Multistep tumor genetic evolution and changes in immunogenicity trigger immune-mediated disease eradication in stage IV melanoma: lessons from a single case.

Durable remissions are observed in 10%-20% of treated patients with advanced metastatic melanoma but the factors associated with long-term complete clinical responses are largely unknown. Here, we report the molecular characteristics of tumor evolution during disease progression along a 9-year clinical course in a patient with advanced disseminated melanoma who received different treatments, including trametinib, ipilimumab, radiation, vemurafenib, surgical tumor debulking and a second ipilimumab course, ultimately achieving complete long-term disease remission.Longitudinal analyses of therapies-resistant metastatic tumors revealed the effects of different treatments on tumor's microenvironment and immunogenicity, ultimately creating a milieu favorable to immunotherapy response. Monitoring of the temporal dynamics of T cells by analysis of the T cell receptor (TCR) repertoire in the tumor and peripheral blood during disease evolution indicated that T-cell clones with common TCR rearrangements, present at low levels at baseline, were maintained and expanded after immunotherapy, and that TCR diversity increased. Analysis of genetic, molecular, and cellular components of the tumor depicted a multistep process in which treatment with kinase inhibitors strongly conditioned the immune microenvironment creating an inflamed milieu converting cold into hot tumors, while ipilimumab impacted and increased the TCR repertoire, a requirement for tumor rejection.Since the optimal sequencing of treatment with antibodies targeting immune checkpoints and kinase inhibitors for advanced melanoma is still clinically debated, this case indicates that immunotherapy success is possible even after progression on targeted therapy.

Genetic Layout of Melanoma Lesions Is Associated with BRAF/MEK-Targeted Therapy Resistance and Transcriptional Profiles.

The genetic landscape of melanoma resistance to targeted therapy with small molecules inhibiting BRAF and MEK kinases is still largely undefined. In this study, we portrayed in detail the somatic alterations of resistant melanoma and explored the associated biological processes and their integration with transcriptional profiles. By targeted next-generation sequencing and whole-exome sequencing analyses, a list of 101 genes showing imbalance in metastatic tumors from patients with a complete/durable response or disease progression during therapy with vemurafenib or with dabrafenib and trametinib was defined. Classification of altered genes in functional categories indicated that the mutational pattern of both resistant tumors and melanoma cell lines was enriched in gene families involved in oncogenic signaling pathways and in DNA repair. Integration of genomic and transcriptomic features showed that the enrichment of mutations in gene sets associated with anabolic processes, chromatin alterations, and IFN-α response determined a significant positive modulation of the same gene signatures at the transcriptional level. In particular, MTORC1 signaling was enriched in tumors from poorly responsive patients and in resistant tumors excised from treated patients. Results indicate that genetic patterns are associated with melanoma resistance to targeted therapy and disclose the underlying key molecular pathways to define drug combinations for improved personalized therapies.

3D tumor explant as a novel platform to investigate therapeutic pathways and predictive biomarkers in cancer patients.

Immunotherapy with immune checkpoint inhibitors can induce durable clinical responses in different human malignancies but the number of responding patients remains globally modest. The limited therapeutic efficacy of ICI depends on multiple factors, among which the immune suppressive features of the tumor microenvironment play a key role. For this reason, experimental models that enable dissection of the immune-hostile tumor milieu components are required to unravel how to overcome resistance and obtain full-fledged anti-tumor immunity. Recent evidence supports the usefulness of 3D ex vivo systems in retaining features of tumor microenvironment to elucidate molecular and immunologic mechanisms of response and resistance to immune checkpoint blockade. In this perspective article we discuss the recent advances in patient-derived 3D tumor models and their potential in support of treatment decision making in clinical setting. We will also share our experience with dynamic bioreactor tumor explant culture of samples from melanoma and sarcoma patients as a reliable and promising platform to unravel immune responses to immune checkpoint inhibitors.

Genetic Variants and Somatic Alterations Associated with MITF-E318K Germline Mutation in Melanoma Patients.

