Cell-in-cell phenomena across the tree of life.

Cells in obligately multicellular organisms by definition have aligned fitness interests, minimum conflict, and cannot reproduce independently. However, some cells eat other cells within the same body, sometimes called cell cannibalism. Such cell-in-cell events have not been thoroughly discussed in the framework of major transitions to multicellularity. We performed a systematic screening of 508 articles, from which we chose 115 relevant articles in a search for cell-in-cell events across the tree of life, the age of cell-in-cell-related genes, and whether cell-in-cell events are associated with normal multicellular development or cancer. Cell-in-cell events are found across the tree of life, from some unicellular to many multicellular organisms, including non-neoplastic and neoplastic tissue. Additionally, out of the 38 cell-in-cell-related genes found in the literature, 14 genes were over 2.2 billion years old, i.e., older than the common ancestor of some facultatively multicellular taxa. All of this suggests that cell-in-cell events may have originated before the origins of obligate multicellularity. Thus, our results show that cell-in-cell events exist in obligate multicellular organisms, but are not a defining feature of them. The idea of eradicating cell-in-cell events from obligate multicellular organisms as a way of treating cancer, without considering that cell-in-cell events are also part of normal development, should be abandoned.

Epigenome and early selection determine the tumour-immune evolutionary trajectory of colorectal cancer.

Immune system control is a major hurdle that cancer evolution must circumvent. The relative timing and evolutionary dynamics of subclones that have escaped immune control remain incompletely characterized, and how immune-mediated selection shapes the epigenome has received little attention. Here, we infer the genome- and epigenome-driven evolutionary dynamics of tumour-immune coevolution within primary colorectal cancers (CRCs). We utilise our existing CRC multi-region multi-omic dataset that we supplement with high-resolution spatially-resolved neoantigen sequencing data and highly multiplexed imaging of the tumour microenvironment (TME). Analysis of somatic chromatin accessibility alterations (SCAAs) reveals frequent somatic loss of accessibility at antigen presenting genes, and that SCAAs contribute to silencing of neoantigens. We observe that strong immune escape and exclusion occur at the outset of CRC formation, and that within tumours, including at the microscopic level of individual tumour glands, additional immune escape alterations have negligible consequences for the immunophenotype of cancer cells. Further minor immuno-editing occurs during local invasion and is associated with TME reorganisation, but that evolutionary bottleneck is relatively weak. Collectively, we show that immune evasion in CRC follows a "Big Bang" evolutionary pattern, whereby genetic, epigenetic and TME-driven immune evasion acquired by the time of transformation defines subsequent cancer-immune evolution.

Testing Adaptive Therapy Protocols Using Gemcitabine and Capecitabine in a Preclinical Model of Endocrine-Resistant Breast Cancer.

Adaptive therapy, an ecologically inspired approach to cancer treatment, aims to overcome resistance and reduce toxicity by leveraging competitive interactions between drug-sensitive and drug-resistant subclones, prioritizing patient survival and quality of life instead of killing the maximum number of cancer cells. In preparation for a clinical trial, we used endocrine-resistant MCF7 breast cancer to stimulate second-line therapy and tested adaptive therapy using capecitabine, gemcitabine, or their combination in a mouse xenograft model. Dose modulation adaptive therapy with capecitabine alone increased survival time relative to MTD but not statistically significantly (HR = 0.22, 95% CI = 0.043-1.1, p = 0.065). However, when we alternated the drugs in both dose modulation (HR = 0.11, 95% CI = 0.024-0.55, p = 0.007) and intermittent adaptive therapies, the survival time was significantly increased compared to high-dose combination therapy (HR = 0.07, 95% CI = 0.013-0.42, p = 0.003). Overall, the survival time increased with reduced dose for both single drugs (p < 0.01) and combined drugs (p < 0.001), resulting in tumors with fewer proliferation cells (p = 0.0026) and more apoptotic cells (p = 0.045) compared to high-dose therapy. Adaptive therapy favors slower-growing tumors and shows promise in two-drug alternating regimens instead of being combined.

Testing Adaptive Therapy Protocols using Gemcitabine and Capecitabine on a Mouse Model of Endocrine-Resistant Breast Cancer.

