Interventional and EBUS cytology in Sweden.

Interventional cytology was first introduced in Sweden in the late 1940ies by Sixten Franzén at the Karolinska University Hospital in Solna, Stockholm. In the early 1950ies, Nils Söderström started using the technique at the University Hospital in Lund. Cytology was successively established as common practice at the pathology departments in Sweden, and e.g. Solna and Lund today have a high rate of cytological samples. Over the years new techniques, such as endobronchial ultrasound (EBUS)-guided fine-needle aspirations, and analyses have been introduced, contributing to the maintained value of cytology as a diagnostic method. In this article, we present a brief history and the current situation of cytology in Sweden with focus on interventional and EBUS cytology.

The long-term prognostic and predictive capacity of cyclin D1 gene amplification in 2305 breast tumours.

BACKGROUND: Use of cyclin D1 (CCND1) gene amplification as a breast cancer biomarker has been hampered by conflicting assessments of the relationship between cyclin D1 protein levels and patient survival. Here, we aimed to clarify its prognostic and treatment predictive potential through comprehensive long-term survival analyses. METHODS: CCND1 amplification was assessed using SNP arrays from two cohorts of 1965 and 340 patients with matching gene expression array and clinical follow-up data of over 15 years. Kaplan-Meier and multivariable Cox regression analyses were used to determine survival differences between CCND1 amplified vs. non-amplified tumours in clinically relevant patient sets, within PAM50 subtypes and within treatment-specific subgroups. Boxplots and differential gene expression analyses were performed to assess differences between amplified vs. non-amplified tumours within PAM50 subtypes. RESULTS: When combining both cohorts, worse survival was found for patients with CCND1-amplified tumours in luminal A (HR = 1.68; 95% CI, 1.15-2.46), luminal B (1.37; 1.01-1.86) and ER+/LN-/HER2- (1.66; 1.14-2.41) subgroups. In gene expression analysis, CCND1-amplified luminal A tumours showed increased proliferation (P < 0.001) and decreased progesterone (P = 0.002) levels along with a large overlap in differentially expressed genes when comparing luminal A and B-amplified vs. non-amplified tumours. CONCLUSIONS: Our results indicate that CCND1 amplification is associated with worse 15-year survival in ER+/LN-/HER2-, luminal A and luminal B patients. Moreover, luminal A CCND1-amplified tumours display gene expression changes consistent with a more aggressive phenotype. These novel findings highlight the potential of CCND1 to identify patients that could benefit from long-term treatment strategies.

Superficial scrapings from breast tumors is a source for biobanking and research purposes.

Breast cancer is a unique tumor disease in terms of the stringent requirement of predictive biomarker assessments. As recommended by current international guidelines, the established markers consist of estrogen receptor (ER), progesterone receptor, human epidermal growth factor and Ki67, and are primarily analyzed by immunohistochemistry. However, new diagnostic methods based on microarray or next-generation sequencing on DNA and mRNA level are gaining ground. These analyses require fresh-frozen tumor tissue that is generally not available from tumors <10 mm in diameter, comprising almost 25% of all resected breast cancer at our department. We here present a simple and standardized method to generate material from small tumors without risking the histopathological examination. Furthermore, we show that the quality of this material is sufficient for subsequent analysis on mRNA, DNA, and epigenetic level. We were also able to use this method for isolation and expansion of cancer stem cells from the majority of tumors. Consequently, researches can be provided with clinically relevant material for translational studies. In conclusion, this method opens up a new possibility for usage of valuable fresh tumor material for research purposes, biobanking, and next-generation sequencing.

Digital droplet PCR TERT promoter mutational screening in fine needle aspiration cytology of thyroid lesions: A highly specific technique for pre-operative identification of high-risk cases.

