Invasive Margin Research Articles

A nomogram based on collagen signature for predicting the immunoscore in colorectal cancer.

The Immunoscore can categorize patients into high- and low-risk groups for prognostication in colorectal cancer (CRC). Collagen plays an important role in immunomodulatory functions in the tumor microenvironment (TME). However, the correlation between collagen and the Immunoscore in the TME is unclear. This study aimed to construct a collagen signature to illuminate the relationship between collagen structure and Immunoscore. A total of 327 consecutive patients with stage I-III stage CRC were included in a training cohort. The fully quantitative collagen features were extracted at the tumor center and invasive margin of the specimens using multiphoton imaging. LASSO regression was applied to construct the collagen signature. The association of the collagen signature with Immunoscore was assessed. A collagen nomogram was developed by incorporating the collagen signature and clinicopathological predictors after multivariable logistic regression. The performance of the collagen nomogram was evaluated via calibration, discrimination, and clinical usefulness and then tested in an independent validation cohort. The prognostic values of the collagen nomogram were assessed using Cox regression and the Kaplan-Meier method. The collagen signature was constructed based on 16 collagen features, which included 6 collagen features from the tumor center and 10 collagen features from the invasive margin. Patients with a high collagen signature were more likely to show a low Immunoscore (Lo IS) in both cohorts (P<0.001). A collagen nomogram integrating the collagen signature and clinicopathological predictors was developed. The collagen nomogram yielded satisfactory discrimination and calibration, with an AUC of 0.925 (95% CI: 0.895-0.956) in the training cohort and 0.911 (95% CI: 0.872-0.949) in the validation cohort. Decision curve analysis confirmed that the collagen nomogram was clinically useful. Furthermore, the collagen nomogram-predicted subgroup was significantly associated with prognosis. Moreover, patients with a low-probability Lo IS, rather than a high-probability Lo IS, could benefit from chemotherapy in high-risk stage II and stage III CRC patients. The collagen signature is significantly associated with the Immunoscore in the TME, and the collagen nomogram has the potential to individualize the prediction of the Immunoscore and identify CRC patients who could benefit from adjuvant chemotherapy.

Novel approaches to targeting gliomas at the leading/cutting edge.

Despite decades of clinical trials and surgical advances, the most common high-grade glioma, glioblastoma (GBM), remains an incurable disease with a dismal prognosis. Because of its infiltrative nature, GBM almost always recurs at the margin, or leading edge, where tumor cells invade the surrounding brain parenchyma. This region of GBMs is unique, or heterogeneous, with its own microenvironment that is different from the tumor bulk or core. The GBM microenvironment at the margin contains immunosuppressive constituents as well as invasive and therapy-resistant tumor cells that are difficult to treat. In addition, the blood-brain barrier remains essentially intact at the infiltrative margin of tumors; further limiting the effectiveness of therapies. The invasive margin creates the greatest challenge for neurosurgeons when managing these tumors. The current paradigm of resection of GBM tumors mainly focuses on resection of the contrast-enhancing component of tumors, while GBMs extend well beyond the contrast enhancement. The infiltrative margin represents a unique challenge and opportunity for solutions that may overcome current limitations in tumor treatments. In this review of the current literature, the authors discuss the current and developing advances focused on the detection and treatment of GBM at the infiltrative margin and how this could impact patient outcomes.

Estimated projection of oral squamous cell carcinoma annual incidence from twenty years registry data: a retrospective cross-sectional study in Indonesia.

The incidence of oral squamous cell carcinoma (OSCC) has not been well documented in Indonesia. Thus, we aimed to analyze trends and clinicopathological profiles of OSCC cases in Indonesia, focusing on differences between age and sex groups. A cross-sectional study was conducted in Indonesia's main referral hospital, analyzing 1,093 registered OSCC cases from 2001 to 2020. Trend analysis was performed using Joinpoint regression analysis to determine the annual percentage change (APC) for overall cases and each case group based on age, sex, and anatomical subsites. APC significance was assessed using a Monte Carlo permutation test. The projection of case numbers for the following 5 years (2021-2025) was estimated using linear/non-linear regression analysis and presented as a mathematical function. The significance of the trend slope was measured using an ANOVA test. Demographic and clinicopathological characteristics of OSCC were analyzed according to age and sex, and their comparative analysis was assessed using Chi-square and its alternatives. The incidence of OSCC in female patients and in the tongue and buccal mucosa showed a positive trend (APC 2.06%; 3.48%; 8.62%, respectively). Moreover, the incidence of OSCC overall, and in women with OSCC, is projected to increase significantly in the next 5 years following the quadratic model. The mean age of patients was 51.09 ± 14.36 years, with male patients being younger than female patients. The male-to-female ratio was 1.15, and 36.5% of these patients were categorized as young (≤45 years old). The tongue was the predominantly affected site. Prominent pathologic characteristics included well-differentiation, keratinization, and grade I of Bryne's (1992) cellular differentiation stage. Most patients presented with advanced staging, lymphovascular invasion, and uninvaded margins. Tumor sites and staging varied according to age, while age and tumor sites differed between sexes. The rising incidence trends of OSCC among Indonesian patients, both in the past and projected future, are concerning and warrant attention. Further research into risk factors should be conducted as preventive measures.

