Cytogenetic and molecular cytogenetic findings in dedifferentiated liposarcoma with neural-like whorling pattern and metaplastic bone formation

Dedifferentiated liposarcoma is a subtype of liposarcoma characterized by juxtaposition of well-differentiated liposarcoma with a nonadipocytic sarcoma. A peculiar form of dedifferentiated liposarcoma has been described, characterized by a nonlipogenic component with a neural-like whorling pattern of growth and metaplastic bone formation. We report the cytogenetic and molecular genetic findings of this peculiar form of dedifferentiation in a retroperitoneal tumor found in a 58-year-old female. The neoplasm had typical histologic findings and a complex karyotype characterized by several numeric and structural chromosomal abnormalities, including the presence of ring and giant rod chromosomes. Molecular cytogenetic studies found high levels of amplification of the MDM2 oncogene, consistent with the amplification of the 12q14 chromosome band, a cytogenetic abnormality commonly found in these tumors. These findings indicate that, despite its unique and peculiar morphologic features, this unusual type of dedifferentiated liposarcoma shares many of the cytogenetic features and molecular genetic abnormalities found in other forms of dedifferentiation. The specific cytogenetics and molecular determinants of these peculiar morphologic findings, however, remain unknown.     

Cytogenetic and molecular genetic analyses of liposarcoma and its soft tissue simulators: recognition of new variants and differential diagnosis

Liposarcoma is one of the most common sarcomas of adults. Its differential diagnosis and accurate subclassification are often problematic; the latter is also important with regard to appropriate treatment and prognosis. We studied a series of 23 liposarcomas that had unusual or previously undescribed features and 10 liposarcoma simulators and correlated the morphologic, cytogenetic, and molecular genetic findings. We found that use of cytogenetic-molecular genetic techniques aids in the distinction between myxoid-round cell liposarcoma and their simulators, chondroid lipoma, myxoid spindle cell-pleomorphic lipoma, cellular intramuscular myxoma, and myxofibrosarcoma. Poorly differentiated forms of round cell liposarcoma lacking morphologic evidence of lipogenesis can also be diagnosed using these techniques; however, the techniques do not aid in distinguishing low-grade myxoid from high-grade round cell liposarcomas. This study also shows that retroperitoneal liposarcomas with myxoid liposarcoma-like zones are part of the morphologic spectrum of well-differentiated-dedifferentiated liposarcoma rather than true myxoid liposarcomas. Perhaps most importantly, our results provide the first molecular genetic evidence that true mixed liposarcomas (mixed well-differentiated and myxoid liposarcoma) do indeed exist. They also unequivocally demonstrate the existence of small, round cell variants of pleomorphic liposarcoma that closely simulate myxoid-round cell liposarcoma.     

Distinction between lipoma and liposarcoma by MDM2 alterations: a case report of simultaneously occurring tumors and review of the literature

We investigated a lipoma and a well-differentiated/dedifferentiated liposarcoma (WD/DDL), occurring simultaneously in one patient for the possible role of p53 and mdm2 in the molecular oncogenesis of liposarcoma and tumor progression. The hypothesis tested was if there is a continuum in the development from lipoma to liposarcoma. Lipoma was characterized by a lack of p53 mutation, p53 LOH and p53 protein expression, as well as by mdm2 amplification and mdm2 protein expression. p53 mutation and p53 LOH were found neither in the well-differentiated nor in the dedifferentiated parts of the liposarcoma. In contrast, mdm2 amplification and an increase in mdm2 protein expression were found to be associated with malignancy and dedifferentiation, whereas p53 protein expression was only slightly increased. These findings indicate that mdm2 constitutes one of the most common targets for molecular aberration in WD/DDL. We suggest that mdm2 is a marker distinguishing between ordinary lipoma and well-differentiated liposarcoma, and that the genesis of these tumors is different.     

