Prognostic impact of translocation t(11;14) and of other cytogenetic abnormalities in patients with AL amyloidosis in the era of contemporary therapies.

OBJECTIVES: Translocation t(11;14) is the most common cytogenetic abnormality in patients with systemic AL amyloidosis with prognostic and therapeutic relevance, which has not been clearly defined in the most recent therapeutic era. METHODS: We assessed its prognostic role in 146 newly-diagnosed patients who received novel agent-based treatment combinations. Event-free survival (EFS), a composite endpoint defined by hematological progression, start of a new treatment-line or death, and overall survival (OS) were the primary endpoints. RESULTS: Half of the patients had at least one FISH abnormality; 40% had t(11;14) which was inversely associated with other cytogenetic abnormalities. At 1, 3, and 6-month landmarks, hematologic response rates were numerically but not statistically higher in the non-t(11;14) group. Patients with t(11;14) were more frequently switched to second-line treatment within 12 months (p = .015). At median follow-up of 31.4 months, t(11;14) was associated with shorter EFS [17.1 (95% CI 3.2-10.6) vs. 27.2 months (95% CI 13.8-40.6), p = .021] and retained its prognostic significance in the multivariate model (HR:1.66, p = .029). The effect on OS was neutral, possibly due to the use of effective salvage therapies. CONCLUSIONS: Our data support the use of targeted therapies for patients with t(11;14) to avoid delays in the achievement of deep hematologic responses.

Combined detection and subclass characteristics analysis of CTCs and CTECs by SE-iFISH in ovarian cancer.

OBJECTIVE: Hematogenous metastasis is essential for the progression of ovarian cancer (OC), and circulating tumor cells (CTCs) are part of the metastatic cascade. However, the detection rate of CTC is low due to the use of less sensitive detection methods. Therefore, this study aimed to detect CTCs and circulating tumorigenic endothelial cells (CTECs) in patients with OC using subtraction enrichment and immunostaining and fluorescence in situ hybridization (SE-iFISH). METHODS: We enrolled a total of 56 subjects, including 20 OC patients and 36 ovarian benign tumor patients. CTCs and CTECs were captured by subtraction enrichment (SE) and counted and classified according to immunofluorescence staining of tumor markers (TMs) carbohydrate antigen 125 (CA125) and human epididymis protein 4 (HE4) combined with fluorescence in situ hybridization (iFISH) of chromosome 8 (Chr8) aneuploidy. The diagnostic value and subtype characteristics of CTCs and CTECs were investigated. RESULTS: The detection rate of CTCs by SE-iFISH was high. Compared with CA125 and HE4, Chr8 aneuploidy was the major identification feature of CTC. CTC counts in OC were statistically higher than those in benign groups. CTC and CTEC with ≥pentaploidy were detected in both groups, illustrating the poor diagnostic value of CTC or CTEC. Distributions of triploid and tetraploid CTC subtypes were significantly different, and combined detection of triploid and tetraploid CTCs showed the best diagnostic value. In contrast, the distribution of CTECs in the OC and benign groups had no statistically significant difference. Small CTCs accounted for over 1/3 of the total CTC count. We also found that small CTCs and CTECs primarily comprised triploid cells, while large CTCs and CTECs mainly comprised pentaploidy and beyond. CONCLUSIONS: The application of SE-iFISH offered a more comprehensive understanding of heterogeneous CTCs and CTECs in OC. Analysis of subclass characteristics of the CTCs and CTECs according to Chr8 aneuploidy and cell size may broaden their potential clinical utility and deepen mechanistic studies in OC.

Evaluation of computational techniques for predicting non-synonymous single nucleotide variants pathogenicity.

