Ansgar Lukowsky

Ultrasound-guided Fine Needle Aspiration Cytology prior to Sentinel Lymph Node Biopsy in Melanoma Patients

BackgroundSentinel lymph node biopsy (SLNB) allows early detection of metastases, thereby enabling early treatment in melanoma patients likely to benefit from adjuvant therapies. This prospective study analyzes the possible benefits of additional ultrasound (US) and fine needle aspiration cytology (FNAC) of sentinel nodes (SN) prior to SLNB.MethodOver a 2-year period 127 melanoma patients with 151 SN were scheduled for SLNB. All SN were initially identified with lymphoscintigraphy, then identified and evaluated by US and the cells aspirated for cytology (FNAC). US findings and FNAC results were compared to surgical findings.ResultsOf 127 patients, 114 had one SN each, 12 had two, and one had three. In vivo US achieved a sensitivity of 79% (95% CI: 62–91%) and a specificity of 72% (95% CI: 62–81%). FNAC showed a sensitivity of 59% (95% CI: 41–76%) and a specificity of 100% (95% CI: 95–100%). The combination of these two in vivo methods achieved an overall sensitivity of 82% (95% CI: 65–93%) and an overall specificity of 72% [95% CI: 62–81%].ConclusionCombined US and FNAC provides important information prior to SLNB in that both procedures identify metastases in the lymph nodes (sensitivity > 80%). Patients with positive FNAC may proceed directly to complete lymph node dissection (cLND) instead of having initial SLNB. Thus, combined US and FNAC may prevent unnecessary anesthesia and surgical management as well reduce costs. In our study 16% (19/121) fewer SLNB procedures were carried out, subsequently replaced by cLND. For patients with a negative combination of in vivo US and FNAC, SLNB remains the best diagnostic option.

Peripheral blood T-cell clonality in mycosis fungoides and nonlymphoma controls.

In mycosis fungoides (MF), T-cell clonality is reported in about 90% of skin and 40% of blood samples. However, identity of blood and cutaneous T-cell clone and prognostic relevance of blood T-cell clonality remain controversial. By PCR/fluorescence fragment analysis with estimation of clonal fragment lengths and relative peak heights, we objectively identified T-cell clonality unrelated to malignant lymphoproliferation in healthy donors (5/38), autoimmune dermatoses (3/8), and nonlymphoma skin cancer (9/39). This T-cell expansion of undetermined significance (TEXUS) was also found in 8/64 MF patients. Dissemination of neoplastic cells into blood, as identified by identical clonal fragment lengths in blood and skin, was detected in 23/64 MF patients. When monitoring for progression at TNM stage for a mean of 45.7 months, univariate analysis identified age of >60 years and detection of a related blood T-cell clone to be of prognostic relevance, whereas detection of TEXUS, sex, TNM stage at initial diagnosis, and detection of a cutaneous T-cell clone were irrelevant. Although multivariate analysis was not possible, further stratification clearly indicated an age of >60 years to be the predominating prognostic factor. In conclusion, investigation of T-cell clonality in skin and blood samples at the initial diagnosis cannot predict the clinical course of MF and the occurrence of TEXUS should be considered when assessing blood T-cell clonality.

Polyclonal expansion of T cells with the TCR Vβ type of the tumour cell in lesions of cutaneous T-cell lymphoma: evidence for possible superantigen involvement

The involvement of superantigens in the pathology of cutaneous T‐cell lymphomas (CTCL) has been suggested before, but without unequivocal evidence for superantigen activity in the patients. Seeking evidence for superantigen activity we analysed clones and microdissected single cells isolated from the epidermis of early‐stage lesions of a CTCL patient for their T‐cell receptor (TCR) Vβ expression and TCR Vγ gene rearrangements. The vast majority of these T cells expressed the TCR Vβ family type of the tumour. From their TCR γ gene rearrangements, however, these cells were polyclonal. The tumour cell clone accounted for about 60% of these cells, about 40% were of heterogeneous origin. This dominance of a single Vβ family in the polyclonally expanded dermal T‐cell populations implies superantigen activity in the CTCL lesions.

A T-cell receptor gamma polymerase chain reaction assay using capillary electrophoresis for the diagnosis of cutaneous T-cell lymphomas.