Liposarcoma: new entities and evolving concepts

Liposarcoma is the most common soft tissue sarcoma and accounts for approximately 20% of all mesenchymal malignancies. In the last decade, the results of several studies have led to the delineation of new variants as well as to the introduction of new concepts, mainly as a result of the fruitful interactions between genetics and pathology. Spindle cell liposarcoma represents an uncommon variant of well-differentiated liposarcoma. It tends to occur in adults and often involves the subcutaneous soft tissue. However, from the observation of a larger number of cases, the anatomic distribution of spindle cell liposarcoma seems to be comparable to that of the other well-differentiated liposarcoma subtypes. Spindle cell liposarcoma tends to recur locally and may dedifferentiate. Morphologically it is composed of a fairly bland neural-like spindle cell proliferation set in a fibrous and/or myxoid background and is associated with an atypical lipomatous component. Great debate has been generated by the introduction of the term atypical lipoma to emphasize the fact that well-differentiated liposarcoma shows risk of local recurrence but no potential for metastasis. In our opinion well-differentiated liposarcoma and atypical lipoma should be considered synonyms that describe lesions identical both morphologically and kayotypically. Dedifferentiated liposarcoma is a distinct type of liposarcoma in which transition from low-grade to high-grade nonlipogenic morphology within a well-differentiated liposarcoma is observed. The transition usually occurs in an abrupt fashion; however, in rare cases it can be more gradual. Recently, it also has been proposed that dedifferentiated liposarcoma should be further classified into low and high grade. Dedifferentiated liposarcoma rarely exhibits heterologous (most often myoid) differentiation. A peculiar "neural-like whorling pattern" of dedifferentiation also has been described recently. Surprisingly, the clinical outcome of dedifferentiated liposarcoma is less aggressive that in other high-grade pleomorphic sarcomas but genetic as well as molecular data exist that may partiallyjustify such a discrepancy. Myxoid and round cell liposarcoma, even if still classified by the World Health Organization as two distinct subtypes, share both clinical and morphologic features. Lesions combining both patterns are very frequent and wide agreement exists in considering round cell liposarcoma as the high-grade counterpart of myxoid liposarcoma. Furthermore, myxoid and round cell liposarcoma share the same characteristic chromosome change. Albeit rare, it has been recently shown that liposarcoma indeed can occur as a primary skin lesion. It often presents clinically as a dome-shaped or polypoid lesion that, histologically, most frequently shows high-grade morphologic features but carries a comparatively good prognosis. Considering currently available data, the most logical classification of liposarcoma is into three main groups: (1) well-differentiated liposarcoma (including adipocytic, sclerosing, inflammatory, spindle-cell, and dedifferentiated variants), characterized by ring or long markers chromosomes derived from the long arm of chromosome 12; (2) myxoid and round cell (poorly differentiated myxoid) liposarcoma, characterized in most cases by a reciprocal translocation t(12;16)(q13;p11); and (3) pleomorphic liposarcoma, characterized by complex karyotypes.     

Distinction Between Lipoma and Liposarcoma by MDM2 Alterations: A Case Report of Simultaneously Occurring Tumors and Review of the Literature

We investigated a lipoma and a well-differentiated/dedifferentiated liposarcoma (WD/DDL), occurring simultaneously in one patient for the possible role of p53 and mdm2 in the molecular oncogenesis of liposarcoma and tumor progression. The hypothesis tested was if there is a continuum in the development from lipoma to liposarcoma. Lipoma was characterized by a lack of p53 mutation, p53 LOH and p53 protein expression, as well as by mdm2 amplification and mdm2 protein expression. p53 mutation and p53 LOH were found neither in the well-differentiated nor in the dedifferentiated parts of the liposarcoma. In contrast, mdm2 amplification and an increase in mdm2 protein expression were found to be associated with malignancy and dedifferentiation, whereas p53 protein expression was only slightly increased. These findings indicate that mdm2 constitutes one of the most common targets for molecular aberration in WD/DDL. We suggest that mdm2 is a marker distinguishing between ordinary lipoma and well-differentiated liposarcoma, and that the genesis of these tumors is different.     

Does parosteal liposarcoma differ from other atypical lipomatous tumors/well-differentiated liposarcomas? A molecular cytogenetic study using combined multicolor COBRA-FISH karyotyping and array-based comparative genomic hybridization

Parosteal adipocytic tumors of the bone are extremely rare. As a result, (cyto-) genetic data on this entity are essentially lacking. In the literature there is debate as to whether these lesions should be classified according to the criteria used in soft-tissue tumor pathology, or if they should be considered a separate bone tumor entity. Here we present a 68-year-old male patient with a tumor in his right upper leg diagnosed as parosteal atypical lipomatous tumor/well-differentiated liposarcoma (ALT/WDLS) on the basis of clinico-radiologic and pathologic findings. Molecular cytogenetic investigations using combined binary ratio labeling fluorescence in situ hybridization and array comparative genomic hybridization showed abnormalities, which are in accordance with the histologic appearance of an atypical lipomatous tumor/well-differentiated liposarcoma. Therefore, on the basis of these molecular cytogenetic investigations, we conclude that parosteal liposarcoma is not a separate entity but should be categorized within the spectrum of soft-tissue ALT/WDLS.     