Multiplex Immunohistochemical Analysis of the Spatial Immune Cell Landscape of the Tumor Microenvironment.

The immune cell landscape of the tumor microenvironment potentially contains information for the discovery of prognostic and predictive biomarkers. Multiplex immunohistochemistry is a valuable tool to visualize and identify different types of immune cells in tumor tissues while retaining its spatial information. Here we provide detailed protocols to analyze lymphocyte, myeloid, and dendritic cell populations in tissue sections. Starting from cutting formalin-fixed paraffin-embedded sections, automatic multiplex staining procedures on an automated platform, scanning of the slides on a multispectral imaging microscope, to the analysis of images using an in-house-developed machine learning algorithm ImmuNet. These protocols can be applied to a variety of tumor specimens by simply switching tumor markers to analyze immune cells in different compartments of the sample (tumor versus invasive margin) and apply nearest-neighbor analysis. This analysis is not limited to tumor samples but can also be applied to other (non-)pathogenic tissues. Improvements to the equipment and workflow over the past few years have significantly shortened throughput times, which facilitates the future application of this procedure in the diagnostic setting.

Mammary analogue secretory carcinoma (MASC) of parotid gland- A case report

Mammary analogue secretory carcinoma is a rare salivary gland carcinoma which shares morphologic and immunohistochemical features with the secretory carcinoma of breast and was first proposed by Skalova in 2010 with translocation of (q13; q25), t (12; 15), subsequently giving ETV6-NTRK3 gene rearrangement. Usually, it is a benign tumor that affects the parotid gland, with slight predilection for males. The present article report a case of secretory carcinoma of right parotid gland with special emphasis on histological feature and immunohistochemistry for diagnostic purpose and its management. Differential diagnosis includes AciCC, ADC-NOS and MEC. In a review by Chiosa et al (2012) in 337 cases of salivary gland malignancy included AciCC, ADC-NOS, MEC, LGCC, non-ITAC, signet ring adenocarcinoma, PLGA and most common malignancy reclassified as MASC were AciCC (11/89), ADC-NOS (14/37), MEC (1/165). Management for MASC includes surgical excision of the lesion (either superficial or total parotidectomy) for local, less aggressive disease like for low-grade malignant salivary gland neoplasms. For more invasive, metastatic disease and positive margins- neck dissection, radiotherapy and chemotherapy are reserved. There is no standard multidisciplinary treatment protocol established due to scarcity of this disease.

ERS: A simple scoring system to predict early recurrence after surgical resection for hepatocellular carcinoma.

Surgical resection (SR) is a potentially curative treatment of hepatocellular carcinoma (HCC) hampered by high rates of recurrence. New drugs are tested in the adjuvant setting, but standardised risk stratification tools of HCC recurrence are lacking. To develop and validate a simple scoring system to predict 2-year recurrence after SR for HCC. 2359 treatment-naïve patients who underwent SR for HCC in 17 centres in Europe and Asia between 2004 and 2017 were divided into a development (DS; n = 1558) and validation set (VS; n = 801) by random sampling of participating centres. The Early Recurrence Score (ERS) was generated using variables associated with 2-year recurrence in the DS and validated in the VS. Variables associated with 2-year recurrence in the DS were (with associated points) alpha-fetoprotein (<10 ng/mL:0; 10-100: 2; >100: 3), size of largest nodule (≥40 mm: 1), multifocality (yes: 2), satellite nodules (yes: 2), vascular invasion (yes: 1) and surgical margin (positive R1: 2). The sum of points provided a score ranging from 0 to 11, allowing stratification into four levels of 2-year recurrence risk (Wolbers' C-indices 66.8% DS and 68.4% VS), with excellent calibration according to risk categories. Wolber's and Harrell's C-indices apparent values were systematically higher for ERS when compared to Early Recurrence After Surgery for Liver tumour post-operative model to predict time to early recurrence or recurrence-free survival. ERS is a user-friendly staging system identifying four levels of early recurrence risk after SR and a robust tool to design personalised surveillance strategies and adjuvant therapy trials.

Immunonutrition supplementation for resectable gastric cancer during standard perioperative chemotherapy (CT) FLOT: A pivotal study, I–SUPPLY.

