Vessel-Guided Mesohepatectomy for Liver Partition and Staged Major Parenchyma-Sparing Hepatectomies with Super-Selective Portal Vein Embolization or Enhanced ALPPS to Achieve R0 Resection for Colorectal Liver Metastases at the Hepatocaval Confluence.

Background. R0 minor parenchyma-sparing hepatectomy (PSH) is feasible for colorectal liver metastases (CRLM) in contact with hepatic veins (HV) at hepatocaval confluence since HV can be reconstructed, but in the case of contact with the first-order glissonean pedicle (GP), major hepatectomy is mandatory. To pursue an R0 parenchyma-sparing policy, we proposed vessel-guided mesohepatectomy for liver partition (MLP) and eventually combination with liver augmentation techniques for staged major PSH. Methods. We analyzed 15 consecutive vessel-guided MLPs for CRLM at the hepatocaval confluence. Patients had a median of 11 (range: 0-67) lesions with a median diameter of 3.5 cm (range: 0.0-8.0), bilateral in 73% of cases. Results. Grade IIIb or more complications occurred in 13%, median hospital stay was 14 (range: 6-62) days, 90-day mortality was 0%. After a median follow-up of 17.5 months, 1-year OS and RFS were 92% and 62%. In nine (64%) patients, MLP was combined with portal vein embolization (PVE) or ALPPS to perform staged R0 major PSH. Future liver remnant (FLR) volume increased from a median of 15% (range: 7-20%) up to 41% (range: 37-69%). Super-selective PVE was performed in three (33%) patients and enhanced ALPPS (e-ALPPS) in six (66%). In two e-ALPPS an intermediate stage of deportalized liver PSH was necessary to achieve adequate FLR volume. Conclusions. Vessel-guided MLP may transform the liver in a paired organ. In selected cases of multiple bilobar CRLM, to guarantee oncological radicality (R0), major PSH is feasible combining advanced surgical parenchyma sparing with liver augmentation techniques when FLR volume is insufficient.

Identification of New Potential Prognostic and Predictive Markers in High-Grade Osteosarcoma Using Whole Exome Sequencing.

Conventional high-grade osteosarcoma (OS) is the most common primary cancer of bone and it typically affects the extremities of adolescents. OS has a complex karyotype, and molecular mechanisms related to carcinogenesis, progression and resistance to therapy are still largely unknown. For this reason, the current standard of care is associated with considerable adverse effects. In this study, our aim was to identify gene alterations in OS patients using whole exome sequencing (WES) to find new potential prognostic biomarkers and therapeutic targets. We performed WES on formalin-fixed paraffin-embedded (FFPE) biopsy materials collected from 19 patients affected by conventional high-grade OS. The clinical and genetic data were analyzed according to response to therapy, presence of metastasis and disease status. By comparing good and poor responders to neoadjuvant therapy, we detected a clear prevalence of mutations in the ARID1A, CREBBP, BRCA2 and RAD50 genes in poor responders that negatively influence the progression-free survival time. Moreover, higher tumor mutational burden values correlated with worse prognosis. The identification of mutations in ARID1A, CREBBP, BRCA2 and RAD50 may support the use of a more specific therapy for tumors harboring these alterations. In particular, BRCA2 and RAD50 are involved in homologous recombination repair, and could thus be used as specific therapy targets of inhibitors of the enzyme Poly ADP Ribose Polymerase (PARP). Finally, tumor mutational burden is found to be a potential prognostic marker for OS.

Raman spectroscopy and topological machine learning for cancer grading.