The human genetic diseases associated with many factors, one of these factors is the non-synonymous Single Nucleotide Variants (nsSNVs) cause single amino acid change with another resulting in protein function change leading to disease. Many computational techniques have been released to expect the impacts of amino acid alteration on protein function and classify mutations as pathogenic or neutral. Here in this article, we assessed the performance of eight techniques; FATHMM, SIFT, Provean, iFish, Mutation Assessor, PANTHER, SNAP2, and PON- P2 using a VaribenchSelectedPure dataset of 2144 pathogenic variants and 3777 neutral variants extracted from the free standard database "Varibench." The first five techniques achieve (45.60-83.75) % specificity, (52.64-94.13) % sensitivity, (51.00-88.90) % AUC, and (49.76-88.24) % ACC on whole dataset, while all eight techniques achieve (36.54-77.88) % specificity, (50.00-75.00) % sensitivity, (51.00-76.40) % AUC, and (25.00-77.78) % ACC on random sample dataset. We also created a Meta classifier (CSTJ48) that combines FATHMM, iFish, and Mutation Assessor. It registers 96.33% specificity, 86.07% sensitivity, 91.20% AUC, and 91.89 ACC. By comparing the results, it's clear that FATHMM gives the highest performance over the seven individual techniques, where it achieves 83.75% and 77.88% specificity, 94.13%, and 75.00% sensitivity, 88.90% and 76.40% AUC, and 88.24% and 77.78% ACC on whole and random sample dataset, respectively. Also, the launched Meta classifier (CSTJ48) is outperforming over all the eight individual tools that compared here.

Circulating Tumor Cells Correlate with Clinicopathological Features and Outcomes in Differentiated Thyroid Cancer.

BACKGROUND/AIMS: As biomarkers, circulating tumor cells (CTCs) from solid tumors can predict metastases and prognoses, and help monitor treatment efficacy. However, conventional CellSearch methods have low sensitivity to differentiated thyroid cancer (DTC) CTCs. In this study, for the first time, we used negative enriching (NE) immunofluorescence-in situ hybridization (iFISH) of chromosome 8 to capture and identify CTCs in DTC patients; and investigated how CTCs correlate with clinicopathological factors and prognosis in DTC patients with distant metastases (DM). METHODS: In this prospective study, we enrolled 72 patients with DTC before they underwent 131I treatment, and 30 healthy controls (HC). Their CTCs were measured in 7.5 ml peripheral blood using the NE-iFISH technique. CTC was defined by the aneuploidy. RESULTS: We detected CTCs in 62 (86.1%) of the 72 subjects with DTC. The mean number of CTCs in patients with DTC with DM (DM+) was significantly higher than in the HC group (P< 0.001) and DTC patients without DM (DM-; P=0.0016). We found CTCs ≥ 5 was significantly associated with DM+ DTC (P=0.009; sensitivity: 64.3%; specificity: 83.8%); CTCs ≥ 7 was related to poor response to 131I treatment (sensitivity: 73.7 %; specificity: 69.6 %), and was also associated with worse prognosis in DM+ DTC (P< 0.001). CONCLUSION: We found CTCs ≥ 5 to be a potential predictive index for DM+ DTC; and CTCs ≥7 as a possible indicator of poor response to 131I treatment and worse prognosis in DM+ DTC.

Circulating tumor cells with karyotyping as a novel biomarker for diagnosis and treatment of nasopharyngeal carcinoma.

BACKGROUND: Circulating tumor cells (CTCs) have been considered great clinical significance in various cancers. However, it remains unknown that how is the role of CTCs in patients with nasopharyngeal carcinoma (NPC). We investigated the value of CTCs enumeration and karyotyping in NPC. METHODS: In the present study, we applied integrated subtraction enrichment and immunostaining-fluorescence in situ hybridization (SE-iFISH) automatic testing system to detect and characterize CTCs of NPC patients. Enumeration and aneuploidy of chromosome 8 in CTCs were examined in various stages of patients with NPC. The changes of CTCs number and karyotyping post to chemotherapy were investigated in NPC. RESULTS: CTCs were detected by SE-iFISH in 46 out of 50 pre-treatment NPC patients, and performed a positive rate of 92.0%. No significant association was found between disease staging and CTCs detection rate. CTCs number constantly increased with TNM stage rising (from stage II to stage IV) no matter in newly diagnosed patients without distant metastasis (M0) and relapsed or distant metastatic patients. The number of CTCs decreased after treatment in patients with partial response (PR), while increased in patients with progressive disease or stable disease (PD/SD). More interestingly, CTCs karyotyping indicated that aneuploidy of chromosome 8 in CTCs was dramatically related to chemotherapeutic efficacy in NPC. Positive correlation was found between CTCs count and plasma EBV DNA level of NPC patients. CONCLUSIONS: CTCs could be detected in various stages of NPC patients using SE-iFISH. CTCs number could indicate the severity degree of disease in NPC. Dynamically monitoring the variations in CTCs number may predict chemotherapy efficacy during treatment. CTCs karyotyping is related to the sensibility of chemotherapy and drug resistance, and karyotyping of CTCs might predict therapeutic efficacy and evaluate chemo-resistance in NPC. CTCs could be used as a monitoring indicator in the fields of treatment, diagnosis and follow-up of NPC.