BACKGROUND: Despite the advent of comprehensive molecular testing in surgical pathology, most centers still rely on the morphological assessment of fine-needle aspiration cytology (FNAC) to triage patients with thyroid nodules for surgery. Subsets of patients could benefit from the inclusion of molecular testing to increase the diagnostic and/or prognostic properties of the cytology analysis, including the assessment of TERT promoter mutations, an event coupled with thyroid malignancy, and poor prognosis. METHODS: In this prospective study, preoperative FNAC material from 65 cases was assessed for TERT promoter hotspot mutations C228T and C250T using the digital droplet PCR (ddPCR) technique on frozen pellets and re-evaluated postoperatively. RESULTS: Our cohort consisted of 15 B-III (23%), 26 B-IV (40%), 1 B-V (2%), and 23 (35%) B-VI lesions according to the Bethesda System for Reporting Thyroid Cytopathology. TERT promoter mutations were detected in 7 cases; 4 papillary thyroid carcinomas (all with preoperative B-VI status), two follicular thyroid carcinomas (one B-IV and one B-V status), and one poorly differentiated thyroid carcinoma (with B-VI status). All mutated cases were verified by mutational analysis of tumor tissue derived from postoperative formalin-fixed paraffin-embedded tissue, while all cases identified as wild-type on FNAC remained wild-type postoperatively. Moreover, the occurrence of a TERT promoter mutation was significantly associated with malignant disease and higher Ki-67 proliferation indices. CONCLUSION: In the present cohort, we found that ddPCR is a highly specific method for detecting high-risk TERT promoter mutations on thyroid FNAC material that could guide different surgical approaches in subsets of indeterminate lesions if reproduced in larger materials.

Proliferation and Immune Response Gene Signatures Associated with Clinical Outcome to Immunotherapy and Targeted Therapy in Metastatic Cutaneous Malignant Melanoma.

Targeted therapy (TT), together with immune checkpoint inhibitors (ICI), has significantly improved clinical outcomes for patients with advanced cutaneous malignant melanoma (CMM) during the last decade. However, the magnitude and the duration of response vary considerably. There is still a paucity of predictive biomarkers to identify patients who benefit most from treatment. To address this, we performed targeted transcriptomics of CMM tumors to identify biomarkers associated with clinical outcomes. Pre-treatment tumor samples from 28 patients with advanced CMM receiving TT (n = 13) or ICI (n = 15) were included in the study. Targeted RNA sequencing was performed using Ion AmpliSeq ™, followed by gene set enrichment analysis (GSEA) using MSigDB's Hallmark Gene Set Collection to identify gene expression signatures correlating with treatment outcome. The GSEA demonstrated that up-regulation of allograft rejection genes, together with down-regulation of E2F and MYC targets as well as G2M checkpoint genes, significantly correlated with longer progression-free survival on ICI while IFNγ and inflammatory response genes were associated with a better clinical outcome on TT. In conclusion, we identify novel genes and their expression signatures as potential predictive biomarkers for TT and ICI in patients with metastatic CMM, paving the way for clinical use following larger validation studies.

Multiplex immune protein profiling of fine-needle aspirates from patients with non-small-cell lung cancer reveals signatures associated with PD-L1 expression and tumor stage.

Biomarker signatures identified through minimally invasive procedures already at diagnosis of non-small-cell lung cancer (NSCLC) could help to guide treatment with immune checkpoint inhibitors (ICI). Here, we performed multiplex profiling of immune-related proteins in fine-needle aspirate (FNA) samples of thoracic lesions from patients with NSCLC to assess PD-L1 expression and identify related protein signatures. Transthoracic FNA samples from 14 patients were subjected to multiplex antibody-based profiling by proximity extension assay (PEA). PEA profiling employed protein panels relevant to immune and tumor signaling and was followed by Qlucore® Omics Explorer analysis. All lesions analyzed were NSCLC adenocarcinomas, and PEA profiles could be used to monitor 163 proteins in all but one sample. Multiple key immune signaling components (including CD73, granzyme A, and chemokines CCL3 and CCL23) were identified and expression of several of these proteins (e.g., CCL3 and CCL23) correlated to PD-L1 expression. We also found EphA2, a marker previously linked to inferior NSCLC prognosis, to correlate to PD-L1 expression. Our identified protein signatures related to stage included, among others, CXCL10 and IL12RB1. We conclude that transthoracic FNA allows for extensive immune and tumor protein profiling with assessment of putative biomarkers of important for ICI treatment selection in NSCLC.

Real-World Diagnostic Accuracy and Use of Immunohistochemical Markers in Lung Cancer Diagnostics.

