Evolutionary histories of breast cancer and related clones.

Recent studies have documented frequent evolution of clones carrying common cancer mutations in apparently normal tissues, which are implicated in cancer development1-3. However, our knowledge is still missing with regard to what additional driver events take place in what order, before one or more of these clones in normal tissues ultimately evolve to cancer. Here, using phylogenetic analyses of multiple microdissected samples from both cancer and non-cancer lesions, we show unique evolutionary histories of breast cancers harbouring der(1;16), a common driver alteration found in roughly 20% of breast cancers. The approximate timing of early evolutionary events was estimated from the mutation rate measured in normal epithelial cells. In der(1;16)(+) cancers, the derivative chromosome was acquired from early puberty to late adolescence, followed by the emergence of a common ancestor by the patient's early 30s, from which both cancer and non-cancer clones evolved. Replacing the pre-existing mammary epithelium in the following years, these clones occupied a large area within the premenopausal breast tissues by the time of cancer diagnosis. Evolution of multiple independent cancer founders from the non-cancer ancestors was common, contributing to intratumour heterogeneity. The number of driver events did not correlate with histology, suggesting the role of local microenvironments and/or epigenetic driver events. A similar evolutionary pattern was also observed in another case evolving from an AKT1-mutated founder. Taken together, our findings provide new insight into how breast cancer evolves.

Time-series blood cytokine profiles correlate with treatment responses in triple-negative breast cancer patients.

BACKGROUND: Triple-negative breast cancer (TNBC) is the most heterogeneous breast cancer subtype. Partly due to its heterogeneity, it is currently challenging to stratify TNBC patients and predict treatment outcomes. METHODS: In this study, we examined blood cytokine profiles of TNBC patients throughout treatments (pre-treatment, during chemotherapy, pre-surgery, and 1 year after the surgery in a total of 294 samples). We analyzed the obtained cytokine datasets using weighted correlation network analyses, protein-protein interaction analyses, and logistic regression analyses. RESULTS: We identified five cytokines that correlate with good clinical outcomes: interleukin (IL)-1α, TNF-related apoptosis-inducing ligand (TRAIL), Stem Cell Factor (SCF), Chemokine ligand 5 (CCL5 also known as RANTES), and IL-16. The expression of these cytokines was decreased during chemotherapy and then restored after the treatment. Importantly, patients with good clinical outcomes had constitutively high expression of these cytokines during treatments. Protein-protein interaction analyses implicated that these five cytokines promote an immune response. Logistic regression analyses revealed that IL-1α and TRAIL expression levels at pre-treatment could predict treatment outcomes in our cohort. CONCLUSION: We concluded that time-series cytokine profiles in breast cancer patients may be useful for understanding immune cell activity during treatment and for predicting treatment outcomes, supporting precision medicine. TRIAL REGISTRATION: The study has been registered with the University Hospital Medical Information Network Clinical Trials Registry ( http://www.umin.ac.jp/ctr/index-j.htm ) with the unique trial number UMIN000023162. The association Japan Breast Cancer Research Group trial number is JBCRG-22. The clinical outcome of the JBCRG-22 study was published in Breast Cancer Research and Treatment on 25 March 2021. https://doi.org/10.1007/s10549-021-06184-w .

Optimization of prediction methods for risk assessment of pathogenic germline variants in the Japanese population.