57 Background: Gastric carcinomas (GC) can be divided into immunogenic and immune-resistant based on the infiltration of the tumour microenvironment (TME) from different immune cells (ICs) such as Natural Killer (NK), activated CD8+, CD3+ and CD4+ memory T cells. High ImmunoScore (IS) (positive cells/mm2 for each marker (CD8+, CD3+, Foxp3+) in the Core Tumour and Invasive Margins) is strongly related to better prognosis of GC patients in terms of Disease-Free Survival and Overall Survival. ICs activation and consequent immunogenic TME requires high nutrient absorption and synthesis accumulation of protein, lipid and nucleotides. However, tumour cells (TCs) promote their own proliferation by stealing the available micronutrients to ICs therefore leading to an immunosuppressive TME phenotype. Methods: This is a translational, prospective, non-randomized study. Primary endpoint is to assess if enteral immune nutrition (EIN) supplementation during the acme of the activity of cytotoxic of perioperative FLOT (docetaxel, oxaliplatin and 5-fluorouracil) in stage II-III GC can revert the TME in favour of ICs over TCs, witnessed by an increase of IS in surgical specimen over biopsy controls. A steady amount of EIN with Arginine, Omega-3-fatty acids, Glutamine and ribonucleic acid, will be added to patient’s diet from day 3 to 8 of each FLOT cycle, twice a day. We plan to use Image analysis software for automatic tumor detection and quantification of CD3+ and CD8+ stained cells whose densities will be converted into IS with pre-defined cutoffs. IS utilizes standardized percentile values (0 to 100%). We identify IS as Low, Intermediate (Int) and High with a mean percentile of 0-25%, &gt;25-70%, &gt;70-100% respectively. Two-group classifications are planned, corresponding to IS Low and Int-High. To better define the kind of tumor infiltrating cells (TILs) and assess the amount of immune suppressive cells, more IHC scoring will be evaluated: helper T-cells CD4+, CD8 T memory (CD 103+), T regulatory (both CD4+ and Foxp3+/CD105+), Macrophages M1 (CD 68+) and M2 (CD 163+). Results: Based on historical controls the rate of Int-High IS (&gt;25-70%, &gt;70% respectively) is about 64%, we expect to find a 20% increase of Int-High IS GC over biopsy control (internal validation cohort). To detect an increase in the percentage of high IS among patients treated with EIN (estimated around 85%) compared to biopsy controls (estimated around 64%) assuming a probability α and β of 0.2, the required sample size will be of 21 patients with resectable GC receiving EIN supplementation plus standard CT. To define the sample size a single proportion test was used. Conclusions: Our hypothesis is that EIN given during perioperative CT might prevent the establishment of an unfavourable TME for ICs in both auxotrophic and non-auxotrophic malignancies, thus leading to an increase of GC immunogenic pattern.

The ability of the eighth edition of the TNM staging system plus minor invasion criteria to predict the biological behaviour of oral cavity carcinomas.

Oral squamous cell carcinoma (OSCC) treatment is based largely on the TNM stage. The eighth edition includes important new prognostic parameters (extranodal extension and depth of invasion), while it does not consider tumour molecular characteristics or minor invasion criteria (perineural and lymphovascular invasion, grading and resection margins). This study evaluated how well the TNM eighth edition predicts the biological behaviour of OSCC, considering survival and risk of locoregional recurrence. Data from 217 patients treated for OSCC were analysed, including epidemiologic characteristics, histological features and treatment. No significant correlations with overall survival or tumour recurrence were found for pT stages and the type of treatment, while different pN stages had significant differences in recurrence, but not in overall survival. We found significant correlations between overall survival and tumour grade and lymphovascular and perineural invasion and a significant correlation between tumour resection margins and the risk of recurrence. The current TNM staging system is a necessary but not sufficient tool for predicting the overall survival and risk of recurrence of OSCC. It could be improved by considering other factors, such as minor invasion criteria and biological markers.

Tumor-associated macrophages trigger MAIT cell dysfunction at the HCC invasive margin

Mucosal-associated invariant T (MAIT) cells represent an abundant innate-like Tcell subtype in the human liver. MAIT cells are assigned crucial roles in regulating immunity and inflammation, yet their role in liver cancer remains elusive. Here, we present a MAIT cell-centered profiling of hepatocellular carcinoma (HCC) using scRNA-seq, flow cytometry, and co-detection by indexing (CODEX) imaging of paired patient samples. These analyses highlight the heterogeneity and dysfunctionality of MAIT cells in HCC and their defective capacity to infiltrate liver tumors. Machine-learning tools were used to dissect the spatial cellular interaction network within the MAIT cell neighborhood. Co-localization in the adjacent liver and interaction between niche-occupying CSF1R+PD-L1+ tumor-associated macrophages (TAMs) and MAIT cells was identified as a key regulatory element of MAIT cell dysfunction. Perturbation of this cell-cell interaction in exvivo co-culture studies using patient samples and murine models reinvigorated MAIT cell cytotoxicity. These studies suggest that aPD-1/aPD-L1 therapies target MAIT cells in HCC patients.