OBJECTIVES: Accurate and reliable diagnostics are crucial as histopathological type influences selection of treatment in lung cancer. The aim of this study was to evaluate real-world accuracy and use of immunohistochemical (IHC) staining in lung cancer diagnostics. MATERIALS AND METHODS: The diagnosis and used IHC stains for small specimens with lung cancer on follow-up resection were retrospectively investigated for a 15-month period at two major sites in Sweden. Additionally, 10 pathologists individually suggested diagnostic IHC staining for 15 scanned bronchial and lung biopsies and cytological specimens. RESULTS: In 16 (4.7%) of 338 lung cancer cases, a discordant diagnosis of potential clinical relevance was seen between a small specimen and the follow-up resection. In half of the cases, there was a different small specimen from the same investigational work-up with a concordant diagnosis. Diagnostic inaccuracy was often related to a squamous marker not included in the IHC panel (also seen for the scanned cases), the case being a neuroendocrine tumor, thyroid transcription factor-1 (TTF-1) expression in squamous cell carcinomas (with clone SPT24), or poor differentiation. IHC was used in about 95% of cases, with a higher number of stains in biopsies and in squamous cell carcinomas and especially neuroendocrine tumors. Pre-surgical transthoracic samples were more often diagnostic than bronchoscopic ones (72-85% vs. 9-53% for prevalent types). CONCLUSIONS: Although a high overall diagnostic accuracy of small specimens was seen, small changes in routine practice (such as consequent inclusion of p40 and TTF-1 clone 8G7G3/1 in the IHC panel for non-small cell cancer with unclear morphology) may lead to improvement, while reducing the number of IHC stains would be preferable from a time and cost perspective.

Signet ring cell variant of follicular thyroid carcinoma: Report of two cases with focus on morphological, expressional and genetic characteristics.

BACKGROUND: Follicular thyroid carcinoma (FTC) is a neoplasm that presents with a micro-follicular growth pattern and a neutrally stained cytoplasm. Seldom, FTCs display unusual morphological characteristics - but given the rarity of these histological subtypes, little is known regarding the underlying genetics and the coupling to patient outcome. CASE PRESENTATION: We present two extremely rare cases of minimally invasive FTC with signet ring cell morphology (SRC-FTC) and describe the cytological, microscopic, immunohistochemical and molecular features for both tumors. Both were male patients, age 71 and 51 respectively. The preoperative cytology for both cases could not pinpoint a clear-cut signet ring cell morphology, but a tendency towards nuclear marginalization was seen. The tumors were 38 mm and 22 mm respectively and displayed evident signet ring cell features in subsets of tumor cells as well as degenerative stromal changes. The tumor cells were positive for TTF1, PAX8 and thyroglobulin, and the proliferation indexes were 4% and 1,9% respectively. Both tumors displayed capsular invasion, but not lymphovascular invasion. The tumors were sequenced for mutations in the TERT promoter and 22 additional cancer-related genes, interestingly; one patient was shown to carry a deleterious intronic variant in PTEN, a tumor suppressor gene coupled to thyroid tumorigenesis and Cowden syndrome. Both patients are alive and well awaiting postoperative radioiodine treatment. CONCLUSIONS: The SRC-FTCs described herein were small, TERT promoter wildtype tumors exhibiting low proliferation, thereby suggesting that these exceedingly rare lesions probably carry a favorable prognosis - although the scarce availability regarding descriptions of this tumor entity nevertheless might justify careful clinical monitoring and mandate investigations in larger case series.

TERT Promoter Mutation Spatial Heterogeneity in a Metastatic Follicular Thyroid Carcinoma: Implications for Clinical Work-Up.

Follicular thyroid carcinoma (FTC) is not routinely diagnosed by a preoperative fine needle aspiration biopsy (FNAB), and the final diagnosis relies on histopathological criteria visible upon microscopic examination of the excised tumor. Several markers have been proposed as helpful in the identification of follicular thyroid tumors with malignant potential and worse prognosis, of which the specific point mutations C250T and C228T in the Telomerase Reverse Transcriptase (TERT) promoter region seem to be particularly promising. We describe a patient presenting with a large pelvic mass, in which a core needle biopsy was consistent with follicular-patterned thyroid tissue positive for a Q61R NRAS mutation and the C228T TERT promoter mutation. Upon clinical investigation, a 60-mm lesion was detected in the right thyroid lobe. The ensuing FNAB was consistent with a follicular thyroid tumor, Bethesda IV, positive for the same NRAS mutation and both the C228T and C250T TERT promoter mutations. A total thyroidectomy was performed, and a widely invasive FTC was diagnosed. Tumor tissue samples from various parts of the primary lesion were investigated for TERT promoter mutations, displaying C228T in three samples and C250T in one. Interestingly, the C228T mutations showed a coupling to areas with high Ki-67 proliferation indexes. Our data indicate that TERT promoter mutations can exhibit spatial heterogeneity in FTCs, with implications for clinical management as well as providing insights into the molecular biology underlying the tumoral etiology.