The MITF-E318K variant has been implicated in genetic predisposition to cutaneous melanoma. We addressed the occurrence of MITF-E318K and its association with germline status of CDKN2A and MC1R genes in a hospital-based series of 248 melanoma patients including cohorts of multiple, familial, pediatric, sporadic and melanoma associated with other tumors. Seven MITF-E318K carriers were identified, spanning every group except the pediatric patients. Three carriers showed mutated CDKN2A, five displayed MC1R variants, while the sporadic carrier revealed no variants. Germline/tumor whole exome sequencing for this carrier revealed germline variants of unknown significance in ATM and FANCI genes and, in four BRAF-V600E metastases, somatic loss of the MITF wild-type allele, amplification of MITF-E318K and deletion of a 9p21.3 chromosomal region including CDKN2A and MTAP. In silico analysis of tumors from MITF-E318K melanoma carriers in the TCGA Pan-Cancer-Atlas dataset confirmed the association with BRAF mutation and 9p21.3 deletion revealing a common genetic pattern. MTAP was the gene deleted at homozygous level in the highest number of patients. These results support the utility of both germline and tumor genome analysis to define tumor groups providing enhanced information for clinical strategies and highlight the importance of melanoma prevention programs for MITF-E318K patients.

Liquid Biopsy and Radiological Response Predict Outcomes Following Discontinuation of Targeted Therapy in Patients with BRAF Mutated Melanoma.

BACKGROUND: Outcomes of patients with metastatic melanoma discontinuing BRAF-targeted therapy for cumulative toxicity after sustained response are unknown. MATERIALS AND METHODS: This retrospective case series analysis conducted at a single Cancer Center in Italy included patients with BRAF mutated metastatic melanoma treated with a BRAF inhibitor as a single agent or in combination with a MEK inhibitor between June 1, 2011 and January 1, 2020 and interrupted treatment due to cumulative toxicity after achieving complete response (CR) or long-lasting partial response (PR; i.e. >12 months). RESULTS: We included 24 patients with a median treatment duration of 59.4 months (95% confidence interval [CI], 55.4-63.4; range, 12-88). CR and PR were achieved in 71% and 29% of patients, respectively. At a median follow-up after treatment discontinuation of 37.8 months (95% CI, 33.7-41.9), the 12-month progression-free survival after discontinuation (dPFS) rate was 70.8% (95% CI 54.8-91.6) and 24-month dPFS rate was 58.3% (95% CI, 41.6-81.8). Baseline patient and tumor characteristics as well as treatment duration and best response did not significantly impact on dPFS. Patients with CR and negative circulating tumor DNA (ctDNA) at time of discontinuation had a significantly improved dPFS compared with patients with either radiological residual disease or ctDNA positivity (p = .007). No patient in CR with undetectable ctDNA experienced progression. CONCLUSION: The risk of progression is high even in patients with sustained sensitivity to BRAF/MEK inhibitors. Integration of liquid biopsy in clinical trials investigating the optimal management of patients with sustained sensitivity to BRAF/MEK inhibitors is warranted. IMPLICATIONS FOR PRACTICE: Outcomes of patients with metastatic melanoma discontinuing BRAF-targeted therapy for cumulative toxicity are unknown. This study analyzed patients with sustained responses (median treatment duration 59.4 months). Twelve- and 24-month progression-free survival following discontinuation were 70.8% and 58.3%, respectively. Complete response and negative circulating tumor DNA at time of discontinuation are promising prognostic biomarkers in this setting.

miR-146a-5p impairs melanoma resistance to kinase inhibitors by targeting COX2 and regulating NFkB-mediated inflammatory mediators.

