi methodology was used to draft and respond to clinically relevant questions. Each statement underwent 3 rounds of voting and reached a consensus level of agreement of ≥80%. RESULTS: The DIRECt group produced 31 statements in 7 areas of interest: diagnosis, risk factors, genetics, pathology-oncology, endoscopy, therapy, and supportive care. There was strong consensus that all individuals younger than 50 should undergo CRC risk stratification and prompt symptom assessment. All newly diagnosed eoCRC patients should receive germline genetic testing, ideally before surgery. On the basis of current evidence, endoscopic, surgical, and oncologic treatment of eoCRC should not differ from later-onset CRC, except for individuals with pathogenic or likely pathogenic germline variants. The evidence on chemotherapy is not sufficient to recommend changes to established therapeutic protocols. Fertility preservation and sexual health are important to address in eoCRC survivors. The DIRECt group highlighted areas with knowledge gaps that should be prioritized in future research efforts, including age at first screening for the general population, use of fecal immunochemical tests, chemotherapy, endoscopic therapy, and post-treatment surveillance for eoCRC patients. CONCLUSIONS: The DIRECt group produced the first consensus recommendations on eoCRC. All statements should be considered together with the accompanying comments and literature reviews. We highlighted areas where research should be prioritized. These guidelines represent a useful tool for clinicians caring for patients with eoCRC.

Unique Patterns of Heterogeneous Mismatch Repair Protein Expression in Colorectal Cancer Unveil Different Degrees of Tumor Mutational Burden and Distinct Tumor Microenvironment Features.

Mismatch repair (MMR) protein expression in colorectal cancer (CRC) cells is usually homogeneously retained or lost. Rare lesions may show a heterogeneous pattern of MMR protein expression. We evaluated MMR protein expression (MLH1, MSH2, MSH6, and PMS2) in 200 CRCs, identifying 3 groups with proficient MMR protein expression (MMRp), deficient MMR protein expression (MMRd), and heterogeneous MMR protein expression (MMRh). MMRh tumors were microdissected on the basis of the expression of the heterogeneous marker. DNA was extracted and subjected to targeted sequencing. RNA was purified from bulk tumors of all MMRh cases and in a control series of 15 MMRp and 10 MMRd CRCs and analyzed using the PanCancer IO 360 Panel (NanoString Technologies). Twenty-nine of the 200 cases (14.5%) were MMRd. Nine cases (4.5%) showed a heterogeneous pattern of MMR expression, with 6 tumors harboring concomitant loss of one of the other MMR proteins, thus featuring areas with double loss at immunohistochemistry (IHC) testing (MMRh double-loss cases). Four of the 6 MMRh double-loss cases were suitable for a separate sequence variant analysis of IHC double-negative and IHC single-negative components of the tumor. In all lesions, both components exhibited a high tumor mutation burden (TMB). Nevertheless, a significant increase in TMB in the double-negative components was observed (mean TMB: negative, 70 mut/Mb vs positive, 59 mut/Mb) because of a higher number of subclonal variants compared with the other component. Comparative gene expression analyses among MMRd, MMRp, and MMRh CRCs highlighted differential gene expression patterns and an increased number of tumor-infiltrating lymphocytes in MMRh lesions, which is also characterized by a substantial population of exhausted CD8+ lymphocytes. We describe a unique subgroup of CRCs showing heterogeneous expression of MMR proteins in a background of concomitant loss of one of the other markers.

Predictive value on advance hodgkin lymphoma treatment outcome of end-of treatment FDG PET/CT in the HD0607 clinical trial.

The Lugano classification for response assessment in lymphoma recommends the use of the 5-point-scale Deauville Score (DS) to assess response evaluation of end-of-treatment FDG-PET/CT (eotPET) in Hodgkin Lymphoma (HL); nevertheless, there is a paucity of data on its accuracy and reproducibility. We focus here on the cohort of advanced stage IIb-IV HL patients enrolled in the HD0607 clinical trial (NCT identifier 00795613) that having had a negative interim PET performed 6 cycles of ABVD (Doxorubicin, Vinblastine, Vincristine and Dacarbazine) and then performed an eotPET. Negative patients were randomized to radiotherapy and no further treatment while positive patients were treated based on local policies. eotPET was re-evaluated independently by two readers evaluated and progression free survival was analysed (PFS). eotPET of 254 patients were analysed. The median follow-up was 43 months. The best receiver operator characteristics cut-off values to distinguish positive and negative patients was 4. The area-under-the-curve was 0.81 (95%CI, 0.70-0.91). Three-years PFS was 0.95 (95% CI 0.90-0.97) in eotPET negative and 0.22 (95% CI 0.11-0.43) in eotPET positive. DS demonstrated a good reproducibility of positivity/negativity between the readers consensus and local site evaluation where the agreement occurred on 95.0% of patients. The present study demonstrates that eotPET is an accurate tool to predict treatment outcome in HL and confirms the appropriateness of the Lugano classification for eotPET evaluation.