Spatial immunophenotypes orchestrate prognosis in triple-negative breast cancer with Miller-Payne grade 4 following neoadjuvant chemotherapy

Some triple-negative breast cancer (TNBC) patients evaluated as Miller-Payne 4 with ypN0 after neoadjuvant chemotherapy (NACT) who have better prognoses should avoid escalation of therapy. We aim to identify these patients by evaluating pretherapeutic spatial distributions of immunophenotypes. Our retrospective study in patients with TNBC assessed as Miller-Payne grade 4/5 with ypN0 showed that Miller-Payne 4 with ypN0 group had poorer 5-year disease-free survival (DFS, 63.8% vs. 83.0%, p = 0.003) and the 5-year overall survival (OS, 71.0% vs. 85.5%, p = 0.007) than Miller-Payne 5 with ypN0 group. High TILs were significantly associated with better DFS and OS in patients with Miller-Payne 4 and ypN0 (both p = 0.016). Spatially, detected by multiplexed ion beam imaging by the time of flight combined with proteomics, tumors assessed as Miller-Payne 4 and ypN0 with good prognosis exhibited an inflamed phenotype, with dominant CD8+ T cells on tumor center, few scattered CD68+ myeloid-derived cells far away from T cells, and deposit of increased activated molecules of lymphocyte. While those with poor prognoses presented excluded phenotypes, with few CD8+ T cells restricted to invasive margins and a high density of CD14+CD68+CD11c+ myeloid cells. A good classifier model based on 29 spatial immunophenotypes was established by the random forest algorithm (AUC = 0.975), for identifying patients with Miller-Payne 4 and ypN0 who had favorable prognoses. We also observed similar signatures in patients with Miller-Payne 5 and ypN0. Taken together, spatial immunophenotypes may assess the prognosis in TNBC patients with Miller-Payne 4 and ypN0 after NACT.

Spatially resolved transcriptomic profiles reveal unique defining molecular features of infiltrative 5ALA-metabolizing cells associated with glioblastoma recurrence

BackgroundSpatiotemporal heterogeneity originating from genomic and transcriptional variation was found to contribute to subtype switching in isocitrate dehydrogenase-1 wild-type glioblastoma (GBM) prior to and upon recurrence. Fluorescence-guided neurosurgical resection utilizing 5-aminolevulinic acid (5ALA) enables intraoperative visualization of infiltrative tumors outside the magnetic resonance imaging contrast-enhanced regions. The cell population and functional status of tumor responsible for enhancing 5ALA-metabolism to fluorescence-active PpIX remain elusive. The close spatial proximity of 5ALA-metabolizing (5ALA +) cells to residual disease remaining post-surgery renders 5ALA + biology an early a priori proxy of GBM recurrence, which is poorly understood.MethodsWe performed spatially resolved bulk RNA profiling (SPRP) analysis of unsorted Core, Rim, Invasive margin tissue, and FACS-isolated 5ALA + /5ALA − cells from the invasive margin across IDH-wt GBM patients (N = 10) coupled with histological, radiographic, and two-photon excitation fluorescence microscopic analyses. Deconvolution of SPRP followed by functional analyses was performed using CIBERSORTx and UCell enrichment algorithms, respectively. We further investigated the spatial architecture of 5ALA + enriched regions by analyzing spatial transcriptomics from an independent IDH-wt GBM cohort (N = 16). Lastly, we performed survival analysis using Cox Proportinal-Hazards model on large GBM cohorts.ResultsSPRP analysis integrated with single-cell and spatial transcriptomics uncovered that the GBM molecular subtype heterogeneity is likely to manifest regionally in a cell-type-specific manner. Infiltrative 5ALA + cell population(s) harboring transcriptionally concordant GBM and myeloid cells with mesenchymal subtype, -active wound response, and glycolytic metabolic signature, was shown to reside within the invasive margin spatially distinct from the tumor core. The spatial co-localization of the infiltrating MES GBM and myeloid cells within the 5ALA + region indicates PpIX fluorescence can effectively be utilized to resect the immune reactive zone beyond the tumor core. Finally, 5ALA + gene signatures were associated with poor survival and recurrence in GBM, signifying that the transition from primary to recurrent GBM is not discrete but rather a continuum whereby primary infiltrative 5ALA + remnant tumor cells more closely resemble the eventual recurrent GBM.ConclusionsElucidating the unique molecular and cellular features of the 5ALA + population within tumor invasive margin opens up unique possibilities to develop more effective treatments to delay or block GBM recurrence, and warrants commencement of such treatments as early as possible post-surgical resection of the primary neoplasm.

