TMBcalc: a computational pipeline for identifying pan-cancer Tumor Mutational Burden gene signatures.

Background: In the precision medicine era, identifying predictive factors to select patients most likely to benefit from treatment with immunological agents is a crucial and open challenge in oncology. Methods: This paper presents a pan-cancer analysis of Tumor Mutational Burden (TMB). We developed a novel computational pipeline, TMBcalc, to calculate the TMB. Our methodology can identify small and reliable gene signatures to estimate TMB from custom targeted-sequencing panels. For this purpose, our pipeline has been trained on top of 17 cancer types data obtained from TCGA. Results: Our results show that TMB, computed through the identified signature, strongly correlates with TMB obtained from whole-exome sequencing (WES). Conclusion: We have rigorously analyzed the effectiveness of our methodology on top of several independent datasets. In particular we conducted a comprehensive testing on: (i) 126 samples sourced from the TCGA database; few independent whole-exome sequencing (WES) datasets linked to colon, breast, and liver cancers, all acquired from the EGA and the ICGC Data Portal. This rigorous evaluation clearly highlights the robustness and practicality of our approach, positioning it as a promising avenue for driving substantial progress within the realm of clinical practice.

Computational Approach for Spatially Fractionated Radiation Therapy (SFRT) and Immunological Response in Precision Radiation Therapy.

The field of precision radiation therapy has seen remarkable advancements in both experimental and computational methods. Recent literature has introduced various approaches such as Spatially Fractionated Radiation Therapy (SFRT). This unconventional treatment, demanding high-precision radiotherapy, has shown promising clinical outcomes. A comprehensive computational scheme for SFRT, extrapolated from a case report, is proposed. This framework exhibits exceptional flexibility, accommodating diverse initial conditions (shape, inhomogeneity, etc.) and enabling specific choices for sub-volume selection with administrated higher radiation doses. The approach integrates the standard linear quadratic model and, significantly, considers the activation of the immune system due to radiotherapy. This activation enhances the immune response in comparison to the untreated case. We delve into the distinct roles of the native immune system, immune activation by radiation, and post-radiotherapy immunotherapy, discussing their implications for either complete recovery or disease regrowth.

Unexpected Transient Glioblastoma Regression in a Patient Previously Treated with Bacillus Calmette-Guérin Therapy: A Case Report and Immunomodulatory Effects Hypothesis.

(1) Background: Glioblastoma multiforme (GBM) is a highly aggressive brain tumor with limited treatment options and poor prognosis. Bacillus Calmette-Guérin (BCG), a live attenuated strain of Mycobacterium bovis, has been used as an immunotherapeutic agent in bladder cancer and has shown non-specific beneficial effects. This report presents a unique case of GBM regression following BCG therapy for bladder cancer, suggesting the potential systemic immunomodulatory effects of BCG on GBM. (2) Case Presentation: A 67-year-old male with a history of bladder cancer treated with BCG presented with neurological symptoms. Imaging revealed two GBM lesions, and surgery was performed to remove one. Subsequently, the patient experienced complete tumor regression after initial stability. (3) Conclusions: This case highlights the potential of BCG or other immunotherapies in GBM treatment and underscores the need for further research. Understanding the immunomodulatory effects of BCG on GBM could lead to innovative therapies for this devastating disease; although, overcoming the immune evasion mechanisms in the brain is a significant challenge. Further investigation is warranted to explore this promising avenue of research.

Anaplastic thyroid cancer cells reduce CD71 levels to increase iron overload tolerance.