Highly effective cancer therapies often face limitations due to acquired resistance and toxicity. Adaptive therapy, an ecologically inspired approach, seeks to control therapeutic resistance and minimize toxicity by leveraging competitive interactions between drug-sensitive and drug-resistant subclones, prioritizing patient survival and quality of life over maximum cell kill. In preparation for a clinical trial in breast cancer, we used large populations of MCF7 cells to rapidly generate endocrine-resistance breast cancer cell line. We then mimicked second line therapy in ER+ breast cancers by treating the endocrine-resistant MCF7 cells in a mouse xenograft model to test adaptive therapy with capecitabine, gemcitabine, or the combination of those two drugs. Dose-modulation adaptive therapy with capecitabine alone increased survival time relative to MTD, but not statistically significant (HR: 0.22, 95% CI 0.043- 1.1 P = 0.065). However, when we alternated the drugs in both dose modulation (HR = 0.11, 95% CI: 0.024 - 0.55, P = 0.007) and intermittent adaptive therapies significantly increased survival time compared to high dose combination therapy (HR = 0.07, 95% CI: 0.013 - 0.42; P = 0.003). Overall, survival time increased with reduced dose for both single drugs (P < 0.01) and combined drugs (P < 0.001). Adaptive therapy protocols resulted in tumors with lower proportions of proliferating cells (P = 0.0026) and more apoptotic cells (P = 0.045). The results show that Adaptive therapy outperforms high-dose therapy in controlling endocrine-resistant breast cancer, favoring slower-growing tumors, and showing promise in two-drug alternating regimens.

Cell-in-cell phenomena across the tree of life.

Cells in obligately multicellular organisms by definition have aligned fitness interests, minimum conflict, and cannot reproduce independently. However, some cells eat other cells within the same body, sometimes called cell cannibalism. Such cell-in-cell events have not been thoroughly discussed in the framework of major transitions to multicellularity. We performed a systematic review of 508 articles to search for cell-in-cell events across the tree of life, the age of cell-in-cell-related genes, and whether cell-in-cell events are associated with normal multicellular development or cancer. Out of the 38 cell-in-cell-related genes found in the literature, 14 genes were over 2.2 billion years old, i.e., older than the common ancestor of some facultatively multicellular taxa. Therefore, we propose that cell-in-cell events originated before the origins of obligate multicellularity. Cell-in-cell events are found almost everywhere: across some unicellular and many multicellular organisms, mostly in malignant rather than benign tissue, and in non-neoplastic cells. Thus, our results show that cell-in-cell events exist in obligate multicellular organisms, but are not a defining feature of them. The idea of eradicating cell-in-cell events from obligate multicellular organisms as a way of treating cancer, without considering that cell-in-cell events are also part of normal development, should be abandoned.

Recoil Measurements in Drosophila Embryos: from Mounting to Image Analysis.

Tension and force propagation play a central role in tissue morphogenesis, as they enable sub- and supra-cellular shape changes required for the generation of new structures. Force is often generated by the cytoskeleton, which forms complex meshworks that reach cell-cell or cell-extracellular matrix junctions to induce cellular rearrangements. These mechanical properties can be measured through laser microdissection, which concentrates energy in the tissue of interest, disrupting its cytoskeleton. If the tissue is undergoing tension, this cut will induce a recoil in the surrounding regions of the cut. This protocol describes how one can perform laser microdissection experiments and subsequently measure the recoil speed of the sample of interest. While we explain how to carry out these experiments in Drosophila embryos, the recoil calibration and downstream analyses can be applied to other types of preparations. Key features Allows measuring tension in live Drosophila embryos with a relatively simple approach. Describes a quick way to mount a high number of embryos. Includes a segmentation-free recoil quantification that reduces bias and speeds up analysis. Graphical overview.

Molecular classification and biomarkers of clinical outcome in breast ductal carcinoma in situ: Analysis of TBCRC 038 and RAHBT cohorts.

Ductal carcinoma in situ (DCIS) is the most common precursor of invasive breast cancer (IBC), with variable propensity for progression. We perform multiscale, integrated molecular profiling of DCIS with clinical outcomes by analyzing 774 DCIS samples from 542 patients with 7.3 years median follow-up from the Translational Breast Cancer Research Consortium 038 study and the Resource of Archival Breast Tissue cohorts. We identify 812 genes associated with ipsilateral recurrence within 5 years from treatment and develop a classifier that predicts DCIS or IBC recurrence in both cohorts. Pathways associated with recurrence include proliferation, immune response, and metabolism. Distinct stromal expression patterns and immune cell compositions are identified. Our multiscale approach employed in situ methods to generate a spatially resolved atlas of breast precancers, where complementary modalities can be directly compared and correlated with conventional pathology findings, disease states, and clinical outcome.