Detection of Circulating Tumor Cells Using Negative Enrichment Immunofluorescence and an In Situ Hybridization System in Pancreatic Cancer.

Pancreatic cancer (PC) is the most lethal type of gastrointestinal cancer, and early detection and monitoring is an urgent problem. Circulating tumor cells (CTCs) are emerging as a non-invasive biomarker for tumor detection. However, the low sensitivity is a main problem in the traditional CellSearch System for detecting CTCs, especially in patients with PC. In this study, we used negative enrichment (NE), immunofluorescence and in situ hybridization (FISH) of chromosome 8 (NE-iFISH) to capture and identify CTCs in PC patients. We showed that the NE-iFISH system exhibited a dramatically high detection rate of CTCs in PC patients (90%). The diagnostic rate of PC reached 97.5% when combining CTCs ≥ 2 and carbohydrate antigen 19-9 (CA19-9) > 37 µmol/L. The 1-year survival in the group of CTCs < 3 was significantly higher than that of CTCs ≥ 3 (p = 0.043). In addition, we analyzed the role of chromosomal instability in CTCs detection. The group of triploid (three hybridization signals of chromosome 8) CTCs ≥ 3 showed a shorter 1-year survival (p = 0.0279) and overall survival (p = 0.0188) than the group with triploid CTCs < 3. Importantly, the triploid CTC number but not the overall CTC counts could be a predictor of chemo-sensitivity. Moreover, circulating tumor microembolus (CTMs) were found in stage IV patients, and were positively related to the poor response to chemotherapy. In conclusion, the NE-iFISH system significantly improved the positive detection rate of CTCs and triploid CTC could be used to predict prognosis or the response to the chemotherapy of PC patients. CTM is a potential indicator of the chemotherapeutic effect in advanced PC patients.

[Detection of Circulating Tumor Cells in Bladder Cancer by Subtraction Enrichment and Immunostaining-fluorescence in situ Hybridization].

OBJECTIVE: To detect circulating tumor cells (CTCs) in peripheral blood from the patients with bladder cancer using subtraction enrichment and immunostaining-fluorescence in situ hybridization (SE-iFISH) and to explore the relation between CTCs detection result and the clinicpathological parameters of tumors. METHODS: Sixteen patients [(66.88±8.97) yr.] with bladder cancerwere included, whlile 10 normal people [(61.90±9.13) yr.] were enrolled as control. Peripheral blood was harvested (6 mL per case) for the detection of CTCs with the method of subtraction enrichment and SE-iFISH, and the correlations of CTCs level to the clinicpathological parameters of tumors were analyzed, including hematuria, size, number, NMIBC or MIBC, stage, grade and pathological type. RESULTS: Positive rates of CTCs in experimental and control group were 43.75% (7/16) and 0% (0/10), respectively. The difference was statistically significant (P=0.022 7). The number of CTCS was not associated with the clinicpathological parameters of bladder cancer, which probably due to small sample size. CONCLUSION: It is applicable to detect CTCs of bladder cancer by SE-iFISH.

Aneuploidy of chromosome 8 in circulating tumor cells correlates with prognosis in patients with advanced gastric cancer.

OBJECTIVE: Previous work indicated that aneuploidy of chromosome 8 in circulating tumor cells (CTCs) correlated with therapeutic efficacy for advanced gastric cancer (AGC) patients. In this follow-up study performed on the same population of AGC patients, we investigated whether and how aneuploidy of chromosome 8 in CTCs correlates with patients' clinical prognosis. METHODS: The prospective study was performed on 31 patients with newly diagnosed AGC. Previously established integrated subtraction enrichment (SE) and immunostaining-fluorescence in situ hybridization (iFISH) platform was applied to identify, enumerate and characterize CTCs. Quantification of CTCs and analysis of their aneuploidy of chromosome 8 were performed on patients before and after therapy. RESULTS: CTCs were measured in 93.5% of AGC patients, and two CTC subtypes with diverse threshold values were identified, multiploid CTCs with the threshold of ≥2 per 7.5 mL and multiploid plus triploid CTCs with the threshold of ≥4, which were found to significantly correlate with poor progression-free survival (PFS) and overall survival (OS). In particular, patients with ≥10% increased multiploid CTCs after an initial 6 weeks of therapy had poor PFS and OS, whereas improved PFS and OS were observed on those who had ≥10% decreased multiploid CTCs. After adjusting for clinically significant factors, ≥10% increased post-therapy multiploid CTCs was the only independent predictor of PFS and OS. CONCLUSIONS: Aneuploidy of CTCs correlates with prognosis of AGC patients. Quantitative comparison monitoring multiploid CTCs before and after therapy may help predict improved or inferior prognosis and chemoresistance.