BACKGROUND: Follicular thyroid cancer (FTC) is a prevalent form of differentiated thyroid cancer, whereas anaplastic thyroid cancer (ATC) represents a rare, fast-growing, undifferentiated, and highly aggressive tumor, posing significant challenges for eradication. Ferroptosis, an iron-dependent cell death mechanism driven by the excessive production of reactive oxygen species and subsequent lipid peroxidation, emerges as a promising therapeutic strategy for cancer. It has been observed that many cancer cells exhibit sensitivity to ferroptosis, while some other histotypes appear to be resistant, by counteracting the metabolic changes and oxidative stress induced by iron overload. METHODS: Here we used human biopsies and in vitro approaches to analyse the effects of iron-dependent cell death. We assessed cell proliferation and viability through MTT turnover, clonogenic assays, and cytofluorimetric-assisted analysis. Lipid peroxidation assay and western blot were used to analyse molecular mechanisms underlying ferroptosis modulation. Two distinct thyroid cancer cell lines, FTC-133 (follicular) and 8505C (anaplastic), were utilized. These cell lines were exposed to ferroptosis inducers, Erastin and RSL3, while simulating an iron overload condition using ferric ammonium citrate. RESULTS: Our evidence suggests that FTC-133 cell line, exposed to iron overload, reduced their viability and showed increased ferroptosis. In contrast, the 8505C cell line seems to better tolerate ferroptosis, responding by modulating CD71, which is involved in iron internalization and seems to have a role in resistance to iron overload and consequently in maintaining cell viability. CONCLUSIONS: The differential tolerance to ferroptosis observed in our study may hold clinical implications, particularly in addressing the unmet therapeutic needs associated with ATC treatment, where resistance to ferroptosis appears more pronounced compared to FTC.

Horizontal Transfer of Malignant Traits and the Involvement of Extracellular Vesicles in Metastasis.

Metastases are responsible for the vast majority of cancer deaths, yet most therapeutic efforts have focused on targeting and interrupting tumor growth rather than impairing the metastatic process. Traditionally, cancer metastasis is attributed to the dissemination of neoplastic cells from the primary tumor to distant organs through blood and lymphatic circulation. A thorough understanding of the metastatic process is essential to develop new therapeutic strategies that improve cancer survival. Since Paget's original description of the "Seed and Soil" hypothesis over a hundred years ago, alternative theories and new players have been proposed. In particular, the role of extracellular vesicles (EVs) released by cancer cells and their uptake by neighboring cells or at distinct anatomical sites has been explored. Here, we will outline and discuss these alternative theories and emphasize the horizontal transfer of EV-associated biomolecules as a possibly major event leading to cell transformation and the induction of metastases. We will also highlight the recently discovered intracellular pathway used by EVs to deliver their cargoes into the nucleus of recipient cells, which is a potential target for novel anti-metastatic strategies.

Intrathoracic neurogenic tumors (ITNs): Management of solid and cystic lesions.

BACKGROUND: Intrathoracic neurogenic tumors (INTs) are derived from nerve tissue and grow within the chest. Preoperative diagnosis can be challenging and only complete surgical exeresis enables confirmation of the suspected diagnosis. Here, we analyzed our experience on management of paravertebral lesions with solid and cystic patterns. METHODS: A monocentric retrospective study was conducted, which included 25 consecutive cases of ITNs in the period from 2010 to 2022. These cases had been surgically treated by thoracoscopic resection alone, or in combination with neurosurgery in the case of dumbbell tumors. The demographic and operative data along with complications were recorded and analyzed. RESULTS: Twenty-five patients were diagnosed with a paravertebral lesion of which 19 (76%) had solid features and six (24%) had cystic features. The most common diagnosis was schwannoma (72%), followed by neurofibroma (20%) and malignant schwannoma (8%). In four cases (12%) the tumor showed an intraspinal extension. None of the patients had recurrence until 6 months of follow-up. Comparison between the VATS and thoracotomy procedures showed that outcome of discharge on the postoperative day, on average, was 2.61 ± 0.5 versus 3.51 ± 0.53, respectively (p-value <0.001). CONCLUSION: The treatment of choice for INTs is complete resection which is tailored to tumor size, location, and extension. In our study, paravertebral tumors with cystic characteristics were not associated with an intraspinal extension and did not show a different behavior from solid tumors.

Introducing Radiotherapy in Metastatic Merkel Cell Carcinoma Patients with Limited Progression on Avelumab: An Effective Step against Primary and Secondary Immune Resistance?