Detection of clonal T-cell receptor γ rearrangements by polymerase chain reaction (TCRγ PCR) followed by high-resolution electrophoresis has now become a valuable tool in the diagnosis of cutaneous T-cell lymphoma (CTCL). The identification of clonal TCRγ PCR products by fluorescent fragment analysi

Evaluation of T-cell clonality in archival skin biopsy samples of cutaneous T-cell lymphomas using the biomed-2 PCR protocol.

Recently, several European centers of lymphoma diagnosis and research developed various polymerase chain reaction (PCR) methods for clonality analysis in suspect T-cell and B-cell proliferations (Biomed-2 Concerted Action). They have mainly been applied to frozen material of systemic B-cell and T-cell malignancies. Thus far, only limited data exist with regard to cutaneous T-cell lymphoma (CTCL) and paraffin-embedded material. Thus, we applied the Biomed-2 T-cell receptor (TCR) γ and TCRβ PCR as well as an in-house TCRγ PCR to a collection of 107 archival skin samples (84 CTCL, 3 systemic TCL and 20 controls). As a result, the Biomed-2 TCRγ PCR revealed 81% clonality, the in-house TCRγ method revealed 86% clonality, and the Biomed-2 TCRβ revealed 78% clonality in CTCL samples generating at least the 300u2009bp fragment in the Biomed-2 control PCR. We found clonal TCRβ rearrangements in 5 of 17 CTCL samples that were polyclonal in the Biomed-2 TCRγ PCR. By combining all Biomed-2 assays, one or more clonal rearrangements were detected in 87% of CTCL and in all 3 systemic TCLs. By combining all TCR PCR assays applied here, clonality was shown in 90% of the CTCL cases. In conclusion, we showed that the Biomed-2 TCR PCR worked well with DNA from paraffin-embedded tissue, revealing a high-clonality detection rate in CTCL, and thus should be highly recommended for routine molecular analysis. In addition, the performance of our in-house TCRγ assay verifies our previously published findings on clonally expanded T-cells in CTCL.

Search for Kaposi's sarcoma-associated virus DNA in hemangioproliferative disorders and cutaneous malignant lymphoma

Recently, a Kaposis sarcoma‐associated herpesvirus (KSHV) was discovered. We evaluated by PCR 14 paraffin‐embedded specimens with the histological diagnosis of endemic, classic and HFV‐associated Kaposis sarcoma (KS) for the presence of the KSHV DNA sequence. In addition, biopsies of adjacent, histologically unaffected skin, peripheral‐blood mononuclear cells (PBMCs) of HIV‐infected KS patients, PBMCs of one classic KS patient, and specimens of patients with hemangioproliferative disorders other than KS as well as samples of cutaneous T‐and B‐cell lymphoma were analyzed for KSHV. In all cases of KS, independent of the KS subtype, KSHV was detected in lesional skin. No KSHV was found in biopsies of the adjacent unaffected skin or PBMCs of HFV‐infected KS patients. We found KSHV in the PBMCs of a patient with classical KS. All specimens of cutaneous T‐and B‐cell lymphomas or lymphomatoid papulosis were negative for KSHV. In addition, the samples with hemangioproliferative disorders other than KS were negative for KSHV. There was one borderline case of KS or acroangiodermatitis that was positive for KSHV. Additional histological sections and clinical evaluation confirmed the diagnosis of classic KS. In summary, the data indicate that PCR for KSHV should be a useful diagnostic tool in cases of hemangioproliferative disorders.

Clonality analysis by T-cell receptor γ PCR and high-resolution electrophoresis in the diagnosis of cutaneous T-cell lymphoma (CTCL)

During T-cell maturation, T-cell receptor (TCR) gene segments rearrange, resulting in a new, unique DNA configuration. The recombined TCR gene loci display a high degree of nucleotide sequence variability. Molecular biological clonality assays focus on this cell-specific DNA pattern. The finding of an identical TCR rearrangement in a large number of T lymphocytes signals a malignant proliferation, although clonality is not always equivalent to malignancy. Thus, detection of clonal TCR gamma rearrangements by polymerase chain reaction (PCR), followed by high-resolution electrophoresis is a valuable tool in the diagnosis of cutaneous and other T-cell lymphomas. For the clonality assay described here, all rearrangements of T cells present in a given sample are amplified by a set of only three TCR gamma-PCRs. The products are investigated by either heteroduplex temperature gradient gel electrophoresis (HD-TGGE) or fluorescent fragment analysis (FFA) on a capillary DNA sequencer (or by both methods), for clonality. Both electrophoresis techniques show highly reproducible results and are comparatively easy to conduct, however, specific instruments are required. Concerning lower detection thresholds, the methods need a minimum of about 1% of clonal T-cells in mixtures with polyclonal T-cells for revealing clonality.

