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Introduction Targeted axillary dissection (TAD) after neoadjuvant chemotherapy (NAC) is a new axillary staging technique that consists of surgical removal of biopsy-proven, positive, clipped axillary nodes (CLN) in addition to the sentinel lymph node biopsy (SLNB), and provides for more conservative axillary surgery1. A question was recently raised whether clipping a positive node for later assessment indeed leads to a management change2. The purpose of our study is to report the feasibility of TAD and to evaluate false negative rate (FNR), impact on surgical ad oncological management. Materials and methods This retrospective, single-institution, study included 73 consecutive women operated between 2019 and 2021 after NAC for cN+ disease confirmed by citology or histology, whose diseased lymph-node was marked with a clip before therapy. At surgery, in case of clinical-radiological complete response, patients underwent TAD (dual mapping with radiocolloid and blue dye + CLN) (n=43), or TAD plus ALND if any residual disease (n=30). The chemoterapeutsic regimen were antracycline/taxane based, with trastuzumab in case of HER2+ tumors. Patients were routinely evaluated with a breast MRI both before and after NAC. Results Clinical and pathological details of the 73 enrolled patients are listed in Table 1. The mean age at diagnosis was 49.53±10 years. Pathologic complete response was achievied in 32 out 73 patients (43.8%) with the greatest rate in HR-/HER2+ tumors (n=10/12; 83.3%). The identification rate of the CLN was 91.8% (68/73), and it was one of SLNs in 68.5% (50/73) of the cases. In cases in which one, two or three or more SLNs were identified, the CLN was in the SLN specimen in 42.9%, 77.8% and 81.8% of cases, respectively. The FNR of the SLN was 18.5% (CI: 4.9-38.1). In 18 cases the CLN was not in the SLN specimen; eleven out of 18 CLNs were positive, leading to ALND, and three of them had additional positive LNs. Only in one case the CLN was negative and the SLN was positive. In 3 cases (4.1%), the CLN was positive in the absence of residual tumour in the breast leading a potential change in the oncological management. Conclusions Removal of CLN after NAC is feasible, allowing de-escalation of surgical management of cN+ women in case of clinical-radiological complete response, as 59% of women avoided a formal ALND. The CLN coincides with SLN in about 70% of cases (more than 80% if three or more SLNs are identified) and reflects the overall status of the axilla in 97.3% of the cases. Adding CLN to SLNB contributes to reduce significantly the FNR of the latter from 18% to 0% (FNR for SLNB =18.5% vs FNR for TAD=0%). Potential changes in surgical (CLN+/SLNs-) and oncological management (CLN+/SLNs- and absence of residual tumor in breast) occurs in 15.1% (11/73) and 4.1% (3/73) of cases, respectively. References 1. Swarnkar PK et al. The Evolving Role of Marked Lymph Node Biopsy (MLNB) and Targeted Axillary Dissection (TAD) after Neoadjuvant Chemotherapy (NACT) for Node-Positive Breast Cancer: Systematic Review and Pooled Analysis. Cancers (Basel) 2021 Mar 26;13(7):1539. 2. Weiss A et al. How Often Does Retrieval of a Clipped Lymph Node Change Adjuvant Therapy Recommendations? A Prospective, Consecutive, Patient Cohort Study. 2022 Jun. Ann Surg Oncol 2022; 29(6): 3764-3771. Table 1: Clinicopathologic characteristics in the cN+ breast cancer patients NAC and clipped Lymph Node NAC Citation Format: Francesca Arienzo, Domenico Campagna, Paola Scavinia, Laura Broglia, Laura Broglia, Alessandra Ascarelli, Helena Colavito, Mirko Montanari, Elena Manna, Tiziana Mastropietro, Emanuele Zarba Meli, Massimo La Pinta, Daniela Musio, Mauro Minelli, Paola Scavina, Michelina Maria Carla Amato, Leopoldo Costarelli. Clipped lymph nodes for cN+ patients decrease false negative rate and lead to potential changes in surgical and oncological management after clinical complete response following neoadjuvant chemotherapy [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-07-27.

Neurofibroma (NF) rarely arises in the retropharyngeal space (RPS) of patients with or without Neurofibromatosis type I (NF-I). The diffuse subtype of NF (DNF) is characterized by an infiltrative growth pattern and typically involves the skin and subcutaneous tissue of the head and neck. We describe the clinic-pathologic features of a DNF involving the RPS of an adult without NF-I. To date, this subtype of NF has never been reported at this site.