The protein expression profile of meningioma cells is associated with distinct cytogenetic tumour subgroups.

AIMS: Limited information exists about the impact of cytogenetic alterations on the protein expression profiles of individual meningioma cells and their association with the clinicohistopathological characteristics of the disease. The aim of this study is to investigate the potential association between the immunophenotypic profile of single meningioma cells and the most relevant features of the tumour. METHODS: Multiparameter flow cytometry (MFC) was used to evaluate the immunophenotypic profile of tumour cells (n = 51 patients) and the Affymetrix U133A chip was applied for the analysis of the gene expression profile (n = 40) of meningioma samples, cytogenetically characterized by interphase fluorescence in situ hybridization. RESULTS: Overall, a close association between the pattern of protein expression and the cytogenetic profile of tumour cells was found. Thus, diploid tumours displayed higher levels of expression of the CD55 complement regulatory protein, tumours carrying isolated monosomy 22/del(22q) showed greater levels of bcl2 and PDGFRß and meningiomas carrying complex karyotypes displayed a greater proliferation index and decreased expression of the CD13 ectoenzyme, the CD9 and CD81 tetraspanins, and the Her2/neu growth factor receptor. From the clinical point of view, higher expression of CD53 and CD44 was associated with a poorer outcome. CONCLUSIONS: Here we show that the protein expression profile of individual meningioma cells is closely associated with tumour cytogenetics, which may reflect the involvement of different signalling pathways in the distinct cytogenetic subgroups of meningiomas, with specific immunophenotypic profiles also translating into a different tumour clinical behaviour.

Presence of Twist1-positive neoplastic cells in the stroma of chromosome-unstable colorectal tumors.

BACKGROUND & AIMS: Cancer cells undergo an epithelial-to-mesenchymal transition (EMT) to become invasive, allowing tumors to progress. However, there is no direct evidence that human cancer cells undergo an EMT. In mouse cancer cells, up-regulation of transcription factor Twist1 was shown to promote an EMT. We searched the stroma of human colorectal tumor samples for TWIST1-positive cells with a mesenchymal phenotype and neoplastic genotype. METHODS: We measured the expression of TWIST1 in human colorectal cancer (CRC) cell lines and examined the effects of overexpression or knockdown in vitro and in mice. We used immunohistochemistry to measure levels of TWIST1 in 201 colorectal tumor samples. In 20 samples, immunostaining was combined with fluorescence in situ hybridization analyses. Levels of TWIST1 messenger RNA (mRNA) were measured in blood samples from 15 patients. RESULTS: TWIST1 was required to maintain the mesenchymal phenotype and invasiveness of the microsatellite-stable CoLo741 cells (which express endogenous TWIST1) and SW480 (expressing transgenic TWIST1). TWIST1 mRNA was not translated in CRC cells with microsatellite instability (HCT116). Syngenic TWIST1-positive colon carcinoma cells (CT26) that invaded tissues surrounding tumors acquired a mesenchymal phenotype. The presence of TWIST1-positive cells in the stroma of human colorectal tumors correlated with microsatellite stability (P = .05), stage IV cancer (P = .02), and disease-free survival time (P < .01). Trisomies of chromosome 7 and/or chromosome 20 were detected in 17 of 20 colorectal tumor samples, each of which contained TWIST1-positive cells with matching chromosomal gains in the tumor stroma (86 of 776 counted cells; 11.1%). No trisomy was observed in TWIST1-negative stromal cells (0 of 1249 cells; P < .001). Levels of TWIST1 mRNA were significantly higher in blood samples from patients with CRC than controls. CONCLUSIONS: The stroma of human colorectal tumors contains TWIST1-positive cancer cells with mesenchymal phenotypes. Patients with CRC have higher levels of TWIST1 mRNA than healthy individuals.

