Evolution of PD-L1 status from primary BC to relapse according to phenotypic evolution from primary BC to metastases. Sankey plots showing the evolution of PD-L1 status from primary breast cancer to relapse, according to phenotypic evolution (stable triple-negative breast cancer, from ER+/HER2- to ER-low, and from ER+/HER2- to triple-negative breast cancer).

Multivariate analysis for overall survival and post relapse survival in the retrospective cohort in patients with ER+/HER2- primary BC. Multivariable Cox proportional hazards analyses for overall survival and post-relapse survival in the retrospective cohort of patients with primary ER+/HER2- breast cancer; hazard ratios (HRs) with 95% confidence intervals and p-values are reported.

Workflow of the study. The flow diagram of the study shows an overview of the two cohorts and the analyses performed.

Clinicopathological features according to ER expression in the TONIC trial cohort Clinicopathological features of patients in the TONIC trial cohort, reported according to ER expression status (ER-low vs ER-0).

Representativeness of Study Participants. Representativeness of study participants in the context of advanced breast cancer, with information on sex, age, race/ethnicity, geography, and overall comparability to the general patient population.

Genes up-regulated or down-regulated in estrogen-receptor (ER)-low (ER 1-9%) vs ER-positive (>10%) by quantitative Significance Analysis of Microarrays (SAM) analysis List of genes significantly up- or down-regulated in tumors that switched from ER+/HER2– to ER-low (1–9%) compared with tumors maintaining a stable ER-positive (≥10%) phenotype, as identified by quantitative Significance Analysis of Microarrays (SAM).

1067 Background: ER expression is one of the main determinants of prognosis in patients (pts) with BC. Phenotypic conversion from ER+ (ER>=10%)/HER2- primary BC towards ER<10%/HER2- advanced BC has a well-known negative impact on outcome. However, in the specific context of phenotypically stable ER+/HER2- BC, the prognostic impact of ER expression in metastases or ER dynamics during disease evolution, remains largely understudied. Methods: We enrolled pts with advanced BC undergoing biopsy of a metastatic site. ER+ was defined as ER>=10%. ER expression was evaluated both as continuous and categorical variable (categories: 10-30%, 30-50%, 50-100%). Overall survival (OS) was the primary study endpoint. Cox multivariable models included covariates associated with OS in univariate analysis. Results: Among 1114 pts, 410 had ER+/HER2- phenotype in both primary and metastatic tumor specimens. In this subgroup, ER expression (both continuous and categoric) in metastases had significant prognostic value: for each 10% lower ER expression, the risk of death increased by 6.7% (p=0.011). Pts with ER 10-30% had significantly worse OS than those with ER 50-100% (HR 0.62, p=0.023), and numerically shorter than ER 30-50% (HR 0.51, p=0.063). The table shows the evolution of ER categories from primary BC to metastases. ER expression dynamics also had prognostic impact. Regarding continuous ER expression changes in paired primary vs. metastatic BC, each 10% ER increase corresponded to 5.9% decrease in the risk of death (p=0.008). Pts whose tumors showed an increased ER expression from 10-30% to 50-100% had the most favorable outcome overall, with better OS compared to pts with persistently low ER levels (HR 6.73, p=0.002), or to pts with decreased ER expression in metastases - particularly pts whose ER levels dropped from 50-100% to 10-30% (HR 2.89, p=014). These pts also had superior OS when compared to pts with persistently high ER levels (HR 2.08, p=0.043). The prognostic impact was preserved at the multivariate analysis (including age, grade, visceral/non-visceral disease, biopsy site). Conclusions: Intratumor ER expression and dynamics may in part explain the prognostic heterogeneity of pts with ER+/HER2- stable phenotype from primary to advanced BC. Pts with lower ER levels in metastases are prognostically disadvantaged. Dynamic changes in ER expression provide additional insights beyond those captured by single-point assessment. Interestingly, pts whose tumors shifted from ER 10–30% to ER 50–100% showed the most favorable prognosis, even outperforming those with consistently high ER levels. Metastases, ER 10-30% 30-50% 50-100% Total Primary BC, ER n % n % n % n % 10-30% 5 1.2 0 0 18 4.4 23 5.6 30-50% 7 1.7 4 1.0 11 2.7 22 5.4 50-100% 24 5.9 21 5.1 320 78.0 364 89 Total 36 8.8 25 6.1 349 85.1 410 100

Most relevant findings from the signature comparisons (analysis of 770 genes) in ER-low and TNBC metastatic samples from the TONIC trial. Boxplots showing differential expression of ESR1 and selected immune-related signatures in ER-low and triple-negative metastatic samples from the TONIC trial.