Inactivation of DNA repair triggers neoantigen generation and impairs tumour growth.

Molecular alterations in genes involved in DNA mismatch repair (MMR) promote cancer initiation and foster tumour progression. Cancers deficient in MMR frequently show favourable prognosis and indolent progression. The functional basis of the clinical outcome of patients with tumours that are deficient in MMR is not clear. Here we genetically inactivate MutL homologue 1 (MLH1) in colorectal, breast and pancreatic mouse cancer cells. The growth of MMR-deficient cells was comparable to their proficient counterparts in vitro and on transplantation in immunocompromised mice. By contrast, MMR-deficient cancer cells grew poorly when transplanted in syngeneic mice. The inactivation of MMR increased the mutational burden and led to dynamic mutational profiles, which resulted in the persistent renewal of neoantigens in vitro and in vivo, whereas MMR-proficient cells exhibited stable mutational load and neoantigen profiles over time. Immune surveillance improved when cancer cells, in which MLH1 had been inactivated, accumulated neoantigens for several generations. When restricted to a clonal population, the dynamic generation of neoantigens driven by MMR further increased immune surveillance. Inactivation of MMR, driven by acquired resistance to the clinical agent temozolomide, increased mutational load, promoted continuous renewal of neoantigens in human colorectal cancers and triggered immune surveillance in mouse models. These results suggest that targeting DNA repair processes can increase the burden of neoantigens in tumour cells; this has the potential to be exploited in therapeutic approaches.

Multimodal treatment with curative intent in a germline BRCA2 mutant metastatic ampullary adenocarcinoma: a case report.

BACKGROUND: Cancers of the Vater ampulla (ampullary cancers, ACs) account for less than 1% of all gastrointestinal tumors. ACs are usually diagnosed at advanced stage, with poor prognosis and limited therapeutic options. BRCA2 mutations are identified in up to 14% of ACs and, differently from other tumor types, therapeutic implications remain to be defined. Here, we report a clinical case of a metastatic AC patient in which the identification of a BRCA2 germline mutation drove a personalized multimodal approach with curative-intent. CASE PRESENTATION: A 42-year-old woman diagnosed with stage IV BRCA2 germline mutant AC underwent platinum-based first line treatment achieving major tumor response but also life-threatening toxicity. Based on this, as well as on molecular findings and expected low impact of available systemic treatment options, the patient underwent radical complete surgical resection of both primary tumor and metastatic lesions. Following an isolated retroperitoneal nodal recurrence, given the expected enhanced sensitivity to radiotherapy in BRCA2 mutant cancers, the patient underwent imaging-guided radiotherapy leading to long-lasting complete tumor remission. After more than 2 years, the disease remains radiologically and biochemically undetectable. The patient accessed a dedicated screening program for BRCA2 germline mutation carriers and underwent prophylactic bilateral oophorectomy. CONCLUSIONS: Even considering the intrinsic limitations of a single clinical report, we suggest that the finding of BRCA germline mutations in ACs should be taken into consideration, together with other clinical variables, given their potential association with remarkable response to cytotoxic chemotherapy that might be burdened with enhanced toxicity. Accordingly, BRCA1/2 mutations might offer the opportunity of personalizing treatment beyond PARP inhibitors up to the choice of a multimodal approach with curative-intent.

Early onset metastatic colorectal cancer in patients receiving panitumumab-based upfront strategy: Overall and sex-specific outcomes in the Valentino trial.