Identification of a signature of evolutionarily conserved stress-induced mutagenesis in cancer.

The clustering of mutations observed in cancer cells is reminiscent of the stress-induced mutagenesis (SIM) response in bacteria. Bacteria deploy SIM when faced with DNA double-strand breaks in the presence of conditions that elicit an SOS response. SIM employs DinB, the evolutionary precursor to human trans-lesion synthesis (TLS) error-prone polymerases, and results in mutations concentrated around DNA double-strand breaks with an abundance that decays with distance. We performed a quantitative study on single nucleotide variant calls for whole-genome sequencing data from 1950 tumors, non-inherited mutations from 129 normal samples, and acquired mutations in 3 cell line models of stress-induced adaptive mutation. We introduce statistical methods to identify mutational clusters, quantify their shapes and tease out the potential mechanism that produced them. Our results show that mutations in both normal and cancer samples are indeed clustered and have shapes indicative of SIM. Clusters in normal samples occur more often in the same genomic location across samples than in cancer suggesting loss of regulation over the mutational process during carcinogenesis. Additionally, the signatures of TLS contribute the most to mutational cluster formation in both patient samples as well as experimental models of SIM. Furthermore, a measure of cluster shape heterogeneity was associated with cancer patient survival with a hazard ratio of 5.744 (Cox Proportional Hazard Regression, 95% CI: 1.824-18.09). Our results support the conclusion that the ancient and evolutionary-conserved adaptive mutation response found in bacteria is a source of genomic instability in cancer. Biological adaptation through SIM might explain the ability of tumors to evolve in the face of strong selective pressures such as treatment and suggests that the conventional 'hit it hard' approaches to therapy could prove themselves counterproductive.

In Silico Investigations of Multi-Drug Adaptive Therapy Protocols.

The standard of care for cancer patients aims to eradicate the tumor by killing the maximum number of cancer cells using the maximum tolerated dose (MTD) of a drug. MTD causes significant toxicity and selects for resistant cells, eventually making the tumor refractory to treatment. Adaptive therapy aims to maximize time to progression (TTP), by maintaining sensitive cells to compete with resistant cells. We explored both dose modulation (DM) protocols and fixed dose (FD) interspersed with drug holiday protocols. In contrast to previous single drug protocols, we explored the determinants of success of two-drug adaptive therapy protocols, using an agent-based model. In almost all cases, DM protocols (but not FD protocols) increased TTP relative to MTD. DM protocols worked well when there was more competition, with a higher cost of resistance, greater cell turnover, and when crowded proliferating cells could replace their neighbors. The amount that the drug dose was changed, mattered less. The more sensitive the protocol was to tumor burden changes, the better. In general, protocols that used as little drug as possible, worked best. Preclinical experiments should test these predictions, especially dose modulation protocols, with the goal of generating successful clinical trials for greater cancer control.

Real-time tracking of bacterial membrane vesicles reveals enhanced membrane traffic upon antibiotic exposure.

Membrane vesicles are ubiquitous carriers of molecular information. A broad understanding of the biological functions of membrane vesicles in bacteria remains elusive because of the imaging challenges during real-time in vivo experiments. Here, we provide a quantitative analysis of the motion of individual vesicles in living microbes using fluorescence microscopy, and we show that while vesicle free diffusion in the intercellular space is rare, vesicles mostly disperse along the bacterial surfaces. Most remarkably, when bacteria are challenged with low doses of antibiotics, vesicle production and traffic, quantified by instantaneous vesicle speeds and total traveled distance per unit time, are significantly enhanced. Furthermore, the enhanced vesicle movement is independent of cell clustering properties but rather is associated with a reduction of the density of surface appendages in response to antibiotics. Together, our results provide insights into the emerging field of spatial organization and dynamics of membrane vesicles in microcolonies.

Pressure-Driven Mitochondrial Transfer Pipeline Generates Mammalian Cells of Desired Genetic Combinations and Fates.