PURPOSE:The purpose of this study was to assess prognosis of estrogen receptor (ER)-low expression and its dynamics in HER2- metastatic breast cancer and to compare sensitivity to nivolumab between ER-low and triple-negative breast cancer (TNBC). EXPERIMENTAL DESIGN:Two cohorts were analyzed: a multicenter cohort of 982 patients with HER2- metastatic breast cancer and one prospective cohort of 110 patients with ER <10%/HER2- metastatic breast cancer enrolled in the TONIC trial (testing nivolumab). Endpoints were overall survival (OS) and post-relapse survival (PRS) in the retrospective cohort and progression-free survival, OS, and clinical benefit rate in the TONIC trial. RESULTS:A total of 7.3% of retrospective cases had ER-low breast cancer, 15 of 110 of the TONIC trial. In the retrospective cohort, patients with ER-low breast cancer had significantly poorer OS (P < 0.001) and numerically shorter PRS (P = 0.230) compared with ER+/HER2- breast cancer and numerically longer OS (P = 0.098) and significantly longer PRS (P = 0.017) compared with TNBC. In the TONIC trial, patients with ER-low breast cancer, compared with TNBC, showed similar response to nivolumab (clinical benefit rate: 20.0% vs. 22.1%; P = 1), progression-free survival (median, 1.7 vs. 2.0 months; P = 0.5), and OS (median, 5.3 vs. 8.6 months; P = 0.3). Among patients with primary ER+/HER2- breast cancer (n = 565), the conversion toward ER-low breast cancer or TNBC at metastasis conferred independent negative prognostic impact both for OS (P = 0.002 and P = 0.001, respectively) and PRS (P = 0.018 and P = 0.001, respectively). CONCLUSIONS:We provided evidence of the prognostic role of ER-low expression and its dynamics in patients with HER2- metastatic breast cancer. We offered insights into sensitivity to anti-PD1 in metastatic breast cancer, showing that patients with ER-low breast cancer have comparable likelihood of responding to nivolumab as those with TNBC.

Distribution of PAM50 intrinsic subtypes across ER-low, TN and ER-low metastatic samples of patients with ER+/HER2- primary tumors. Donut plots showing the distribution of PAM50 intrinsic subtypes in metastatic samples from patients with ER+/HER2- primary breast cancer, according to phenotypic evolution (conversion to ER-low, conversion to triple-negative, or stable ER+/HER2-).

1100 Background: Although 1% as ER cutoff to discriminate ER+/HER2- from triple-negative (TN) BC serves the purpose of maximizing access to endocrine therapy, it may be suboptimally informative in terms of prognosis and immune checkpoint inhibitor (ICI) benefit. In the early setting, we reported that ER-low BC (ER 1-9%/HER2-) is immunologically and prognostically similar to TN (ER 0%) [Massa, 2023]. Here we focus on MBC and aim to assess prognosis of ER-low BC, including the conversion from ER+(≥10%)/HER2- to ER-low at relapse. We also compared ER-low and TN in terms of outcome after ICI, and TILs. Methods: We included two cohorts of MBC patients (pts): a multicentric retrospective cohort and pts with ER<10%/HER2- MBC enrolled in the TONIC trial testing nivolumab +/- induction therapy. All pts had ER and HER2 status assessed on metastatic tumor lesion; in the retrospective cohort ER and HER2 status on primary tumor was also available. Survival endpoints in the retrospective cohort: overall survival (OS, from BC diagnosis), post-relapse survival (PRS, from BC relapse). Exploratory endpoints from the TONIC trial: clinical benefit rate (CBR), OS and PFS (both from randomization). TILs were assessed on MBC samples (both cohorts) and on matched primary tumor (retrospective cohort). Results: We included 1112 pts in the retrospective cohort: the prevalence of ER-low BC was 3.6% in primary tumor and 5.9% in MBC lesions. In the TONIC trial, 15/110 pts had ER-low MBC. In the retrospective cohort, ER-low and TN MBC showed similar outcome, and significantly unfavorable vs ER+/HER2- MBC (Table). Among pts with ER+/HER2- primary BC, 6.7% converted to ER-low and 14.7% to TN at relapse. ER+/HER2- BC pts converting to ER-low had similar outcome than those converted to TN, and significantly poorer than pts with stable ER+/HER2- (Table). In the TONIC trial, ER-low MBC pts, compared to TN, showed similar response to ICI (CBR: 20.0% vs 22.1%, p=1), similar PFS and numerically worse OS (Table). TIL levels on MBC samples were similar between ER-low and TN both in the retrospective (median: 6% vs 5%, p=0.83) and the TONIC cohorts (median: 5.5% vs 5.0%, p=0.56) and were higher than ER+/HER2- (median: 3%). Conclusions: We confirmed that ER-low and TN MBC show similar poor outcome and the shift from ER+/HER2- to ER-low has a comparable unfavorable prognostic impact compared to conversion to TN. Our results support the hypothesis that ER-low and TN are immunologically akin and similarly prone to respond to ICI. [Table: see text]