Anti-EGFRs plus doublet chemotherapy is considered the optimal upfront option for RAS/BRAF wild-type left-sided metastatic colorectal cancer (mCRC). Early-onset (EO) mCRC has an increasing incidence and its prognostic/predictive role and management is debatable. We performed a post hoc analysis of Valentino study, that randomized RAS wild-type mCRC patients to two panitumumab-based maintenance regimens after FOLFOX/panitumumab induction. We assessed the safety and efficacy outcomes in patients stratified for age (<50/≥50 years old). We assessed progression-free survival (PFS), overall survival (OS), response rate (ORR), rate of treatment-related and panitumumab-related adverse events (AEs) and quality of life (QoL). In 229 patients enrolled, 35 (15%) had EO mCRC, with a higher rate of female sex (P = .020) and lower rate of primary tumor resection (P = .001). Median PFS and OS were 10.9 vs 10.8 months (P = .593) and 28.1 vs 27.5 months (P = .865) in patients <50 and ≥50 years old, respectively, with no significant impact of maintenance arm. ORR and disease control rate were 74% vs 65% (P = .337) and 97% vs 81% (P = .013) in patients <50 or ≥50 years old. In younger patients, a trend for increased chemotherapy-related AEs (peculiarly anemia) was shown, while significantly decreased EGFR-related hypomagnesemia and increased skin rash were reported. No significant differences in treatment intensity or QoL were observed. In patients with EO mCRC and RAS wild-type status, we found no differences in terms of survival outcomes based on age when selecting maintenance strategies. Management of treatment-related AEs should consider the differential toxicity profile of age and sex.

Liquid biopsies to monitor and direct cancer treatment in colorectal cancer.

Colorectal cancer (CRC) is one of the most prevalent and deadly cancers worldwide. Despite recent improvements in treatment and prevention, most of the current therapeutic options are weighted by side effects impacting patients' quality of life. Better patient selection towards systemic treatments represents an unmet clinical need. The recent multidisciplinary and molecular advancements in the treatment of CRC patients demand the identification of efficient biomarkers allowing to personalise patient care. Currently, core tumour biopsy specimens represent the gold-standard biological tissue to identify such biomarkers. However, technical feasibility, tumour heterogeneity and cancer evolution are major limitations of this single-snapshot approach. Genotyping circulating tumour DNA (ctDNA) has been addressed as potentially overcoming such limitations. Indeed, ctDNA has been retrospectively demonstrated capable of identifying minimal residual disease post-surgery and post-adjuvant treatment, as well as spotting druggable molecular alterations for tailoring treatments in metastatic disease. In this review, we summarise the available evidence on ctDNA applicability in CRC. Then, we review ongoing clinical trials assessing how liquid biopsy can be used interventionally to guide therapeutic choice in localised, locally advanced and metastatic CRC. Finally, we discuss how its widespread could transform CRC patients' management, dissecting its limitations while suggesting improvement strategies.

Benefit from upfront FOLFOXIRI and bevacizumab in BRAFV600E-mutated metastatic colorectal cancer patients: does primary tumour location matter?

BACKGROUND: Recent data suggest that BRAFV600E-mutated metastatic colorectal cancer (mCRC) patients with right-sided tumours and ECOG-PS = 0 may achieve benefit from the triplet regimen differently than those with left-sided tumours and ECOG-PS > 0. METHODS: The predictive impact of primary sidedness and ECOG-PS was evaluated in a large real-life dataset of 296 BRAFV600E-mutated mCRC patients treated with upfront triplet or doublet ± bevacizumab. Biological differences between right- and left-sided BRAFV600E-mutated CRCs were further investigated in an independent cohort of 1162 samples. RESULTS: A significant interaction effect between primary sidedness and treatment intensity was reported in terms of both PFS (p = 0.010) and OS (p = 0.003), with a beneficial effect of the triplet in the right-sided group and a possible detrimental effect in the left-sided. No interaction effect was observed between ECOG-PS and chemo-backbone. In the MSS/pMMR population, a consistent trend for a side-related subgroup effect was observed when FOLFOXIRI ± bevacizumab was compared to oxaliplatin-based doublets±bevacizumab (p = 0.097 and 0.16 for PFS and OS, respectively). Among MSS/pMMR tumours, the BM1 subtype was more prevalent in the right-sided group (p = 0.0019, q = 0.0139). No significant differences were observed according to sidedness in the MSI-H/dMMR population. CONCLUSIONS: Real-life data support the use of FOLFOXIRI ± bevacizumab only in BRAFV600E-mutated mCRC patients with right-sided tumours.

