Advancing the PD-L1 CPS test in metastatic TNBC: Insights from pathologists and findings from a nationwide survey.

Pembrolizumab has received approval as a first-line treatment for unresectable/metastatic triple-negative breast cancer (mTNBC) with a PD-L1 combined positive score (CPS) of ≥ 10. However, assessing CPS in mTNBC poses challenges. Firstly, it represents a novel analysis for breast pathologists. Secondly, the heterogeneity of PD-L1 expression in mTNBC further complicates the assessment. Lastly, the lack of standardized assays and staining platforms adds to the complexity. In KEYNOTE trials, PD-L1 expression was evaluated using the IHC 22C3 pharmDx kit as a companion diagnostic test. However, both the 22C3 pharmDx and VENTANA PD-L1 (SP263) assays are validated for CPS assessment. Consequently, assay-platform choice, staining conditions, and scoring methods can significantly impact the testing outcomes. This consensus paper aims to discuss the intricacies of PD-L1 CPS testing in mTNBC and provide practical recommendations for pathologists. Additionally, we present findings from a nationwide Italian survey elucidating the state-of-the-art in PD-L1 CPS testing in mTNBC.

[Covid-19 emergency and telemedicine: multifamily psychoeducational intervention in a forensic setting.] / Emergenza covid-19 e telemedicina: intervento psicoeducativo multifamiliare in contesto forense.

AIM: The aim is to describe remote multifamily psychoeducational intervention led by REMS "Castore" team (an Italian health care facility for offenders who suffer from mental disorders and socially dangerous) in ASL Roma 5, a local health authority, during covid-19 emergency. METHODS: The applied theoretical models have been the integrated psychoeducational intervention by I. Falloon and the multifamily psychoeducational intervention based on problem solving by F. Veltro. The multifamily psychoeducational intervention was attempted by inpatients' relatives from June to August 2020 with 8 weekly meetings of 90' each led by two psychiatric rehabilitation therapists and healthcare experts. Six families were involved in the study and its members were assessed by questionnaire on family problems, Brief-COPE and general health questionnaire tests. Users were assessed by Level of Expressed Emotion Scale test for their expressed emotion. RESULTS: Data analysis highlighted the general low level of subjective and objective burden for family members in association with a high perception of that support provided by REMS. Furthermore, the study has shown those coping approaches oriented to the research of practical solutions, acceptance of events and assertive communication. DISCUSSION: The low subjective and objective burden may be attributed to implicit security conditions in REMS and the absence of tasks mainly carried out by experts. Those coping styles oriented to more practical than emotional approaches suggest an emotional hyper control or stigma perception leading to isolation and loneliness. CONCLUSIONS: The multifamily psychoeducational intervention has allowed a trustworthy relationship with REMS. Considering that the families involved in the study have never benefited by other psychoeducational interventions before, their early involvement appears to be a potential tool for preventing and managing crisis as well as minimizing recidivism.

Myeloma Cells Deplete Bone Marrow Glutamine and Inhibit Osteoblast Differentiation Limiting Asparagine Availability.

Multiple myeloma (MM) cells consume huge amounts of glutamine and, as a consequence, the amino acid concentration is lower-than-normal in the bone marrow (BM) of MM patients. Here we show that MM-dependent glutamine depletion induces glutamine synthetase in stromal cells, as demonstrated in BM biopsies of MM patients, and reproduced in vitro by co-culturing human mesenchymal stromal cells (MSCs) with MM cells. Moreover, glutamine depletion hinders osteoblast differentiation of MSCs, which is also severely blunted by the spent, low-glutamine medium of MM cells, and rescued by glutamine restitution. Glutaminase and the concentrative glutamine transporter SNAT2 are induced during osteoblastogenesis in vivo and in vitro, and both needed for MSCs differentiation, pointing to enhanced the requirement for the amino acid. Osteoblastogenesis also triggers the induction of glutamine-dependent asparagine synthetase (ASNS), and, among non-essential amino acids, asparagine rescues differentiation of glutamine-starved MSCs, by restoring the transcriptional profiles of differentiating MSCs altered by glutamine starvation. Thus, reduced asparagine availability provides a mechanistic link between MM-dependent Gln depletion in BM and impairment of osteoblast differentiation. Inhibition of Gln metabolism in MM cells and supplementation of asparagine to stromal cells may, therefore, constitute novel approaches to prevent osteolytic lesions in MM.