Approximately a half of breast tumors classified as HER2-negative exhibit HER2-low-positive expression. We recently described a high instability of HER2-low-positive expression from primary breast cancer (BC) to relapse. Previous studies reporting discordance in HER2 status between baseline biopsy and residual disease (RD) in patients undergoing neoadjuvant treatment did not include the HER2-low-positive category. The aim of this study is to track the evolution of HER2-low-positive expression from primary BC to RD after neoadjuvant treatment. Patients undergoing neoadjuvant treatment with available baseline tumor tissue and matched samples of RD (in case of no pCR) were included. HER2-negative cases were sub-classified as HER2-0 or HER2-low-positive (IHC 1+ or 2+ and ISH negative). Four-hundred forty-six patients were included. Primary BC phenotype was: HR-positive/HER2-negative 23.5%, triple-negative (TN) 35%, HER2-positive 41.5%. HER2-low-positive cases were 55.6% of the HER2-negative cohort and were significantly enriched in the HR-positive/HER2-negative vs. TN subgroup (68.6% vs. 46.8%, p = 0.001 χ2 test). In all, 35.3% of non-pCR patients (n = 291) had a HER2-low-positive expression on RD. The overall rate of HER2 expression discordance was 26.4%, mostly driven by HER2-negative cases converting either from (14.8%) or to (8.9%) HER2-low-positive phenotype. Among HR-positive/HER2-negative patients with HER2-low-positive expression on RD, 32.0% and 57.1% had an estimated high risk of relapse according to the residual proliferative cancer burden and CPS-EG score, respectively. In conclusion, HER2-low-positive expression showed high instability from primary BC to RD after neoadjuvant treatment. HER2-low-positive expression on RD may guide personalized adjuvant treatment for high-risk patients in the context of clinical trials with novel anti-HER2 antibody-drug conjugates.

Liver cancer represents the third leading cause of cancer-related death worldwide. Cholangiocarcinoma (CCA) is the second most common type of liver cancer after hepatocellular carcinoma, accounting for 10-15% of all primary liver malignancies. Both the incidence and mortality of CCA have been steadily increasing during the last decade. Moreover, most CCAs are diagnosed at an advanced stage, when therapeutic options are very limited.CCA may arise from any tract of the biliary system and it is classified into intrahepatic, perihilar, and distal CCA, according to the anatomical site of origin. This topographical classification also reflects distinct genetic and histological features, risk factors, and clinical outcomes. This review focuses on histopathology of CCA, its differential diagnoses, and its diagnostic pitfalls.

Autoimmune hepatitis (AIH) is a relatively rare non-resolving chronic liver disease, which mainly affects women. It is characterized by hypergammaglobulinemia, circulating autoantibodies, interface hepatitis on liver histology and a favourable response to immunosuppression. The putative mechanism for the development of autoimmune hepatitis is thought to be the interaction between genetic predisposition, environmental triggers and failure of the native immune system. AIH still remains a major diagnostic and therapeutic challenge, mainly because it is a very heterogeneous disease. Prompt and timely diagnosis is crucial since, if left untreated, AIH has a high mortality rate. Histological demonstration of hepatitis is required for the diagnosis of AIH and, therefore, liver biopsy is mandatory in the initial diagnostic work-up, before treatment. In this review, we summarize the histological features of AIH with the main aim of highlighting the most important clinical-pathological hallmarks useful in the routine diagnostic practice.

