Pamela M. Ward

Cell detachment and metastasis

SummaryCancer cell detachment in three distinct and critical parts of the metastatic cascade is discussed.1.The detachment of cancer cells from their parent tumors is an initial early event in metastasis. The site of detachment with respect to proximity to blood vessels may determine the initial dissemination route. Many factors affect cell detachment; we specifically consider the effects of growth-rate, necrosis, enzyme activity, and stress on cell release in terms of metastasis-promoting mechanisms.2.Detachment is also discussed in relation to active cancer cell locomotion, where localized detachment from the substratum is a prerequisite for translatory movement. The importance of active cell movement in tissue invasion has only recently been assessed, and, in the case of at least some human malignant melanomas, a zone of actively moving cancer cells is believed to precede the growing body of the tumor.3.The secondary release of cancer cells from temporary arrest sites at the vascular endothelium consequent upon intravascular dissemination is also a major area of investigation. Circulating cancer cells arrest at vascular endothelium or are impacted in small vessels, however, most are released into the circulation and subsequently perish. The blood stream is a hostile environment, and it is probable that cancer cells are sufficiently damaged in translocation by hemodynamic trauma and humoral factors such that they easily detach or are ‘sheared-off’ the vascular endothelium by blood flow. Another possibility is that in some cases they are processed by ‘first organ encounters’ and perish before or shortly after arriving in a second organ. Animal studies have shown that, following intravenous injection, 60–100% of the injected dose of viable cancer cells are initially arrested in the lungs, but very few remain after 24hr. As it is only those retained cells which produce tumors, the mechanisms involved in this secondary release, which occurs in all organs so far examined, are critical to any understanding of the metastatic cascade and metastatic inefficiency.The arrest of cancer cells at the vascular endothelium and their subsequent release have been associated with the presence of platelets, and the deposition of fibrin and manipulation of platelet-aggregating mechanisms and fibrinolysis are discussed in terms of their antimetastatic effects. The role of the reticulo-endothelial system, natural killer cells, and polymorphs is discussed in relation to cancer cell clearance from blood vessels and also to inherent cancer cell properties which may act to inhibit their metastasis.Although detachment of cancer cells from a primary tumor may be regarded as metastasis promoting, secondary release of cancer cells may be associated with inhibition of metastasis.

ABERRANT ANTIGEN EXPRESSION DETECTED BY MULTIPARAMETER THREE COLOR FLOW CYTOMETRY IN INTERMEDIATE AND HIGH GRADE B-CELL LYMPHOMAS

The aberrant expression of antigens (Ag) in lymphoproliferative disorders may cause a diagnostic problem when single parameter immunohistochemical assays are performed on frozen or paraffin sections because coexpression by relevant cells is not determined. This aberrant expression also raises the question as to whether mixed lineage (biphenotypic) lymphoid proliferations exist. Marrow (6) and extramedullary (20) tissues from 26 patients with diffuse, intermediate and high grade, B-cell lymphomas (IWF E=1, F=1, G=19, H=1 and J=4) were analyzed with 19 markers using 3-color flow cytometry. The percentages (%) of patients with double Ag coexpression in at least 20% of the CD19+ or CD20+ lymphoma cells were: stem cell (SC) Ag: CD10 = 58 and CD34 = 15; T-cell Ag: CD2 = 38, CD5 = 19 and CD7 = 19; myeloid (My) Ag: CD13 = 19 and CD33 = 8. The corresponding % with unusual triple Ag coexpression in at least 10% of the CD19+ B-cells were SC+T+ Ag: CD10CD2 = 50, CD10CD5 = 27, CD10CD7 = 38, CD34CD2 = 31, CD34CD5 = 19 and CD34CD7 = 27; T+T+ Ag: CD2CD5 = 35, CD2CD7 = 42 and CD5CD7 = 31; T+My+ Ag: CD2CD13 = 35 and CD2CD33 = 12; and My+My+ Ag: CD13CD33 = 12. Ten of 12 lymphomas tested showed clonal immunoglobulin (Ig) heavy chain gene rearrangements in the absence of clonal T-cell receptor (TCR) gene rearrangements. None (0%) of the My Ag positive cases showed immunoreactivity for myeloperoxidase. We conclude that the anomalous T and My Ag expression seen in the above B-cell lymphomas is not indicative of mixed lineage proliferation but represents the aberrant expression of these antigens by the malignant cells.