In the last decade, Raman Spectroscopy is establishing itself as a highly promising technique for the classification of tumour tissues as it allows to obtain the biochemical maps of the tissues under investigation, making it possible to observe changes among different tissues in terms of biochemical constituents (proteins, lipid structures, DNA, vitamins, and so on). In this paper, we aim to show that techniques emerging from the cross-fertilization of persistent homology and machine learning can support the classification of Raman spectra extracted from cancerous tissues for tumour grading. In more detail, topological features of Raman spectra and machine learning classifiers are trained in combination as an automatic classification pipeline in order to select the best-performing pair. The case study is the grading of chondrosarcoma in four classes: cross and leave-one-patient-out validations have been used to assess the classification accuracy of the method. The binary classification achieves a validation accuracy of 81% and a test accuracy of 90%. Moreover, the test dataset has been collected at a different time and with different equipment. Such results are achieved by a support vector classifier trained with the Betti Curve representation of the topological features extracted from the Raman spectra, and are excellent compared with the existing literature. The added value of such results is that the model for the prediction of the chondrosarcoma grading could easily be implemented in clinical practice, possibly integrated into the acquisition system.

Surgical treatment of recurrent retroperitoneal sarcoma in its different patterns: A 15-years' two-centers experience.

INTRODUCTION: There is limited data available regarding the role of surgery in the treatment of retroperitoneal sarcoma (RPS) recurrences. We herein report the short- and mid-term outcomes of patients who underwent surgical treatment of RPS recurrences at two Italian centers over a 15-years' experience. MATERIALS AND METHODS: From January 2005 to January 2020, 33 patients underwent surgical treatment of isolated locally recurrent RPS (LR group), locally recurrent RPS associated with the presence of distant recurrence (LR + DM group), and distant-only recurrent RPS (DM group). Only procedures performed to obtain a macroscopically radical treatment with curative intent were included. Data regarding pre-, intra-, post-operative course, and follow-up, collected in an Institutional database, were retrospectively analyzed, and compared. RESULTS: LR-group was composed of 15 patients, LR + DM group of 9 patients, and DM group of 9 patients. During the follow-up, 78.5% of the LR group, 77.8% of the DM group and 100% of the LR + DM group (p = 0.244) experienced a second recurrence. 7/11 (63.6%) patients in the LR group, 2/7 (28.5%) patients in the DM-group, and 0/9 (0.0%) patients in the LR + DM group underwent to almost one further local treatments of their recurrences (p = 0.010). No differences in the mean disease-free survival (p = 0.127), overall survival (OS) (p = 0.165) was reported among the three groups. Repeated surgery was an independent factor affecting survival in multivariate analysis (p = 0.01). CONCLUSIONS: A surgical treatment of RPS recurrences should always be taken into consideration, also in metastatic patients and/or in those who have already undergone surgery for previous RPS recurrence, because this approach may offer survival benefits.

Zebrafish Patient-Derived Xenograft Model to Predict Treatment Outcomes of Colorectal Cancer Patients.

The use of zebrafish embryos for personalized medicine has become increasingly popular. We present a co-clinical trial aiming to evaluate the use of zPDX (zebrafish Patient-Derived Xenografts) in predicting the response to chemotherapy regimens used for colorectal cancer patients. zPDXs are generated by xenografting tumor tissues in two days post-fertilization zebrafish embryos. zPDXs were exposed to chemotherapy regimens (5-FU, FOLFIRI, FOLFOX, FOLFOXIRI) for 48 h. We used a linear mixed effect model to evaluate the zPDX-specific response to treatments showing for 4/36 zPDXs (11%), a statistically significant reduction of tumor size compared to controls. We used the RECIST criteria to compare the outcome of each patient after chemotherapy with the objective response of its own zPDX model. Of the 36 patients enrolled, 8 metastatic colorectal cancer (mCRC), response rate after first-line therapy, and the zPDX chemosensitivity profile were available. Of eight mCRC patients, five achieved a partial response and three had a stable disease. In 6/8 (75%) we registered a concordance between the response of the patient and the outcomes reported in the corresponding zPDX. Our results provide evidence that the zPDX model can reflect the outcome in mCRC patients, opening a new frontier to personalized medicine.

Chondroblastoma-like osteosarcoma: a clinicopathological and molecular study of a rare osteosarcoma variant.