PURPOSE: To investigate the ability of radiotherapy (RT) to prolong progression-free survival (PFS) and to report treatment-related toxicities among oligoprogressive metastatic Merkel cell carcinoma (mMCC) patients on avelumab. METHODS: We retrospectively collected clinical data on mMCC patients who underwent radiotherapy for limited progression on avelumab. Patients were categorized as primary or secondary immune refractory depending on the time of onset of resistance to immunotherapy (at the first or subsequent follow-up visits after avelumab initiation). Pre- and post-RT PFS were calculated. Overall survival (OS) from the first progression treated with RT was also reported. Radiological responses and toxicities were evaluated according to the irRECIST criteria and RTOG scoring system, respectively. RESULTS: Eight patients, including five females, with a median age of 75 years, met our inclusion criteria. The median gross tumor and clinical target volumes at first progression on avelumab were 29.85 cc and 236.7 cc, respectively. The treatment sites included lymph node, skin, brain, and spine metastases. Four patients received more than one course of RT. Most patients were treated with palliative radiation doses (mainly 30 Gy in 3 Gy/day fractions). Two patients were treated with stereotactic RT. Five/eight patients were primary immune refractory. The objective response rate at the first post-RT assessment was 75%, whereas no local failure was reported. The median pre-RT PFS was 3 months. The pre-RT PFS was 37.5% at 6 months and 12.5% at 1 year. The median post-RT PFS was not reached. The post-RT PFS was 60% at 6 months and 1 year. The post-RT OS was 85.7% at 1 year and 64.3% at 2 years. No relevant treatment-related toxicity was observed. After a median follow-up of 18.5 months, 6/8 patients are still alive and continuing on avelumab therapy. CONCLUSIONS: Adding radiotherapy to mMCC patients with limited progression on avelumab seems to be safe and effective in prolonging the successful use of immunotherapy, regardless of the type of immune refractoriness.

Forecasting Individual Patients' Best Time for Surgery in Colon-Rectal Cancer by Tumor Regression during and after Neoadjuvant Radiochemotherapy.

The standard treatment of locally advanced rectal cancer is neoadjuvant chemoradiotherapy before surgery. For those patients experiencing a complete clinical response after the treatment, a watch-and-wait strategy with close monitoring may be practicable. In this respect, the identification of biomarkers of the response to therapy is extremely important. Many mathematical models have been developed or used to describe tumor growth, such as Gompertz's Law and the Logistic Law. Here we show that the parameters of those macroscopic growth laws, obtained by fitting the tumor evolution during and immediately after therapy, are a useful tool for evaluating the best time for surgery in this type of cancer. A limited number of experimental observations of the tumor volume regression, during and after the neoadjuvant doses, permits a reliable evaluation of a specific patient response (partial or complete recovery) for a later time, and one can evaluate a modification of the scheduled treatment, following a watch-and-wait approach or an early or late surgery. Neoadjuvant chemoradiotherapy effects can be quantitatively described by applying Gompertz's Law and the Logistic Law to estimate tumor growth by monitoring patients at regular intervals. We show a quantitative difference in macroscopic parameters between partial and complete response patients, reliable for estimating the treatment effects and best time for surgery.

Colorectal cancer-derived small extracellular vesicles induce TGFß1-mediated epithelial to mesenchymal transition of hepatocytes.

BACKGROUND: Metastatic disease is the major cause of cancer-related deaths. Increasing evidence shows that primary tumor cells can promote metastasis by preparing the local microenvironment of distant organs, inducing the formation of the so-called "pre-metastatic niche". In recent years, several studies have highlighted that among the tumor-derived molecular components active in pre-metastatic niche formation, small extracellular vesicles (sEVs) play a crucial role. Regarding liver metastasis, the ability of tumor-derived sEVs to affect the activities of non-parenchymal cells such as Kupffer cells and hepatic stellate cells is well described, while the effects on hepatocytes, the most conspicuous and functionally relevant hepatic cellular component, remain unknown. METHODS: sEVs isolated from SW480 and SW620 CRC cells and from clinical samples of CRC patients and healthy subjects were used to treat human healthy hepatocytes (THLE-2 cells). RT-qPCR, Western blot and confocal microscopy were applied to investigate the effects of this treatment. RESULTS: Our study shows for the first time that TGFß1-carrying CRC_sEVs impair the morphological and functional properties of healthy human hepatocytes by triggering their TGFß1/SMAD-dependent EMT. These abilities of CRC_sEVs were further confirmed by evaluating the effects elicited on hepatocytes by sEVs isolated from plasma and biopsies from CRC patients. CONCLUSIONS: Since it is known that EMT of hepatocytes leads to the formation of a fibrotic environment, a well-known driver of metastasis, these results suggest that CRC_sEV-educated hepatocytes could have an active and until now neglected role during liver metastasis formation.

