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BACKGROUND: Single antigen beads (SAB) are used for monitoring HLA antibodies in pretransplant and posttransplant patients despite the discrepancy between virtual and actual crossmatch results and transplant outcomes. This discrepancy can be attributed to the presence of conformational variants of HLA-I on SAB, assessment of which would increase the concordance between SAB and flow cytometry crossmatch (FCXM) results, thus enabling improved organ accessibility for the waiting list patients and a better prediction of antibody-mediated rejection. METHODS: The conformational variants were examined on HLA-I beads, iBeads, acid-/alkali-treated beads, and T cells using HLA-I monoclonal antibodies (W6/32, TFL-006, and heavy chain (HC)-10). RESULTS: The affinity of the monoclonal antibodies against HLA-I beads confirmed the presence and heterogeneous density of peptide-associated β2-microglobulin–associated HLA HC (pepA-β2aHC), peptide-free-β2aHC (pepF-β2aHC), and β2-free HC (β2fHC) on every single antigen-coated bead. In contrast, iBeads harbor a high density of pepA-β2aHC, low density of pepF-β2aHC, and are lacking β2fHC. The FCXM analyses confirmed the prevalence of pepA-β2aHC, but not pepF-β2aHC or β2fHC on resting T cells. CONCLUSIONS: The strength of a donor-specific antibody should be assessed with a bead-specific mean fluorescence intensity cutoff based on TFL-006 reactivity against HLA-I beads, and HC-10 against iBeads, where the β2fHC or pepF-β2aHC normalized donor-specific antibody level would reveal the true anti–pepA-β2aHC reactivity associated with positive FCXM.

Personalized vaccine, recognized after the failure of allogenic melanoma whole cell and lysate vaccine phase III trials, involves culturing cells from a patient's own tumor within a short duration and with less passages but with optimized expression of tumor-associated antigens (TAAs). Its feasibility is established by comparing pure cell lines generated from fresh and cryopreserved tissues (n=164) of patients with lymph node (LN) and distant metastases. Stable cell lines (from 67% of specimens) are subcultured after cryopreserving them. Pure cell lines established after eliminating fibroblasts (from 96% of the cell lines) include those from LN (69%), soft tissues including cutaneous (60%), liver (64%), lung (75%), bone (80%), brain (75%), and other sites (73%). Within 3.5 months, stable cell lines (or =50 million cells) are established from initiating the cell culture. For LN metastases, the duration differs significantly (P20.05) between fresh (1.4-3.4 months) and cryopreserved (2.4-4.7 months) tissues. The expression of TAAs varies as follows: Tyrosinase (81%)Melan-A (80%)HMB45/gp-100 (75%)Mel-5/TRP-1 (65%)MAGE-1 (47%)S-100 (28%). The number of TAAs per cell line differs between early (7) and late (7) passages. Among late passage cell lines, lesser percentage of cell lines express three to six antigens pointing out that early passage (7) cell lines may be needed for antigen-targeted immunotherapy. This study provides a protocol for establishing cell lines within 2-5 months for personalized vaccine therapy for nodal and organ metastatic melanoma patients.

The authors tested the hypothesis that the therapeutic destruction of residual tumors may be reflected in the level of serum total gangliosides (sTG). The sTG level was evaluated in 17 patients with in-transit melanoma and 70 patients with resected regional node metastasis, who have received a polyvalent therapeutic melanoma cell vaccine. The treatment response was determined by regression of in-transit metastases or by overall survival after resection. sTG levels were measured, blinded, before and after immunotherapy. The mean sTG level of the in-transit melanoma patients increased from 18.57 +/- 3.18 mg/dL pretreatment to 23.7 +/- 5.5 mg/dL between weeks 2 and 16 after initiation of treatment (p(2) < 0.0001). By week 24, the level had returned to its prevaccine level in the seven clinical responders (18.1 +/- 2.3 mg/dL vs. 20.4 +/- 3.2 mg/dL; p(2) < 0.050) but remained higher than its prevaccine level in the 10 nonresponders (23.3 +/- 5.1 mg/dl vs. 17.2 +/- 2.7 mg/dL). Similarly, the sTG level of the patients with nodal metastases increased between weeks 2 and 16 after the first vaccine treatment; by week 24, it had returned to pretreatment level in patients who survived more than 56 weeks but remained significantly elevated (p(2) < 0.01) in patients who survived less than 56 weeks. The sTG level increased between weeks 2 and 16 in all vaccine recipients and returned to prevaccine level by week 24 in all who showed measurable regression of in-transit melanoma (7 of 17 patients) or improved overall survival (53 of 70 patients). The data suggest that sTG level could be a potential tool for assessing the response to immunotherapy in melanoma patients by week 24.