OBJECTIVE: Chondroblastoma-like osteosarcoma (CBLOS) is a rare and poorly understood variant of OS. We examined the clinicopathological, immunohistochemical and molecular features of six CBLOSs to highlight the differences with conventional high-grade OS (CHGOS) and CB, including CB with aggressive features. METHODS: We performed histone 3.3 mutation analysis by gene sequencing and/or immunohistochemistry in all cases, while whole exome sequencing (WES) was performed on two CB-like osteosarcomas and 11 conventional high-grade OS. RESULTS: CBLOSs were predominantly localised at acral sites and involved mainly male subjects with a mean age of 29 years. One patient who had metastases at presentation died of disease, while another patient who developed multiple local recurrences and lung metastases was alive with no evidence of disease (ANED) at 294 months. The remaining patients were ANED after a mean interval of 70.8 months. Histologically, all CBLOS presented aggressive features, including nuclear atypia and infiltrative growth. Immunohistochemistry with H3F3 K36M mutant antibody was negative in all CBLOSs, and none of the five tumours tested by gene sequencing had H3F3B mutations. Conversely, all CBs presented the H3F3B K36M variant and were positive for immunostaining with the H3F3 K36M antibody. Two CBLOSs analysed by WES differed in amount and type of mutation from 11 cases of CHGOS. Moreover, CBLOSs showed lower copy number alteration (CNA) score values than CHGOSs. CONCLUSIONS: CBLOS presents a different genetic background and a less aggressive clinical behaviour in comparison with CHGOS. Search of the H3F3B K36M mutation is useful in the differential diagnosis with CB.

In Pancreatic Adenocarcinoma Alpha-Synuclein Increases and Marks Peri-Neural Infiltration.

α-Synuclein (α-syn) is a protein involved in neuronal degeneration. However, the family of synucleins has recently been demonstrated to be involved in the mechanisms of oncogenesis by selectively accelerating cellular processes leading to cancer. Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal human cancers, with a specifically high neurotropism. The molecular bases of this biological behavior are currently poorly understood. Here, α-synuclein was analyzed concerning the protein expression in PDAC and the potential association with PDAC neurotropism. Tumor (PDAC) and extra-tumor (extra-PDAC) samples from 20 patients affected by PDAC following pancreatic resections were collected at the General Surgery Unit, University of Pisa. All patients were affected by moderately or poorly differentiated PDAC. The amount of α-syn was compared between tumor and extra-tumor specimen (sampled from non-affected neighboring pancreatic areas) by using in situ immuno-staining with peroxidase anti-α-syn immunohistochemistry, α-syn detection by using Western blotting, and electron microscopy by using α-syn-conjugated immuno-gold particles. All the methods consistently indicate that each PDAC sample possesses a higher amount of α-syn compared with extra-PDAC tissue. Moreover, the expression of α-syn was much higher in those PDAC samples from tumors with perineural infiltration compared with tumors without perineural infiltration.

A murine mesenchymal stem cell model for initiating events in osteosarcomagenesis points to CDK4/CDK6 inhibition as a therapeutic target.

Osteosarcoma is a high-grade bone-forming neoplasm, with a complex genome. Tumours frequently show chromothripsis, many deletions, translocations and copy number alterations. Alterations in the p53 or Rb pathway are the most common genetic alterations identified in osteosarcoma. Using spontaneously transformed murine mesenchymal stem cells (MSCs) which formed sarcoma after subcutaneous injection into mice, it was previously demonstrated that p53 is most often involved in the transformation towards sarcomas with complex genomics, including osteosarcoma. In the current study, not only loss of p53 but also loss of p16Ink4a is shown to be a driver of osteosarcomagenesis: murine MSCs with deficient p15Ink4b, p16Ink4a, or p19Arf transform earlier compared to wild-type murine MSCs. Furthermore, in a panel of nine spontaneously transformed murine MSCs, alterations in p15Ink4b, p16Ink4a, or p19Arf were observed in eight out of nine cases. Alterations in the Rb/p16 pathway could indicate that osteosarcoma cells are vulnerable to CDK4/CDK6 inhibitor treatment. Indeed, using two-dimensional (n = 7) and three-dimensional (n = 3) cultures of human osteosarcoma cell lines, it was shown that osteosarcoma cells with defective p16INK4A are sensitive to the CDK4/CDK6 inhibitor palbociclib after 72-hour treatment. A tissue microarray analysis of 109 primary tumour biopsies revealed a subset of patients (20-23%) with intact Rb, but defective p16 or overexpression of CDK4 and/or CDK6. These patients might benefit from CDK4/CDK6 inhibition, therefore our results are promising and might be translated to the clinic.