Generating mammalian cells with desired mitochondrial DNA (mtDNA) sequences is enabling for studies of mitochondria, disease modeling, and potential regenerative therapies. MitoPunch, a high-throughput mitochondrial transfer device, produces cells with specific mtDNA-nuclear DNA (nDNA) combinations by transferring isolated mitochondria from mouse or human cells into primary or immortal mtDNA-deficient (ρ0) cells. Stable isolated mitochondrial recipient (SIMR) cells isolated in restrictive media permanently retain donor mtDNA and reacquire respiration. However, SIMR fibroblasts maintain a ρ0-like cell metabolome and transcriptome despite growth in restrictive media. We reprogrammed non-immortal SIMR fibroblasts into induced pluripotent stem cells (iPSCs) with subsequent differentiation into diverse functional cell types, including mesenchymal stem cells (MSCs), adipocytes, osteoblasts, and chondrocytes. Remarkably, after reprogramming and differentiation, SIMR fibroblasts molecularly and phenotypically resemble unmanipulated control fibroblasts carried through the same protocol. Thus, our MitoPunch "pipeline" enables the production of SIMR cells with unique mtDNA-nDNA combinations for additional studies and applications in multiple cell types.

Large scale, robust, and accurate whole transcriptome profiling from clinical formalin-fixed paraffin-embedded samples.

Transcriptome profiling can provide information of great value in clinical decision-making, yet RNA from readily available formalin-fixed paraffin-embedded (FFPE) tissue is often too degraded for quality sequencing. To assess the clinical utility of FFPE-derived RNA, we performed ribo-deplete RNA extractions on > 3200 FFPE slide samples; 25 of these had direct FFPE vs. fresh frozen (FF) replicates, 57 were sequenced in 2 different labs, 87 underwent multiple library analyses, and 16 had direct microdissected vs. macrodissected replicates. Poly-A versus ribo-depletion RNA extraction methods were compared using transcriptomes of TCGA cohort and 3116 FFPE samples. Compared to FF, FFPE transcripts coding for nuclear/cytoplasmic proteins involved in DNA packaging, replication, and protein synthesis were detected at lower rates and zinc finger family transcripts were of poorer quality. The greatest difference in extraction methods was in histone transcripts which typically lack poly-A tails. Encouragingly, the overall sequencing success rate was 81%. Exome coverage was highly concordant in direct FFPE and FF replicates, with 98% agreement in coding exon coverage and a median correlation of whole transcriptome profiles of 0.95. We provide strong rationale for clinical use of FFPE-derived RNA based on the robustness, reproducibility, and consistency of whole transcriptome profiling.

Expression of metalloproteinase-9 in patients with mild and severe forms of gastroesophageal reflux disease.

BACKGROUND: Gastroesophageal reflux disease develops when the stomach contents causes troublesome symptoms and complications. Mild forms are non-erosive and erosive esophagitis, and severe forms are Barrett's esophagus and Esophageal adenocarcinoma. Matrix metalloproteinases are endopeptidases that can degrade components of the extracellular matrix, they play an important role in tumor invasion as well as in metastasis. OBJECTIVE: To correlate the expression of metalloproteinase 9 (MMP-9) in esophageal biopsies from patients with mild and severe forms of Gastroesophageal reflux disease. METHOD: Cross-sectional study. The expression of MMP-9 was determined in biopsies of esophageal tissue of patients with mild and severe GRD. The included variables were age, sex, diagnosis, smoking and alcoholic habits, body mass index (BMI) and expression of MMP-9. Descriptive statistics was performed, Kappa for concordance in diagnosis as well as X2. RESULTS: There were 50 patients, 32 (64%) men and 18 (36%) women, mean age 52.13 ± 14.75 years of age. 12 (24%) with smoking and 7 (14%) with alcoholism. Average BMI was 26.71 ± 4.07 kg/m2 (15 to 33); 40 (80%) with obesity. The inter observer concordance for histopathological diagnosis was 1.0 and 0.84 for esophagitis degrees. 27 (54%) patients had esophagitis, 16 (32%) Barrett's esophagus and 7 (14%) esophageal cancer. There was expression of MMP-9 in four patients with esophagitis, five with Barrett's esophagus and five with esophageal cancer. Statistical significance was found between the expression of MMP-9 and smoking (p = 0.011) and histopathological diagnosis (p = 0.052). CONCLUSIONS: The expression of MMP-9 is most common in severe forms compared to the mild forms of GRD.