Introduction: Primary sarcoma of the vulva is an extremely rare entity, representing only 1%–3% of all vulvar malignant neoplasms. Among sarcomas, leiomyosarcoma (LMS) is the most prevalent histologic variant. Due to the rarity of LMS, guidelines are lacking and phase III trials have not been carried out, so clinical management is based on local clinical practice and physician experience. Case presentation: Here, we described a case of primary LMS of the vulva and its successful management, with the adoption of neoadjuvant chemotherapy and surgery. We report a case of a 74-year-old woman with 12.5 cm vulvar LMS. The patient received three cycles of neoadjuvant chemotherapy with a partial response. Radical vulvectomy with vulvar reconstruction with V-F flap was carried out. Surgical margins were negative. Three additional cycles of adjuvant chemotherapy were delivered. Results: One year after treatment, the patient was disease-free. Conclusion: There are no approved therapeutic protocols for this rare neoplasia. Surgery is the mainstay of treatment. However, it is not always feasible, so neoadjuvant chemotherapy was delivered for downstaging the vulvar lesion. We suppose that neoadjuvant chemotherapy has optimized the possibilities of radical surgery. Despite the anectodical nature of this case presentation, neoadjuvant chemotherapy seems a valid therapeutic option for managing patients with bulky vulvar sarcoma. Further large collaborative studies are warranted to identify the best therapeutic option for these patients.

Backgrounds The organization of minor salivary glands (MSG) infiltrates, in patients with Sjögren’s syndrome (SS), associates with disease severity and progression. Aberrant regulation of lymphocyte autophagy is involved in autoimmunity, and in previous work, we provided the first evidence of upregulated autophagy in CD4+ T cells infiltrating SS MSG. The aim of this study was to further explore autophagy in SS infiltrating and circulating lymphocytes and to investigate its role in disease histopathological progression. Methods After collection of 20 SS MSG, the presence of lymphocyte aggregates (foci) and the formation of germinal center (GC)-like structures were observed by H&E and confirmed by immunohistochemistry. The expression of autophagy-related genes, Atg5 and MAP1LC3A, was detected by RT-PCR on microdissected salivary gland tissue and control tonsils. In MSG and tonsils, autophagic lymphocytes were identified by the detection of the autophagosome protein LC3B visualized as LC3 puncta staining by immunofluorescence. Peripheral blood autophagy was assessed by flow cytometry in SS and healthy controls (HC). Results Real-time PCR demonstrated higher expression in the autophagy genes Atg5 and MAP1LC3A in MSG GCs as compared to both small foci (p = 0.0075, p = 0.0002) and GCs from tonsils (p = 0.0001, p = 0.0037). In MSG, LC3 puncta staining was detectable on both CD3+ and CD20+ lymphocytes; in tonsils, LC3 puncta was almost undetectable on all lymphocytes. Compared to HC (n = 20), flow cytometry did not reveal any increase of autophagy in SS circulating lymphocytes (n = 30). Conclusions In SS MSG, lymphocytes’ autophagy is a feature of infiltrating T and B cells and is associated with histological severity. Interestingly, in MSG aberrant regulation of autophagy is detectable in GC-like structures possibly indicating its involvement in the development and persistence of the autoimmune process within the lesions.