Predicting pathogenic germline variants (PGVs) in breast cancer patients is important for selecting optimal therapeutics and implementing risk reduction strategies. However, PGV risk factors and the performance of prediction methods in the Japanese population remain unclear. We investigated clinicopathological risk factors using the Tyrer-Cuzick (TC) breast cancer risk evaluation tool to predict BRCA PGVs in unselected Japanese breast cancer patients (n = 1,995). Eleven breast cancer susceptibility genes were analyzed using target-capture sequencing in a previous study; the PGV prevalence in BRCA1, BRCA2, and PALB2 was 0.75%, 3.1%, and 0.45%, respectively. Significant associations were found between the presence of BRCA PGVs and early disease onset, number of familial cancer cases (up to third-degree relatives), triple-negative breast cancer patients under the age of 60, and ovarian cancer history (all P < .0001). In total, 816 patients (40.9%) satisfied the National Comprehensive Cancer Network (NCCN) guidelines for recommending multigene testing. The sensitivity and specificity of the NCCN criteria for discriminating PGV carriers from noncarriers were 71.3% and 60.7%, respectively. The TC model showed good discrimination for predicting BRCA PGVs (area under the curve, 0.75; 95% confidence interval, 0.69-0.81). Furthermore, use of the TC model with an optimized cutoff of TC score ≥0.16% in addition to the NCCN guidelines improved the predictive efficiency for high-risk groups (sensitivity, 77.2%; specificity, 54.8%; about 11 genes). Given the influence of ethnic differences on prediction, we consider that further studies are warranted to elucidate the role of environmental and genetic factors for realizing precise prediction.

Transaortic cryoablation of uncommon atrioventricular nodal reentrant tachycardia after Senning and Rastelli operations.

A 36-years-old woman with a congenital corrected transposition of the great arteries, who underwent Senning and Rastelli operations, was admitted with a symptomatic supraventricular tachycardia. During an electrophysiological study, uncommon atrioventricular (AV) nodal reentrant tachycardia was induced. The coronary veins and coronary sinus did not connect to the systemic venous atrium. The His bundle electrogram (HBE) was recorded at the anterior septum of the mitral valve via the aorta. The target of ablation was the site of the earliest atrial activation during the tachycardia, 5 mm posterior to the AV node, and a successful cryoablation was performed using a transaortic approach. Both the antegrade and retrograde conduction of the slow AV nodal pathway was eliminated.

Transvenous shock-only implantable cardioverter defibrillator after an atrio-pulmonary Fontan surgery.

A 42-year-old woman with tricuspid atresia who underwent a Fontan surgery (atrio-pulmonary connection) was admitted to our hospital due to symptomatic ventricular tachycardia (VT). A defibrillation lead was implanted in a distal site of a coronary vein since there was no usual entry to the ventricle. Ventricular pacing was impossible due to the high threshold, however, good sensing was obtained. Three years later, she felt palpitations and a subsequent shock therapy while climbing stairs. The cardioverter data showed that an appropriate cardioversion therapy successfully converted VT to normal rhythm.

High-resolution imaging mass spectrometry combined with transcriptomic analysis identified a link between fatty acid composition of phosphatidylinositols and the immune checkpoint pathway at the primary tumour site of breast cancer.

BACKGROUND: The fatty acid (FA) composition of phosphatidylinositols (PIs) is tightly regulated in mammalian tissue since its disruption impairs normal cellular functions. We previously found its significant alteration in breast cancer by using matrix-assisted laser desorption and ionisation imaging mass spectrometry (MALDI-IMS). METHODS: We visualised the histological distribution of PIs containing different FAs in 65 primary breast cancer tissues using MALDI-IMS and investigated its association with clinicopathological features and gene expression profiles. RESULTS: Normal ductal cells (n = 7) predominantly accumulated a PI containing polyunsaturated FA (PI-PUFA), PI(18:0/20:4). PI(18:0/20:4) was replaced by PIs containing monounsaturated FA (PIs-MUFA) in all non-invasive cancer cells (n = 12). While 54% of invasive cancer cells (n = 27) also accumulated PIs-MUFA, 46% of invasive cancer cells (n = 23) accumulated the PIs-PUFA, PI(18:0/20:3) and PI(18:0/20:4). The accumulation of PI(18:0/20:3) was associated with higher incidence of lymph node metastasis and activation of the PD-1-related immune checkpoint pathway. Fatty acid-binding protein 7 was identified as a putative molecule controlling PI composition. CONCLUSIONS: MALDI-IMS identified PI composition associated with invasion and nodal metastasis of breast cancer. The accumulation of PI(18:0/20:3) could affect the PD-1-related immune checkpoint pathway, although its precise mechanism should be further validated.

Long-Term Efficacy of Implantable Cardioverter Defibrillator in Repaired Tetralogy of Fallot　- Role of Anti-tachycardia Pacing.