ANTECEDENTES: La enfermedad por reflujo gastroesofágico (ERGE) se desarrolla cuando el contenido estomacal ocasiona síntomas molestos o complicaciones. Las formas leves son esofagitis no erosiva y erosiva; las graves, esófago de Barrett y adenocarcinoma esofágico. Las metaloproteinasas de la matriz degradan componentes de la matriz extracelular, y tienen un papel importante en la invasión tumoral y la metástasis. OBJETIVO: Relacionar la expresión de la metaloproteinasa-9 (MMP-9) en biopsias esofágicas de pacientes con formas leves y graves de ERGE. MÉTODO: Estudio transversal. Se determinó la expresión de MMP-9 en biopsias esofágicas de pacientes con ERGE grave y leve. Las variables fueron edad, sexo, diagnóstico, tabaquismo, alcoholismo, índice de masa corporal (IMC) y expresión de MMP-9. Se realizó estadística descriptiva, concordancia para el diagnóstico y prueba de ji al cuadrado. RESULTADOS: 50 pacientes, 32 (64%) hombres y 18 (36%) mujeres, con edad media de 52.13 ± 14.75 años. Doce (24%) fumadores y 7 (14%) con alcoholismo. El IMC promedio fue de 26.71 ± 4.07 kg/m2 (rango: 15-33); 40 (80%) eran obesos. La concordancia entre observadores para el diagnóstico histopatológico fue de 1.0, y de 0.84 para esofagitis. Veintisiete (54%) tuvieron esofagitis, 16 (32%) esófago de Barrett y 7 (14%) cáncer de esófago. Hubo expresión de MMP-9 en cuatro pacientes con esofagitis, cinco con esófago de Barrett y cinco con cáncer esofágico. Encontramos diferencia estadísticamente significativa entre la expresión de MMP-9 y el tabaquismo (p = 0.011) y el diagnóstico histopatológico (p = 0.052). CONCLUSIONES: La expresión de MMP-9 es más frecuente en las formas graves que en las leves de ERGE.

Ancient genes establish stress-induced mutation as a hallmark of cancer.

Cancer is sometimes depicted as a reversion to single cell behavior in cells adapted to live in a multicellular assembly. If this is the case, one would expect that mutation in cancer disrupts functional mechanisms that suppress cell-level traits detrimental to multicellularity. Such mechanisms should have evolved with or after the emergence of multicellularity. This leads to two related, but distinct hypotheses: 1) Somatic mutations in cancer will occur in genes that are younger than the emergence of multicellularity (1000 million years [MY]); and 2) genes that are frequently mutated in cancer and whose mutations are functionally important for the emergence of the cancer phenotype evolved within the past 1000 million years, and thus would exhibit an age distribution that is skewed to younger genes. In order to investigate these hypotheses we estimated the evolutionary ages of all human genes and then studied the probability of mutation and their biological function in relation to their age and genomic location for both normal germline and cancer contexts. We observed that under a model of uniform random mutation across the genome, controlled for gene size, genes less than 500 MY were more frequently mutated in both cases. Paradoxically, causal genes, defined in the COSMIC Cancer Gene Census, were depleted in this age group. When we used functional enrichment analysis to explain this unexpected result we discovered that COSMIC genes with recessive disease phenotypes were enriched for DNA repair and cell cycle control. The non-mutated genes in these pathways are orthologous to those underlying stress-induced mutation in bacteria, which results in the clustering of single nucleotide variations. COSMIC genes were less common in regions where the probability of observing mutational clusters is high, although they are approximately 2-fold more likely to harbor mutational clusters compared to other human genes. Our results suggest this ancient mutational response to stress that evolved among prokaryotes was co-opted to maintain diversity in the germline and immune system, while the original phenotype is restored in cancer. Reversion to a stress-induced mutational response is a hallmark of cancer that allows for effectively searching "protected" genome space where genes causally implicated in cancer are located and underlies the high adaptive potential and concomitant therapeutic resistance that is characteristic of cancer.

Management of chronic unstable acromioclavicular joint injuries.

The acromioclavicular joint represents the link between the clavicle and the scapula, which is responsible for the synchronized dynamic of the shoulder girdle. Chronic acromioclavicular joint instability involves changes in the orientation of the scapula, which provokes cinematic alterations that might result in chronic pain. Several surgical strategies for the management of patients with chronic and symptomatic acromioclavicular joint instability have been described. The range of possibilities includes anatomical and non-anatomical techniques, open and arthroscopy-assisted procedures, and biological and synthetic grafts. Surgical management of chronic acromioclavicular joint instability should involve the reconstruction of the torn ligaments because it is accepted that from three weeks after the injury, these structures may lack healing potential. Here, we provide a review of the literature regarding the management of chronic acromioclavicular joint instability. LEVEL OF EVIDENCE: Expert opinion, Level V.

Management of unstable distal third clavicle fractures: clinical and radiological outcomes of the arthroscopy-assisted conoid ligament reconstruction and fracture cerclage with sutures.