Background: Frailty is a condition characterized by the reduction of the individual’s homeostatic reserves, leading to an increased vulnerability to stressors and an increased risk of unfavourable events. The aging of the population and the consequent need to implement new paradigms of care and assistance, have given this tool a growing interest in many medical disciplines1. In rheumatology, however, the interest is still limited2. The Frailty Index (FI), developed on an arithmetical model of deficit accumulation, is an accurate tool for assessing frailty, providing an estimate of the biological aging. Objectives: Creation of a FI to be used in clinical practice in patients with Sjogren’s syndrome (SS) and evaluation of the correlations with patient‘s age, duration of illness, activity and disease damage at baseline and in the following 5 years. Methods: The FI is composed by a checklist of non-predefined variables (deficits) constituted by symptoms, signs, diseases, disabilities, and laboratory findings. The deficits must meet these criteria: age-related; associated with negative outcomes; multidimensional (referring to different domains of the health status); present in at least 1%, but not more than 80% of the sample. To each variable is assigned the value of 0 (= no deficit) or 1 (= deficit). The FI is the ratio between deficits presented by the individual and the total number of deficits considered, thus providing a measure of frailty ranging between 0 (no frailty) and 1 (maximum of frailty)3. A FI was developed for patients with SS consisting of 43 items (17 comorbidities, 14 signs and symptoms, 5 disabilities and 7 laboratory findings). Statistical analysis was performed with Spearman’s test for correlation assessment, the Mann Whitney test for comparing non-parametric variables was used. Results: FI was administered to a first small group of 30 female consecutive patients recruited as outpatients at the clinic dedicated to SS. The average age was 57.2 yrs, mean age at diagnosis 52.7 yrs and average disease duration 4.7 yrs. At the time of completing the FI, the average disease activity (ESSDAI) was 3.4, the mean value of the damage (SjSDDI) 1.6 and the average score of FI equal to 0.21. A statistically significant correlation between FI and age has been reported (p = 0.017). No significant correlations between frailty and duration, activity and disease damage have been highlighted at the moment. Conclusion: For the first time a FI was developed for patients with SS consisting of 43 items. The data shows a relationship between age andFI. The correlation is statistically significant, similarly to what is reported in the literature for other conditions. This confirms that FI is indeed an objective marker of aging and even though the sample population is young (average age = 57.2 years), FI maintains its main properties. This tool can be used to assess the health status of patients, making it possible to identify those at greater risk of trajectories or unfavourable outcomes. It is currently being administered the FI to patients with SS whose clinical course will be evaluated in the next 5 years (complications, mortality, hospitalization, institutionalization and disability). References [1] Canevelli M, et al, Promoting the Assessment of Frailty in the Clinical Approach to Cognitive Disorders, Front. Aging Neurosc 2017 [2] Rockwood MR, et al, FI to Measure Health Status in People with SSc, J Rheum 2014 [3] Searle SD, et al, A standard procedure for creating a FI, BMC Geriatrics2008 Disclosure of Interests: None declared

Background: Autophagy is a lysosome mediated catabolic process that promotes cell survival during stress conditions. Aberrant regulation of autophagy is involved in the pathogenesis of a widening spectrum of autoimmune disorders: in particular, activation of autophagy pathway is implicated in aberrant survival of activated T cells. We recently demonstrated that autophagy is up-regulated in CD4+ T lymphocytes from patients with Sjogren’s Syndrome (SS) both in peripheral blood and tissue. Moreover, aberrant autophagy correlates with systemic disease activity. Little is known on the role of autophagy in B cells homeostasis in SS. Objectives: To investigate the activation of the autophagy pathway in lymphocytes infiltrating salivary gland in SS and peripheral blood from patients with SS. Methods: Frozen SS minor salivary glands (MSG) from 20 patients were sectioned and evaluated with immunohistochemistry (IHC) by staining for CD3+, CD20+, and CD21+. MSG sections, sequential to IHC, were stained by Cresyl Violet and microdissected (Laser Capture Microdissection) in order to isolate and collect small infiltrates, large CD21- infiltrates, and large CD21+ infiltrates (GC-like structures). Microdissected GC from frozen human tonsils were used as control. The expression of autophagy genes Atg5 and MAP1LC3II (expressed as 2^deltaCT normalized to GADPH) was determined by qPCR on microdissected tissue. For studies on peripheral blood, 19 SS patients and 11 healthy controls were enrolled, and autophagy, expressed as the ratio between Mean Fluorescence Intensity and the isotopic control (rMFI), was evaluated by Cyto-ID® Autophagy detection kit in T lymphocytes (CD4+ and CD8+) and B lymphocytes (CD19+) using a FACSCalibur cytometer. Results: Expression of the autophagy genes Atg5 and MAP1LC3 was significantly higher in the GCs from SS salivary glands compared to control tonsils (p Immunophenotyping studies revealed activation of the autophagy pathway in both circulating CD19+ and CD4+ lymphocytes from SS (p=0.04 and p=0.03 compared to HC); conversely, no pathway activation was detected in CD8+ cells. Of note, in patients with SS activation of autophagy was most marked in circulating CD19+ B cells compared the other subsets [(CD19+ vs CD4+ (p Conclusion: Our findings revealed that the autophagy pathway is aberrantly activated in lymphocytes infiltrating salivary glands of SS patients. Of note, progressively higher expression levels of autophagy genes mirrored the severity and extension of inflammatory infiltrates. Immunophenotyping studies revealed that activation of autophagy is preeminent in B cells. Thus, activation of autophagy emerges as a feature of both infiltrating and circulating SS lymphocytes, and as mechanism possibly implicated in the activation and survival o?f autoreactive cells. Disclosure of Interests: Serena Colafrancesco: None declared, Antonina Minniti: None declared, Roberta Priori: None declared, Marta Vomero: None declared, cristiana barbati: None declared, Francesca Arienzo: None declared, Valentina Iannizzotto: None declared, Elena Pipi: None declared, Joana Campos: None declared, Saba Nayar: None declared, Francesco Ciccia Grant/research support from: CELGENE, PFIZER, Consultant for: UCB, NOVARTIS, CELGENE, PFIZER, LILLY, Paid instructor for: UCB, NOVARTIS, CELGENE, PFIZER, LILLY, JANSSEN, Speakers bureau: UCB, NOVARTIS, CELGENE, PFIZER, LILLY, JANSSEN, MSD, ROCHE, AMGEN, Francesca Barone Grant/research support from: GlaxoSmithKline, Roche, UCB Pharma, Actelion, ONO Pharmaceutical, Consultant for: GlaxoSmithKline, Roche, Actelion, ONO Pharmaceutical, Guido Valesini: None declared, cristiano alessandri: None declared