BACKGROUND: Tetralogy of Fallot (TOF) is one of the common congenital heart diseases (CHD) in implantable cardioverter defibrillator (ICD) recipients, but few studies have reported the long-term outcomes of and the anti-tachycardia pacing (ATP) efficacy in repaired TOF.Methods and Results:Twenty-one repaired TOF patients with an ICD implanted between April 2003 and March 2015 were investigated retrospectively. ICD therapy and clinical outcome were analyzed. Mean patient age was 39±11 years; 62% were male; and mean age at repair surgery was 9.4±6.8 years. During a median follow-up of 5.6 years (range, 2.6-8.4 years), no patients died. Appropriate ATP were delivered in 11 patients (52%), with appropriate shocks in 5 patients (24%) and inappropriate shocks in 5 patients (24%). The success rate of ATP was 98% for fast ventricular tachycardia (VT; cycle length ≤320 ms) and 98% for slow VT (cycle length >320 ms). ATP effectiveness increased from 81.5% with the first ATP attempt to 93.7% with the second ATP attempt, to 97.5% with the third ATP attempt, and to 98.6% with the fourth or successive ATP attempt (P<0.0001, Cochran-Armitage trend test). CONCLUSIONS: ATP was highly effective in repaired TOF regardless of VT cycle length. Multiple ATP attempts could have an important role in VT termination, and the novel subcutaneous ICD without ATP capability should be used carefully.

A homeobox protein, NKX6.1, up-regulates interleukin-6 expression for cell growth in basal-like breast cancer cells.

Among breast cancer subtypes, basal-like breast cancer is particularly aggressive, and research on the molecules involved in its pathology might contribute to therapy. In this study, we found that expression of NKX6.1, a homeobox transcription factor, is higher in basal-like breast cancer than in other subtypes. In loss-of-function experiments on basal-like breast cancer cell lines, NKX6.1-depleted cells exhibited reduced cell growth. Because cytokine interleukin-6 (IL-6) is expressed in basal-like breast cancer, and increases cell growth, we analyzed expression levels of IL6, an IL-6 gene, and observed reduced IL6 expression in NKX6.1-depleted cells. In a reporter assay, IL6 promoter activity was reduced by loss of NKX6.1 function. A pull-down assay showed that NKX6.1 binds to the proximal region in IL6 promoter. These results indicate that NKX6.1 directly up-regulates IL6 expression. To investigate further, we established cells with forced expression of IL-6. We observed that exogenous IL-6 expression restored the reduced cell growth of NKX6.1-depleted cells. Furthermore, orthotopic xenografts showed that NKX6.1-depleted cells lost the capacity for tumor formation. We therefore conclude that NKX6.1 is a factor for IL-6-regulated growth and tumor formation in basal-like breast cancer. Our findings facilitate profound understanding of basal-like breast cancer, and the development of suitable therapy.

[Possible predictors of discontinuation of basal-flow of postoperative patient-controlled analgesia].

BACKGROUND: The purpose of this study was to identify possible predictors of discontinuation of basal-flow of postoperative patient-controlled analgesia. METHODS: We reviewed postoperative pain assessment records by the postoperative pain service team from April 2010 to July 2011 in which surgical patients were provided with intravenous or epidural patient-controlled analgesia (IV-PCA or Epi-PCA). From these data, we extracted cases with discontinuation of basal-flow of PCA, and candidate variables such as patients' characteristics, preoperative and intraoperative variables were assessed. Predictors with significant univariate association (P < 0.20) with the primary outcome were used to construct multivariable logistic regression models. RESULTS: We enrolled 685 patients for IV-PCA and 606 for Epi-PCA and obtained discontinuation groups (105 and 73 cases, respectively) with this cohort data. Results of multivariate analysis showed female, non-laparotomy, low body weight, and non-droperidol as independent risk factors for IV-PCA and low body weight, no-co-existing disease, and gastrointestinal surgery for Epi-PCA. There were no significant differences in pain intensity between discontinuation and non-discontinuation cases. The primary cause of discontinuation was PONV for IV-PCA and hypotension for Epi-PCA, respectively. CONCLUSIONS: We should apply IV-PCA for female slender surgical patients undergoing non-laparotomy with great caution and provide prevention for PON. We should pay attention to incidence of postoperative hypotenion when we administer Epi-PCA to slender gastrointestinal surgical patients without co-existing disease.