Promiscuous translocations of chromosome arm 17q in human neuroblastomas

Deletions of chromosome arm 1p and amplification of the MYCN oncogene are well-recognized genetic changes in neuroblastoma cells. Technical difficulties in cytogenetic analysis of this tumour have hampered the recognition of other recurring abnormalities, but recent use of molecular cytogenetic techniques has indicated significant involvement of chromosome arm 17q. In primary tumours and in cell lines, a recurrent unbalanced translocation t(1p;17q) has been identified by fluorescence in situ hybridization. We confirm the occurrence of this translocation in primary tumours and, in addition, we describe seven new structural rearrangements all of which result in gain of 17q in tumour cells. These rearrangements involved chromosome arms 9p, 10q, 11p, 14q, and 16q. Triplication of the 17q arm was seen in one case. The 17q breakpoint was most commonly q21. All these 17q changes were found in near-diploid tumours. We have also reviewed the literature for neuroblastoma karyotypes involving 17q abnormalities; taken in conjunction with our findings this indicates a remarkable promiscuity of translocation partners, with more than 20 different chromosome regions involved in 17q translocations. Genes Chromosom. Cancer 19:143–149, 1997 © 1997 Wiley-Liss Inc.     

Consistent cytogenetic aberrations in hepatoblastoma: A common pathway of genetic alterations in embryonal liver and skeletal muscle malignancies?

Cytogenetic analyses of four consecutive hepatoblastomas revealed near-diploid stemline karyotypes with relatively simple chromosome aberrations. Cytogenetic abnormalities shared by each tumor included trisomy for all of part of chromosome 2 and trisomy for chromosome 20. In two cases, partial trisomy for chromosome 2 resulted from direct duplication of long arm material with the shortest region of overlap being 2q23-2q35. In one tumor, each metaphase also contained a variable number of double minute chromosomes found not to derive from NMYC amplification. Interestingly, trisomy for 2q and trisomy 20 are also characteristic events in pediatric embryonal rhabdomyosarcomas. Furthermore, others have reported loss of heterozygosity for the short arm of chromosome 11 in both hepatoblastoma and childhood embryonal rhabdomyosarcoma, and both these malignant diseases are associated with Beckwith-Wiedemann syndrome. These cytogenetic and molecular findings suggest a parallel pathway of genetic steps in the initiation and/or progression of tumors of embryonal liver and skeletal muscle.     

DISTINCT mdm2/p53 EXPRESSION PATTERNS IN LIPOSARCOMA SUBGROUPS: IMPLICATIONS FOR DIFFERENT PATHOGENETIC MECHANISMS

Recent findings have indicated that TP53 inactivation in sarcomas may result from mutation and/or deletion of the TP53 gene or, alternatively, from binding to the MDM2 gene products. To investigate further a possible role of the two genes in sarcomas, 24 large and deep-seated lipomas and 74 liposarcomas of various subtypes were analysed for mdm2 and p53 overexpression by immunocytochemistry. Nineteen cases of the same series were also molecularly analysed for both MDM2 gene amplification and TP53 mutations, and a further ten cases for non-random chromosomal abnormalities. In the retroperitoneal well-differentiated–dedifferentiated (WD–DD) group, 15/16 WD and 8/8 DD liposarcomas displayed the mdm2+/p53+ phenotype, consistent with MDM2 gene amplification in the absence of TP53 mutations. In the non-retroperitoneal WD–DD group, 5/11 WD liposarcomas also retained the mdm2+/p53+ phenotype whereas all DD liposarcomas showed an immunophenotype and, when assessed, a genotype consistent with mutant TP53. Null mdm2 immunophenotype, coupled with evidence of a specific chromosome translocation t(12;16), was constantly observed in both the usual and the cellular subtypes of myxoid liposarcoma, three cases of which also showed TP53 alterations at the genetic or protein level. Neither mdm2 nor p53 overexpression was observed in the lipomas. The results show the existence of three main pathogenetically distinct groups of liposarcoma. The first retroperitoneal WD–DD group, which represents a novel class of tumours within a single histological category of sarcoma, where MDM2-mediated inactivation of p53 could be related to the pathogenetic mechanism. The second is the non-retroperitoneal WD–DD group, where the TP53 mutations appear to correlate with the dedifferentiation process. The third is the myxoid group, which is characterized by its own unique cytogenetic profile and never shows any involvement of TP53 or MDM2 genes. As for diagnostic significance, the absence of mdm2 and p53 reactivity in lipomas seems to represent a useful marker for differential diagnosis from lipoma-like WD liposarcomas. © 1997 by John Wiley & Sons, Ltd.     