Detailing the ultrastructure's increase of prion protein in pancreatic adenocarcinoma.

BACKGROUND: Recent evidences have shown a relationship between prion protein (PrPc) expression and pancreatic ductal adenocarcinoma (PDAC). Indeed, PrPc could be one of the markers explaining the aggressiveness of this tumor. However, studies investigating the specific compartmentalization of increased PrPc expression within PDAC cells are lacking, as well as a correlation between ultrastructural evidence, ultrastructural morphometry of PrPc protein and clinical data. These data, as well as the quantitative stoichiometry of this protein detected by immuno-gold, provide a significant advancement in understanding the biology of disease and the outcome of surgical resection. AIM: To analyze quantitative stoichiometry and compartmentalization of PrPc in PDAC cells and to correlate its presence with prognostic data. METHODS: Between June 2018 and December 2020, samples from pancreatic tissues of 45 patients treated with pancreatic resection for a preoperative suspicion of PDAC at our Institution were collected. When the frozen section excluded a PDAC diagnosis, or the nodules were too small for adequate sampling, patients were ruled out from the present study. Western blotting was used to detect, quantify and compare the expression of PrPc in PDAC and control tissues, such as those of non-affected neighboring pancreatic tissue of the same patient. To quantify the increase of PrPc and to detect the subcellular compartmentalization of PrPc within PDAC cells, immuno-gold stoichiometry within specific cell compartments was analyzed with electron microscopy. Finally, an analysis of quantitative PrPc expression according to prognostic data, such as cancer stage, recurrence of the disease at 12 mo after surgery and recurrence during adjuvant chemotherapy was made. RESULTS: The amount of PrPc within specimen from 38 out of 45 patients was determined by semi-quantitative analysis by using Western blotting, which indicates that PrPc increases almost three-fold in tumor pancreatic tissue compared with healthy pancreatic regions [242.41 ± 28.36 optical density (OD) vs 95 ± 17.40 OD, P < 0.0001]. Quantitative morphometry carried out by using immuno-gold detection at transmission electron microscopy confirms an increased PrPc expression in PDAC ductal cells of all patients and allows to detect a specific compartmentalization of PrPc within tumor cells. In particular, the number of immuno-gold particles of PrPc was significantly higher in PDAC cells respect to controls, when considering the whole cell (19.8 ± 0.79 particles vs 9.44 ± 0.45, P < 0.0001). Remarkably, considering PDAC cells, the increase of PrPc was higher in the nucleus than cytosol of tumor cells, which indicates a shift in PrPc compartmentalization within tumor cells. In fact, the increase of immuno-gold within nuclear compartment exceeds at large the augment of PrPc which was detected in the cytosol (nucleus: 12.88 ± 0.59 particles vs 5.12 ± 0.32, P < 0.0001; cytosol: 7.74. ± 0.44 particles vs 4.3 ± 0.24, P < 0.0001). In order to analyze the prognostic impact of PrPc, we found a correlation between PrPc expression and cancer stage according to pathology results, with a significantly higher expression of PrPc for advanced stages. Moreover, 24 patients with a mean follow-up of 16.8 mo were considered. Immuno-blot analysis revealed a significantly higher expression of PrPc in patients with disease recurrence at 12 mo after radical surgery (360.71 ± 69.01 OD vs 170.23 ± 23.06 OD, P = 0.023), also in the subgroup of patients treated with adjuvant CT (368.36 ± 79.26 OD in the recurrence group vs 162.86 ± 24.16 OD, P = 0.028), which indicates a correlation with a higher chemo-resistance. CONCLUSION: Expression of PrPc is significantly higher in PDAC cells compared with control, with the protein mainly placed in the nucleus. Preliminary clinical data confirm the correlation with a poorer prognosis.

