The clinical relevance of molecular staging for melanoma.

The presence of metastatic disease in the regional nodal basin is the most important prognostic indicator for patients with malignant melanoma. The metastatic status of the sentinel lymph node (SLN), defined as the first node in the basin to drain a primary tumor, has been shown to represent that of the entire basin. Since routine histologic examination of lymph nodes often underestimates the presence of micrometastatic disease, a more sensitive assay for detecting tumor cells is needed. We have previously shown that a molecular assay based on the reverse transcriptase polymerase chain reaction (RT-PCR) was able to define a population of patients at higher risk for both recurrence and death, compared with routine H&E histology. Recently, we have compared "molecular staging" of patients by RT-PCR with conventional S-100 immunohistochemistry (IHC) staining of the SLNs. In these studies, SLN specimens were bivaled, and half of each specimen was examined by routine histology, including both H&E and S-100 IHC. The other half of each specimen was analyzed by a nested RT-PCR assay. H&E histology alone detected metastatic disease in 36 of 233 (16%) patients tested. Serial sectioning and IHC detected micrometastatic disease in another 16 patients, thus increasing the proportion of patients with nodal disease to 22%. RT-PCR detected micrometastatic disease in 114 of 181 patients who were negative by conventional methods, further increasing the proportion of patients with evidence of nodal disease to 70% overall. The clinical significance of these findings is still uncertain. The value of additional therapy (including elective lymph node dissection and interferon therapy) for patients who are positive only by the molecular method is currently being investigated by the national multi-center Sunbelt Melanoma Trial.

Neo-self antigens and the expansion of B-1 cells: lessons from atherosclerosis-prone mice.

The pathogenesis of atherosclerosis involves an inflammatory process that is modulated by the immune system, and within these complex responses we have discerned a possible role for an archetypic B-1 clone. We speculate that due to their immunogenicity and in vivo distribution the "neo"-self determinants created in oxidatively modified LDL are highly stimulatory for certain B-1 cell clones. These neo-self determinants, which can be created chemically, by somatic processes, may in fact represent the molecular analogues of somatic maturation, or even aging. These changes, including those on non-protein antigens induced by oxidative metabolism, amongst others, create neo-determinants against which the host no doubt can not develop rigorous B-cell tolerance. The onset of expression of these oxidative neo-determinants relatively late in development may well serve a useful function for the highly evolved mammalian immune system, as targeting by evolutionarily selected B-1 clones may facilitate the amplification of other useful antibody-mediated physiologic functions. As in the case of the T15 clone, these antibodies may aid in protection against common microbial pathogens. Hence we postulate that during the evolution of the adaptive immune system the neo-self antigenic milieu may have been exploited for the natural selection of primordial clonal specificities. The T15 B-1 clone may then illustrate a common paradigm in which there has been natural selection based on utility for the defense of the individual from environmental threats, as well as for possible "housekeeping" role(s) and the maintenance of cellular homeostasis.

Clinical relevance of molecular staging for melanoma: comparison of RT-PCR and immunohistochemistry staining in sentinel lymph nodes of patients with melanoma.

OBJECTIVE: To determine the clinical significance of a molecular assay based on the reverse transcriptase polymerase chain reaction (RT-PCR) for the presence of micrometastatic melanoma cells in sentinel lymph nodes (SLNs). SUMMARY BACKGROUND DATA: Routine histologic examination of lymph nodes often underestimates the presence of micrometastatic disease. The authors have previously shown that an RT-PCR assay designed to detect melanocyte-specific expression of the tyrosinase gene could be used to define a population of patients at higher risk for both recurrence and death compared with routine hematoxylin and eosin (H&E) histology. In this study, the authors used the tyrosinase RT-PCR assay in a patient population examined by a more detailed histologic analysis, including S-100 immunohistochemistry. METHODS: Patients underwent lymphatic mapping and SLN biopsy. SLN specimens were bivalved, and half of each specimen was serially sectioned and examined by routine H&E histology and S-100 immunohistochemistry. The other half of each specimen was analyzed by a nested RT-PCR assay. RESULTS: Hematoxylin and eosin histology detected metastatic disease in 36 (16%) of the 233 patients tested. S-100 immunohistochemistry detected micrometastatic disease in another 16 patients, and 114 (63%) of 181 patients with histology-negative nodes had positive findings on RT-PCR. There were significant differences between PCR-positive and PCR-negative patient groups in Breslow thickness, Clark level, and the presence of ulceration of the primary tumor, factors that have been shown to correlate with recurrence and survival. CONCLUSIONS: These results suggest that RT-PCR can increase the sensitivity of detection of metastatic melanoma cells in SLNs over the current standard methods, including H&E histology and S-100 immunohistochemistry. Further long-term follow-up is needed to detect actual differences in recurrence and overall survival.