[A patient with primary breast cancer who responded remarkably well to neoadjuvant chemotherapy with FEC100 followed by Abraxane].

A 49-year-old woman visited our hospital presentind with a right breast lump. She underwent core needle biopsy, and her disease was diagnosed as breast cancer(invasive ductal carcinoma, ER slightly positive, PgR and HER2 negative). We chose neoadjuvant chemotherapy because the tumor size was over 3 cm in diameter with a histological grade III, and she asked to have her breast conserved. Because she had an allergy to alcohol, we treated her with FEC100 followed by Abraxane(260mg/ m2)q3W for 4 courses. After chemotherapy, she received breast conserving therapy. During the treatment with Abraxane, the patient was very well and showed no major side effects except for grade 3 neutropenia was found on an outpatient basis. After chemotherapy, breast MRI detected no invasive lesion. Pathological examination showed pCR. We concluded that Abraxane was a good option as neoadjuvant chemotherapy for early breast cancer.

Periportal hepatocyte proliferation at midgestation governs maternal glucose homeostasis in mice.

The maternal liver is challenged by metabolic demands throughout pregnancy. However, hepatocyte dynamics and their physiological significance in pregnancy remain unclear. Here, we show in mice that hepatocyte proliferation is spatiotemporally regulated in each liver lobular zone during pregnancy, with transient proliferation of periportal and pericentral hepatocytes during mid and late gestation, respectively. Using adeno-associated virus (AAV)-8-mediated expression of the cell cycle inhibitor p21 in hepatocytes, we show that inhibition of hepatocyte proliferation during mid, but not late, gestation impairs liver growth. Transcriptionally, genes involved in glucose/glycogen metabolism are downregulated in late pregnancy when midgestational hepatocyte proliferation is attenuated. In addition, hepatic glycogen storage is abolished, with concomitant elevated blood glucose concentrations, glucose intolerance, placental glycogen deposition, and fetal overgrowth. Laser capture microdissection and RNA-seq analysis of each liver lobular zone show zone-specific changes in the transcriptome during pregnancy and identify genes that are periportally expressed at midgestation, including the hyaluronan-mediated motility receptor (Hmmr). Knockdown of Hmmr in hepatocytes by AAV8-shHmmr suppresses periportal hepatocyte proliferation at midgestation and induces impaired hepatic glycogen storage, glucose intolerance, placental glycogen deposition and fetal overgrowth. Our results suggest that periportal hepatocyte proliferation during midgestation is critical for maternal glycogen metabolism and fetal size.

Impact of preoperative electrophysiological intervention on occurrence of peri/postoperative supraventricular tachycardia following Fontan surgery.

BACKGROUND: Little is known about the effects of preoperative electrophysiological study (EPS) and catheter ablation (CA) in Fontan surgery candidates with supraventricular tachycardia (SVT). OBJECTIVE: The purpose of this study was to investigate the clinical impact of EPS-guided intervention in Fontan surgery candidates with preceding SVT events. METHODS: A total of 109 consecutive patients with a history of SVT before Fontan surgery were divided into 3 groups: 44 in whom EPS with CA was attempted (CA group); 21 in whom EPS without CA was attempted (EPS group); and 44 in whom EPS was not performed (N group). The incidence and diagnosis of SVT, acute success rate of CA, and risk factors of peri/postoperative SVT were retrospectively investigated. RESULTS: The total incidence of SVT within 1 year after Fontan surgery was 34% (n = 37), with 91% of cases occurring within 1 month. Among the 71 SVT incidences diagnosed with EPS, 31 were atrioventricular reentrant tachycardias (AVRTs) involving twin atrioventricular nodes, 12 were atrioventricular nodal reentrant tachycardias, 12 were atrial tachycardias, 7 were orthodromic AVRTs via the accessory pathway, 7 were atrial flutters, and 2 were junctional tachycardias. The acute success rate of CA was 91% (48/53). The rate of peri/postoperative atrioventricular reciprocating SVT was significantly lower in the CA group than in the N or EPS group (11% vs 43% or 43%; P <.05). No/unsuccessful CA significantly increased the risk of peri/postoperative SVT in multivariate analysis (odds ratio 4.43; 95% confidence interval 1.69-11.59). CONCLUSION: Preoperative CA reduces peri/postoperative SVT occurrence in Fontan surgery candidates at high risk for SVT.