BACKGROUND: Targeted therapy with BRAF and MEK inhibitors has improved the survival of patients with BRAF-mutated metastatic melanoma, but most patients relapse upon the onset of drug resistance induced by mechanisms including genetic and epigenetic events. Among the epigenetic alterations, microRNA perturbation is associated with the development of kinase inhibitor resistance. Here, we identified and studied the role of miR-146a-5p dysregulation in melanoma drug resistance. METHODS: The miR-146a-5p-regulated NFkB signaling network was identified in drug-resistant cell lines and melanoma tumor samples by expression profiling and knock-in and knock-out studies. A bioinformatic data analysis identified COX2 as a central gene regulated by miR-146a-5p and NFkB. The effects of miR-146a-5p/COX2 manipulation were studied in vitro in cell lines and with 3D cultures of treatment-resistant tumor explants from patients progressing during therapy. RESULTS: miR-146a-5p expression was inversely correlated with drug sensitivity and COX2 expression and was reduced in BRAF and MEK inhibitor-resistant melanoma cells and tissues. Forced miR-146a-5p expression reduced COX2 activity and significantly increased drug sensitivity by hampering prosurvival NFkB signaling, leading to reduced proliferation and enhanced apoptosis. Similar effects were obtained by inhibiting COX2 by celecoxib, a clinically approved COX2 inhibitor. CONCLUSIONS: Deregulation of the miR-146a-5p/COX2 axis occurs in the development of melanoma resistance to targeted drugs in melanoma patients. This finding reveals novel targets for more effective combination treatment. Video Abstract.

A proposal of an updated classification for pelvic relapses of rectal cancer to guide surgical decision-making.

BACKGROUND AND OBJECTIVES: Selection of patients affected by pelvic recurrence of rectal cancer (PRRC) who are likely to achieve a R0 resection is mandatory. The aim of this study was to propose a classification for PRRC to predict both radical surgery and disease-free survival (DFS). METHODS: PRRC patients treated at the National Cancer Institute of Milan (Italy) were included in the study. PRRC were classified as S1, if located centrally (S1a-S1b) or anteriorly (S1c) within the pelvis; S2, in case of sacral involvement below (S2a) or above (S2b) the second sacral vertebra; S3, in case of lateral pelvic involvement. RESULTS: Of 280 reviewed PRRC patients, 152 (54.3%) were evaluated for curative surgery. The strongest predictor of R+ resection was the S3 category (OR, 6.37; P = .011). Abdominosacral resection (P = .012), anterior exenteration (P = .012) and extended rectal re-excision (P = .003) were predictive of R0 resection. S3 category was highly predictive of poor DFS (HR 2.53; P = .038). DFS was significantly improved after R0 surgery for S1 (P < .0001) and S2 (P = .015) patients but not for S3 cases (P = .525). CONCLUSIONS: The proposed classification allows selection of subjects candidates to curative surgery, emphasizing that lateral pelvic involvement is the main predictor of R+ resection and independently affects the DFS.

Rectus Abdominis Myofascial Flap for Vaginal Reconstruction After Pelvic Exenteration.

BACKGROUND: Several techniques for vaginal reconstruction after pelvic exenteration such as myocutaneous and myoperitoneal flaps are available. However, the use of a myofascial flap has not been previously described. Thus, the objective of this article is to present our experience of vaginal reconstruction with rectus abdominis myofascial (RAMF) flap. METHODS: Between May 2008 and March 2017, 16 patients underwent anterior, posterior, or total pelvic exenteration with RAMF flap vaginal reconstruction. Patient records were systematically reviewed; demographic, clinic and pathologic, operative details, flap-related and non-flap-related complications, and risk factors for wound healing are reported. Quality of life and sexual function were also investigated. RESULTS: Eleven (68.8%) of 16 patients died during the follow-up (29.1 ± 25 months), whereas 5 (31.3%) are still alive. Early complications were reported in 7 patients (43.8%), with 2 (12.5%) flap-related and 5 (31.3%) non-flap-related complications. Similarly, late complications were reported in 5 patients (31.3%), with 2 (12.5%) flap-related and 3 (18.8%) non-flap-related complications. Quality of life measured by SF-36 (Survey Short Form 36) significantly improved at 12-month follow-up in comparison with baseline (physical component summary 31.5 ± 4.8 vs 26.8 ± 2.9; P = 0.027; mental component summary 29.5 ± 6.0 vs 25.9 ± 2.0; P = 0.042). CONCLUSIONS: This study demonstrates for the first time that RAMF flap vaginal reconstruction after pelvic exenteration is an efficacious and safe technique. Furthermore, it is associated with a significant improvement of quality of life and sexual function in those women who had sexual intercourse before surgery.