Reinduction of an Anti-EGFR-based First-line Regimen in Patients with RAS Wild-type Metastatic Colorectal Cancer Enrolled in the Valentino Study.

BACKGROUND: In patients with RAS/BRAF wild-type metastatic colorectal cancer (mCRC), growing evidence supports anti-epidermal growth factor receptor (EGFR) retreatment, whereas little is known on the outcomes of anti-EGFR-based reinduction therapy during the upfront strategy. METHODS: We included patients enrolled in the Valentino study who had disease progression and received at least one dose of post-progression therapy. The Kaplan-Meier method and Cox proportional hazards regression were used for the survival analysis. When comparing the outcomes of anti-EGFR-based reinduction versus any second line, a propensity score-based matching was used. RESULTS: Liver-limited/single site of disease (P < .001 and P = .002), left-sidedness (P = .029), surgery of metastases (P = .003), early tumor shrinkage, and deeper responses (P = .018 and P = .036) were associated with the use of anti-EGFR-based reinduction versus any other second line. All patients treated with reinduction had an anti-EGFR-free interval of at least 3 months. In the propensity score-matched population, progression-free survival (PFS) was similar in the 2 treatment groups, the overall survival (OS) was significantly longer for patients treated with reinduction (P = .029), and the response rate was higher in patients treated with reinduction (P = .033). An oxaliplatin-free interval ≥12 months, left-sidedness, and molecular hyperselection beyond RAS/BRAF were associated with significantly better outcomes after anti-EGFR-based reinduction. CONCLUSIONS: Reinduction strategies with anti-EGFR-based regimens are commonly used in clinical practice. Our data highlight the importance of clinical-molecular selection for re-treatments and the need for prospective strategy trials in selected populations.

Assessment of brain tumors by magnetic resonance dynamic susceptibility contrast perfusion-weighted imaging and computed tomography perfusion: a comparison study.

PURPOSE: To investigate the association and agreement between magnetic resonance dynamic susceptibility contrast perfusion-weighted imaging (DSC-PWI) and computed tomography perfusion (CTP) in determining vascularity and permeability of primary and secondary brain tumors. MATERIAL AND METHODS: DSC-PWI and CTP studies from 97 patients with high-grade glioma, low-grade glioma and solitary brain metastasis were retrospectively reviewed. Normalized cerebral blood flow (nCBF), cerebral blood volume (nCBV), capillary transfer constant (nK2) and permeability surface area product (nPS) values were obtained. Variables among groups were compared, and correlation and agreement between DSC-PWI and CTP were tested. RESULTS: All DSC-PWI and CTP parameters were higher in high-grade than in low-grade gliomas (p < 0.01 and p < 0.001). Metastases had greater DSC-PWI nCBV (p < 0.05), nCTP-CBF (p < 0.05), nCTP-CBV (p < 0.01) and nCTP-PS (p < 0.0001) than low-grade gliomas and more elevated nCTP-PS (p < 0.01) than high-grade gliomas. The correlation was strong between DSC-PWI nCBF and CTP nCBF (r = 0.79; p < 0.00001) and between DSC-PWI nCBV and CTP nCBV (r = 0.83; p < 0.00001), weaker between DSC-PWI nK2 and CTP nPS (r = 0.29; p < 0.01). Bland-Altman plots indicated that the agreement was strong between DSC-PWI nCBF and CTP nCBF, good between DSC-PWI nCBV and CTP nCBV and poorer between DSC-PWI nK2 and CTP nPS. CONCLUSION: DSC-PWI and CTP CBF and CBV maps were comparable and interchangeable in the assessment of tumor vascularity, unlike DSC-PWI K2 and CTP PS maps that were more discordant in the analysis of tumor permeability. CTP could be an alternative method to quantify tumor neoangiogenesis when MRI is not available or when the patient does not tolerate it.

MicroRNAs Expression in Response to rhNGF in Epithelial Corneal Cells: Focus on Neurotrophin Signaling Pathway.