Bovine pestivirus is a new alternative virus for multiple myeloma oncolytic virotherapy.

BACKGROUND: The oncolytic viruses have shown promising results for the treatment of multiple myeloma. However, the use of human viruses is limited by the patients' antiviral immune response. In this study, we investigated an alternative oncolytic strategy using non-human pathogen viruses as the bovine viral diarrhea virus (BVDV) that were able to interact with CD46. METHODS: We treated several human myeloma cell lines and non-myeloma cell lines with BVDV to evaluate the expression of CD46 and to study the effect on cell viability by flow cytometry. The possible synergistic effect of bortezomib in combination with BVDV was also tested. Moreover, we infected the bone marrow mononuclear cells obtained from myeloma patients and we checked the BVDV effect on different cell populations, defined by CD138, CD14, CD3, CD19, and CD56 expression evaluated by flow cytometry. Finally, the in vivo BVDV effect was tested in NOD-SCID mice injected subcutaneously with myeloma cell lines. RESULTS: Human myeloma cells were selectively sensitive to BVDV treatment with an increase of cell death and, consequently, of apoptotic markers. Consistently, bone marrow mononuclear cells isolated from myeloma patients treated with BVDV, showed a significant selective decrease of the percentage of viable CD138+ cells. Interestingly, bortezomib pre-treatment significantly increased the cytotoxic effect of BVDV in myeloma cell lines with a synergistic effect. Finally, the in vitro data were confirmed in an in vivo myeloma mouse model showing that BVDV treatment significantly reduced the tumoral burden compared to the vehicle. CONCLUSIONS: Overall, our data indicate, for the first time, a direct oncolytic effect of the BVDV in human myeloma cells suggesting its possible use as novel alternative anti-myeloma virotherapy strategy.

Management of a periductal stromal tumor in a young woman: Our breast unit experience.

Periductal stromal tumor (PDST) is a rare biphasic tumor of the breast that exhibits low-grade malignancy and intermediate behavior. It is characterized by proliferation of atypical spindle cells surrounding benign mammary ducts and infiltrating adjacent adipose tissue. PDST is distinguished from phyllodes tumor by its lack of leaf-like architecture; however, it is still unclear whether PDST is a separate entity or a certain spectrum of phyllodes tumor. Phyllodes tumors constitute a group of rare epithelial lesions of the breast which mainly develops at around 40-50 years. The histologic characteristics to be considered are many and often heterogeneous in the same lesion which makes interpretation in needle biopsy material difficult. Most phyllodes tumors have a benign nature, with a high rate of postsurgical recurrence. In the malignant form, metastases are described by distant hematogenous route; its indolent behavior implies a tight surgical management with precise excision of the lesion even if there is not, however, a unanimous consent on the parameters of accuracy of the margins.

Self-healing CD30- T-clonal proliferation of the tongue: report of an extremely rare case.

BACKGROUND: The etiology of traumatic ulcerative granulomas with stromal eosinophilia (TUGSE) is not clear, traumatic irritation having advocated as the most likely cause. TUGSEs are typically self-limiting slow-healing lesions of the oral mucosa with unclear pathogenesis, commonly manifesting as a rapidly developing, long-lasting ulcer. CASE PRESENTATION: Here we report a controversial case of a self-healing lesion of the tongue in a 57 year-old woman. A clonal T-cell proliferation and CD30 negative immunohistochemical (IHC) profile could be documented. DISCUSSION AND CONCLUSION: In view of the very peculiar clinical and histological features, a retrospective diagnosis of a TUGSE with scarce eosinophilic infiltrate (possibly in regression), displaying CD30- T-clonal proliferation was eventually rendered. The patient did not report signs of recurrence after a 3-year follow-up period.

Bone Marrow CX3CL1/Fractalkine is a New Player of the Pro-Angiogenic Microenvironment in Multiple Myeloma Patients.