Electrochemotherapy: a good idea in recurrent basal cell carcinoma treatment.

Basal cell carcinoma (BCC) is the most common skin cancer in Caucasian population. Surgical excision is the gold standard treatment for BCC. Secondary surgical approach in patients presenting surgical scars after multiple surgical excisions or submitted to radiotherapy without obtaining a safe clearance margin, should be avoided. In recent years the use of electrochemotherapy (ECT) in head and neck cancers using intravenous bleomycin was specifically addressed and BCC resulted to be among tumors with the best response to ECT. We report a case of a recurrent nasal BCC treated with ECT with a complete regression of lesions after 8 weeks and a disease free period of 7 months. ECT is a good palliative treatment not affecting survival rate but improving quality of life in patients.

miRNA profiling in colorectal cancer highlights miR-1 involvement in MET-dependent proliferation.

Altered expression of miRNAs is associated with development and progression of various human cancers by regulating the translation of oncogenes and tumor suppressor genes. In colorectal cancer, these regulators complement the Vogelstein multistep model of pathogenesis and have the potential of becoming a novel class of tumor biomarkers and therapeutic targets. Using quantitative real-time PCR, we measured the expression of 621 mature miRNAs in 40 colorectal cancers and their paired normal tissues and identified 23 significantly deregulated miRNAs. We subsequently evaluated their association with clinical characteristics of the samples and presence of alterations in the molecular markers of colorectal cancer progression. Expression levels of miR-31 were correlated with CA19-9 and miR-18a, miR-21, and miR-31 were associated with mutations in APC gene. To investigate the downstream regulation of the differentially expressed miRNAs identified, we integrated putative mRNA target predictions with the results of a meta-analysis of seven public gene expression datasets of normal and tumor samples of colorectal cancer patients. Many of the colorectal cancer deregulated miRNAs computationally mapped to targets involved in pathways related to progression. Here one promising candidate pair (miR-1 and MET) was studied and functionally validated. We show that miR-1 can have a tumor suppressor function in colorectal cancer by directly downregulating MET oncogene both at RNA and protein level and that reexpression of miR-1 leads to MET-driven reduction of cell proliferation and motility, identifying the miR-1 downmodulation as one of the events that could enhance colorectal cancer progression.

Coffee, decaffeinated coffee, tea, and pancreatic cancer risk: a pooled-analysis of two Italian case-control studies.

To evaluate the association between coffee, decaffeinated coffee, and tea consumption and pancreatic cancer risk in a pooled analysis of two Italian case-control studies, between 1983 and 2008, we conducted two case-control studies in Northern Italy, including a total of 688 pancreatic cancer cases and 2204 hospital controls with acute, non-neoplastic diseases. We computed multivariate odds ratios (ORs) and 95% confidence intervals (CIs) for coffee drinking (mostly espresso and mocha), adjusting for age, sex, center, year of interview, education, body mass index, tobacco smoking, alcohol drinking, and diabetes. Compared with coffee nondrinkers, the multivariate OR for coffee drinkers was 1.34 (95% CI: 1.01-1.77). However, there was no trend in risk with respect to dose and duration. The OR for an increment of one cup per day was 1.05 (95% CI: 0.98-1.11). There was no heterogeneity in strata of age, sex, and other covariates, including tobacco smoking. No association emerged for decaffeinated coffee (for drinkers the OR was 0.87, 95% CI: 0.60-1.26, compared with decaffeinated coffee nondrinkers) or tea (for tea drinkers the OR was 0.92, 95% CI: 0.75-1.14). The lack of relationship with dose and duration weighs against a causal association between coffee and pancreatic cancer, which is in agreement with most evidence on the issue.

Distal clearance margin of 1 cm or less: a safe distance in lower rectum cancer surgery.