One-lung ventilation in obese patients undergoing thoracoscopic lobectomy for lung cancer.

BACKGROUND: We evaluated the safety and feasibility of one-lung ventilation in obese patients undergoing thoracoscopic lobectomy and whether obesity affected peri- and postoperative outcomes. METHODS: This was a retrospective single center study including consecutive patients undergoing thoracoscopic lobectomy between October 2019 and February 2022. Obese patients were statistically compared to a control group to evaluate any differences in relation to one-lung ventilation and peri- and postoperative outcomes. RESULTS: Our study population included 111 patients; of these, 26 (23%) were included in the obese group, while 85 (77%) were included within the nonobese group. To obtain one-lung ventilation in nonobese patients, a double-lumen tube was more frequently used than a single-lumen tube with bronchial blocker (61% vs. 39%; p = 0.02), while in obese patients a single-lumen tube with bronchial blocker was used more than a double-lumen tube (81% vs. 19%, p = 0.001). Intergroup comparison showed that a double-lumen tube was the preferred method in nonobese patients, while a single-lumen tube with bronchial blockers was the strategy of choice in obese patients (p = 0.0002). Intubation time was longer in the obese group than in the nonobese group (94.0 ± 6.1 vs. 85.0 ± 7.0 s; p = 0.0004) and failure rate of first attempt at intubation was higher in the obese group (23% vs. 5%; p = 0.01). Obesity was not associated with increased intra-, peri- and postoperative complications and/or mortality. CONCLUSIONS: One-lung ventilation is a feasible and safe procedure also in obese patients and obesity did not negatively affect peri- and postoperative outcomes after lung resection.

Lactate modulates microglia polarization via IGFBP6 expression and remodels tumor microenvironment in glioblastoma.

Lactic acidosis has been reported in solid tumor microenvironment (TME) including glioblastoma (GBM). In TME, several signaling molecules, growth factors and metabolites have been identified to induce resistance to chemotherapy and to sustain immune escape. In the early phases of the disease, microglia infiltrates TME, contributing to tumorigenesis rather than counteracting its growth. Insulin-like Growth Factor Binding Protein 6 (IGFBP6) is expressed during tumor development, and it is involved in migration, immune-escape and inflammation, thus providing an attractive target for GBM therapy. Here, we aimed at investigating the crosstalk between lactate metabolism and IGFBP6 in TME and GBM progression. Our results show that microglia exposed to lactate or IGFBP6 significantly increased the Monocarboxylate transporter 1 (MCT1) expression together with genes involved in mitochondrial metabolism. We, also, observed an increase in the M2 markers and a reduction of inducible nitric oxide synthase (iNOS) levels, suggesting a role of lactate/IGFBP6 metabolism in immune-escape activation. GBM cells exposed to lactate also showed increased levels of IGFBP6 and vice-versa. Such a phenomenon was coupled with a IGFBP6-mediated sonic hedgehog (SHH) ignaling increase. We, finally, tested our hypothesis in a GBM zebrafish animal model, where we observed an increase in microglia cells and igfbp6 gene expression after lactate exposure. Our results were confirmed by the analysis of human transcriptomes datasets and immunohistochemical assay from human GBM biopsies, suggesting the existence of a lactate/IGFBP6 crosstalk in microglial cells, so that IGFBP6 expression is regulated by lactate production in GBM cells and in turn modulates microglia polarization.

Fraisinib: a calixpyrrole derivative reducing A549 cell-derived NSCLC tumor in vivo acts as a ligand of the glycine-tRNA synthase, a new molecular target in oncology.