C-X3-C motif chemokine ligand 1 (CX3CL1)/fractalkine is a chemokine released after cleavage by two metalloproteases, ADAM metallopeptidase domain 10 (ADAM10) and ADAM metallopeptidase domain 17 (ADAM17), involved in inflammation and angiogenesis in the cancer microenvironment. The role of the CX3CL1/ C-X3-C motif chemokine receptor 1(CX3CR1) axis in the multiple myeloma (MM) microenvironment is still unknown. Firstly, we analyzed bone marrow (BM) plasma levels of CX3CL1 in 111 patients with plasma cell disorders including 70 with active MM, 25 with smoldering myeloma (SMM), and 16 with monoclonal gammopathy of undetermined significance (MGUS). We found that BM CX3CL1 levels were significantly increased in MM patients compared to SMM and MGUS and correlated with BM microvessel density. Secondly, we explored the source of CX3CL1 in MM and BM microenvironment cells. Primary CD138⁺ cells did not express CXC3L1 but up-regulated its production by endothelial cells (ECs) through the involvement of tumor necrosis factor alpha (TNFα). Lastly, we demonstrated the presence of CX3CR1 on BM CD14⁺CD16⁺ monocytes of MM patients and on ECs, but not on MM cells. The role of CX3CL1 in MM-induced angiogenesis was finally demonstrated in both in vivo chick embryo chorioallantoic membrane and in vitro angiogenesis assays. Our data indicate that CX3CL1, present at a high level in the BM of MM patients, is a new player of the MM microenvironment involved in MM-induced angiogenesis.

Neurofibromatosis type I and multiple myeloma coexistence: A possible link?

The association between Neurofibromatosis type I (NF1) and multiple myeloma (MM), a plasma cell, dyscrasia is very rare. Here we put to the attention of the scientific community two new cases. The first one is a patient with active MM whereas the second with smoldering MM. Both patients present typical features of NF1 but skeletal alterations were present only in the second case including dysplasia, marked scoliosis and osteoporosis. MM osteolytic lesions were absent in both patients. In addition to the clinical diagnosis of NF1, a molecular testing for NF1 gene mutations has been performed finding that patient one was heterozygous for the c.6855C>A (Tyr2285Ter) mutation, while patient two was heterozygous for the c.7838dupC (Lys2614GlufsTer20) mutation. The two mutations were diagnosed both in genomic DNA from peripheral blood and from MM cells. The potential link between NF1 mutation and the increased risk of MM is discussed in the report.

A Rare Case of Systemic AL Amyloidosis with Muscle Involvement: A Misleading Diagnosis.

Muscle involvement in AL amyloidosis is a rare condition, and the diagnosis of amyloid myopathy is often delayed and underdiagnosed. Amyloid myopathy may be the initial manifestation and may precede the diagnosis of systemic AL amyloidosis. Here, we report the case of a 73-year-old man who was referred to our center for a monoclonal gammopathy of undetermined significance (MGUS) diagnosed since 1999. He reported a progressive weakness of proximal muscles of the legs with onset six months previously. Muscle biopsy showed mild histopathology featuring alterations of nonspecific type with a mixed myopathic and neurogenic involvement, and the diagnostic turning point was the demonstration of characteristic green birefringence under cross-polarized light following Congo red staining of perimysial vessels. Transmission electron microscopy (TEM) confirmed amyloid fibrils around perimysial vessels associated with collagen fibrils. A stepwise approach to diagnosis and staging of this disorder is critical and involves confirmation of amyloid deposition, identification of the fibril type, assessment of underlying amyloidogenic disorder, and evaluation of the extent and severity of amyloidotic organ involvement.

Expression of CD38 in myeloma bone niche: A rational basis for the use of anti-CD38 immunotherapy to inhibit osteoclast formation.