PURPOSE: The purpose of this study was to investigate the prognostic role of distal clearance margin (DCM) in lower rectum cancer surgery. MATERIALS AND METHODS: Two-hundred-three cancer patients underwent total rectal resection, possibly followed by adjuvant chemoradiotherapy. DCM was classified as positive or negative (<1, > or =1 cm) and investigated with multivariable proportional hazard models. RESULTS: A total of 52 deaths, 19 local relapses, 40 distant metastases, and three second primaries were observed as first events. Five-year survival with positive, negative <1, or negative > or =1 cm DCM was 51%, 81%, and 69%, respectively (p = 0.018). The difference was significant between positive and negative DCM (p = 0.031), not between negative <1 and > or =1 cm (p = 0.106). Local and distant 5-year incidences according to DCM were 30%, 8%, and 8% (p = 0.006) and 38%, 26%, and 19% (p = 0.857), respectively. CONCLUSIONS: DCM, but not tumor size, is a prognostic factor after sphincter-saving surgery, which is safe whenever a negative margin is achieved.

Simultaneous idiopathic segmental infarction of the great omentum and acute appendicitis: a rare association.

Idiopathic segmental infarction of the greater omentum is an uncommon cause of acute abdomen. The etiology is still unclear and the symptoms mimic acute appendicitis. Its presentation simultaneously with acute appendicitis is still more infrequent. We present a case of a 47-year old woman without significant previous medical history, admitted with an acute abdomen, in which the clinical diagnosis was acute appendicitis and in whom an infarcted segment of right side of the greater omentum was also found at laparotomy. As the etiology is unknown, we highlighted some of the possible theories, and emphasize the importance of omental infarction even in the presence of acute appendicitis as a coincident intraperitoneal pathological condition.

The art of decision-making in surgery. To what extent does economics influence choice?

Traditionally most surgeons have paid little attention to the costs of healthcare treatments. With the increase in the number of efficacious surgical alternatives, a distinct scarcity of available resources has emerged. Since the Eighties, the impact of surgical expenditure has been increasing everywhere. The causes are: medical progress, increased life expectancy, escalating costs and decreasing revenues. The surgeon has been increasingly forced to weigh up theories, doctrines and techniques of economics and management. This created new problems of choice. In any event, the surgeon's decision-making could lead to negative consequences if the primary concern is with the financial constraints and he is prompted simply to act rather than to achieve his therapeutic goal. In conclusion, although the impact of economic considerations is inevitable in the choice of surgery, the terms and methods involved in the process are rather ambiguous. In other words, surgeons face with the dilemma of the patient to whom the economist denies treatment. To be aware of this issue is the first step, but there is still much more to do in order to define the terms of action.

Quantitative analysis of plasma DNA in colorectal cancer patients: a novel prognostic tool.

Extracellular DNA in the plasma or serum of cancer patients has been recently proposed as a source of analyzable cancer-related gene sequences (qualitative approach). Furthermore, patients with different tumor types show high levels of cell-free circulating DNA both in plasma and serum (quantitative approach) at the time of surgery. Our aim was to verify whether the level of cell-free DNA in plasma might help in detecting recurrences during follow-up of colorectal cancer (CRC) patients. We studied 70 patients undergoing surgery for primary CRC. Plasma samples were obtained at the time of surgery and during follow-up. The cell-free circulating DNA in plasma was quantified by the Dipstick Kit method. At the time of surgery, in all patients, cell-free DNA levels in plasma were about 25 times higher in comparison with 20 healthy donors. In contrast, the carcinoembryonic antigen (CEA) value of this cohort of patients was altered in only about 37% of cases. During follow-up, cell-free DNA levels decreased progressively in tumor-free patients, while it increased in those developing recurrences or metastases. The results were further supported by qualitative analysis of circulating tumor-specific DNA, such as K-Ras mutations and p16(INK4a) promoter hypermethylation. These preliminary data confirm that plasma tumor DNA levels (i) are significantly higher in patients with CRC, (ii) decrease progressively in the follow-up period in tumor-free patients, and (iii) increase in patients with recurrence or metastasis. We suggest, therefore, that the quantification of plasma cell-free DNA might represent a useful tool for monitoring of CRC and, prospectively, for identifying high-risk individuals.

Reconstitution of human telomerase reverse transcriptase expression rescues colorectal carcinoma cells from in vitro senescence: evidence against immortality as a constitutive trait of tumor cells.