Career situation of first and presenting author Post-doctoral fellow. Introduction Sjogren’s syndrome (SS) is an autoimmune condition characterised by systemic B cell activation, autoantibody production and ectopic germinal centers (GC) formation within salivary gland (SG). The extent of SG infiltrate has been proposed as biomarker of disease severity. Plasma levels of CXCL13 correlate with GC activity in animal models and disease severity in SS, suggesting its potential use as a surrogate serum marker to monitor local B cell activation. Objectives To evaluate the potential role of CXCL13 as biomarker of SG pathology in two independent SS cohorts. Methods 109 patients with SS were recruited at Sapienza University of Rome (Italy) (n=60), or at Queen Elizabeth Hospital in Birmingham and Barts Health NHS Trust in London (n=49). Both sera and matched paraffin-embedded minor SG biopsy were available. Sicca (n=57) and healthy subjects (HS) (n=19) sera were used as control. CXCL13 gene expression was also assessed in 25 frozen SGs. Results CXCL13 serum levels were higher in SS patients [90.3 (84.2) pg/ml], compared to both sicca [61.9 (38.6) pg/ml), p=0.0005] and HS [36.5 (40.18) pg/ml, p Conclusions Our data foster the use of CXCL13 to monitor the extent of local pathology in SS and its validation in longitudinal clinical studies. Both serum and tissue expression of CXCL13 correlate with SS histological severity, suggesting a major role of this chemokine in SG lymphocytes recruitment and organization. Disclosure of Interest None declared.

Objectives SS is an autoimmune condition characterized by systemic B-cell activation, autoantibody production and ectopic germinal centres’ formation within the salivary gland (SG). The extent of SG infiltrate has been proposed as a biomarker of disease severity. Plasma levels of CXCL13 correlate with germinal centres’ activity in animal models and disease severity in SS, suggesting its potential use as a surrogate serum marker to monitor local B-cell activation. The aim of this study was to evaluate the potential role of CXCL13 as a biomarker of SG pathology in two independent SS cohorts. Methods 109 patients with SS were recruited at Sapienza University of Rome (Italy) (n = 60), or at Queen Elizabeth Hospital in Birmingham and Barts Health NHS Trust in London (n = 49). Both sera and matched minor SG biopsy were available. Sicca (n = 57) and healthy subjects’ (n = 19) sera were used as control. Results CXCL13 serum level was higher in SS patients compared with controls. Correlations between its serum levels and a series of histomorphological parameters, including size of the aggregates and the presence germinal centres', were observed. Conclusion Our data foster the use of CXCL13 to monitor the extent of local pathology in SS and its validation in longitudinal clinical studies.