Sequential and hierarchical chromosomal changes and chromosome instability are distinct features of high hyperdiploid pediatric acute lymphoblastic leukemia.

BACKGROUND: Pathogenesis of the non-random accumulation of extra chromosomes in the low and high hyperdiploid (HeL, HeH) pre-B pediatric acute lymphoblastic leukemia (B-pALL) is largely unknown, and has been clarified with respect only to tetrasomic chromosomes. We analyzed the hierarchy of changes in chromosome number and chromosomal instability, as well as clonal heterogeneity and evolution, in the untreated bone marrow cell samples from 214 B-pALL patients. PROCEDURE: Applying relocation, 2 × 4 color interphase fluorescence in situ hybridization was used to detect copy number alterations (CNAs) of the most commonly involved chromosomes, 4, 6, 10, 14, 17, 18, 21, and X. This approach allowed us to acquire a dataset correlated for all eight parameters. RESULTS: Based on chromosome number, an average of 6.9 and 10.2, whereas according to unique constellation 15.3 and 26.7 subclones could be identified in the HeL and HeH subgroups, respectively. Cluster analysis revealed the order of CNAs to chromosomes was highly conserved, and network analysis indicated changes in chromosome number were sequential for 80-90% of all numerical aberrations. Significant chromosome instability was revealed in both subgroups of leukemia. CONCLUSIONS: Data generated using this new approach indicate that chromosomal instability, which causes heterogeneity in the subclonal landscape, and the sequential changes to chromosome numbers, are both determining factors in the pathomechanism of the hyperdiploid B-pALL. These new observations could prompt research into the mitotic machinery of leukemic cells to identify new therapeutic targets for treating this disease.

Tracking circulating PD-L1-positive cells to monitor the outcome of patients with gastric cancer receiving anti-HER2 plus anti-PD-1 therapy.

Dual blockade of HER2 and PD-1/PD-L1 is the most promising regimen for HER2-positive patients with gastric cancer (GC); PD-L1 combined positive score, rather than HER2 status, indicates potential benefit. Circulating tumor cells (CTCs) and circulating endothelial cells (CECs) derived from the tumor microenvironment provide platforms for the dynamic evaluation of PD-L1 expression. Whether PD-L1 positive CTCs/CECs (PD-L1+CTCs/CECs) can serve as biomarkers for evaluating the efficacy of combination therapy remains unknown. Therefore, this study investigated PD-L1 expression and heterogeneous karyotypic features of CTCs/CECs and their involvement in the clinical response to treatment in 72 patients with advanced GC by applying a pre-established surface molecule-independent subtraction enrichment (SE)-iFISH strategy. In the captured PD-L1 positive cells, there were 42.80% and 57.20% of CTCs and CECs, respectively. PD-L1+ CTCs were pre-therapeutically detected in 0% (0/11) of HER2-negative patients and 14.75% (9/61) of HER2-positive patients. The presence of baseline PD-L1+CTCs was relevant to inferior prognosis (mPFS: 14.40 months vs 5.00 months, P = 0.065); post-treatment PD-L1+ CECs were associated with longer irPFS (immunotherapeutic-related PFS) (mPFS: 15.57 months vs 6.73 months, P = 0.053). Further dynamic karyotype-based profiling of PD-L1+ CTCs/CECs indicated that multiploidy and triploidy were the dominant subtypes of baseline PD-L1+ CTCs, and that triploidy was specifically associated with therapeutic resistance. Intratherapeutically detected multiploid PD-L1+ CECs demonstrated a superior clinical response; triploidy and tetraploidy contributed to acquired resistance. The karyotypic features of PD-L1+CTCs/CECs should be dynamically profiled in patients with GC treated with anti-HER2 plus anti-PD-1 therapy. Triploid-PD-L1+ CTCs and multiploid-PD-L1+ CECs are potential indicators of therapeutic response.

Biphasic co-detection of melanoma aneuploid tumor cells and tumor endothelial cells in guidance of specifying the field cancerized surgical excision margin and administering immunotherapy.