Family History, Reproductive, and Lifestyle Risk Factors for Fibroadenoma and Breast Cancer.

BACKGROUND: To understand which breast cancer (BC) risk factors also increase the risk of fibroadenoma and investigate whether these factors have the same effect in BC patients with previous fibroadenoma. METHODS: Using multistate survival analysis on a large dataset (n = 58 322), we examined the effects of BC risk factors on transitions between three states: event-free, biopsy-confirmed fibroadenoma, and BC. Hazard ratios and corresponding 95% confidence intervals associated with covariate effects were estimated. Median follow-up time was 25.3 years. RESULTS: The mean ages at diagnosis of fibroadenoma and BC were 42.6 and 48.3 years, respectively. Participant characteristics known to increase the risk of BC were found to increase the risk of fibroadenoma (family history of BC and higher education). Participant characteristics known to confer protective effects for BC (older age at menarche, more children, and larger childhood body size) were found to reduce fibroadenoma risk. The effect sizes associated with the direct transitions from event-free to fibroadenoma and BC were generally not different for the covariates tested. Age at fibroadenoma diagnosis was associated with the transition from fibroadenoma to BC (hazard ratioper year increase = 1.07 [95% confidence interval = 1.03 to 1.12]). CONCLUSION: We showed that biopsy-confirmed fibroadenomas shared many risk factors with BC. More work is needed to understand the relationships between fibroadenoma and BC to identify women who are at high risk of developing BC after a fibroadenoma diagnosis.

Prognostic value of Ki67 analysed by cytology or histology in primary breast cancer.

AIMS: The accuracy of biomarker assessment in breast pathology is vital for therapy decisions. The therapy predictive and prognostic biomarkers oestrogen receptor (ER), progesterone receptor, HER2 and Ki67 may act as surrogates to gene expression profiling of breast cancer. The aims of this study were to investigate the concordance of consecutive biomarker assessment by immunocytochemistry on preoperative fine-needle aspiration cytology versus immunohistochemistry (IHC) on the corresponding resected breast tumours. Further, to investigate the concordance with molecular subtype and correlation to stage and outcome. METHODS: Two retrospective cohorts comprising 385 breast tumours with clinicopathological data including gene expression-based subtype and up to 10-year overall survival data were evaluated. RESULTS: In both cohorts, we identified a substantial variation in Ki67 index between cytology and histology and a switch between low and high proliferation within the same tumour in 121/360 cases. ER evaluations were discordant in only 1.5% of the tumours. From cohort 2, gene expression data with PAM50 subtype were used to correlate surrogate subtypes. IHC-based surrogate classification could identify the correct molecular subtype in 60% and 64% of patients by cytology (n=63) and surgical resections (n=73), respectively. Furthermore, high Ki67 in surgical resections but not in cytology was associated with poor overall survival and higher probability for axillary lymph node metastasis. CONCLUSIONS: This study shows considerable differences in the prognostic value of Ki67 but not ER in breast cancer depending on the diagnostic method. Furthermore, our findings show that both methods are insufficient in predicting true molecular subtypes.

Mandatory chromosomal segment balance in aneuploid tumor cells.

BACKGROUND: Euploid chromosome balance is vitally important for normal development, but is profoundly changed in many tumors. Is each tumor dependent on its own structurally and numerically changed chromosome complement that has evolved during its development and progression? We have previously shown that normal chromosome 3 transfer into the KH39 renal cell carcinoma line and into the Hone1 nasopharyngeal carcinoma line inhibited their tumorigenicity. The aim of the present study was to distinguish between a qualitative and a quantitative model of this suppression. According to the former, a damaged or deleted tumor suppressor gene would be restored by the transfer of a normal chromosome. If so, suppression would be released only when the corresponding sequences of the exogenous normal chromosome are lost or inactivated. According to the alternative quantitative model, the tumor cell would not tolerate an increased dosage of the relevant gene or segment. If so, either a normal cell derived, or, a tumor derived endogenous segment could be lost. METHODS: Fluorescence in Situ Hybridization based methods, as well as analysis of polymorphic microsatellite markers were used to follow chromosome 3 constitution changes in monochromosomal hybrids. RESULTS: In both tumor lines with introduced supernumerary chromosomes 3, the copy number of 3p21 or the entire 3p tended to fall back to the original level during both in vitro and in vivo growth. An exogenous, normal cell derived, or an endogenous, tumor derived, chromosome segment was lost with similar probability. Identification of the lost versus retained segments showed that the intolerance for increased copy number was particularly strong for 3p14-p21, and weaker for other 3p regions. Gains in copy number were, on the other hand, well tolerated in the long arm and particularly the 3q26-q27 region. CONCLUSION: The inability of the cell to tolerate an experimentally imposed gain in 3p14-p21 in contrast to the well tolerated gain in 3q26-q27 is consistent with the fact that the former is often deleted in human tumors, whereas the latter is frequently amplified. The findings emphasize the importance of even minor changes in copy number in seemingly unbalanced aneuploid tumors.