PURPOSE: Nerve growth factor efficacy was demonstrated for corneal lesions treatment, and recombinant human NGF (rhNGF) was approved for neurotrophic keratitis therapy. However, NGF-induced molecular responses in cornea are still largely unknown. We analyzed microRNAs expression in human epithelial corneal cells after time-dependent rhNGF treatment. METHODS: Nearly 700 microRNAs were analyzed by qRT-PCR. MicroRNAs showing significant expression differences were examined by DIANA-miRpath v.3.0 to identify target genes and pathways. Immunoblots were performed to preliminarily assess the strength of the in silico results. RESULTS: Twenty-one microRNAs (miR-26a-1-3p, miR-30d-3p, miR-27b-5p, miR-146a-5p, miR-362-5p, mir-550a-5p, mir-34a-3p, mir-1227-3p, mir-27a-5p, mir-222-5p, mir-151a-5p, miR-449a, let7c-5p, miR-337-5p, mir-29b-3p, miR-200b-3p, miR-141-3p, miR-671-3p, miR-324-5p, mir-411-3p, and mir-425-3p) were significantly regulated in response to rhNGF. In silico analysis evidenced interesting target genes and pathways, including that of neurotrophin, when analyzed in depth. Almost 80 unique target genes (e.g., PI3K, AKT, MAPK, KRAS, BRAF, RhoA, Cdc42, Rac1, Bax, Bcl2, FasL) were identified as being among those most involved in neurotrophin signaling and in controlling cell proliferation, growth, and apoptosis. AKT and RhoA immunoblots demonstrated congruence with microRNA expression, providing preliminary validation of in silico data. CONCLUSIONS: MicroRNA levels in response to rhNGF were for the first time analyzed in corneal cells. Novel insights about microRNAs, target genes, pathways modulation, and possible biological responses were provided. Importantly, given the putative role of microRNAs as biomarkers or therapeutic targets, our results make available data which might be potentially exploitable for clinical applications.

Trastuzumab deruxtecan (DS-8201) in patients with HER2-expressing metastatic colorectal cancer (DESTINY-CRC01): a multicentre, open-label, phase 2 trial.

BACKGROUND: HER2 amplification has been identified in 2-3% of patients with colorectal cancer, although there are currently no approved HER2-targeted therapies for colorectal cancer. We aimed to study the antitumour activity and safety of trastuzumab deruxtecan (an antibody-drug conjugate of humanised anti-HER2 antibody with topoisomerase I inhibitor payloads) in patients with HER2-expressing metastatic colorectal cancer. METHODS: DESTINY-CRC01 is an open-label, phase 2 study that recruited patients from 25 clinics and hospitals in Italy, Japan, Spain, the UK, and the USA. Eligible patients had centrally confirmed HER2-expressing metastatic colorectal cancer that had progressed on two or more previous regimens (HER2-targeted therapies other than trastuzumab deruxtecan permitted), were aged 18 years or older (≥20 years in Japan), had an Eastern Cooperative Oncology Group score of 0 or 1, and had RAS and BRAFV600E wild-type tumours. Patients were enrolled into one of three cohorts by HER2 expression level: cohort A (HER2-positive, immunohistochemistry [IHC] 3+ or IHC2+ and in-situ hybridisation [ISH]-positive), cohort B (IHC2+ and ISH-negative), or cohort C (IHC1+). Patients received 6·4 mg/kg trastuzumab deruxtecan intravenously every 3 weeks until disease progression, unacceptable adverse events, withdrawal of consent, or death. The primary endpoint was confirmed objective response rate in cohort A by independent central review which was assessed in the full analysis set and safety was assessed in the safety analysis set. Both the full analysis set and the safety analysis set included all patients who received one or more doses of trastuzumab deruxtecan. This ongoing trial is registered with ClinicalTrials.gov, number NCT03384940. FINDINGS: Between Feb 23, 2018, and July 3, 2019, 78 patients were enrolled in the study (53 in cohort A, seven in cohort B, and 18 in cohort C), all of whom received at least one dose of study drug. For the 53 (68%) patients with HER2-positive tumours (cohort A), a confirmed objective response was reported in 24 (45·3%, 95% CI 31·6-59·6) patients after a median follow-up of 27·1 weeks (IQR 19·3-40·1). Grade 3 or worse treatment-emergent adverse events that occurred in at least 10% of all participants were decreased neutrophil count (17 [22%] of 78) and anaemia (11 [14%]). Five patients (6%) had adjudicated interstitial lung disease or pneumonitis (two grade 2; one grade 3; two grade 5, the only treatment-related deaths). INTERPRETATION: Trastuzumab deruxtecan showed promising and durable activity in HER2-positive metastatic colorectal cancer refractory to standard treatment, with a safety profile consistent with that reported in previous trastuzumab deruxtecan trials. Interstitial lung disease and pneumonitis are important risks requiring careful monitoring and prompt intervention. FUNDING: Daiichi Sankyo.