Non-alcoholic fatty liver disease (NAFLD) encompasses a spectrum of different conditions which are characterized by hepatic steatosis in the absence of secondary causes. It is currently the most common chronic liver disease worldwide, and its estimated prevalence is about 1.5-6.5%. The only histological finding of steatosis ("simple" steatosis) represents the uncomplicated form of NAFLD, while non-alcoholic steatohepatitis (NASH) is its inflammatory subtype associated with disease progression to cirrhosis and hepatocellular carcinoma (HCC), and represents the major indication for liver transplantation. NASH is still a diagnostic and therapeutic challenge for clinicians and liver biopsy is currently the only accepted method to reliably distinguish NASH from "simple" steatosis. From the histological perspectives, NAFLD and NASH continue to be an area of active interest for pathologists, with a specific focus on better methods of evaluation, morphologic clues to pathogenesis, and predictors of fibrosis progression. This review focuses on histopathology of NAFLD in adults, with the aim to provide a practical diagnostic approach useful in the clinical routine.

About a half of HER2-negative breast cancer (BC) show HER2-low expression that can be targeted by new antibody-drug conjugates. The main aim of this study is to describe the evolution of HER2 expression from primary BC to relapse by including HER2-low category in both primary and recurrent BC samples. Patients with matched primary and relapse BC samples were included. HER2 was evaluated according to ASCO/CAP recommendations in place at the time of diagnosis. A cutoff of >10% cells staining for HER2-positivity was applied. HER2-negative cases were sub-classified as HER2-low (IHC = 1 + /2+ and ISH not amplified), or HER2-0 (IHC-0). 547 patients were included. The proportion of HER2-low cases was 34.2% on the primary tumor and 37.3% on the relapse samples. Among HER2-negative cases, HER2-low status was more frequent in HR-positive vs triple-negative tumors (47.3% vs 35.4% on primary tumor samples, 53.8% vs 36.2% on relapse samples). The overall rate of HER2 discordance was 38.0%, mostly represented by HER2-0 switching to HER2-low (15%) and HER2-low switching to HER2-0 (14%). Among patients with a primary HER2-negative tumor, the rate of HER2 discordance was higher in HR-positive/HER2-negative vs triple-negative cases (45.5% vs 36.7% p = 0.170). This difference was mostly driven by cases switching from HER2-0 to HER2-low. HER2-low expression is highly unstable during disease evolution. Relapse biopsy in case of a primary HER2-0 tumor may open new therapeutic opportunities in a relevant proportion of patients.

Intratumoral immune infiltrate was recently reported in gastrointestinal stromal tumors (GISTs). However, the tumor-intrinsic factors that dictate GIST immunogenicity are still largely undefined. To shed light on this issue, a large cohort (82 samples) of primary untreated GISTs, representative of major clinicopathological variables, was investigated by an integrated immunohistochemical, transcriptomic, and computational approach. Our results indicate that tumor genotype, location, and malignant potential concur to shape the immunogenicity of primary naive GISTs. Immune infiltration was greater in overt GISTs compared with that in lesions with limited malignant potential (miniGISTs), in KIT/PDGFRA-mutated tumors compared with that in KIT/PDGFRA WT tumors, and in PDGFRA-mutated compared with KIT-mutated GISTs. Within the KIT-mutated subset, a higher degree of immune colonization was detected in the intestine. Immune hot tumors showed expression patterns compatible with a potentially proficient but curbed antigen-specific immunity, hinting at sensitivity to immunomodulatory treatments. Poorly infiltrated GISTs, primarily KIT/PDGFRA WT intestinal tumors, showed activation of Hedgehog and WNT/β-catenin immune excluding pathways. This finding discloses a potential therapeutic vulnerability, as the targeting of these pathways might prove effective by both inhibiting pro-oncogenic signals and fostering antitumor immune responses. Finally, an intriguing anticorrelation between immune infiltration and ANO1/DOG1 expression was observed, suggesting an immunomodulatory activity for anoctamin-1.