Array-CGH and multipoint FISH to decode complex chromosomal rearrangements.

BACKGROUND: Recently, several high-resolution methods of chromosome analysis have been developed. It is important to compare these methods and to select reliable combinations of techniques to analyze complex chromosomal rearrangements in tumours. In this study we have compared array-CGH (comparative genomic hybridization) and multipoint FISH (mpFISH) for their ability to characterize complex rearrangements on human chromosome 3 (chr3) in tumour cell lines. We have used 179 BAC/PAC clones covering chr3 with an approximately 1 Mb resolution to analyze nine carcinoma lines. Chr3 was chosen for analysis, because of its frequent rearrangements in human solid tumours. RESULTS: The ploidy of the tumour cell lines ranged from near-diploid to near-pentaploid. Chr3 locus copy number was assessed by interphase and metaphase mpFISH. Totally 53 chr3 fragments were identified having copy numbers from 0 to 14. MpFISH results from the BAC/PAC clones and array-CGH gave mainly corresponding results. Each copy number change on the array profile could be related to a specific chromosome aberration detected by metaphase mpFISH. The analysis of the correlation between real copy number from mpFISH and the average normalized inter-locus fluorescence ratio (ANILFR) value detected by array-CGH demonstrated that copy number is a linear function of parameters that include the variable, ANILFR, and two constants, ploidy and background normalized fluorescence ratio. CONCLUSION: In most cases, the changes in copy number seen on array-CGH profiles reflected cumulative chromosome rearrangements. Most of them stemmed from unbalanced translocations. Although our chr3 BAC/PAC array could identify single copy number changes even in pentaploid cells, mpFISH provided a more accurate analysis in the dissection of complex karyotypes at high ploidy levels.

Evaluation of exome sequencing to estimate tumor burden in plasma.

Accurate estimation of systemic tumor load from the blood of cancer patients has enormous potential. One avenue is to measure the presence of cell-free circulating tumor DNA in plasma. Various approaches have been investigated, predominantly covering hotspot mutations or customized, patient-specific assays. Therefore, we investigated the utility of using exome sequencing to monitor circulating tumor DNA levels through the detection of single nucleotide variants in plasma. Two technologies, claiming to offer efficient library preparation from nanogram levels of DNA, were evaluated. This allowed us to estimate the proportion of starting molecules measurable by sequence capture (<5%). As cell-free DNA is highly fragmented, we designed and provide software for efficient identification of PCR duplicates in single-end libraries with a varying size distribution. On average, this improved sequence coverage by 38% in comparison to standard tools. By exploiting the redundant information in PCR-duplicates the background noise was reduced to ∼1/35,000. By applying our optimized analysis pipeline to a simulation analysis, we determined the current sensitivity limit to ∼1/2400, starting with 30 ng of cell-free DNA. Subsequently, circulating tumor DNA levels were assessed in seven breast- and one prostate cancer patient. One patient carried detectable levels of circulating tumor DNA, as verified by break-point specific PCR. These results demonstrate exome sequencing on cell-free DNA to be a powerful tool for disease monitoring of metastatic cancers. To enable a broad implementation in the diagnostic settings, the efficiency limitations of sequence capture and the inherent noise levels of the Illumina sequencing technology must be further improved.

Microenvironment-Dependent Phenotypic Changes in a SCID Mouse Model for Malignant Mesothelioma.