Metastatic melanoma to regional lymph nodes.

There is an epidemic of melanoma in the United States and throughout most parts of the word. Recent advancements in the management of this disease has provided the patient with more options. The emerging technology of lymphatic mapping and sentinel node biopsy results in a more conservative, less morbid procedure to obtain nodal staging information. At the same time, providing the pathologist the 1-2 nodes from the basin most likely to contain metastatic disease, allows for a more detailed examination of the sentinel lymph node. This more detailed examination may include serial sectioning, immunohistochemical staining or even molecular biology techniques based on RT-PCR to provide more accurate staging. National trials are ongoing to examine the clinical relevance of the disease that is detected and the 'upstaging' that occurs with more sensitive assays for occult metastases.

Molecular staging for melanoma and breast cancer.

Despite increased sensitivity of PCR techniques, routine H&E histology and, in some cases, immunohistochemistry remain the gold standards for the detection of micrometastatic disease. Highly sensitive and specific molecular assays such as RT-PCR provide an ideal way to detect micrometastatic disease in tissues or blood at risk for metastases. RT-PCR has been shown to increase detection of micrometastases, especially in patients with breast cancer and melanoma. These assays have the potential to provide valuable tumor staging and progression information and thus determine the need for further surgery, adjuvant chemotherapy, and antigen-specific immunotherapy. As investigators gain more experience using molecular assays, the results of these assays will be more likely to guide clinical staging and decision making.

Cutaneous lymphatic drainage in patients with grossly involved nodal basins.

BACKGROUND: The development of lymphatic mapping techniques has facilitated the identification of the sentinel lymph node (SLN), the first node in the regional basin into which cutaneous lymphatics flow from a particular skin area. Previous studies have shown that SLN histology reflects the histology of the entire basin, because melanoma metastases progress in an orderly fashion, involving the SLN before higher nodes in the basin become involved with metastatic disease. It is uncertain whether these orderly cutaneous lymphatic flow patterns are maintained in grossly involved basins. Lymphatic mapping was performed in a population of melanoma patients with clinically palpable lymphadenopathy to address this question. We aimed to determine whether the presence of gross nodal disease in the basin alters lymphatic flow into that basin so that lymphatic mapping techniques are not applicable, and, in patients referred with a grossly involved basin, whether preoperative lymphoscintigraphy should be performed to identify other regional basins at risk for metastases. METHODS: Eight patients presented with grossly palpable disease in the regional basin and underwent preoperative lymphoscintigraphy. All patients with palpable disease and all basins indicated by lymphoscintigraphy to be at risk were dissected. Three patients presented with clinically palpable nodes at the time of diagnosis, and five developed nodal disease on clinical follow-up after undergoing initial wide local excision only. A total of 10 basins in the eight patients were dissected. Of these, eight of the basins had grossly palpable regional nodal disease, and the other two basins were identified by preoperative lymphoscintigraphy as being at risk for metastases. The SLN was identified with intraoperative mapping, harvested, and submitted to pathology. Complete therapeutic lymph node dissections were performed following the SLN harvest in the basins with grossly palpable disease. SLN biopsy alone was performed in the two basins that did not have clinically palpable adenopathy but showed cutaneous lymphatic flow from the scintigram. RESULTS: Sixteen SLNs were harvested from these eight basins with grossly palpable disease, and 14 (87.5%) contained tumor. In each case, one of the SLNs was the grossly palpable node, and in six of the basins (75%) it was the only site of melanoma metastases. An additional 190 higher level, non-SLNs were removed, 32 (16.8%) of which contained microscopic foci of metastatic melanoma (P = .015). The null hypothesis that melanoma nodal metastasis is a random event is rejected. Two patients with trunk melanoma primary sites were identified to have other basins at risk for metastatic disease on lymphoscintigraphy. SLN biopsies were performed in these two patients, and one had microscopic nodal disease in the SLN. CONCLUSIONS: These data support the fact that cutaneous lymphatic drainage patterns are maintained in patients with grossly involved basins, thus buttressing the idea that the SLN is the node most likely to develop metastatic disease. Gross disease in the basin does not significantly alter cutaneous lymphatic flow into the regional basin, as the sentinel lymph node identified under these circumstances is the same as with the grossly involved node. Preoperative lymphoscintigraphy in patients who present with grossly involved nodes in one basin may identify other regional basins with micrometastatic disease and deserves further study in this setting.