Introduction: Neuroischemic (NI) diabetic foot (DF) is an advanced complication leading to limb amputations and cardiac mortality. We contributed to demonstrate that bone marrow (BM) T2D neuropathy impairs vascular regenerative hematopoietic cell mobilization. We here hypothesize that autonomic neuropathy (DAN) and pathological orthostatic hypotension (reducing systolic and diastolic blood pressure -BP at least 30 and 10 mm Hg) convert to cardiovascular death in T2D patients with NI-DF. Subjects and Methods: We performed DAN and postural hypotension tests in 204 surgical-treated neuropathic T2D without (87 N) or with (117 NI) critical limb ischemia (pO2 <30 mm Hg). In peripheral blood (PB) (204) and BM (65) samples we evaluated: percentage of haematopoietic precursor CD34+, VEGF receptor KDR+ (PreC) and neurokinin-1 receptor on BM-CD34+ cells (BM-NK1R+preC) by flow cytometry; circulating P substance neuropeptide by ELISA. Results: We observed 46 NI cardiac deaths (DD) and 158 alive patients (AD) after prospective 5 years. DD were older but comparable for diabetes age, BMI, HbA1c, post-revascularization oximetry and TUC lesion grade. All T2D had autonomic failure but only DD were orthostatically hypotensive (-BP -35±2 and -21± 3 mm Hg p<0.01 vs. AD) with different BM/PB preC incremental ratio (8.8±4 vs. 1.5 ±0.3 106 events p<0.0003 vs. AD), similar circulating P levels, reduced BM-NK1R+preC (14.5±8.5 vs. 36.4±5 % p<0.04). Both -BP correlate with BM/PB preC (p< 0.04) but not with BM-NK1R+preC. Conclusions: We prospectively confirm that autonomic failure impacts on vascular homeostasis and cardiac mortality in T2D with prevalent NI-DF. Now we demonstrate that pathological orthostatic hypotension, a simple clinical measure, could contribute to persistent ischemic bone marrow signal in neuroreceptor reduced BM-derived stem cells, worsening BM vascular regenerative capacity and cardiac performance. Disclosure M. Sambataro: None. L. Sambado: None. G. Spinetti: None. E. Seganfreddo: None. E. Trevisiol: None. M. Marcon: None. P. Stefani: None. A. Furlan: None. M. Cacciatore: None. A. Paccagnella: None. Funding Italian Diabetic Patients' Association of Treviso and Oderzo

Diabetic neuropathy is the most common complication of diabetes and is frequently associated with foot ischemia and infection, but its pathogenesis is controversial. We hypothesized that proinsulin expression in peripheral blood mononuclear cells is a process relevant to this condition and could represent a link among hyperglycemia, nerve susceptibility, and diabetic foot lesions. We assessed proinsulin expression by using flow cytometry in dendritic cells from control participants and patients with type 2 diabetes with or without peripheral neuropathy or accompanied by diabetic foot. Among 32 non-neuropathic and 120 neuropathic patients with type 2 diabetes, we performed leg electromyography and found average sensory sural nerve conduction velocities of 48 ± 4 and 30 ± 4 m/s, respectively ( P < 0.03). Of those with neuropathy, 42 were without lesions, 39 had foot lesions, and 39 had neuroischemic foot lesions (allux oximetry <30 mmHg). In this well-defined diabetic population, but not in nondiabetic participants, a progressively increasing level of peripheral blood dendritic cell proinsulin expression was detected, which directly correlated with circulating TNF-α levels ( P < 0.002) and multiple conduction velocities of leg nerves ( P < 0.05). These results are consistent with the hypothesis that, in type 2 diabetes, proinsulin-expressing blood cells, possibly via their involvement in innate immunity, may play a role in diabetic peripheral neuropathy and foot lesions.-Sambataro, M., Sambado, L., Trevisiol, E., Cacciatore, M., Furlan, A., Stefani, P. M., Seganfreddo, E., Durante, E., Conte, S., Della Bella, S., Paccagnella, A., dei Tos, A. P. Proinsulin-expressing dendritic cells in type 2 neuropathic diabetic patients with and without foot lesions.