Coronary microvascular function is impaired in patients with cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy.

BACKGROUND AND PURPOSE: Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a rare inherited disease caused by NOTCH3 gene mutations. Although the main clinical features reflect brain injury, CADASIL is a systemic microangiopathy, and cardiac involvement has been observed but not systematically assessed. We aimed to study the prevalence and severity of coronary microvascular dysfunction (CMD) in CADASIL patients. METHODS: Seventeen patients with genetically confirmed CADASIL, aged <60 years (mean age 40 ± 9 years), with ≤1 cardiovascular risk factor underwent neurological and neuropsychological evaluation, 3T brain magnetic resonance imaging (MRI), 12-lead electrocardiography (ECG), standard echocardiography, and measurement of myocardial blood flow at rest (resting MBF) and of maximal myocardial blood flow following Regadenoson infusion (Reg-MBF) by 13 NH3 positron emission tomography (PET). Coronary flow reserve (CFR) was defined as Reg-MBF/resting MBF. PET results were compared to those of 15 healthy controls who were age and sex matched. RESULTS: Twelve patients (71%) presented migraine, none (53%) had psychiatric disturbances, and one (6%) had a previous stroke. None had cognitive impairment or ECG or echocardiography abnormalities. Both Reg-MBF and CFR were blunted in CADASIL patients compared with controls (Reg-MBF 2.46 ± 0.54 vs. 3.09 ± 0.44 ml/g/min, respectively; p < 0.01; CFR 2.74 ± 0.36 vs. 3.28 ± 0.66, respectively, p < 0.01). No correlations were found between Reg-MBF values and neuropsychological performance or cerebral lesion burden on MRI. CONCLUSIONS: CADASIL patients exhibit blunted CFR due to CMD, which can be severe and is independent of the severity of brain lesion load and cognitive performances. CADASIL is a systemic microcirculation disease, and active surveillance of cardiac symptoms should be considered in these patients.

Concomitant semi-quantitative and visual analysis improves the predictive value on treatment outcome of interim 18F-fluorodeoxyglucose/positron-emission tomography in advanced Hodgkin lymphoma.

BACKGROUND: Qualitative assessment using the Deauville five-point Scale (DS) is the gold standard for interim and end-of treatment positron-emission tomography (PET) interpretation in lymphoma. In the present study we assessed the reliability and the prognostic value of different semi- quantitative parameters in comparison with DS for interim PET (iPET) interpretation in Hodgkin lymphoma (HL). METHODS: A cohort of 82 out of 260 patients with advanced stage HL enrolled in the International Validation Study (IVS), scored as 3 to 5 by the expert panel was included in the present report. Two nuclear medicine physicians blinded to patient history, clinical data and treatment outcome reviewed independently the iPET using the following parameters: DS, SUVmax, SUVpeak of the most active lesion, Qmax (ratio of SUVmax of the lesion to liver SUVmax) and Qres (ratio of SUVpeak of the lesion to liver SUVmean). The optimal sensitivity, specificity, positive and negative predictive value to predict treatment outcome was calculated for all the above parameters with the receiver operator characteristics analysis. RESULTS: The prognostic value of all parameters were similar, the best cut-off value being 4 for DS (area under the curve [AUC], 0.81 95% CI: 0.72-0.90), 3.81 for SUVmax (AUC 0.82 95% CI: 0.73-0.91), 3.20 for SUVpeak (AUC 0.86 95% CI: 0.77-0.94), 1.07 for Qmax (AUC 0.84 95% CI: 0.75-0.93) and 1.38 for Qres (AUC 0.84 95% CI: 0.75-0.93). The reproducibility of different parameters was similar as the inter-observer variability measured with Cohen's kappa were 0.93 (95% CI: 0.84-1.01) for the DS, 0.88 (0.77-0.98) for SUVmax, 0.82 (0.70-0.95) for SUVpeak, 0.85 (0.74-0.97) for Qres and 0.78 (0.65-0.92) for Qmax. Due to the high specificity of SUVpeak (0.87) and to the good sensitivity of DS (0.86), upon the use of both parameters the positive predictive value increased from 0.65 of the DS alone to 0.79. When both parameters were positive in iPET, 3-years Failure-Free Survival (FFS) was significantly lower compared to patients whose iPET was interpreted with qualitative parameters only (DS 4 or 5): 21% vs. 35%. On the other hand, the FFS of patients with negative results was not significantly different (88% vs. 86%). CONCLUSIONS: In this study we demonstrated that, combining semi-quantitative parameters with SUVpeak to a pure qualitative interpretation key with DS, it is possible to increase the positive predictive value of iPET and to identify with higher precision the patients subset with a very dismal prognosis. However, these retrospective findings should be confirmed prospectively in a larger patient cohort.