The progression of melanoma nodal metastasis is dependent on tumor thickness of the primary lesion.

BACKGROUND: Recent results of several clinical trials using the technique of intraoperative lymphatic mapping and sentinel lymph node (SLN) biopsy confirm the validity of the concept of there being an order to the progression of melanoma nodal metastases. This report reviews the H. Lee Moffitt Cancer Center experience with this procedure, one of the largest series described to date. These data demonstrate that the involvement of the SLNs, as well as higher-echelon nodes, is directly proportional to the melanoma tumor thickness, as measured by the method of Breslow. METHODS: The investigators at the H. Lee Moffitt Cancer Center retrospectively reviewed their experience using lymphatic mapping and SLN biopsies in the treatment of malignant melanoma. All eligible patients with primary malignant melanomas underwent preoperative and intraoperative mapping of the lymphatic drainage of their primary sites, along with SLN biopsies. All patients with positive SLNs underwent complete regional basin nodal dissection. For 20 consecutive patients with one positive SLN, all of the nodes from the complete lymphadenectomy were serially sectioned and examined by S-100 immunohistochemical analysis, to detect additional metastatic disease. RESULTS: Six hundred ninety-three patients consented to undergo lymphatic mapping and SLN biopsy. The SLNs were successfully identified and collected for 688 patients, yielding a 99% success rate. One hundred patients (14.52%) showed evidence of nodal metastasis. The rates of SLN involvement for primary tumors with thicknesses of <0.76 mm, 0.76-1.0 mm, 1.0-1.5 mm, 1.5-4.0 mm, and >4.0 mm were 0%, 5.3%, 8%, 19%, and 29%, respectively. Eighty-one patients underwent complete lymph node dissection after observation of a positive SLN, and only six patients with positive SLNs demonstrated metastatic disease beyond the SLN (7.4%). The tumor thicknesses for these six patients ranged from 2.8 to 6.0 mm. No patient with a tumor thickness of <2.8 mm was found to have evidence of metastatic disease beyond the SLN in complete lymph node dissection. All 20 patients with a positive SLN for whom all of the regional nodes were serially sectioned and examined by S-100 immunohistochemical analysis failed to show additional positive nodes. CONCLUSIONS: These results suggest that regional lymph node involvement may be dependent on the thickness of the primary tumor. As the primary tumor thickness increases, so does the likelihood of involvement of SLNs and higher regional nodes in the basin beyond the positive SLNs.

The BCL-6 proto-oncogene controls germinal-centre formation and Th2-type inflammation.