The prognostic and predictive role of tumor-infiltrating lymphocytes (FoxP3 + and CD8 +) and tumor-associated macrophages in early HER2 + breast cancer

PurposeIn HER2-positive (HER2 +) breast cancer, tumor-infiltrating lymphocytes (TILs) and tumor-associated macrophages (TAMs) may influence the efficacy of the HER2-antibody trastuzumab and the patient’s outcome. In this HER2 + patient cohort, our aim was to study the numbers of FoxP3 + regulatory TILs and CD8 + cytotoxic TILs, their correlations with CD68 + and CD163 + TAMs, and the prognostic and predictive value of the studied factors.MethodsWe evaluated 139 non-metastatic HER2 + breast cancer patients operated between 2001 and 2008. The FoxP3+TIL count (FoxP3+TILs) was assessed using the hotspot method, and the CD8 + TIL count (CD8+mTILs) utilizing a digital image analysis from invasive margin areas. The ratios between CD8+mTILs and FoxP3+TILs as well as CD8+mTILs and TAMs were calculated.ResultsFoxP3 + TILs and CD8 + mTILs correlated positively with each other (p<0.001). FoxP3+TILs had a positive correlation with CD68+and CD163+TAMs (p≤0.038), while CD8 + mTILs correlated only with CD68+TAMs (p<0.001). In the HER2 + and hormone receptor-positive Luminal B subgroup, high numbers of FoxP3+TILs were associated with shorter disease-free survival (DFS) (54% vs. 79%, p = 0.040). The benefit from adjuvant trastuzumab was extremely significant among patients with a high CD8 + mTILs/CD68 + TAMs ratio, with overall survival (OS) 84% vs. 33% (p = 0.003) and breast cancer-specific survival (BCSS) 88% vs. 48% (p = 0.009) among patients treated with or without trastuzumab, respectively.ConclusionIn the HER2 + Luminal B subgroup, high FoxP3 + TILs were associated with shorter DFS. A high CD8 + mTILs/CD68 + TAMs ratio seems to associate with impressive efficacy of trastuzumab.

The tumor–stroma ratio and the immune microenvironment improve the prognostic prediction of pancreatic ductal adenocarcinoma

Tumor-infiltrating immune cells and fibroblasts are significant components of the tumor microenvironment (TME) of pancreatic ductal adenocarcinoma (PDAC), and they participate in tumor progression as closely as tumor cells. However, the relationship between the features of the TME and patient outcomes and the interactions among TME components are still unclear. In this study, we evaluated the PDAC TME in terms of the quantity and location of cluster of differentiation (CD)4+ T cells, CD8+ T cells, macrophages, stromal maturity, and tumor-stroma ratio (TSR), as evaluated by immunohistochemical staining of serial whole-tissue sections from 116 patients with PDAC. The density of T cells and macrophages (mainly activated macrophages) was significantly higher at the invasive margins (IMs) than at the tumor center (TC). CD4+ T cells were significantly association with all the other tumor-associated immune cells (TAIs) including CD8, CD68 and CD206 positive cells. Tumors of the non-mature (intermediate and immature) stroma type harbored significantly more CD8+ T cells at the IMs and more CD68+ macrophages at the IMs and the TC. The density of CD4+, CD8+, and CD206+ cells at the TC; CD206+ cells at the IMs; and tumor-node-metastasis (TNM) staging were independent risk factors for patient outcomes, and the c-index of the risk nomogram for predicting the survival probability based on the TME features and TNM staging was 0.772 (95% confidence interval: 0.713–0.832). PDAC harbored a significantly immunosuppressive TME, of which the IMs were the hot zones for TAIs, while cells at the TC were more predictive of prognosis. Our results indicated that the model based on the features of the TME and TNM staging could predict patient outcomes.