Acral Dedifferentiated Chondrosarcoma: Report of a Case Arising in the Proximal Phalanx of the Fourth Finger.

Dedifferentiated chondrosarcoma is a well-recognized entity, but its occurrence in the distal extremities is exceedingly rare. We present the case of a 49-year-old woman who experienced local recurrence of an "enchondroma" of the proximal phalanx of the fourth finger of the left hand, which had been initially treated with intralesional curettage at another hospital 4 years before, and 1 year before for a local recurrence. The imaging findings indicated an aggressive behavior, and an incisional biopsy showed a highly cellular proliferation of spindle and pleomorphic elements without evidence of matrix production intermixed with few fragments of a well-differentiated cartilaginous neoplasm with bland cellular atypia, focal nuclear hyperchromatism, and binucleation. An isocitrate dehydrogenase 2 R172S mutation was detected. The final diagnosis was dedifferentiated chondrosarcoma. Despite amputation of the fourth finger, the patient developed lung metastases and further local relapse. Recurrent cartilaginous tumors of the extremities should not be underestimated and should be followed in view of the possible acquisition of aggressive clinical behavior.

A 13th-century cystic echinococcosis from the cemetery of the monastery of Badia Pozzeveri (Lucca, Italy).

OBJECTIVE: To differentially diagnose a calcified formation recovered from a 13th century AD grave from the Tuscan monastery of Badia Pozzeveri, Lucca, Italy. MATERIALS: A calcified formation from the thoraco-abdominal region of a skeleton buried in the monastery cemetery. METHODS: Cone Beam Computed Tomography, Scanning Electron Microscope and Energy Dispersive X-Ray Spectroscopy. RESULTS: A hollow, calcified ovoid formation was identified as typical of a hydatid cyst, permitting the diagnosis of cystic echinococcosis in a 35-45year-old female. CONCLUSIONS: The study reveals the circulation of the parasite Echinococcus granulosus in the region of Lucca in late medieval Tuscany. SIGNIFICANCE: This finding is the fourth case of cystic echinococcosis from an archaeological context in Italy and provides insight into environmental conditions that appear to have affected members of a community, irrespective of social status. LIMITATIONS: Caution and the application of multiple analyses must be exercised in the differential diagnosis to discriminate among calcified formations. SUGGESTIONS FOR FURTHER RESEARCH: Analysis of stable isotopes of the calcified formation, such as 15N and 13C, in order to compare them with isotopic values of the host individual and to further confirm the parasitic origin of the find.

Diffuse bone and soft tissue angiomatosis with GNAQ mutation.

We describe a unique case of skeletal and extraskeletal angiomatosis complicated by Kasabach-Merritt syndrome. The patient was a 3-year-old boy, who presented with involvement of both femurs and left tibia, as well as with soft tissue lesions of the left thigh. At birth, multiple hemangiomas of the soft tissues of the frontal and parietal scalp had been identified, together with a space-occupying lesion of the lung. Histologically, the skeletal and soft tissue lesions consisted of a proliferation of thin-walled, dilated blood vessels, with an endothelial lining devoid of atypia and exhibiting immunoreactivity for CD31 and CD34, while podoplanin and GLUT1 were negative. Whole exome sequencing performed on samples from the lesion of the femur, the tibia and the skin of the thigh, showed a GNAQ (c.286A>T:p.T96S) variant in all specimens, that was confirmed with digital droplet PCR. This case expands the clinical and pathologic spectrum of vascular proliferations showing similar molecular biology, characterized by GNAQ, GNA11 or GNA14 mutations.

Dedifferentiated soft tissue leiomyosarcoma with heterologous osteosarcoma component: case report and review of the literature.