Background Ectopic production of the lymphoid chemokines CXCL13 and CXCL12 has been described in tertiary lymphoid structures (TLS) that harbour in the salivary glands of patients with Sjogren’s Syndrome (pSS). Whilst CXCL13 expression correlates with clinical features, its potential role as a biomarker to monitor the organisation and severity of the salivary gland infiltrate has been hampered by the lack of sensitive tools to describe TLS extent and features. Objectives To investigate CXCL13 and CXCL12 serum and tissue expression and to find any possible association with clinical, histological and laboratory features. Methods We studied histological features of the minor salivary glands (MSG) and sera of respectively fifty and seventy (table 1) unselected consecutive patients with pSS (AECG criteria). Concentration of CXCL13 and CXCL12 were evaluated by ELISA in patient sera and eleven healthy controls (HC). Paraffin embedded MSG were studied by haematoxylin/eosin and anti-CD3, anti-CD20, anti-CD21 staining. Images analysis was used to calculate focus score (FS), mean foci area, percentage of infiltration (%i), segregated foci (%SF),%GCs and lymphoepitelial lesions (%LEL). GCs from MSG and tonsils were microdissected and quantitative PCR was used to test CXCL12 and CXCL13 transcripts. Results Histological analysis unveiled strong correlations between the mean foci area with the%i and the presence of SF; positive correlations were also observed between the%i and both the FS and%SF. This was significantly higher in patients exhibiting SF. The% of SF positively correlated with FS, presence of%GC and%LEL that also correlated with the%i and the%SF (image). Mean CXCL13 and CXCL12 serum levels were significantly higher in pSS compared to HC [(124.12±119.73 pg/ml vs 8.9±15.4 pg/ml (p=0.001) and 34.6±54.2 pg/ml vs 2.5±8.3 pg/ml (p=0.05), respectively]. CXCL13 was significantly higher in patients with SF, with GCs and LEL and correlated with the mean foci area, the%i, the FS and the percentage of LEL. Higher CXCL13 levels were associated with the presence of antibodies and other biological findings including hyperglobulinemia. Higher CXCL13 levels were also able to discriminate patients with lymphoma (p=0.009). CXCL12 levels correlated with the FS,%i and% of LEL. Transcript analysis showed no difference in the expression of CXCL13 between MSG and tonsil GC, whilst CXCL12 was found significantly higher in MSG (p Conclusions Our results suggest the utility to expand the parameters of histologic evaluation of MSG, whilst reinforcing the role of the FS as reliable instrument to reflect the severity of inflammation. Analysis of MSG infiltration and foci segregation was able to identify subjects with increased proliferative risk. We demonstrated that serum CXCL13, is a biomarker of histological severity and is able to stratify patients with lymphoma. The high levels of CXCL12 in MSG GC suggest a differential biology of TLS in the SG, probably implicated in aberrant B cell clone survival. Disclosure of Interest None declared

Background Minor salivary gland (MSG) biopsy represents an useful tool not only for the diagnosis of primary Sjogren9s Syndrome (pSS) but also to evaluate patients prognosis. Recognition of germinal centers (GCs) by hematoxilin eosin (HE) and/or IHC staining for follicular dendritic cells (FDC) detection is mandatory, representing a risk factor for lymphoma development. Focus score (FS) is one of the main instrument to quantify MSG impairment, nonetheless quality information regarding the type of infiltrate such as the entity, structure and localization, are lacking. Objectives Aim of this study is to find any association of specific histological features of MSG from patients with pSS with the principal clinical and laboratory features. Moreover, to investigate the utility of histological parameters, other than FS or GCs, for characterizing patients. Methods Patients with pSS were enrolled in our SS clinic, and clinical/laboratory data (table) referring to the time of MSG biopsy, gathered on a dedicated database. MSG, removed for diagnostic purposes, were preserved as paraffin embedded tissue, then cut and sequentially stained by HE monoclonal mouse anti-CD20 (lymphocytes B); monoclonal mouse anti-CD21 (FDC)]. Images were collected by Zeiss Axio Scan and analysed (ZEN software) as follows: FS calculation, mean foci area, percentage of infiltration, presence of segregated foci (SF) (specifically, clear evidence of T and B cells area by CD3-CD20 double staining), GCs and lymphoepithelial lesions (LELs) detection. Results 53 MSG from patients with pSS were collected and analysed. Patients clinical and laboratory data are reported in table. FS positively correlated with the percentage of infiltration (p Conclusions The FS was associated with the presence of GCs and LELs, as well as with more organized infiltrates characterized by segregation in T and B areas (SF), thus representing an useful tool which mirrors the risk of lymphoma. From our study, the qualitative characteristics of the biopsy, including SF, percentage of infiltration or the mean foci area, appear to be strictly linked. Moreover, their association with the presence of GCs and LELs supports the importance to consider also these features during histological examination. The lack of correlation between histological parameters and clinical/laboratory features might reveal a weaker connection between histological findings and specific SS phenotypes except for the relationship between glandular swelling and GCs which confirms how this clinical aspect should be considered as a risk factor for lymphoma development. Disclosure of Interest None declared