Translating imaging traits of mass-forming intrahepatic cholangiocarcinoma into the clinic: From prognostic to therapeutic insights

Background & AimsThe progress toward clinical translation of imaging biomarkers for mass-forming intrahepatic cholangiocarcinoma (MICC) is slower than anticipated. Questions remain on the biologic behaviour underlying imaging traits. We developed and validated imaging-based prognostic systems for resected MICCs with an appraisal of the tumour immune microenvironment (TIME) underpinning patient-specific imaging traits. MethodsBetween January 2009 and December 2019, a total of 322 patients who underwent dynamic computed tomography/magnetic resonance imaging and curative-intent resection for MICC at three hepatobiliary institutions were retrospectively recruited, divided into training (n = 193) and validation (n = 129) datasets. Two radiological and clinical scoring (RACS) systems, one integrating preoperative variables and one integrating preoperative and postoperative variables, were developed using Cox regression analysis. We then prospectively analysed the TIME of tissue samples from 20 patients who met study criteria from January 2021 to December 2021 using multiplexed immunofluorescence. ResultsPreoperative and postoperative MICC-RACS systems built on carbohydrate antigen 19-9, albumin, tumour number, radiological/pathological nodal status, pathological necrosis, and three radiological traits (arterial enhancement pattern, tumour boundary, and capsular retraction) demonstrated good performance in predicting disease-specific (C-statistic >0.80) and disease-free (C-statistic >0.75) survival that outperformed rival models and staging systems across study cohorts (P <0.05 for all). Patients with MICC-RACS score of 0–2 (low risk), 3–5 (medium risk), and ≥6 (high risk) had incrementally worse prognosis after surgery. Significant differences in spatial distribution and infiltration level of immune cells were identified between arterial enhancement patterns. Enhanced infiltration of immunosuppressive regulatory T cells and M2-like macrophages at the invasive margin were noted in tumours with distinct boundary and capsular retraction, respectively. ConclusionsOur MICC-RACS systems are simple but powerful prognostic tools that may facilitate the understanding of spatially distinct TIMEs and patient-tailored immunotherapy approach. Impact and ImplicationsThe progress toward clinical translation of imaging biomarkers for mass-forming intrahepatic cholangiocarcinoma (MICC) is slower than anticipated. Questions remain on the biologic behaviour of MICC underlying imaging traits. In this study, we proposed novel and easy-to-use tools, built on radiological and clinical features, that demonstrated good performance in predicting the prognosis either before or after surgery and outperformed rival models/systems across major imaging modalities. The characteristic radiological traits integrated into prognostic systems (arterial enhancement pattern, tumour boundary, and capsular retraction) were highly correlated with heterogeneous tumour-immune microenvironments, thereby renovating treatment paradigms for this difficult-to-treat disease.

Tumor-infiltrating lymphocytes and tumor-associated macrophages as potential predictors of lymph node metastases in major salivary gland cancers.

The purpose of this study is to define if tumor-infiltrating lymphocytes (TILs) and tumor-associated macrophages (TAMs) could represent potential predictors of lymph node metastases (LNM) in salivary gland cancers (SGC). A selected number of immunohistochemical markers related to TILs (CD3, CD4, CD68, and FOXP3) and TAMs (CD68 and CD163) were investigated on major salivary gland cancers. TIL and TAM densities were measured on digital images using the open-source QuPath both in the tumor interior (TI) and invasive margin (IM). Correlation with pathologic N classification and follow-up clinical data was investigated. A total of 25 consecutive patients (men: 11; median age: 62.0) were included. Densities of CD3+ IM (OR = 7.7, 95% CI 1.2-51.2), CD8+ TI (OR = 7.7, 95% CI 1.2-51.2), CD8+ IM (OR = 7.7, 95% CI 1.2-51.2), FOXP3+ TI (OR = 24.0, 95% CI 2.2-255.9), CD68+ TI (OR = 7.7, 95% CI 1.2-51.2), and CD163+ IM (OR = 7.7, 95% CI 1.2 - 51.2), and the Immunoscore CD8/CD3 (OR = 1.9, 95% CI 1.1-3.4) were significantly associated with LNM (p < 0.05). CD3+ TI density was significantly associated with tumor recurrence and death (HR = 5.8, 95% CI 1.5-22.6; p < 0.05). A high density of specific TIL and TAM subpopulations might be correlated with a higher probability of LNM in SGC.

Adenosquamous cell carcinoma of the head and neck: a retrospective single institution series.

Head and neck adenosquamous cell carcinoma (HN-ASCC) is a rare, aggressive neoplasm, with limited data reported in the literature. The aim of this study was to assess tumour behaviour and prognostic factors impacting overall survival (OS) in a retrospective, single institution series. A retrospective study on patients affected by HN-ASCC who were treated surgically between 2002 and 2019 at the Department of Otorhinolaryngology - Head and Neck Surgery of the University of Brescia was conducted. Demographics, clinical data, OS, and relative prognostic factors were analysed. The study included 32 patients, with a median age of 66 years, mostly males (84.4%) and untreated (68.8%). Adjuvant treatments followed surgery in 28.1% of patients. Compared to conventional SCC, ASCC showed a higher proportion of cases arising in the larynx (40.6%); no difference was found in other features. Advanced (pT3-4) local stage at presentation (p = 0.023), perineural invasion (PNI, p = 0.01), and positive margins (p = 0.007) were independent negative prognostic factors for OS. HN-ASCC is a rare, aggressive cancer, most frequently arising in the larynx of elderly males, usually diagnosed in an advanced local stage. OS is generally poor, affected by local advanced stage, PNI, and positive resection margins.