Background and purpose: Lung cancer is the leading cause of death in both men and women, constituting a major public health problem worldwide. Non-small-cell lung cancer accounts for 85%-90% of all lung cancers. We propose a compound that successfully fights tumor growth in vivo by targeting the enzyme GARS1. Experimental approach: We present an in-depth investigation of the mechanism through which Fraisinib [meso-(p-acetamidophenyl)-calix(4)pyrrole] affects the human lung adenocarcinoma A549 cell line. In a xenografted model of non-small-cell lung cancer, Fraisinib was found to reduce tumor mass volume without affecting the vital parameters or body weight of mice. Through a computational approach, we uncovered that glycyl-tRNA synthetase is its molecular target. Differential proteomics analysis further confirmed that pathways regulated by Fraisinib are consistent with glycyl-tRNA synthetase inhibition. Key results: Fraisinib displays a strong anti-tumoral potential coupled with limited toxicity in mice. Glycyl-tRNA synthetase has been identified and validated as a protein target of this compound. By inhibiting GARS1, Fraisinib modulates different key biological processes involved in tumoral growth, aggressiveness, and invasiveness. Conclusion and implications: The overall results indicate that Fraisinib is a powerful inhibitor of non-small-cell lung cancer growth by exerting its action on the enzyme GARS1 while displaying marginal toxicity in animal models. Together with the proven ability of this compound to cross the blood-brain barrier, we can assess that Fraisinib can kill two birds with one stone: targeting the primary tumor and its metastases "in one shot." Taken together, we suggest that inhibiting GARS1 expression and/or GARS1 enzymatic activity may be innovative molecular targets for cancer treatment.

Combined minimally invasive para-esophageal hernia repair and lobectomy for lung cancer.

Preexisting para-esophageal hernia may increase the risk of postoperative complications after lobectomy for lung cancer. A combined laparoscopic hernia repair and thoracoscopic lobectomy may be performed to manage hernia and lung cancer concurrently. This approach possibly prevented complications from the presence of hernia after lobectomy.

Dynamical Synergy of Drug Combinations during Cancer Chemotherapy.

Synergistic drug combinations often provide effective strategies to increase treatment efficacy and, during therapy, it is a time-dependent process. Data for colorectal and lung cancer in vivo were used for the phenomenological study of dynamical synergy during treatments. The proposed approach takes into consideration tumor regrowth by macroscopic laws. The time dependencies of synergistic drug combinations are analyzed by different parametric indicators. The cumulative effects of the single therapy and drug combinations are quantitatively well described and related to the cumulative doses. In conclusion, the analysis of dynamical synergy during chemotherapy has to take into account the effects of the drug doses and the tumor regrowth, which can provide a reliable description of the synergistic time dependence.

Chitinase domain containing 1 increase is associated with low survival rate and M0 macrophages infiltrates in colorectal cancer patients.

Colorectal cancer (CRC) is one of the most common cancers in the world. Here, we undertook an analysis of microarray datasets consisting of colon biopsies of healthy subjects and of patients affected by CRC, in order to analyze the expression levels of Chitinase domain-containing protein 1 (CHID1) and to correlate them with the clinical data available in the datasets. Analysis of expression levels showed a significant increase of CHID1 in CRC biopsies compared to the mucosa of healthy subjects. Patients' stratification by TNM staging revealed significant increases in CHID1 expression levels as the disease progressed. Furthermore, we found that mutated BRAF patients exhibit higher levels of CHID1 expression. Patients with a poor surviving prognosis at 5 years expressed high levels of CHID1 compared to wild-type. The histochemical analysis carried out by the Human Protein Atlas web tool documented moderate to strong-intensity staining detection of CHID1 protein in CRC biopsies. Furthermore, CRC patients were selected and clustered into two groups, high and low CHID1 expression levels (HCEL and LCEL). We obtained two signatures, the genes significant positive (GSPC-CHID1) and negative (GSNC-CHID1) correlated to CHID1 expression levels. The genomic deconvolution analysis between the GSPC-CHID1, GSNC-CHID1, and 17 cell immunological signatures, highlighted the potential infiltration of Macrophages M0 in HCEL patients, and potential infiltration of Macrophages M1 cells in LCEL patients. In addition, the signature GSPC-CHID1 expressed unfavorable genes to the CRC patient's survival. Mirror results were obtained for the GSNC-CHID1 signature. From the outcome of our investigation, it is possible to conclude that HCEL are associated with an unfavorable prognosis for CRC patients.

Did conversion to thoracotomy during thoracoscopic lobectomy increase post-operative complications and prejudice survival? Results of best evidence topic analysis.