Personalized therapeutic strategies in HER2-driven gastric cancer.

BACKGROUND: Trastuzumab is the only approved targeted therapy in patients with HER2-amplified metastatic gastric cancer (GC). Regrettably, in clinical practice, only a fraction of them achieves long-term benefit from trastuzumab-based upfront strategy. To advance precision oncology, we investigated the therapeutic efficacy of different HER2-targeted strategies, in HER2 "hyper"-amplified (≥ 8 copies) tumors. METHODS: We undertook a prospective evaluation of HER2 targeting with monoclonal antibodies, tyrosine kinase inhibitors and antibody-drug conjugates, in a selected subgroup of HER2 "hyper"-amplified gastric patient-derived xenografts (PDXs), through the design of ad hoc preclinical trials. RESULTS: Despite the high level of HER2 amplification, trastuzumab elicited a partial response only in 2 out of 8 PDX models. The dual-HER2 blockade with trastuzumab plus either pertuzumab or lapatinib led to complete and durable responses in 5 (62.5%) out of 8 models, including one tumor bearing a concomitant HER2 mutation. In a resistant PDX harboring KRAS amplification, the novel antibody-drug conjugate trastuzumab deruxtecan (but not trastuzumab emtansine) overcame KRAS-mediated resistance. We also identified a HGF-mediated non-cell-autonomous mechanism of secondary resistance to anti-HER2 drugs, responsive to MET co-targeting. CONCLUSION: These preclinical randomized trials clearly indicate that in HER2-driven gastric tumors, a boosted HER2 therapeutic blockade is required for optimal efficacy, leading to complete and durable responses in most of the cases. Our results suggest that a selected subpopulation of HER2-"hyper"-amplified GC patients could strongly benefit from this strategy. Despite the negative results of clinical trials, the dual blockade should be reconsidered for patients with clearly HER2-addicted cancers.

The genomic landscape of response to EGFR blockade in colorectal cancer.

Colorectal cancer is the third most common cancer worldwide, with 1.2 million patients diagnosed annually. In late-stage colorectal cancer, the most commonly used targeted therapies are the monoclonal antibodies cetuximab and panitumumab, which prevent epidermal growth factor receptor (EGFR) activation. Recent studies have identified alterations in KRAS and other genes as likely mechanisms of primary and secondary resistance to anti-EGFR antibody therapy. Despite these efforts, additional mechanisms of resistance to EGFR blockade are thought to be present in colorectal cancer and little is known about determinants of sensitivity to this therapy. To examine the effect of somatic genetic changes in colorectal cancer on response to anti-EGFR antibody therapy, here we perform complete exome sequence and copy number analyses of 129 patient-derived tumour grafts and targeted genomic analyses of 55 patient tumours, all of which were KRAS wild-type. We analysed the response of tumours to anti-EGFR antibody blockade in tumour graft models and in clinical settings and functionally linked therapeutic responses to mutational data. In addition to previously identified genes, we detected mutations in ERBB2, EGFR, FGFR1, PDGFRA, and MAP2K1 as potential mechanisms of primary resistance to this therapy. Novel alterations in the ectodomain of EGFR were identified in patients with acquired resistance to EGFR blockade. Amplifications and sequence changes in the tyrosine kinase receptor adaptor gene IRS2 were identified in tumours with increased sensitivity to anti-EGFR therapy. Therapeutic resistance to EGFR blockade could be overcome in tumour graft models through combinatorial therapies targeting actionable genes. These analyses provide a systematic approach to evaluating response to targeted therapies in human cancer, highlight new mechanisms of responsiveness to anti-EGFR therapies, and delineate new avenues for intervention in managing colorectal cancer.