Profile of immunoglobulin heavy chain variable gene repertoires and highly selective detection of malignant clonotypes in acute lymphoblastic leukemia.

The predominant B cell immunoglobulin heavy chain variable gene (IgH‐V) usage and the uniquely rearranged, clonotype‐specific variable‐diversity‐joining region gene (VDJ) sequences were identified in patients with B cell acute lymphoblastic leukemia (B‐ALL) using a novel DNA‐based gene amplification strategy. The approach allows a thorough and sensitive determination of the number of clonal leukemic IgH rearrangements and their precise V gene usage. This strategy may be applied in the detection of minimal residual disease, in surveillance after induction of disease‐free states, and in analyzing the effectiveness of purging autologous bone marrow of malignant clones. An initial primary polymerase chain reaction (PCR), directed by an IgH‐J generic primer and a complement of family‐specific IgH‐V primers, defined the major B cell IgH‐V gene usage. Use of an IgH‐J generic primer supplanted the use of a constant region primer anchor and thus eliminated the need to target mRNA by the traditional RNA reverse transcription–PCR amplification method. Monoclonality of rearranged VDJ bands was further substantiated by high‐resolution denaturant gel electrophoretic analysis. The predominant amplified bands were subcloned and sequenced. By sequencing through VDJ juxtaposed regions, that is, the third complementarity‐determining region, clonotype‐specific primers were developed and used in a secondary clonotype primer‐directed PCR (CPD‐PCR) to detect, with extreme sensitivity and specificity, a unique B cell clone. Analysis of the products of the CPD‐PCR permitted the detection of a single malignant cell among 1 million polyclonal cells and superseded the constraints of prior studies that have provided a limited evaluation of family variable gene repertoire usage. Leukemic clonal rearrangements were detected in 100% of the eight cases of pediatric and two cases of adult B‐ALL studied. Two or more clonal IgH‐VDJ amplified sequences were observed in 50% of the B‐ALL bone marrows analyzed. In two cases, clonotype‐specific oligodeoxynucleotide primers, derived from B‐ALL VDJ sequences, directed the secondary CPD‐PCR, and disease activity was monitored after chemotherapy and allogeneic bone marrow transplantation. J. Leukoc. Biol. 57: 856–864; 1995.

Extra copies of chromosome 2 are a recurring aberration in ALK-negative lymphomas with anaplastic morphology

The purpose of this study was to evaluate fluorescence in situ hybridization abnormalities of the 2p23 anaplastic lymphoma kinase (ALK) gene loci in lymphomas with anaplastic morphology. We studied 24 anaplastic large cell lymphomas (ALCL) classified by World Health Organization criteria [17 primary nodal/systemic (10 ALK+, 7 ALK−), seven primary cutaneous], and 17 additional non-Hodgkins lymphomas [one ALK+ B-lineage lymphoma, 14 ALK− diffuse large B-cell lymphomas (seven anaplastic variants, five nonanaplastic, two secondary CD30+), two follicular lymphomas]. ALK− lymphomas with anaplastic morphology showed extra nonrearranged anaplastic lymphoma kinase gene loci (P=0.004) due to trisomy 2 irrespective of the following factors: B or T/null phenotype (P=0.315), diagnostic categories of systemic or cutaneous ALCL or the above-mentioned B-cell lymphomas (P=0.131), and CD30 positivity by immunohistochemistry (P=1.000). Trisomy 2 was absent in all ALK+ lymphomas (P=0.009), which showed rearranged ALK gene loci (P<0.001). Whether trisomy 2 is a primary or secondary event that leads to ALK− lymphomas cannot be determined from this study. Its presence in secondary B-cell lymphomas suggests that trisomy 2 may be a secondary cytogenetic aberration in lymphomas in general. Further investigation of this finding is necessary to further our understanding of the heterogeneous group of ALK− lymphomas.