Genetic and clinical landscape of breast cancers with germline BRCA1/2 variants.

The genetic and clinical characteristics of breast tumors with germline variants, including their association with biallelic inactivation through loss-of-heterozygosity (LOH) and second somatic mutations, remain elusive. We analyzed germline variants of 11 breast cancer susceptibility genes for 1,995 Japanese breast cancer patients, and identified 101 (5.1%) pathogenic variants, including 62 BRCA2 and 15 BRCA1 mutations. Genetic analysis of 64 BRCA1/2-mutated tumors including TCGA dataset tumors, revealed an association of biallelic inactivation with more extensive deletions, copy neutral LOH, gain with LOH and younger onset. Strikingly, TP53 and RB1 mutations were frequently observed in BRCA1- (94%) and BRCA2- (9.7%) mutated tumors with biallelic inactivation. Inactivation of TP53 and RB1 together with BRCA1 and BRCA2, respectively, involved LOH of chromosomes 17 and 13. Notably, BRCA1/2 tumors without biallelic inactivation were indistinguishable from those without germline variants. Our study highlights the heterogeneity and unique clonal selection pattern in breast cancers with germline variants.

Combined Cohesin-RUNX1 Deficiency Synergistically Perturbs Chromatin Looping and Causes Myelodysplastic Syndromes.

STAG2 encodes a cohesin component and is frequently mutated in myeloid neoplasms, showing highly significant comutation patterns with other drivers, including RUNX1. However, the molecular basis of cohesin-mutated leukemogenesis remains poorly understood. Here we show a critical role of an interplay between STAG2 and RUNX1 in the regulation of enhancer-promoter looping and transcription in hematopoiesis. Combined loss of STAG2 and RUNX1, which colocalize at enhancer-rich, CTCF-deficient sites, synergistically attenuates enhancer-promoter loops, particularly at sites enriched for RNA polymerase II and Mediator, and deregulates gene expression, leading to myeloid-skewed expansion of hematopoietic stem/progenitor cells (HSPC) and myelodysplastic syndromes (MDS) in mice. Attenuated enhancer-promoter loops in STAG2/RUNX1-deficient cells are associated with downregulation of genes with high basal transcriptional pausing, which are important for regulation of HSPCs. Downregulation of high-pausing genes is also confirmed in STAG2-cohesin-mutated primary leukemia samples. Our results highlight a unique STAG2-RUNX1 interplay in gene regulation and provide insights into cohesin-mutated leukemogenesis. SIGNIFICANCE: We demonstrate a critical role of an interplay between STAG2 and a master transcription factor of hematopoiesis, RUNX1, in MDS development, and further reveal their contribution to regulation of high-order chromatin structures, particularly enhancer-promoter looping, and the link between transcriptional pausing and selective gene dysregulation caused by cohesin deficiency.This article is highlighted in the In This Issue feature, p. 747.

A dog with acute myelomonocytic leukemia.

A three-year-old dog with marked leukocytosis, lymphadenopathy, and diarrhea showed an increase in unidentified blasts in the peripheral blood, and they were proliferated in the bone marrow. The dog was diagnosed with myelomonocytic leukemia (M4) because the blast cells were demonstrated by cytochemical staining to be both myeloid and monocytic cells. Although the dog was treated with a multi-combination chemotherapy and induction therapy using vitamin K2, it died on day 47 after the first admission. This case is the first report of M4 in Japan.