Structural alterations of the promoter region of the BCL-6 proto-oncogene represent the most frequent genetic alteration associated with non-Hodgkin lymphoma, a malignancy often deriving from germinal-centre B cells. The BCL-6 gene encodes a zinc-finger transcriptional repressor normally expressed in both B cells and CD4+ T cells within germinal centres, but its precise function is unknown. We show that mice deficient in BCL-6 displayed normal B-cell, T-cell and lymphoid-organ development but have a selective defect in T-cell-dependent antibody responses. This defect included a complete lack of affinity maturation and was due to the inability of follicular B cells to proliferate and form germinal centres. In addition, BCL-6-deficient mice developed an inflammatory response in multiple organs characterized by infiltrations of eosinophils and IgE-bearing B lymphocytes typical of a Th2-mediated hyperimmune response. Thus, BCL-6 functions as a transcriptional switch that controls germinal centre formation and may also modulate specific T-cell-mediated responses. Altered expression of BCL-6 in lymphoma represents a deregulation of the pathway normally leading to B cell proliferation and germinal centre formation.

The absence of the transcription activator TFE3 impairs activation of B cells in vivo.

TFE3 is a ubiquitously expressed member of the TFE3/mi family of basic helix loop helix zipper transcription factors. TFE3 binds to muE3 sites located in the immunoglobulin heavy-chain (IgH) intronic enhancer, heavy-chain variable region promoters, the Ig kappa intronic enhancer, and regulatory sites in other genes. To understand the role of TFE3 in Ig expression and lymphoid development, we used embryonic stem (ES) cell-mediated gene targeting and RAG2-/- blastocyst complementation to generate mice which lack TFE3 in their B and T lymphocytes. TFE3- ES cells fully reconstitute the B- and T-cell compartments, giving rise to normal patterns of IgM+ B220+ B cells and CD4+ and CD8+ T cells. However, TFE3- B cells show several defects consistent with poor B-cell activation. Serum IgM levels are reduced twofold and IgG and IgA isotypes are reduced three- to sixfold in the TFE3- chimeras even though in vitro, the TFE3- splenocytes secrete normal levels of all isotypes in response to lipopolysaccharide activation. Peripheral TFE3- B cells also show reduced surface expression of CD23 and CD24 (heat-stable antigen).

Lymphatic Mapping for Melanoma: Long-term Results of Regional Nodal Sampling With Radioguided Surgery.

BACKGROUND: Lymphatic mapping and sentinel lymph node (SLN) biopsy are new techniques used in the surgical treatment of patients with malignant melanoma. These procedures have the potential to change the surgical treatment of the disease to provide a more rational approach to adjuvant therapy. METHODS: A prospective database of melanoma patients undergoing lymphatic mapping and SLN biopsy was reviewed to identify prognostic factors for overall and disease-free survival in this patient population. RESULTS: Five-year overall and disease-free survival was 92.3% and 79.0%, with a median follow-up of 17 months. The number of histologically positive SLNs was the most powerful predictor of overall and disease-free survival. Patients with no histologically positive SLNs had a five-year overall and disease-free survival of 97.9% and 93.3%, respectively. Tumor ulceration and Clark level greater than or equal to III were the significant prognostic factors for survival. CONCLUSIONS: The use of lymphatic mapping and SLN biopsy effectively stages patients with primary cutaneous melanoma. Additionally, the presence of histologically positive SLNs is the most powerful indicator of overall and disease-free survival for these patients.

Abnormal peripheral lymphocyte function in c-abl mutant mice.

The proto-oncogene c-abl encodes a tyrosine kinase that is hypothesized to function in proliferation-stimulatory signaling pathways. Previous work on mice homozygous for targeted mutations in the c-abl gene (ablml and abl2 mutant strains) has demonstrated multiple defects, including a susceptibility to infections that results in a high mortality rate after weaning. FACS analysis of the hemopoietic system of c-abl mutants demonstrated variable reductions in B and T lymphocytes in adult bone marrow, thymus, spleen, and peripheral blood. In addition, bone marrow from mutants showed a decreased ability to respond to interleukin-7. We further found that B cells from ablm1 mice had a reduced ability to respond to lipopolysaccharide (decreased to 10% of control response) that was dependent on the culture conditions and the tissue of origin of B cells. Peripheral blood from the mutants also had a reduced response to the T cell mitogen concanavalin A. Immune response in ablm1 mice as determined by the mixed lymphocyte response and the sheep red blood cell plaque-forming assay was grossly normal. These findings suggest that although specific signaling pathways in lymphocytes may involve c-Abl, the immune system can function in the absence of a normal c-abl gene product.

