Second-harmonic generation microscopy represents an important tool to evaluate extracellular matrix collagen structure, which undergoes changes during cancer progression. Thus, it is potentially relevant to assess breast cancer development. We propose the use of second-harmonic generation images of tumor stroma selected on hematoxylin and eosin-stained slides to evaluate the prognostic value of collagen fibers analyses in peri and intratumoral areas in patients diagnosed with invasive ductal breast carcinoma. Quantitative analyses of collagen parameters were performed using ImageJ software. These parameters presented significantly higher values in peri than in intratumoral areas. Higher intratumoral collagen uniformity was associated with high pathological stages and with the presence of axillary lymph node metastasis. In patients with immunohistochemistry-based luminal subtype, higher intratumoral collagen uniformity and quantity were independently associated with poorer relapse-free and overall survival, respectively. A multivariate response recursive partitioning model determined 12.857 and 11.894 as the best cut-offs for intratumoral collagen quantity and uniformity, respectively. These values have shown high sensitivity and specificity to differentiate distinct outcomes. Values of intratumoral collagen quantity and uniformity exceeding the cut-offs were strongly associated with poorer relapse-free and overall survival. Our findings support a promising prognostic value of quantitative evaluation of intratumoral collagen by second-harmonic generation imaging mainly in the luminal subtype breast cancer.

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Introduction:In breast cancer (BC), desmoplastic reaction, assembled primarily by fibroblasts, is associated with unfavorable prognosis, but the reason of this fact remains still unclear. In this context, nonlinear optics microscopy, including Fluorescence Lifetime Imaging Microscopy (FLIM), has provided advancement in cellular metabolism research. In this paper, our purpose is to differentiate BC cells metabolism with or without contact to desmoplastic reaction. Formalin fixed, paraffin embedded samples were used at different points of hematoxylin stained sections. Methodology:Sections from 14 patients with invasive ductal breast carcinoma were analyzed with FLIM methodology to NAD(P)H and FAD fluorescence lifetime on a Confocal Upright LSM780 NLO device (Carl Zeiss AG, Germany).Quantification of the fluorescence lifetime and fluorescence intensity was evaluated by SPC Image software (Becker &Hickl) and ImageJ (NIH), respectively. Optical redox ratio was calculated by dividing the FAD fluorescence intensity by NAD(P)H fluorescence intensity. Data value for FLIM measurements and fluorescence intensities were calculated using Wilcoxon test; p< 0.05 was considered significant. Results: BC cells in contact with desmoplastic reaction presented a significantly lower NAD(P)H and FAD fluorescence lifetime. Furthermore, optical redox ratio was also lower in these tumor cells. Conclusion: Our results suggest that contact of BC cells with desmoplastic reaction increase their metabolic activity, which might explain the adverse prognosis of cases associated with higher peritumoral desmoplastic reaction.

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Introduction: Sepsis in febrile neutropenia (FN) is a life threatening condition, and a health problem of increasing proportions. Although multiple organ dysfunction syndrome (MODS) frequently precedes death in patients with sepsis, the ultimate mechanisms responsible for organ dysfunction and tissue damage in sepsis are yet to be determined. Currently, tissue damage is attributed to an exacerbated response of the immune and hemostatic systems, mediated by endothelial cells, platelets and neutrophils. Of note, recent evidence demonstrated that neutrophils, platelets and fibrin participate in this response by mediating neutrophil extracellular traps (NET) formation, and promoting the hemostatic containment of infectious foci. In animal models, down-regulation of NET formation, coagulation and platelet activation are usually associated with deficiencies in pathogen clearance. Unfortunately, activation of hemostasis and NET formation could potentially contribute to tissue damage by a process called "immunethrombosis". Although the increase of sepsis severity in patients with severe neutropenia is well described, the mechanisms of sepsis-associated tissue damage in the context of severe neutropenia/thrombocytopenia are yet to be determined. Methods: In order to investigate the mechanisms of tissue damage in the context of severe neutropenia/thrombocytopenia, we compiled clinical data from two different prospective sepsis cohorts (A, neutropenic; n=129; and B, non-neutropenic; n=30) followed at our Institution. In addition, we reviewed histopathological data from 16 autopsies of individuals with hematological malignancies and septic shock from our institution (cohort C; n=16). H&E-stained slides from liver, kidneys and lungs were systematically analyzed by one investigator, and reviewed by 2 experienced pathologists, all of them blind to the presence or absence of neutropenia and thrombocytopenia. In each organ, we characterized (as present or absent) three main organic lesions: thrombi in microvessels, microorganism colonies, and inflammatory infiltrate (mononuclear and polymorphonuclear). Inflammation was graded as weak or intense, and only considered when there was no neoplastic infiltration. Results: Median ages of patients from cohorts A and B were respectively 46.0 years (13-78), and 59.4 years (22-85); P<0.0001. In cohort A, neutrophil counts were < 100/mcl in 55.8% of patients and between 100-500/mcl in 42.6%. Platelet counts were also lower in cohort A (30,126 vs 213,933/mcl; P<0.0001). Median SOFA scores (at admission) were 4 (0-15) and 5 (0-17); P=0.3 in neutropenic and non-neutropenic patients respectively, and sepsis-related mortality was 22.5% and 10.3% in the same groups (P=0.19). Among patients with a higher SOFA score, mortality was higher in neutropenic patients (100% vs 33.3%; p=0.04). The frequencies of clinically-evident infection foci were 60% in cohort A and 100% in cohort B (P=0.0001). In contrast, positive blood cultures were present in 38% of neutropenic, but in only 3.3% of non-neutropenic patients (P<0.0001). The autopsy-based study included 10 patients with lymphoma and 6 with acute leukemia. The cause of death was septic shock in all of them, and three patients presented severe neutropenia (<500/mcl). The main histological findings are shown in table 1. The only neutropenic patient with microthrombi presented AML-M3 and leukostasis. Using H&E staining, no bacterial colonies were found in any slide. Conclusions: as expected, septic patients with severe neutropenia presented a worse outcome compared to non-neutropenic patients in our cohort. In addition, the lower frequency of clinically-defined infectious foci, coupled with a strikingly higher frequency of positive blood cultures, suggest that severe neutropenia and thrombocytopenia could impair pathogen containment and clearance. Severe neutropenia/thrombocytopenia was compatible with inflammatory infiltrates and microvascular thrombosis in lungs, although the latter was only observed in a patient with leukostasis and promyelocityc acute leukemia. Table 1. Histological findings in autopsies of neutropenic and non-neutropenic patients Microthrombi Inflammation (weak/intense) Lungs Kidney Liver Lungs Kidney Liver Neutropenic 33% 33% 0% 50%/50% 0%/0% 0%/0% Non-neutropenic 56.25% 25% 6.25% 70%/20% 34%/9% 67%/0% Disclosures No relevant conflicts of interest to declare.