P26 Risk factors for complications in cutaneous squamous cell carcinoma and proposal of a simplified follow-up strategy, making use of available healthcare resources

Abstract Cutaneous squamous cell carcinoma (cSCC) is a common malignancy, generally cured by surgical excision. A minority of such tumours recur, metastasize or even cause death. The 2021 British Association of Dermatologists (BAD) guidelines outline high-risk features that are used to guide the follow-up of patients after a primary cSCC. The aims of this study were to identify histological features of cSCC that are significantly associated with the development of complications, and to compare the sensitivity, specificity and number of appointments required to follow-up patients using different follow-up criteria. In total, 556 cases of primary invasive cSCCs excised between January 2014 and March 2018 were included and followed-up for a minimum of 4 years. Five per cent (n = 26) of tumours were associated with at least one cSCC-related complication, namely, local recurrence 3.8% (n = 21), nodal metastasis 1.6% (n = 9), distant metastasis 0.4% (n = 2) and disease-specific death 0.7% (n = 4). The initial diagnosed complication was local recurrence in 20 of 26 cases. Only two patients had the first complication beyond 2 years of the original cSCC. Factors that were statistically significantly associated with the development of a complication (P &amp;lt; 0.001) are larger diameter, larger depth of invasion, tumour invasion beyond the dermis, perineural invasion (irrespective of size), lymphovascular invasion, gross bone erosion and deep margin &amp;lt; 1 mm. Differentiation and peripheral margin were not significantly associated with complications. Therefore, it may be proposed that patients with tumours with at least one of the following risk factors be followed-up: diameter &amp;gt; 40 mm; depth of invasion &amp;gt; 6 mm; invasion beyond the dermis; presence of perineural or lymphovascular invasion or deep margin &amp;lt; 1 mm. In adopting these criteria, 739 appointments in 156 patients would be required over 2 years (4 monthly during the first year and 6 monthly during the second year). Twenty of the patients who developed a complication would be followed-up (sensitivity 76.9%, specificity 69.4%). Using the BAD guidelines, up to four more patients with complications would have been followed-up (sensitivity 92.3%, specificity 44.7%), but this would have required a total of 1556 follow-up visits in 242 patients. Given the limited resources, patient burden and the fact that local recurrence is most often the first complication, it may be reasonable to offer routine follow-up only to patients who have at least one of the proposed criteria. Coupled with patient education, this strategy could greatly reduce the number of follow-up visits required. Such a strategy may allow resources to be utilized to reduce waiting times, particularly for 2-week wait referrals, decreasing the delay in new skin cancer diagnoses.

DS08 (P26) Risk factors for complications in cutaneous squamous cell carcinoma and proposal of a simplified follow-up strategy: making use of available healthcare resources

Abstract Cutaneous squamous cell carcinoma (cSCC) is a common malignancy, generally cured by surgical excision. A minority of such tumours recur, metastasize or even cause death. The 2021 British Association of Dermatologists (BAD) guidelines outline high-risk features that are used to guide the follow-up of patients after a primary cSCC. The aims of this study were to identify histological features of cSCC that are significantly associated with the development of complications and to compare the sensitivity, specificity and number of appointments required to follow-up patients using different follow-up criteria. In total, 556 cases of primary invasive cSCCs excised between January 2014 and March 2018 were included and followed-up for a minimum of 4 years. Five per cent (n = 26) of tumours were associated with at least one cSCC-related complication, namely, local recurrence 3.8% (n = 21), nodal metastasis 1.6% (n = 9), distant metastasis 0.4% (n = 2) and disease-specific death 0.7% (n = 4). The initial diagnosed complication was local recurrence in 20 of 26 cases. Only two patients had the first complication beyond 2 years of the original cSCC. Factors that were statistically significantly associated with the development of a complication (P &amp;lt; 0.001) were larger diameter, larger depth of invasion, tumour invasion beyond the dermis, perineural invasion (irrespective of size), lymphovascular invasion, gross bone erosion and deep margin &amp;lt; 1 mm. Differentiation and peripheral margin were not significantly associated with complications. Therefore, it may be proposed that patients with tumours with at least one of the following risk factors be followed-up: diameter &amp;gt; 40 mm, depth of invasion &amp;gt; 6 mm, invasion beyond the dermis, presence of perineural or lymphovascular invasion or deep margin &amp;lt; 1 mm. In adopting these criteria, 739 appointments in 156 patients would be required over 2 years (4-monthly during the first year and 6-monthly during the second year). Twenty of the patients who developed a complication would be followed-up (sensitivity 76.9%, specificity 69.4%). Using the BAD guidelines, up to four more patients with complications would have been followed-up (sensitivity 92.3%, specificity 44.7%), but this would have required a total of 1556 follow-up visits in 242 patients. Given the limited resources, patient burden and the fact that local recurrence is most often the first complication, it may be reasonable to offer routine follow-up only to patients who have at least one of the proposed criteria. Coupled with patient education, this strategy could greatly reduce the number of follow-up visits required. Such a strategy may allow resources to be utilized to reduce waiting times, particularly for 2-week wait referrals, decreasing the delay in new skin cancer diagnoses.