The potential complications related to unplanned conversion to thoracotomy remains a major concern in thoracoscopic lobectomy and may limit the wide adoption of this strategy. We reviewed the literature from 1990 until February 2022, analyzing all papers comparing successful thoracoscopic lobectomy versus converted thoracoscopic lobectomy and/or upfront thoracotomy lobectomy to establish whether unplanned conversion negatively affected outcomes. Thirteen studies provided the most applicable evidence to evaluate this issue. Conversion to thoracotomy was reported to occur in up to 23% of cases (range, 5%-16%). Vascular injury, calcified lymph nodes, and dense adhesions were the most common reasons for conversion. Converted thoracoscopic lobectomy compared to successful thoracoscopic lobectomy was associated with longer operative time and hospital stay in all studies, with higher postoperative complication rates in seven studies, and with higher perioperative mortality rates in four studies. No significant differences were found between converted thoracoscopic lobectomy and upfront thoracotomy lobectomy. Five studies evaluated long-term survival, and in all papers conversion did not prejudice survival. Surgeons should not fear unplanned conversion during thoracoscopic lobectomy, but to avoid unexpected conversion that may negatively impact surgical outcome, a careful selection of patients is recommended-especially for frail patients.

Ex Vivo Irradiation of Lung Cancer Stem Cells Identifies the Lowest Therapeutic Dose Needed for Tumor Growth Arrest and Mass Reduction In Vivo.

Radiotherapy represents a first-line treatment for many inoperable lung tumors. New technologies offer novel opportunities for the treatment of lung cancer with the administration of higher doses of radiation in smaller volumes. Because both therapeutic and toxic treatment effects are dose-dependent, it is important to identify a minimal dose protocol for each individual patient that maintains efficacy while decreasing toxicity. Cancer stem cells sustain tumor growth, promote metastatic dissemination, and may give rise to secondary resistance. The identification of effective protocols targeting these cells may improve disease-free survival of treated patients. In this work, we evaluated the existence of individual profiles of sensitivity to radiotherapy in patient-derived cancer stem cells (CSCs) using both in vitro and in vivo models. Both CSCs in vitro and mice implanted with CSCs were treated with radiotherapy at different dose intensities and rates. CSC response to different radiation doses greatly varied among patients. In vitro radiation sensitivity of CSCs corresponded to the therapeutic outcome in the corresponding mouse tumor model. On the other side, the dose administration rate did not affect the response. These findings suggest that in vitro evaluation of CSCs may potentially predict patients' response, thus guiding clinical decision.

Advanced or Metastatic Cutaneous Squamous Cell Carcinoma: The Current and Future Role of Radiation Therapy in the Era of Immunotherapy.

Radiation therapy (RT) is an effective therapeutic option for small localized cutaneous squamous cell carcinoma (cSCC) among patients who are not eligible for or refuse surgery. RT also has a defined role as an adjuvant treatment in cases of adverse features that predispose to tumor recurrence after local excision. Since the development of cSCC is often a late consequence of chronic sun exposure, its occurrence is more common among elderly patients whose comorbidities may contraindicate surgical procedures. These could be impeded not only by frail medical conditions but also by technical issues. Indeed, an aggressive locoregional behavior of cSCC may culminate in unresectability due to widespread invasion of neighboring tissues. Moreover, cSCC could develop distant metastases. Both locally advanced and metastatic cSCCs carry a poor prognosis. In these scenarios, recent discoveries of tumor molecular targets are promoting the use of promising systemic therapies, especially immunotherapy, over RT. However, the results from using immunotherapy and, even more so, of chemotherapy are still not optimal. By contrast, advances in radiation delivery equipment can safely treat even large and complex-shaped cSCC targets in challenging body sites. In addition, RT could also have a role in metastatic cSCC settings by enhancing the effectiveness of concomitant immunotherapy. The aim of this review is to summarize and comment on the body of literature about the use of radiotherapy for operable and inoperable locally advanced cSCCs and for metastatic ones in an attempt to define its current and future role.

Tumor Volume Regression during and after Radiochemotherapy: A Macroscopic Description.

Tumor volume regression during and after chemo and radio therapy is a useful information for clinical decisions. Indeed, a quantitative, patient oriented, description of the response to treatment can guide towards the modification of the scheduled doses or the evaluation of the best time for surgery. We propose a macroscopic algorithm which permits to follow quantitatively the time evolution of the tumor volume during and after radiochemotherapy. The method, initially validated with different cell-lines implanted in mice, is then successfully applied to the available data for partially responding and complete recovery patients.