An optimum safety excision margin (EM) delineated by precise demarcation of field cancerization along with reliable biomarkers that enable predicting and timely evaluating patients' response to immunotherapy significantly impact effective management of melanoma. In this study, optimized biphasic "immunofluorescence staining integrated with fluorescence insitu hybridization" (iFISH) was conducted along the diagnosis-metastasis-treatment-cellular MRD axis to longitudinally co-detect a full spectrum of intact CD31- aneuploid tumor cells (TCs), CD31+ aneuploid tumor endothelial cells (TECs), viable and necrotic circulating TCs (CTCs) and circulating TECs (CTECs) expressing PD-L1, Ki67, p16 and Vimentin in unsliced specimens of the resected primary tumor, EM, dissected sentinel lymph nodes (SLNs) and peripheral blood in an early-stage melanoma patient. Numerous PD-L1+ aneuploid TCs and TECs were detected at the conventional safety EM (2 cm), quantitatively indicating the existence of a field cancerized EM for the first time. Contrary to highly heterogeneous PD-L1 expression and degrees of Chr8 aneuploidy in TCs and TECs in the primary lesions as well as CTCs and CTECs in peripheral blood, almost all TCs and TECs in SLNs and EM were homogeneously PD-L1+ haploid cells. Dynamic monitoring and cellular MRD assessment revealed that, in contrast to PD-L1+ CTCs being responsive to the immune checkpoint inhibitor (ICI-anti-PD-1), multiploid (≥pentasomy 8) PD-L1+ and Ki67+ CTECs were respectively resistant to ICI-sensitized T cells. In therapeutically stressed lymphatic and hematogenous metastatic cascades, stratified phenotypic and karyotypic profiling of iFISH tissue and liquid biopsied TCs, TECs, CTCs and CTECs in future large-cohort studies will enable appropriate re-specification of the optimal safety EM and distribution mapping of in-depth characterized, subcategorized target cells to help illustrate their metastatic relevance, ultimately improving risk stratification and clinical intervention of tumor progression, metastases, therapy resistance and cancer relapse.

Molecular Cytogenetic Study of the NF2 Gene Deletion in Meningioma in Sudanese Patients.

Meningioma is the second most common adult central nervous system tumor. Mutations and/or deletions within the tumor suppressor gene neurofibromatosis type 2 (NF2) are associated with meningioma development and progression. We studied 29 meningioma samples by cytogenetic analysis and interphase fluorescence in situ hybridization (I-FISH) using a locus-specific probe for the NF2 gene region. We detected loss of the NF2 gene in all samples except for one. In 10 of the 29 samples, karyotypic analyses confirmed the I-FISH results and revealed additional numerical and/or structural rearrangements in nine of them. Our study confirmed: i) the limited role of banding cytogenetics in assessing chromosomal rearrangements in meningioma, as this tumor is hard to be grown in cell culture; ii) we could show that two-color I-FISH is well-suited for NF2-deletion screening. Our results were in accordance with those of comparable studies, even though the frequency of 97.0% of meningiomas with NF2 deletions is exceptionally high in the studied Sudanese patients.

Longitudinal detection of subcategorized CD44v6+ CTCs and circulating tumor endothelial cells (CTECs) enables novel clinical stratification and improves prognostic prediction of small cell lung cancer: A prospective, multi-center study.

Current management of small cell lung cancer (SCLC) remains challenging. Effective biomarkers are needed to subdivide patients presenting distinct treatment response and clinical outcomes. An understanding of heterogeneous phenotypes of aneuploid CD31- circulating tumor cells (CTCs) and CD31+ circulating tumor endothelial cells (CTECs) may provide novel insights in the clinical management of SCLC. In the present translational and prospective study, increased cancer metastasis-related cell proliferation and motility, accompanied with up-regulated mesenchymal marker vimentin but down-regulated epithelial marker E-cadherin, were observed in both lentivirus infected SCLC and NSCLC cells overexpressing the stemness marker CD44v6. Aneuploid CTCs and CTECs expressing CD44v6 were longitudinally detected by SE-iFISH in 120 SCLC patients. Positive detection of baseline CD44v6+ CTCs and CD44v6+ CTECs was significantly associated with enhanced hepatic metastasis. Karyotype analysis revealed that chromosome 8 (Chr8) in CD44v6+ CTCs shifted from trisomy 8 towards multiploidy in post-therapeutic patients compared to pre-treatment subjects. Furthermore, the burden of baseline CD44v6+ CTCs (t0) or amid the therapy (t1-2), the ratio of baseline CD31+ CTEC/CD31- CTC (t0), and CTC-WBC clusters (t0) were correlated with treatment response and distant metastases, particularly brain metastasis, in subjects with limited disease (LD-SCLC) but not in those with extensive disease (ED-SCLC). Multivariate survival analysis validated that longitudinally detected CD44v6+/CD31- CTCs was an independent prognostic factor for inferior survival in SCLC patients. Our study provides evidence for the first time that comprehensive analyses of CTCs, CTECs, and their respective CD44v6+ subtypes enable clinical stratification and improve prognostic prediction of SCLC, particularly for potentially curable LD-SCLC.