Although in vitro establishment of new colorectal carcinoma (CRC) cell lines is an infrequent event, we have observed that primary cultures of CRC can be repeatedly and reproducibly initiated following in vitro plating of tumor-derived epithelial cells. These cultures, however, usually display a short life span as they undergo a limited number of cell passages before entering a state of irreversible growth arrest. In this study, we show that short-lived CRC primary cultures lack constitutive telomerase activity and undergo a senescence process characterized by progressive telomere shortening. Moreover, transduction of these cells with a retroviral vector encoding human telomerase reverse transcriptase (hTERT) is sufficient to reconstitute telomerase activity and allow immortalization. Detailed molecular characterization of hTERT-immortalized CRC cell lines confirms their individual tumor origin by showing expression of colonic epithelial differentiation markers, such as cytokeratin-20 (CK20), full match with class I and class II human leukocyte antigen genotyping of autologous B-lymphoblastoid cells, and presence of somatic mutations in key cancer genes (KRAS2, APC) identical to those of the corresponding autologous original tumor tissues. Moreover, functional characterization of hTERT-immortalized CRC cell lines shows that they have a transformed phenotype, being able to form colonies in soft agar and tumors in severe combined immunodeficient mice. Most interestingly, immunohistochemical analysis of original tumor tissues indicates that short-lived CRC primary cultures, although hTERT-negative in vitro, derive from hTERT-positive tumors. Taken together, our data show that, in a least subset of CRC, biochemical pathways involved in maintenance of telomere length, such as telomerase, are not activated in a constitutive way in all tumor cells.

Sphincter-saving surgery for low rectal cancer. The experience of the National Cancer Institute, Milano.

The treatment of tumors of the distal rectum continues to be a matter of great controversy among oncologic surgeons. There are increasingly promising indications that functionally conservative surgery may be a valid therapeutic alternative to conventional therapy in patients with tumours of the lower rectum, traditionally treated by abdomino-perineal resection and definitive colostomy. Many points are presently under evaluation and we want to discuss some of the most relevant topics that are now permitting to change the guide lines of therapy of this disease. Our view of the problem is based on a personal experience cumulated in fourteen years of activity in a specialized unit and this paper reports the main results of a complex and diversified study carried out during this period at the National Cancer Institute of Milan.

Human tumor-derived heat shock protein 96 mediates in vitro activation and in vivo expansion of melanoma- and colon carcinoma-specific T cells.

Heat shock proteins (hsp) 96 play an essential role in protein metabolism and exert stimulatory activities on innate and adaptive immunity. Vaccination with tumor-derived hsp96 induces CD8(+) T cell-mediated tumor regressions in different animal models. In this study, we show that hsp96 purified from human melanoma or colon carcinoma activate tumor- and Ag-specific T cells in vitro and expand them in vivo. HLA-A*0201-restricted CD8(+) T cells recognizing Ags expressed in human melanoma (melanoma Ag recognized by T cell-1 (MART-1)/melanoma Ag A (Melan-A)) or colon carcinoma (carcinoembryonic Ag (CEA)/epithelial cell adhesion molecule (EpCAM)) were triggered to release IFN-gamma and to mediate cytotoxic activity by HLA-A*0201-matched APCs pulsed with hsp96 purified from tumor cells expressing the relevant Ag. Such activation occurred in class I HLA-restricted fashion and appeared to be significantly higher than that achieved by direct peptide loading. Immunization with autologous tumor-derived hsp96 induced a significant increase in the recognition of MART-1/Melan-A(27-35) in three of five HLA-A*0201 melanoma patients, and of CEA(571-579) and EpCAM(263-271) in two of five HLA-A*0201 colon carcinoma patients, respectively, as detected by ELISPOT and HLA/tetramer staining. These increments in Ag-specific T cell responses were associated with a favorable disease course after hsp96 vaccination. Altogether, these data provide evidence that hsp96 derived from human tumors can present antigenic peptides to CD8(+) T cells and activate them both in vitro and in vivo, thus representing an important tool for vaccination in cancer patients.