Radial and longitudinal margins in surgery of perihilar cholangiocarcinoma: When R1 definition is associated with different prognosis

BackgroundInfiltrated margins of resection (R1) and lymph node invasion are dominant negative predictors of survival in patients with a resectable perihilar cholangiocarcinoma. Less clear is whether survival predictors stratify differently between R0 (tumor-free margins) and R1 patients and whether the prognosis of the latter patients is influenced by the pattern of neoplastic infiltration (ie, radial versus longitudinal infiltration). We retrospectively evaluated a series of reported resected perihilar cholangiocarcinoma to obtain insights on the predictive power of these histologic features. MethodsThe study includes 264 patients with perihilar cholangiocarcinoma treated between 2004 and 2019 in our center and followed up for >18 months. There were 176 patients with R0 (66.6%) and 88 patients with R1 (33.3%), 31 with radial infiltration only, 30 with longitudinal infiltration only, and 27 with both infiltration patterns. In all patients, the criteria for resection was the absence of metastatic involvement (ie, distant organ metastases, liver metastases, and lymph node metastases beyond the hepatoduodenal ligament). Histopathologic specimens of the resected tumors were centrally reviewed by a pathologist unaware of the clinical outcomes. ResultsThree- and 5-year long-term survival were significantly better in R0 (respectively) compared to R1 patients (55% and 42% vs 42% and 18%, respectively, P < .05). In R1 patients with radial infiltration only and those with radial + longitudinal infiltration, both disease-free and overall survival were worse than those with longitudinal infiltration only (median disease-free survival of 18 and 23 months, respectively, P < .05, median overall survival of 33 and 39 months, respectively, P < .05). At multivariable analysis, nodal status, side of hepatectomy, grading, and presence of radial margin infiltration were associated with long-term outcome. ConclusionRadial infiltration of resection margins enhances the negative prognostic value of R1 margins in perihilar cholangiocarcinoma patients and should specifically be accounted for in the prediction of the outcome of adjuvant therapy.

LAG3 in gastric cancer: it’s complicated

PurposeLymphocyte activation gene 3 (LAG3) is thought to contribute to T cell exhaustion within the tumor microenvironment of solid tumors. This study aimed to analyze the spatial distribution of LAG3 + cells in relation to clinicopathological and survival data in a large set of 580 primary resected and neoadjuvantly treated gastric cancers (GC).MethodsLAG3 expression was evaluated in tumor center and invasive margin using immunohistochemistry and whole-slide digital image analysis. Cases were divided into LAG3-low and LAG3-high expression groups based on (1) median LAG3 + cell density, (2) cut-off values adapted to cancer-specific survival using Cutoff Finder application.ResultsSignificant differences in spatial distribution of LAG3 + cells were observed in primarily resected GC, but not in neoadjuvantly treated GC. LAG3 + cell density showed evident prognostic value at following cut-offs: in primarily resected GC, 21.45 cells/mm2 in tumor center (17.9 vs. 10.1 months, p = 0.008) and 208.50 cells/mm2 in invasive margin (33.8 vs. 14.7 months, p = 0.006); and in neoadjuvantly treated GC, 12.62 cells/mm2 (27.3 vs. 13.2 months, p = 0.003) and 123.00 cells/mm2 (28.0 vs. 22.4 months, p = 0.136), respectively. Significant associations were found between LAG3 + cell distribution patterns and various clinicopathological factors in both cohorts. In neoadjuvantly treated GC, LAG3 + immune cell density was found to be an independent prognostic factor of survival (HR = 0.312, 95% CI 0.162–0.599, p < 0.001).ConclusionIn this study, a higher density of LAG3 + cells was associated with favorable prognosis. Current results support the need for extended analysis of LAG3. Differences in the distribution of LAG3 + cells should be considered, as they could influence clinical outcomes and treatment responses.Graphical abstract