Comprehensive Atlas of Circulating Rare Cells Detected by SE-iFISH and Image Scanning Platform in Patients With Various Diseases.

Objective: Circulating rare cells (CRCs) are known as a crucial nucleated cellular response to pathological conditions, yet the landscape of cell types across a wide variety of diseases lacks comprehensive understanding. This study aimed at detecting and presenting a full spectrum of highly heterogeneous CRCs in clinical practice and further explored the characterization of CRC subtypes in distinct biomarker combinations and aneuploid chromosomes among various disease groups. Methods: Peripheral blood was obtained from 2,360 patients with different cancers and non-neoplastic diseases. CRC capture and identification were accomplished using a novel platform integrating subtraction enrichment and immunostaining-fluorescence in situ hybridization (SE-iFISH) strategy with a high-throughput automated image scanning system, on which hemocyte, tumor, epithelial, endothelial, mesenchymal, and stemness biomarkers were immunostained and displayed simultaneously. Double chromosome enumeration probe (CEP8 and CEP12) co-detection was performed on isolated CRCs from an extended trial for two chromosome ploidy patterns. Results: A comprehensive atlas categorizing the diverse CRCs into 71 subtypes outlining was mapped out. The presence of epithelial-mesenchymal transition (EMT) or endothelial-mesenchymal transition (EndoMT), the cells with progenitor property, hematologic CRCs expressing multiple biomarkers, CRCs at "naked nuclei" status, and the rarely reported aneuploid mesenchymal epithelial-endothelial fusion cluster were described. Circulating tumor cells (CTCs) were detected in 2,157 (91.4%) patients; the total numbers of CTCs and circulating tumor-derived endothelial cells (CTECs) were relatively higher in several digestive system cancer types and non-neoplastic infectious diseases (p < 0.05). Co-detection combining CEP8 and CEP12 showed a higher diagnostic specificity on account of 57.27% false negativity of CRC detection through a single probe of CEP8. Conclusions: The alternative biomarkers and chromosomes to be targeted by SE-iFISH and the image scanning platform, along with the comprehensive atlas, offer insight into the heterogeneity of CRCs and reveal potential contributions to specific disease diagnosis and therapeutic target cell discovery.

Case report: Post-therapeutic laryngeal carcinoma patient possessing a high ratio of aneuploid CTECs to CTCs rapidly developed de novo malignancy in pancreas.

Effectively evaluating therapeutic efficacy, detecting minimal residual disease (MRD) after therapy completion, and predicting early occurrence of malignancy in cancer patients remain as unmet imperative clinical demands. This article presents a case of a laryngeal carcinoma patient who had a surgical resection and complete post-operative chemoradiotherapy in combination with the targeted therapy, then rapidly developed pancreatic adenocarcinoma. Detected by SE-iFISH, the patient had a substantial amount of 107 non-hematological aneuploid circulating rare cells including 14 circulating tumor cells (CTCs, CD31-/CD45-) and 93 circulating tumor endothelial cells (CTECs, CD31+/CD45-) with a high ratio of CTECs/CTCs > 5 upon finishing post-surgical combination regimens. Positive detection of those aneuploid non-hematological circulating rare cells was five months prior to subsequent plasma CA19-9 increasing and ten months before the de novo pancreatic cancer was diagnosed by medical imaging modalities. Besides previously reported clinical utilities of co-detection of aneuploid CD31- CTCs and CD31+ CTECs in real-time evaluation of therapeutic efficacy, longitudinal monitoring of emerging treatment resistance and adequate detection of MRD, a large cohort study is necessary to further investigate whether, and how, a high ratio of MRD CTECs to CTCs may function as an appropriate index forecasting either occurrence or metastatic distant recurrence of malignancy in post-therapeutic cancer patients.

IgD multiple myeloma: biology, diagnosis, and treatment.