INTRODUCTION: Surgical treatment is indicated for the management of Neer type IIB fractures of the distal third of the clavicle. The aim of this study was to assess the clinical and radiological outcomes, in cases of unstable distal third clavicle fractures managed by means of an arthroscopy-assisted conoid ligament reconstruction and fracture cerclage with sutures. METHODS: Nine patients with unstable distal third clavicle fractures (Neer type IIB) managed arthroscopically by means of a conoid ligament reconstruction and fracture cerclage with sutures, between 2008 and 2012, were included. The QoL was evaluated at the last follow-up visit, by means of the Health Survey questionnaire (SF36), the visual analogue scale (VAS) for pain, the Disabilities of the Arm, Shoulder and Hand (DASH) questionnaire, the constant score, and a Global Satisfaction scale (from 0 to 10). The mean time from fracture fixation until radiological union, the development of hardware loosening, nonunion, infections, and hardware skin discomfort were evaluated. RESULTS: The mean age was 36 [21-48] years old. The mean [range] time from surgery until the last follow-up visit was 49 [46-52] months. Values of the questionnaires assessed at the last follow-up visit were: (1) physical SF36 score (50.72 ± 6.88); (2) mental SF36 score (50.92 ± 11.65); (3) VAS for pain (1.86 ± 1.35); (4) DASH questionnaire (11.97 ± 7.03); (5) constant score (89.67 ± 8.55), and (6) Global Satisfaction (8.17 ± 0.98). The mean time elapsed from fracture fixation to radiological union was 8.41 ± 3.26 months. Hardware loosening was observed in none of the patients. Nonunion was observed in 11.11% (1/9) of the patients. Hardware skin discomfort was observed in 11.11% (1/9) of the patients. CONCLUSION: Patients with unstable distal third clavicle fractures managed by means of an arthroscopy-assisted conoid ligament reconstruction and fracture cerclage with sutures may have good clinical and radiological outcomes, with no need for a second surgical procedure to remove any metal hardware. LEVEL OF EVIDENCE: Therapeutic; case series, Level IV.

Prevalence of remaining horizontal instability in high-grade acromioclavicular joint injuries surgically managed.

PURPOSE: To determine the prevalence of remaining horizontal instability in high-grade acromioclavicular joint (ACJ) injuries surgically managed by means of four different surgical strategies and to assess its relation to the clinical outcomes and the quality of life. METHODS: In this multicentric non-randomized retrospective study, 53 patients with high-grade ACJ injuries surgically managed (by means of open or arthroscopic surgery) were clinically and radiographically assessed at 24 months or more after shoulder surgery. The presence of post-surgical remaining horizontal instability was evaluated by means of Alexander or axillary X-ray views. The study population was divided into two groups: patients with evidence of post-surgical remaining horizontal instability and patients without evidence of post-surgical remaining horizontal instability at the last follow-up visit. The relationship between remaining horizontal instability and the quality-of-life questionnaires was analyzed. RESULTS: 18.87% (10/53) of the Alexander or axillary X-rays views showed post-surgical remaining horizontal instability at the last follow-up visit (INSTAB-group). Results of the questionnaires were: (1) physical SF36 score (INSTAB-group 57.02 ± 3.17 and NO-INSTAB-group 57.66 ± 3.30, p = 0.583); (2) mental SF36 score (INSTAB-group 53.95 ± 3.98 and NO-INSTAB-group 55.71 ± 3.30, p = 0.150); (3) NRS for pain (INSTAB-group 1.30 ± 1.49 and NO-INSTAB-group 0.83  ± 1.08, p = 0.260); (4) DASH questionnaire (INSTAB-group 5.27 ± 5.42 and NO-INSTAB-group 3.06 ± 2.30, p = 0.049); (5) Constant score (INSTAB-group 93.4 ± 3.5 and NO-INSTAB-group 94.83  ± 4.3, p = 0.333); and Global satisfaction (INSTAB-group 8.7  ± 0.95 and NO-INSTAB-group 8.64 ± 1.03, p = 0.874). CONCLUSION: Independently of the type of procedure, post-surgical remaining horizontal instability was present in almost one-fifth of the patients, and this group of patients showed a significantly worse DASH score. The addition of an acromioclavicular augmentation might have to be considered, taking into account that its absence may have a negative impact in terms of shoulder disabilities. LEVEL OF EVIDENCE: Level IV, prognostic case series.