It is known that multiple myeloma (MM) cells express CD38 and that a recently developed human anti-CD38 monoclonal antibody Daratumumab mediates myeloma killing. However, the expression of CD38 and other functionally related ectoenzymes within the MM bone niche and the potential effects of Daratumumab on bone cells are still unknown. This study firstly defines by flow cytometry and immunohistochemistry the expression of CD38 by bone marrow cells in a cohort of patients with MM and indolent monoclonal gammopathies. Results indicate that only plasma cells expressed CD38 at high level within the bone niche. In addition, the flow cytometry analysis shows that CD38 was also expressed by monocytes and early osteoclast progenitors but not by osteoblasts and mature osteoclasts. Indeed, CD38 was lost during in vitro osteoclastogenesis. Consistently, we found that Daratumumab reacted with CD38 expressed on monocytes and its binding inhibited in vitro osteoclastogenesis and bone resorption activity from bone marrow total mononuclear cells of MM patients, targeting early osteoclast progenitors. The inhibitory effect was not observed from purified CD14+ cells, suggesting an indirect inhibitory effect of Daratumumab. Interestingly, all-trans retinoic acid treatment increased the inhibitory effect of Daratumumab on osteoclast formation. These observations provide a rationale for the use of an anti-CD38 antibody-based approach as treatment for multiple myeloma-induced osteoclastogenesis.

Cutaneous localization in multiple myeloma in the context of bortezomib-based treatment: how do myeloma cells escape from the bone marrow to the skin?

The skin is a possible site of extramedullary localization in multiple myeloma (MM) patients; however, the mechanisms involved in this process are poorly understood. We describe the case of a refractory MM patient who developed a cutaneous localization under bortezomib treatment and we further expanded observations in other eight MM patients. We focused on the expression of genes involved in plasma cell skin homing, including CCR10, which was highly expressed. Moreover, we observed a lack of CXCR4 surface expression and the down-regulation of ICAM1/CD54 throughout the progression of the disease, suggesting a possible mechanism driving the escape of MM cells from the bone marrow into the skin.

Dependence on glutamine uptake and glutamine addiction characterize myeloma cells: a new attractive target.

The importance of glutamine (Gln) metabolism in multiple myeloma (MM) cells and its potential role as a therapeutic target are still unknown, although it has been reported that human myeloma cell lines (HMCLs) are highly sensitive to Gln depletion. In this study, we found that both HMCLs and primary bone marrow (BM) CD138(+) cells produced large amounts of ammonium in the presence of Gln. MM patients have lower BM plasma Gln with higher ammonium and glutamate than patients with indolent monoclonal gammopathies. Interestingly, HMCLs expressed glutaminase (GLS1) and were sensitive to its inhibition, whereas they exhibited negligible expression of glutamine synthetase (GS). High GLS1 and low GS expression were also observed in primary CD138(+) cells. Gln-free incubation or treatment with the glutaminolytic enzyme l-asparaginase depleted the cell contents of Gln, glutamate, and the anaplerotic substrate 2-oxoglutarate, inhibiting MM cell growth. Consistent with the dependence of MM cells on extracellular Gln, a gene expression profile analysis, on both proprietary and published datasets, showed an increased expression of the Gln transporters SNAT1, ASCT2, and LAT1 by CD138(+) cells across the progression of monoclonal gammopathies. Among these transporters, only ASCT2 inhibition in HMCLs caused a marked decrease in Gln uptake and a significant fall in cell growth. Consistently, stable ASCT2 downregulation by a lentiviral approach inhibited HMCL growth in vitro and in a murine model. In conclusion, MM cells strictly depend on extracellular Gln and show features of Gln addiction. Therefore, the inhibition of Gln uptake is a new attractive therapeutic strategy for MM.

Concomitant KIT/BRAF and PDGFRA/BRAF mutations are rare events in gastrointestinal stromal tumors.