BCR/ABL amplification in chronic myelocytic leukemia blast crisis following imatinib mesylate administration

The onset of accelerated phase or blast crisis of chronic myelocytic leukemia (CML) is usually associated with the acquisition of new chromosome abnormalities in addition to the t(9;22)(q34;q11) that is characteristic of the chronic phase CML. We describe the cytogenetic and molecular genetic findings in two cases of myelocytic blast crisis of CML, one occurring 6 months after commencing treatment with the ABL-specific tyrosine kinase inhibitor imatinib mesylate (STI571, Glivec, or Gleevec) and the second treated with imatinib mesylate for established blast crisis. In both cases, multiple secondary cytogenetic abnormalities were observed at transformation, with homogeneously staining regions that were shown to contain BCR/ABL amplification by fluorescence in situ hybridization appearing after imatinib mesylate administration. BCR/ABL amplification is emerging as an important mechanism of acquired resistance to imatinib mesylate.     

Rearrangement of chromosomal region 8q11-13 in lipomatous tumours: correlation with lipoblastoma morphology

Cytogenetics is of considerable value when diagnosing lipomatous tumours, as different tumour types have different more or less specific chromosomal abnormalities. One such entity is lipoblastoma, which is a benign lipomatous tumour that often exhibits rearrangements of chromosome bands 8q11-13, and the gene PLAG1 has been implicated as the target of these chromosomal changes. All lipomatous tumours karyotyped at the Norwegian Radium Hospital were reviewed, looking for rearrangements of 8q11-13. Five tumours exhibiting chromosomal abnormalities affecting this region were found. Only one of them was morphologically diagnosed as a lipoblastoma, two being classified as lipomas, one as a hibernoma, and one as a well-differentiated liposarcoma. The two tumours successfully analysed with bacterial artificial chromosomes (BACs) covering the gene PLAG1 showed involvement of this gene in the rearrangement. The findings raise the question as to what extent the diagnosis lipoblastoma should be based on histopathological or cytogenetic/molecular data or a combination thereof. When karyotypic information from this series was combined with available literature data, it was found that the sensitivity of 8q11-13 rearrangements for diagnosing lipoblastomas when found in a lipomatous tumour was 77% and that the corresponding specificity was 98%. The validity of these calculations of the diagnostic information provided by the cytogenetic findings is, of course, totally dependent on the morphological diagnosis made in each case. Regardless of what the precise phenotypic diagnosis was, it is suggested that lipomatous tumours with 8q11-13 rearrangement constitute a distinct pathogenetic entity. When selective therapies tailor-made against the specific pathogenetic rearrangement become available, it will become mandatory to pay more attention to the genetic constitution of the tumour cells than to their phenotypic appearance.     

Neuroblastoma tumour genetics: clinical and biological aspects

Neuroblastoma tumour cells show complex combinations of acquired genetic aberrations, including ploidy changes, deletions of chromosome arms 1p and 11q, amplification of the MYCN oncogene, and-most frequently-gains of chromosome arm 17q. Despite intensive investigation, the fundamental role of these features in neuroblastoma initiation and progression remains to be understood. Nonetheless, great progress has been made in relating tumour genetic abnormalities to tumour behaviour and to clinical outcome; indeed, neuroblastoma provides a paradigm for the clinical importance of tumour genetic abnormalities. Knowledge of MYCN status is increasingly being used in treatment decisions for individual children, and the clinical value of 1p and 17q data as adjuncts or refinements in risk stratification is under active investigation. Reliable detection of these molecular cytogenetic features should be regarded as mandatory for all new cases at presentation.     