B-1 cells: unique origins and functions.

B-1 cells represent a distinct population of B lymphocytes with unique phenotypic, developmental and functional characteristics. We present evidence that the expression of MHC class II antigens differentiates two distinct developmental pathways which define the fetal-type (FT) and adult-type B-cell lineages. Further in-vivo and in-vitro analyses suggest that B-1 cells are derived primarily if not exclusively from the FT lineage. Combining these results with other studies suggesting that signalling plays a role in the development of B-1 cells, we propose a modified dual lineage model for the generation of B-1 cells. In this model fetal-type B cells are uniquely 'born' with the capacity to be B-1 cells, however, they must be properly educated (i.e. receive the appropriate signals) before they can be 'made' functional, phenotypic B-1 cells. With respect to the function of B-1 cells, we have used PerC/BM allotype chimeric animals to investigate the capacity of B-1 cells to participate in the formation of germinal centers. In these mice we were unable to detect any significant involvement of B-1 cells in the generation of germinal centers. These results are discussed in the context of the known characteristics of B-1 responses including the low frequency of hypermutation and isotype class switching.

Bone marrow B lymphocyte development in c-abl-deficient mice.

Mice homozygous for a mutation in the c-abl tyrosine kinase gene have multiple defects including high postnatal mortality, runting, morphological abnormalities, susceptibility to infections, and reductions in lymphocytes and their precursors. FACS analysis of bone marrow from mutant mice demonstrates variable reductions in pro-B and pre-B cells. While the numbers of cells in these populations are profoundly reduced in some mutants (16 and 1.2% of control pro-B and pre-B cells, respectively), normal levels are found in other individuals. In the affected mutants, some reductions are observed in many stages of B cell development. The response of B cell precursors to the cytokine interleukin-7 is variably affected while that of several other cytokines (stem cell factor, interleukin-3, GM-CSF, G-CSF, and erythropoietin) is normal in c-abl mutants. The population defects caused by the c-abl mutation can be recreated in normal mice by the transfer of adult bone marrow but, surprisingly, not fetal liver. These studies demonstrate that c-Abl signaling pathways may play a role in the earliest stages of B cell development in a developmental stage-specific manner. In spite of these variable abnormalities, however, the hemopoietic system of c-abl mutant animals is surprisingly intact.

Ig gamma 2b transgenes promote B cell development but alternate developmental pathways appear to function in different transgenic lines.

Analysis of B cell development in three strains of gamma 2b transgenic mice shows that the gamma 2b H chain can replace the microH chain in promoting B cell differentiation. The 348C line produces 90% gamma 2b-only B cells and 10% B cells; which co-express gamma 2b and endogenous sIgM and sIgD. These IgG2b+ B cells develop into mature, recirculating CD23+ B cells. The 343-1 and gamma 2b-T15 transgenic mice produce sIgMhigh:sIgDlow:CD23- B cells that generally co-express the gamma 2b transgene-encoded H chain. Such B cells are either developmentally arrested immature B cells or arise from B-1 (CD5) progenitors. The gamma 2b-T15 mice can produce gamma 2b-only CD23+ B cells following inactivation of the endogenous mu locus, whereas 343-1 mice fail to develop B cells. Thus, gamma 2b H chains: 1) can act alone to promote the development of mature B cells, 2) synergize with microH chains for allelic exclusion, and 3) vary in their influence on B cell development in different transgenic mouse strains.

Murine CD8+ T cells that specifically delete autologous CD4+ T cells expressing V beta 8 TCR: a role of the Qa-1 molecule.

Interactions mediated by TCRs expressed on different T cell subsets may play a role in immunoregulation. To investigate this idea, we studied the regulation of superantigen-induced TCR V beta-restricted responses. We asked whether the in vivo regulation of CD4+ V beta 8+ T cells following SEB injection is controlled by CD8+ T cells. We found that in mice deficient in CD8+ T cells, the down-regulation of CD4+ V beta 8+ T cells below baseline is not observed. Moreover, following SEB administration, CD8+ T cells emerge that preferentially kill subpopulations of activated CD4+ V beta 8+ but not CD4+ V beta 8- T cells in vitro. This TCR V beta-specific cytotoxicity is dependent on beta 2-microglobulin and is inhibited by antisera specific for Qa-1 but not by antibody to MHC class Ia. These data suggest the idea that the specificity of immune regulation may involve CD8+ T cell recognition of TCR V beta determinants and Qa-1 molecules expressed on CD4+ T cells.