AIM: The BRAF mutation is a rare pathogenetic alternative to KIT/PDGFRA mutation in GIST and causes Imatinib resistance. A recent description of KIT and BRAF mutations co-occurring in an untreated GIST has challenged the concept of their being mutually exclusive and may account for ab initio resistance to Imatinib, even in the presence of Imatinib-sensitive KIT mutations. BRAF sequencing is generally limited to KIT/PDGFRA wild-type cases. Hence, the frequency of concomitant mutations may be underestimated. METHODS: We screened for KIT (exon 9, 11 ,13 ,17), PDGFRA (exon 12,14, 18) and BRAF (exon 15) mutations a series of 407 GIST. Additionally, we evaluated the BRAF V600E mutation-specific antibody, VE1, as a surrogate for V600E mutation, on a series of 313 GIST (24 on whole sections, 288 cases on tissue array), including 6 cases molecularly ascertained to carry the BRAF V600E mutation. RESULTS: No concomitant KIT/BRAF or PDGFRA/BRAF mutations were detected. BRAF mutation was detected only in one case, wild-type for KIT/PDGFRA. All the 6 BRAF-mutant cases stained positive with the VE1 antibody. A weak VE1 expression was observed in 14/287 (4.9%) BRAF wild-type cases, as observed also in 2/6 BRAF-mutant cases. Overall in our series, sensitivity and specificity of the VE1 antobody were 100% and 95.1%, respectively. CONCLUSIONS: The concomitance of BRAF mutation with either KIT or PDGFRA mutation is rare in GIST. In these tumors, moderate/strong VE1 immunoreactivity is a valuable surrogate for molecular analysis. Instead, genotyping is warranted in the presence of weak VE1 staining.

The Proteasome Inhibitor Bortezomib Maintains Osteocyte Viability in Multiple Myeloma Patients by Reducing Both Apoptosis and Autophagy: A New Function for Proteasome Inhibitors.

Multiple myeloma (MM) is characterized by severely imbalanced bone remodeling. In this study, we investigated the potential effect of proteasome inhibitors (PIs), a class of drugs known to stimulate bone formation, on the mechanisms involved in osteocyte death induced by MM cells. First, we performed a histological analysis of osteocyte viability on bone biopsies on a cohort of 37 MM patients with symptomatic disease. A significantly higher number of viable osteocytes was detected in patients treated with a bortezomib (BOR)-based regimen compared with those treated without BOR. Interestingly, both osteocyte autophagy and apoptosis were affected in vivo by BOR treatment. Thereafter, we checked the in vitro effect of BOR to understand the mechanisms whereby BOR maintains osteocyte viability in bone from MM patients. We found that osteocyte and preosteocyte autophagic death was triggered during coculturing with MM cells. Our evaluation was conducted by analyzing either autophagy markers microtubule-associated protein light chain 3 beta (LC3B) and SQSTM1/sequestome 1 (p62) levels, or the cell ultrastructure by transmission electron microscopy. PIs were found to increase the basal levels of LC3 expression in the osteocytes while blunting the myeloma-induced osteocyte death. PIs also reduced the autophagic death of osteocytes induced by high-dose dexamethasone (DEX) and potentiated the anabolic effect of PTH(1-34). Our data identify osteocyte autophagy as a new potential target in MM bone disease and support the use of PIs to maintain osteocyte viability and improve bone integrity in MM patients.

Unraveling the contribution of ectoenzymes to myeloma life and survival in the bone marrow niche.

The bone marrow provides a protected environment for generating a vast array of cell types. Bones are thus a dynamic source of structural components and soluble factors used either locally or at a distance from their site of production. We discuss the role of ectoenzymes in the bone niche where human myeloma grows. Selected ectoenzymes have been tested for their ability to promote production of substrates involved in signaling, synthesis of growth factors and hormones, and modulation of the immune response. Because of the difficulty of simultaneously tracking all these activities, we narrow our focus to events potentially influencing synthesis of adenosine (ADO), an important regulator of multiple biological functions, including local immunological tolerance. Our working hypothesis, to be discussed and partially tested herein, is that CD38, and likely BST1/CD157--both NAD(+) -consuming enzymes, are active in the myeloma niche and lead a discontinuous chain of ectoenzymes whose final products are exploited by the neoplastic plasma cell as part of its local survival strategy. Coadjuvant ectoenzymes include PC-1/CD203a, CD39, and CD73, which control the production of ADO. Results discussed here and from ongoing experiments indicate that the myeloma niche hosts the canonical, as well as alternative, pathways of ADO generation. Other possibilities are presented and discussed.