IgD multiple myeloma is uncommon. Patients generally present at a younger age and have shorter progression free and overall survivals (OSs). Its rarity has inhibited development of a specific risk stratification system or informed best treatment protocols. We present interphase fluorescence in situ hybridization results from a group of 29 cases. These showed evidence of a decreased male to female ratio, decreased OS in patients aged 70 and over, better outcomes in those with kappa light chain restriction, and CD56 positive patients had longer survivals than those lacking CD56. We discuss the biology of IgD multiple myeloma, the need for prospective studies, and challenges for improvements in diagnosis and treatment. We suggest an International Register to accelerate development of best practice guidelines for diagnosis, risk stratification, and treatment.

Clonal heterogeneity and rates of specific chromosome gains are risk predictors in childhood high-hyperdiploid B-cell acute lymphoblastic leukemia.

B-cell acute lymphoblastic leukemia (B-ALL) is the commonest childhood cancer. High hyperdiploidy (HHD) identifies the most frequent cytogenetic subgroup in childhood B-ALL. Although hyperdiploidy represents an important prognostic factor in childhood B-ALL, the specific chromosome gains with prognostic value in HHD-B-ALL remain controversial, and the current knowledge about the hierarchy of chromosome gains, clonal heterogeneity and chromosomal instability in HHD-B-ALL remains very limited. We applied automated sequential-iFISH coupled with single-cell computational modeling to identify the specific chromosomal gains of the eight typically gained chromosomes in a large cohort of 72 primary diagnostic (DX, n = 62) and matched relapse (REL, n = 10) samples from HHD-B-ALL patients with either favorable or unfavorable clinical outcome in order to characterize the clonal heterogeneity, specific chromosome gains and clonal evolution. Our data show a high degree of clonal heterogeneity and a hierarchical order of chromosome gains in DX samples of HHD-B-ALL. The rates of specific chromosome gains and clonal heterogeneity found in DX samples differ between HHD-B-ALL patients with favorable or unfavorable clinical outcome. In fact, our comprehensive analyses at DX using a computationally defined risk predictor revealed low levels of trisomies +18+10 and low levels of clonal heterogeneity as robust relapse risk factors in minimal residual disease (MRD)-negative childhood HHD-B-ALL patients: relapse-free survival beyond 5 years: 22.1% versus 87.9%, P < 0.0001 and 33.3% versus 80%, P < 0.0001, respectively. Moreover, longitudinal analysis of matched DX-REL HHD-B-ALL samples revealed distinct patterns of clonal evolution at relapse. Our study offers a reliable prognostic sub-stratification of pediatric MRD-negative HHD-B-ALL patients.

Detection of circulating rare cells benefitted the diagnosis of malignant solitary pulmonary nodules.

INTRODUCTION: Solitary pulmonary nodules (SPNs) are challenging in differentiating between benignancy and malignancy. Therefore, more effective non-invasive biomarkers are urgently needed. The purpose of this investigation was to examine whether circulating rare cells (CRCs) could facilitate the differentiation between benign and malignant SPNs as well as its sensitivity and specificity. METHODS: 164 patients diagnosed with SPNs, 24 healthy volunteers, and 25 patients diagnosed with advanced-stage lung cancer were included. CT/PET-CT images, serum tumor markers, and biopsy results were collected. The CRCs were examined using subtraction enrichment and immunostaining-fluorescence in situ hybridization (SE-iFISH) and their relationship with malignant or benign SPNs was analyzed. RESULTS: The total CRC numbers from patients with malignant SPNs diagnosed by biopsy were significantly greater compared to those with benign SPNs (P < 0.0001), but not significantly different from patients with advanced lung cancer (P > 0.05). The total CRCs, with a cut-off value of 21.5 units, showed 67.6% sensitivity and 73.3% specificity [area under curve (AUC) 95% CI, 0.778 (0.666-0.889)] in discriminating benign and malignant SPNs and the triploid CRCs exhibited a high positive likelihood ratio of 8.4, which suggested that CRCs appeared to have a distinct advantage in discriminating benign and malignant SPNs compared to CT/PET-CT images and serum tumor markers and could be a potential screening indicator for lung cancer in the high-risk population. CONCLUSIONS: SE-iFISH could effectively detect CRCs including circulating tumor cells (CTCs) and circulating tumor-derived endothelial cells (CTECs) and the detection of CRCs could benefit the differentiation of patients with benign and malignant SPNs.