De novo development and self-replenishment of B cells.

Previous studies distinguished two murine B cell lineages: the conventional lineage, which comprises the majority of B cells, and the Ly-1 B lineage (B-1a), which represents a small percentage of total adult B cells. A third subset, B-1b cells, shares many properties with B-1a cells, including the characteristic ability to self-replenish, but does not express Ly-1 (CD5). Reconstitution studies presented here show that (i) although the B220- population in adult spleen and bone marrow contains very little progenitor activity for B-1a cells, it can reconstitute roughly half the normal number of B-1b cells; (ii) B-1 progenitors present in adult bone marrow and spleen function at low levels in adult animals; (iii) peritoneal B-1 cells (principally B-1b) that develop following bone marrow transfer, like B-1 cells from normal animals, are capable of substantial self-replenishment; and (iv) conventional B cells do not expand (self-replenish) in adoptive recipients, although they can persist for long periods. Collectively, these progenitor and self-replenishment characteristics provide a developmental base for distinguishing B-1a, B-1b and conventional B cells.

CD43 (S7) expression identifies peripheral B cell subsets.

CD43 (leukosialin) expression has previously been demonstrated on the surface of developing B cells in mouse bone marrow and on plasma cells induced in vitro, but not on peripheral B cells in spleen. Here we show that CD43, as recognized by mAb S7, is indeed expressed on a small population of splenic B cells. Flow cytometric phenotyping of normal mice and radiation chimeras reveals that CD43/S7 is expressed on virtually all (> 90 to 95%) splenic B-1 cells and the majority of peritoneal B-1 cells, but not on conventional B cells. The expression of CD43/S7, in conjunction with other cell surface markers, clearly distinguishes B-1 cells from follicular, marginal zone, and immature B cells in the unstimulated adult spleen and permits further phenotyping of these subsets. The phenotype of splenic and peritoneal B-1 cells in normal BALB/c and BAB/25 mice is essentially identical with the exception that all peritoneal B-1 cells express CD11b (Mac-1) and some lack CD43/S7 and heat stable Ag (as detected by the mAb 53-10) expression. Although splenic B-1, marginal zone, and immature B cells share many phenotypic characteristics, these studies show that, in addition to CD43, they differ with respect to the expression levels of a variety of Ags including heat stable Ag, B220, and the B cell activation Ag B7.

Major histocompatibility complex class II expression distinguishes two distinct B cell developmental pathways during ontogeny.

All mature B cells coexpress major histocompatibility complex (MHC) class II molecules, I-A and I-E, which are restriction elements required for antigen presentation to CD4+ T cells. However, the expression of class II during the early stages of B cell development has been unclear. We demonstrate here that there is a difference in the expression of class II during murine B cell development in the fetal liver and adult bone marrow (BM). These differences define two distinct B cell developmental pathways. The Fetal-type (FT) pathway is characterized by pre-B and immature IgM+ B cells generated in the fetal liver which initially lack all class II expression. In contrast, the Adult-type (AT) pathway is typified by B cells developing in the adult BM which express class II molecules from the pre-B cell stage. In vitro stromal cell cultures of sorted fetal liver and adult BM pro-B cells indicated that the difference in I-A expression during B cell development is intrinsic to the progenitors. In addition, we show that FT B cell development is not restricted to the fetal liver but occurs in the peritoneal cavities, spleens, liver, and BM of young mice up to at least 1 mo of age. The AT B cell development begins to emerge after birth but is, however, restricted to the BM environment. These findings indicate that there are two distinct B cell developmental pathways during ontogeny, each of which could contribute differentially to the immune repertoire and thus the functions of B cell subsets and lineages.