BACKGROUND: Soft tissue dedifferentiated leiomyosarcoma with heterologous osteosarcomatous component is an extremely rare entity described in only few cases in the literature. CASE PRESENTATION: We report the case of a 65-year-old male patient who, after initial inadequate surgery of a tumor of the left forearm, developed local recurrence that was treated with neoadjuvant chemotherapy, surgery and postoperative radiation therapy. Histologically the tumor showed an abrupt separation of two different patterns. One component consisted of interlacing fascicles of spindle cells with cigar-shaped nuclei strongly positive for smooth muscle actin, desmin and H-caldesmon. The other component consisted of a high-grade pleomorphic sarcoma with osteoid and chondroid matrix production, which positive for SATB2. Thus, a final diagnosis of dedifferentiated leiomyosarcoma was rendered. Fifteen months after treatment, the patient presented further local and distant relapse with pulmonary metastases and died 23 months after the first presentation. DISCUSSION AND CONCLUSIONS: Dedifferentiated leiomyosarcoma is a highly malignant neoplasm with a poor outcome. Extensive sampling of soft tissue leiomyosarcomas is recommended to detect possible dedifferentiated areas as they represent a crucial prognostic parameter.

Contribution of Raman Spectroscopy to Diagnosis and Grading of Chondrogenic Tumors.

In the last decade, Raman Spectroscopy has demonstrated to be a label-free and non-destructive optical spectroscopy able to improve diagnostic accuracy in cancer diagnosis. This is because Raman spectroscopic measurements can reveal a deep molecular understanding of the biochemical changes in cancer tissues in comparison with non-cancer tissues. In this pilot study, we apply Raman spectroscopy imaging to the diagnosis and grading of chondrogenic tumors, including enchondroma and chondrosarcomas of increasing histologic grades. The investigation included the analysis of areas of 50×50 µm2 to approximately 200×200 µm2, respectively. Multivariate statistical analysis, based on unsupervised (Principal Analysis Components) and supervised (Linear Discriminant Analysis) methods, differentiated between the various tumor samples, between cells and extracellular matrix, and between collagen and non-collagenous components. The results dealt out basic biochemical information on tumor progression giving the possibility to grade with certainty the malignant cartilaginous tumors under investigation. The basic processes revealed by Raman Spectroscopy are the progressive degrading of collagen type-II components, the formation of calcifications and the cell proliferation in tissues ranging from enchondroma to chondrosarcomas. This study highlights that Raman spectroscopy is particularly effective when cartilaginous tumors need to be subjected to histopathological analysis.

A Splenic IgG4+ Sclerosing Angiomatoid Nodular Transformation (SANT) Treated by Hemisplenectomy: A Radiologic, Histochemical, and Immunohistochemical Study.

INTRODUCTION: Sclerosing angiomatoid nodular transformation (SANT) is a rare benign lesion of unknown origin for which total splenectomy is the standard treatment. CASE PRESENTATION: A 54-year-old man with a history of recurrent pancreatitis, bicuspid aortic valve, and aortic dissection underwent abdominal ultrasound, Computed tomography and magnetic resonance imaging, which revealed a 6-cm hypoechoic splenic mass diagnosed as cavernous hemangioma. Owing to his relevant past history, he was considered eligible for emisplenectomy and not for total excision, which is associated with long-term risks, especially infections. RESULTS: Histologic examination revealed several nodules of varying size separated by sclerotic stroma with scattered inflammatory cells rich in IgG4+ in a background of splenic red pulp. Immunohistochemical stains showed a characteristic panel for CD34, CD31, and CD8. CONCLUSIONS: The diagnosis of SANT should be considered in any patient presenting with a splenic lesion containing an angiomatoid or inflammatory component. The only method able to establish a correct diagnosis is histologic and immunohistochemical evaluation. Complete splenectomy is generally considered the best approach. However, if the patient is at high risk of infection and localization of the lesion allows for selective devascularization of the affected part of the spleen, the lesion could be removed by hemisplenectomy. In some patients SANT is related to high blood levels of IgG4. Thus, corticosteroids might be useful for treating IgG4+ SANT and for preventing other IgG4-related diseases.