Neuroblastoma -Clinical Applications of Molecular Parameters

At least two genetic events have been identified which are characteristic of certain neuroblastomas. These are loss of a critical region on the short arm of chromosome 1, and amplification of the MYCN proto-oncogene. Our studies suggest that the two genetic events may be related, and that loss of heterozygosity (LOH) for chromosome 1p may precede the development of amplification. RAS gene mutations appear to be rare, and no other oncogene has been shown to be consistently activated by amplification or other mechanism. LOH for chromosome 14q has been identified recently, but its frequency and significance is not clear. Based on flow cytometric analysis of DNA content, tumour cytogenetics and molecular studies, three distinct genetic subsets of neuroblastomas are emerging. The first is characterized by a hyperdiploid or near-triploid modal karyotype, with few if any cytogenetic rearrangements. These patients are generally less than one year of age with localized disease and a good prognosis. The second group is characterized by a near-diploid or near-tetraploid karyotype, with no consistent rearrangement identified to date. They are generally older patients with more advanced stages of disease that progress slowly and are ultimately fatal. The third group is characterized by a near-diploid or tetraploid karyotype, with deletions or LOH for chromosome 1p, amplification of MYCN, or both. These patients are generally older with advanced stages of disease which is rapidly progressive. Thus, genetic analysis of neuroblastoma cells by karyotype, flow cytometry and determination of MYCN copy number provides information that has prognostic significance and can more appropriately direct the choice of treatment. A better understanding of these genetic abnormalities and the biochemical pathways that they effect may provide insights into malignant transformation and progression, as well as provide targets for future therapeutic approaches.     

Correlation of cytogenetic analysis and loss of heterozygosity studies in human diffuse astrocytomas and mixed oligo-astrocytomas.

The aims of this study were to correlate cytogenetic studies and molecular genetic loss of heterozygosity (LOH) analyses in human astrocytomas and mixed oligo-astrocytomas, and to locate putative tumor suppressor genes on chromosome 10. Paired blood and tumor samples from 53 patients were analyzed. The tumors included 45 diffuse astrocytomas (39 grade 4, 4 grade 3, and 2 grade 2), 1 astroblastoma, and 7 mixed oligo-astrocytomas (2 grade 4, 4 grade 3, and 1 grade 2). By cytogenetic analyses the most common numeric chromosome abnormalities were +7, -10, -13, -14, -17, +19, -22, and -Y. The most common structural abnormalities involved chromosome arms 1p, 1q, 5p, and 9p. By LOH and dosage analysis the most common molecular genetic abnormalities were of chromosome arms 5p, 6p, 7q, 9p, 10p, 10q, 13q, 14q, 17p, and 19p. When the results of all methods were combined, the most commonly abnormal chromosomes were, in descending frequency, 10, Y, 17, 7, 13, and 9. In 80 percent of cases the cytogenetic and molecular genetic studies were concordant. LOH studies were more sensitive in detecting loss of genetic material than cytogenetic analyses and accounted for 60% of the discordant results. When there were structural abnormalities, such as translocations or inversions, cytogenetic analysis was more sensitive in detecting an abnormality than molecular genetic studies. In addition to the 24 tumors which appeared to lose an entire copy of chromosome 10, there were 10 tumors with molecular genetic or cytogenetic evidence of loss of only a portion of chromosome 10. The genetic analyses of these tumors suggest that there are 2 regions on chromosome 10 that may contain potential tumor suppressor genes. One lies distal to locus D10S22 from 10q22 to 10qter, and the other lies proximal to locus TST1 on the 10q arm near the centromere or on the 10p arm.     

Cytogenetic and molecular cytogenetic findings of intimal sarcoma

Intimal sarcomas are exceptionally rare tumors that arise from the tunica intima of large vessels. Most intimal sarcomas are high-grade tumors that exhibit fibroblastic or myofibroblastic differentiation. We report the cytogenetic findings of a tumor from a 57-year-old man. The tumor had a pleomorphic and spindle-cell morphology, and it also exhibited a complex karyotype that was characterized by several numeric and structural chromosomal abnormalities. Molecular cytogenetic analysis showed amplification of MDM2, SAS, and CDK4, but not of HMGA2, ATF1, or DDIT3, which supported the findings of a previous comparative genomic hybridization study. Further studies are needed to determine whether the cytogenetic abnormalities found in this case are recurrent events for this poorly characterized malignancy.     