Impact of Molecular Staging Methods in Primary Melanoma: Reverse-Transcriptase Polymerase Chain Reaction (RT-PCR) of Ultrasound-Guided Aspirate of the Sentinel Node Does Not Improve Diagnostic Accuracy, But RT-PCR of Peripheral Blood Does Predict Survival

PURPOSEnThis study analyzes (1) the value of tyrosinase reverse-transcriptase polymerase chain reaction (RT-PCR) of aspirates obtained by ultrasound-guided fine-needle aspiration cytology (US-FNAC) of sentinel nodes (SNs) in patients with melanoma before sentinel lymph node biopsy (SLNB) and (2) the value of RT-PCR of blood samples of all SLNB patients.nnnPATIENTS AND METHODSnBetween 2001 and 2003, 127 patients with melanoma (median Breslow depth, 2.1 mm) underwent SLNB. FNAC was performed in all SNs of all patients pre- and post-SLNB. The aspirates were partly shock-frozen for RT-PCR and were partly used for standard cytology. Peripheral blood was collected at the time of SLNB and at every outpatient visit thereafter.nnnRESULTSnThirty-four (23%) of 120 SNs were positive for melanoma. SN involvement was predicted by US-FNAC with a sensitivity of 82% and a specificity of 72%. Additional tyrosinase RT-PCR revealed the same sensitivity of 82% and a specificity of 72%. At a median follow-up time of 40 months from first blood sample, peripheral-blood RT-PCR was a significant independent predictor of disease-free survival (DFS) and overall survival (OS; P < .001).nnnCONCLUSIONnUS-FNAC is highly accurate and eliminates the need for SLNB in 16% of all SLNB patients. RT-PCR of the aspirate or excised SN does not improve sensitivity or specificity. RT-PCR of blood samples predicts DFS and OS.

Biosynthesis of proinsulin in islets of langerhans of the carp (Cyprinus carpio)

Abstract Islets of Langerhans from carp were incubated with [U- 14 C]leucine at 15 °C for 2 h. About 3–4% of the newly synthesized protein was proinsulin. The identity of the newly synthesized protein was proved by: (1) its high specific radioactivity, (2) a molecular weight of 10 000, (3) co-chromatography with porcine insulin after tryptic digestion, (4) cleavage of the tryptic digest into two chains which co-chromatograph with the A- and B-chains of porcine insulin and (5) by the failure of cleavage of the original, non-trypsinated protein into two chains. A conversion of proinsulin into insulin was not observed.

An approach to the sensitivity of temperature-gradient gel electrophoresis in the detection of clonally expanded T-cells in cutaneous T-cell lymphoma.

Detection of T‐cell clonality by polymerase chain reaction (PCR) and high‐resolution electrophoresis facilitates differentiation of early stages of cutaneous T‐cell lymphoma (CTCL) from benign T‐cell‐rich dermatoses. However, data regarding the sensitivity of the various electrophoresis techniques differ remarkably. In the present study, the capacity of heteroduplex (HD)‐loaded temperature‐gradient gel electrophoresis (TGGE) to detect clonally expanded T‐cells was assessed systematically and modifications to the procedure were defined. Using our standard protocol, HD‐TGGE detected clonal T‐cell receptor (TCR)‐gamma PCR products, generated from the Jurkat cell line, down to a total of 2 ng/μL (14 ng) DNA. However, slowly migrating single strands of the clonal PCR product reduced the amount of the clonality indicating homoduplices. To overcome this single‐strand formation, thus decreasing the detection limit, the urea concentration in the gel and the temperature ramp for the HD‐formation were altered, as well as the temperature gradient in the gel. Application of the modified protocol resulted in a tenfold lower detection limit of 0.15 ng/μL (1.05 ng) DNA in the clonal band. The sensitivity of the adapted HD‐TGGE was investigated by dilution experiments using the well established T‐cell lines Jurkat, Molt‐4, MyLa and SeAx. By these approaches clonal PCR products diluted in nonclonal PCR products were detectable down to concentrations of 5—10%. Comparably, in the case of mixtures of clonal in nonclonal DNA the detection limit reached 5—10% clonal DNA. However, by dilution of clonal cells in nonclonal peripheral blood mononuclear cells, which corresponds to in vivo conditions, a lower detection limit of approximately 1—5% was observed.