Metachronous seeding of lymph node metastases in rats bearing the MT-100-TC mammary carcinoma: The effect of elective lymph node dissection

SummaryFollowing the introduction of cancer cells into the lymphatic system, metastases in ‘down-stream’ lymph nodes often appear in a sequential manner. This could be due to synchronous seeding of the in-line nodes with progressively diminishing numbers of tumorigenic cancer cells, or alternatively, by discrete, stepwise (metachronous) seeding of ‘down-stream’ nodes by ‘up-stream’ nodal metastases acting as ‘generalizing’ sites. Metachronous seeding to local lymph nodes is potentially curable by elective lymph node dissection; synchronous seeding is not.Synchronous versus metachronous seeding of lymph node metastases was investigated using the MT-100-TC mammary carcinoma injected into the hind foot-webs of rats. When the primary tumor was removed by amputation one week after injection, 1/15 animals survived; in contrast, removal of the draining popliteal lymph node in addition to the primary lesion, resulted in 8/19 long-term survivors. At this time, occult metastases detectable by bioassay butnot by conventional histology, were present in all draining popliteal nodes and in 60 percent of lungs. The fact that some amputees were cured when the popliteal node was removed, indicated the metachronous nature of nodal metastases in this system. Further, recurrence of nodal and lung metastases in those amputees in which the popliteal node was left intact, identified the popliteal node as a ‘generalizing’ site. By the time popliteal node involvement was evident by conventional histology, micrometastases were present in ‘down-stream’ nodes, and accordingly, removal of the popliteal node and the primary lesion at this time was not curative.

The relationship between lymphogenous and hematogenous metastasis in rats bearing the MT-100-TC mammary carcinoma

Temporal and quantitative aspects of lymphogenous and hematogenous metastasis were examined using the rat MT-100-TC mammary carcinoma injected into the hind feet of syngeneic rats. Metastases first appeared in the draining popliteal nodes and then progressed in an invariable pattern to regional and then distal nodes: ‘skipped’ negative nodes within a chain of positive nodes were not observed. Metastatic progression in the lymphatic system occurred metachronously, with nodal metastases acting as ‘generalizing’ sites for ‘downstream’ nodes. Perturbation of lymph flow was apparent when nodes were involved with tumor, and resulted in retrograde seeding of contralateral nodes. Lung involvement was first observed by ectopic bioassay in 50 per cent of animals after 1 and 2 weeks of primary tumor growth; in contrast, all animals had popliteal involvement after 1 week. These results indicate that lymph nodes and lungs are not seeded synchronously, and the lungs are seeded after nodal metastases. Thus, a phase of metastasis has been identified, during which resection of the primary tumor and local nodes may well be curative in the 50 per cent of cases in which the disease is confined to these sites.

Molecular Genetic Methods in Diagnosis and Treatment

During the past 20 yr, molecular biology and molecular genetics have provided techniques and procedures that allow the precise diagnoses of leukemias and lymphomas (for a review, see ref. 1). These methodologies have provided powerful, robust, and precise characterizations of genetic mutations and gene rearrangements (both normal and abnormal) that have been applied to the diagnosis and treatment of these malignancies. In this chapter we discuss methodologies currently utilized within the molecular diagnostic laboratory of cancer centers and major academic hospitals. We introduce these methodologies in both general terms and within the context of a differential diagnosis of leukemia/lymphoma. The long-term goal of the molecular hematologist/oncologist is to describe all mutations that occur within a given type or subtype of leukemia/lymphoma. This involves the discovery of new somatic and possibly hereditary mutations that contribute to the genesis of these hematologic malignancies. However, for this information to be useful in treatment regimens, therapies that target or affect these growth-dysregulating mutations must be found. Because diagnosis of mutation and gene rearrangement in the hematologic malignancies is far ahead of therapeutic modalities, we also discuss new research tools that will have impact on the experimental hematologist.