BACKGROUND AND AIMS: Malignant mesothelioma is an aggressive, therapy-resistant tumor. Mesothelioma cells may assume an epithelioid or a sarcomatoid phenotype, and presence of sarcomatoid cells predicts poor prognosis. In this study, we investigated differentiation of mesothelioma cells in a xenograft model, where mesothelioma cells of both phenotypes were induced to form tumors in severe combined immunodeficiency mice. METHODS: Xenografts were established and thoroughly characterized using a comprehensive immunohistochemical panel, array comparative genomic hybridization (aCGH) of chromosome 3, fluorescent in situ hybridization, and electron microscopy. RESULTS: Epithelioid and sarcomatoid cells gave rise to xenografts of similar epithelioid morphology. While sarcomatoid-derived xenografts had higher growth rates, the morphology and expression of differentiation-related markers was similar between xenografts derived from both phenotypes. aCGH showed a convergent genotype for both xenografts, resembling the original aggressive sarcomatoid cell sub-line. CONCLUSION: Human mesothelioma xenografts from sarcomatoid and epithelioid phenotypes converged to a similar differentiation state, and genetic analyses suggested that clonal selection in the mouse microenvironment was a major contributing factor. This thoroughly characterized animal model can be used for further studies of molecular events underlying tumor cell differentiation.

Adaptation of a commercial fluorescent in situ hybridization test to the diagnosis of malignant cells in effusions.

In effusion cytology, adjuvant techniques are often needed for the differentiation of reactive proliferating mesothelial cells and malignant cells. In the case of malignancy the further challenge is to distinguish metastatic tumors from the primary malignant mesothelioma. Fluorescence in situ hybridization (FISH) of cells in interphase is an accurate method to monitor the genetic status of cells, detecting aneuploid signals and gene deletions. Moreover, it has been proposed that a homozygous deletion of the p16(INK4A) gene could more specifically identify malignant mesothelial cells among the exfoliated cells. The first objective of this study was to adapt the commercial FISH-test, UroVysion originally designed for the cytological diagnosis of bladder cancer, to the analysis of cells in effusions. The second objective was to test the clinical utility of the test. Sixty-eight pleural effusions were evaluated. The cytological diagnosis was malignant in 29 cases, inconclusive in 24 cases and benign in 15 cases. The independently verified final diagnoses were mesothelioma in 21 cases, metastatic cancer in 29 and benign in 18 cases. The algorithm for aneuploidy distinguished almost all tested malignant conditions from benign ones, also those with inconclusive cytology. The 9p21 locus, carrying the p16(INK4A) gene, was homozygously deleted in two of the metastatic cancers, while this was seen in 12 of the 21 malignant mesotheliomas. Thus the commercial UroVysion-test can be used to accurately distinguish malignant and reactive cells in effusions, particularly when cytology is inconclusive. The test may also indicate presence of MM.

Segmental duplications and evolutionary plasticity at tumor chromosome break-prone regions.

We have previously found that the borders of evolutionarily conserved chromosomal regions often coincide with tumor-associated deletion breakpoints within human 3p12-p22. Moreover, a detailed analysis of a frequently deleted region at 3p21.3 (CER1) showed associations between tumor breaks and gene duplications. We now report on the analysis of 54 chromosome 3 breaks by multipoint FISH (mpFISH) in 10 carcinoma-derived cell lines. The centromeric region was broken in five lines. In lines with highly complex karyotypes, breaks were clustered near known fragile sites, FRA3B, FRA3C, and FRA3D (three lines), and in two other regions: 3p12.3-p13 ( approximately 75 Mb position) and 3q21.3-q22.1 ( approximately 130 Mb position) (six lines). All locations are shown based on NCBI Build 36.1 human genome sequence. The last two regions participated in three of four chromosome 3 inversions during primate evolution. Regions at 75, 127, and 131 Mb positions carry a large ( approximately 250 kb) segmental duplication (tumor break-prone segmental duplication [TBSD]). TBSD homologous sequences were found at 15 sites on different chromosomes. They were located within bands frequently involved in carcinoma-associated breaks. Thirteen of them have been involved in inversions during primate evolution; 10 were reused by breaks during mammalian evolution; 14 showed copy number polymorphism in man. TBSD sites showed an increase in satellite repeats, retrotransposed sequences, and other segmental duplications. We propose that the instability of these sites stems from specific organization of the chromosomal region, associated with location at a boundary between different CG-content isochores and with the presence of TBSDs and "instability elements," including satellite repeats and retroviral sequences.