Ring chromosome formation as a novel escape mechanism in patients with inverted duplication and terminal deletion

Ring chromosomes are rare cytogenetic findings and are associated at phenotypic level with mental retardation and congenital abnormalities. Features specific for ring chromosome syndromes often overlap with the features of terminal deletions for the corresponding chromosomes. Here, we report a case of a ring chromosome 14 which was identified by conventional cytogenetics and shown to have a terminal deletion and an additional inverted duplication with a triplication by using large insert clone and oligo array-comparative genomic hybridization (array-CGH), fluorescence in situ hybridization (FISH) and multiplex ligation-dependent probe amplification (MLPA). The combination of an inverted duplication with a terminal deletion in a ring chromosome is of special interest for the described syndromes of chromosome 14. The presented findings might explain partly overlapping clinical features described in terminal deletion, duplication and ring chromosome 14 cases, as these rearrangements can be easily overlooked when performing GTG-banding only. Furthermore, we suggest that ring chromosome formation can act as an alternative chromosome rescue next to telomere healing and capture, particularly for acrocentric chromosomes. To our knowledge, this is the first time an inverted duplication with a terminal deletion in a ring chromosome is identified and characterized using high-resolution molecular karyotyping. Systematic evaluation of ring chromosomes by array-CGH might be especially useful in distinguishing cases with a duplication/deletion from those with a deletion only.     

Cytogenetic findings in blastoid mantle cell lymphoma

A subset of mantle cell lymphoma (MCL) tumors has blastoid morphology, and a number of morphologic variants of blastoid MCL have been described in the literature. In this report, we document the cytogenetic findings in 27 cases of blastoid MCL. Conventional cytogenetic analyses were performed on bone marrow aspirates involved by MCL from 27 patients. There were 14 men and 13 women with a median age of 63 years (range, 40-79 years). Diagnostic tissue biopsy and bone marrow specimens were reviewed, and cases were divided into 2 morphologic groups: classic (12 cases) and pleomorphic (15 cases), as defined in the World Health Organization classification. All tumors had an immunophenotype compatible with MCL, were positive for cyclin D1, and carried the t(11;14). Twenty-four cases had complex karyotypes with 3 or more chromosomal abnormalities in addition to the t(11;14). In classic blastoid MCL, abnormalities of chromosomes 13, 18, and 8 were most common. In pleomorphic blastoid MCL, abnormalities of chromosomes 13, 17, and 3 were most frequent. Chromosome 22 abnormalities were detected exclusively in the pleomorphic group. Tumors in which the neoplastic cells showed prominent nucleoli had a significantly higher frequency of chromosome 17 abnormalities (P = 0.03). We conclude that blastoid MCL tumors often show complex cytogenetic aberrations. Some abnormalities correlate with morphologic features, suggesting that morphologic variants of blastoid MCL may arise via different molecular pathways.     

Acute leukemias: a paradigm for the integration of new technologies in diagnosis and classification.

Acute leukemias (involving the myeloid and lymphoid lineages) and the related myelodysplastic and myeloproliferative syndromes are an extremely heterogeneous group of clonal neoplastic disorders. Traditionally, these diseases were classified solely by morphologic and cytochemical criteria, as originally championed in the 1976 French-American-British Group proposal. Since 1976, however, phenomenal scientific advances have provided new insights into the biology and genetics of the acute leukemias and myeloproliferative/myelodysplastic diseases. Immunophenotyping approaches have begun to supplant more traditional cytochemical assays for lineage determination, and cytogenetic and molecular genetic studies led to the identification and cloning of the genes involved in a large number of the recurrent genetic abnormalities in these diseases. New automated molecular technologies, reviewed in this article, now allow rapid and sensitive detection of these genetic abnormalities in leukemic cells. Such immunophenotyping and molecular genetic information is now considered crucial for both diagnostic and therapeutic decision-making. As our scientific knowledge and diagnostic sophistication increases, classification schemes for these disorders based solely on morphologic features are increasingly seen as unsatisfactory. This review highlights progress in the development of new morphologic classification schemes for the acute leukemias that integrate critical clinical, biologic, and genetic features.