Successful catheter ablation of ventricular tachycardia in a patient with congenitally corrected transposition of great arteries after double switch operation.

Ventricular tachycardia (VT) may cause sudden death late after repair of congenital heart disease. Radiofrequency catheter ablation (CA) of VT can be effective but may be hampered by hypertrophied myocardium or prosthetic material. A 33-year-old man with congenitally corrected transposition of the great arteries (ccTGA) had undergone a double switch operation (DSO) with the combined Mustard and Rastelli procedures when he was 10 years old. He developed sustained VT 16 years after the surgery. An electrophysiological study was performed using a 3D mapping system. During the VT, abnormal fragmented potentials were identified in the right ventricular side near the patch of ventricular septal defect. Radiofrequency energy was delivered at the site with changes in the QRS morphology. Electroanatomical mapping of the left ventricle was performed via a retrograde transaortic approach. Successful ablation of the fractionated potentials was likewise achieved on the left side. We report, to our knowledge, the first case of a successful radiofrequency CA of VT in a patient with ccTGA after a DSO. A slow conduction zone, which was proved to be part of the tachycardia substrate, existed around the patch of ventricular septal defect. Fragmented activities during VT were recorded from both ventricles. The tachycardia circuit was eliminated after ablating the right and left sides of the ventricular septal defect patch.

Pattern of RECK CpG methylation as a potential marker for predicting breast cancer prognosis and drug-sensitivity.

The membrane-anchored glycoprotein RECK negatively regulates multiple metalloproteinases and is frequently downregulated in tumors. Forced RECK expression in cancer cells results in suppression of tumor angiogenesis, invasion, and metastasis in xenograft models. A previous methylome study on breast cancer tissues detected inverse correlation between RECK CpG methylation (in an intron-1 region) and relapse-free survival. In this study, we focused on another region of the RECK CpG island (a promoter/exon-1 region) and found an inverse correlation between its methylation and RECK-inducibility by an HDAC inhibitor, MS275, among a panel of breast cancer cell lines (n=15). In clinical samples (n=62), RECK intron-1 methylation was prevalent among luminal breast cancers as reported previously (26 of 38 cases; 68%) and particularly enriched in tumors of the ER+PR- subclass (10 of 10 cases) and of higher histological grades (Grade 2 and 3; 28 of 43 cases; P=0.006). In about a half of these cases, promoter/exon-1 methylation was absent, and hence, RECK may be inducible by certain drugs such as MS275. Our results indicate the value of combined use of two RECK methylation markers for predicting prognosis and drug-sensitivity of breast cancers.

[Difficult airway management during emergency tracheostomy in a patient with severe rheumatoid arthritis].

We experienced difficult airway management in a 65-year-old woman with acute dyspnea due to bilateral recurrent nerve palsy suffering from severe rheumatoid arthritis for fifty years. Her cervical spine was ankylosed and could not be extended at all. Tracheostomy was planned under local anesthesia because of difficulty of endotracheal intubation, possibility of airway obstruction and laryngeal edema. In this condition, the surgical area was narrow and difficult to approach. The surgical bleeding and blood-aspiration into the tracheostomy site occurred followed by airway obstruction. A rigid tracheal tube could not be inserted through the tracheal incision and SpO2 decreased to 81%. We inserted a percutaneous cricothyroidotomy cannula through the tracheal incision and superimposed HFJV on her spontaneous ventilation. Assisting the ventilation in this way finally, a spiral endtracheal tube was inserted and her oxygenation became stable.

Sudden cardiac arrest associated with an anomalous aortic origin of the left coronary artery from the opposite sinus of valsalva.

A 13-year-old boy was brought to our hospital after recovering from ventricular fibrillation that occurred after an episode of chest pain during training with his soccer team. Subsequent 64-slice multidetector computed tomography revealed the left coronary artery arising from the right sinus of Valsalva, which coursed between the ascending aorta and root of the main pulmonary artery. Surgical correction including unroofing of the left coronary ostium and pulmonary artery translocation was performed successfully. One year later, he remained asymptomatic and was back on his soccer team.