Intratumor genetic heterogeneity and head and neck cancer relapse.

BACKGROUND: Head and neck squamous cell carcinomas are treated by surgery, radiotherapy (RT), chemoradiotherapy (CRT) or combinations thereof, but locoregional recurrences (LRs) occur in 30-40% of treated patients. We have previously shown that in approximately half of the LRs after CRT, cancer driver mutations are not shared with the index tumor. AIM: To investigate two possible explanations for these genetically unrelated relapses, treatment-induced genetic changes and intratumor genetic heterogeneity. METHODS: To investigate treatment-induced clonal DNA changes, we compared copy number alterations (CNAs) and mutations between primary and recurrent xenografted tumors after treatment with (C)RT. Intratumor genetic heterogeneity was studied by multi-region sequencing on DNA from 31 biopsies of 11 surgically treated tumors. RESULTS: Induction of clonal DNA changes by (C)RT was not observed in the xenograft models. Multi-region sequencing demonstrated variations in CNA profiles between paired biopsies of individual tumors, with copy number heterogeneity scores varying from 0.027 to 0.333. In total, 32 cancer driver mutations could be identified and were shared in all biopsies of each tumor. Remarkably, multi-clonal mutations in these same cancer driver genes were observed in 6 of 11 tumors. Genetically distinct heterogeneous cell cultures could also be established from single tumors, with different biomarker profiles and drug sensitivities. CONCLUSION: Intratumor genetic heterogeneity at the level of the cancer driver mutations might explain the discordant mutational profiles in LRs after CRT, while there are no indications in xenograft models that these changes are induced by CRT.

[The formation of infliximab and anti-infliximab immune complexes as an explanation for non-responding to infliximab treatment of rheumatoid arthritis: observational study in 4 patients]. / Het ontstaan van immuuncomplexen van infliximab en anti-infliximab als een verklaring voor het falen van infliximabtherapie bij reumatoïde artritis: observationeel onderzoek bij 4 patiënten.

OBJECTIVE: To investigate the in vivo mechanism of non-responding to infliximab treatment of patients with rheumatoid arthritis (RA) and the role of anti-infliximab antibodies by using radiolabeled infliximab. DESIGN: Descriptive and comparative study. METHOD: Two responding and two non-responding RA patients were infused with radiolabeled infliximab. Subsequently imaging investigations and serum analysis were performed at set times. RESULTS: The scintigrams showed that the labelled infliximab was mainly present in the blood until 24 h after infusion. There was a trend of faster blood clearance and higher liver and spleen uptake of 99mTc-infliximab in one non-responding patient. Labelled infliximab was taken up by inflamed joints. The anti-infliximab level was high (1008 and 1641 U/ml) in the non-responders and low or not detectable in the responders. Sucrose gradients of serum revealed antibody complexes in both non-responders. Various sizes of antibody complexes, including very large ones, were observed in one non-responder who developed a serious infusion reaction. CONCLUSION: Infliximab-anti-infliximab immune complexes were found to form in RA non-responders due to the presence of significant quantities of anti-infliximab. This finding may partly explain the failure of the infliximab treatment.

Imaging and serum analysis of immune complex formation of radiolabelled infliximab and anti-infliximab in responders and non-responders to therapy for rheumatoid arthritis.

BACKGROUND: Many patients with rheumatoid arthritis are currently successfully treated with infliximab (anti-tumour necrosis factor); however, about 30% of the patients do not respond to infliximab. One of the postulated hypotheses of not responding is the fast clearance of infliximab due to the development of infliximab-anti-infliximab complexes. OBJECTIVE: To investigate the in vivo mechanism of not responding and the role of human anti-chimeric antibodies (HACAs) by using radiolabelled infliximab. METHODS: Two responding and two non-responding patients with rheumatoid arthritis, infused with radiolabelled infliximab, were investigated by both imaging and serum analysis. RESULTS: Images showed predominant presence of infliximab in blood up to 24 h, with a trend of faster blood clearance and of higher liver/spleen uptake in a non-responding patient. Clinically inflamed joints showed uptake of the drug. The HACA level in the non-responders was high (1641 and 1008 U/ml), but low or not detectable in responders. Sucrose gradients of serum showed antibody complexes in both non-responders. Various sizes of antibody complexes, including very large ones, were observed in a non-responder who developed a serious infusion reaction. CONCLUSION: Formation of infliximab-anti-infliximab complexes were found in non-responders due to the presence of large amounts of HACA. This finding, supported by both imaging and serum analysis data, may explain failure of infliximab treatment.

Preparation and evaluation of (89)Zr-Zevalin for monitoring of (90)Y-Zevalin biodistribution with positron emission tomography.

PURPOSE: To evaluate whether (89)Zr can be used as a PET surrogate label for quantification of (90)Y-ibritumomab tiuxetan ((90)Y-Zevalin) biodistribution and dosimetry before myeloablative radioimmunotherapy. METHODS: Zevalin was labelled with (89)Zr by introducing N-succinyldesferal (N-sucDf) as a second chelate. For comparison of the in vitro stability of (89)Zr-Zevalin and (88)Y-Zevalin (as a substitute for (90)Y), samples were incubated in human serum at 37 degrees C up to 6 days. Biodistribution of (89)Zr-Zevalin and (88)Y-Zevalin was assessed at 24, 48, 72 and 144 h p.i. by co-injection in nude mice bearing the non-Hodgkin's lymphoma (NHL) xenograft line Ramos. The clinical performance of (89)Zr-Zevalin-PET was evaluated via a pilot imaging study in a patient with NHL, who had undergone [(18)F]FDG-PET 2 weeks previously. RESULTS: Modification of Zevalin with N-sucDf resulted in an N-sucDf-to-antibody molar ratio of 0.83+/-0.04. After radiolabelling and purification, the radiochemical purity and immunoreactivity of (89)Zr-Zevalin always exceeded 95% and 80%, respectively. (89)Zr-Zevalin showed the same stability in serum as (88)Y-Zevalin, with a radiochemical purity >95% during a period of 6 days. The co-injected (89)Zr-Zevalin and (88)Y-Zevalin conjugates showed a very similar biodistribution, except for liver and bone accumulation at 72 and 144 h p.i., which was significantly higher for (89)Zr than for (88)Y. PET images obtained after injection of (89)Zr-Zevalin showed clear targeting of all known tumour lesions. CONCLUSION: (89)Zr-Zevalin and (88)Y-Zevalin showed a very similar biodistribution in mice, implying that (89)Zr-Zevalin-PET might be well suited for prediction of (90)Y-Zevalin biodistribution in a myeloablative setting.

Characterization of CD44v6 isoforms in head-and-neck squamous-cell carcinoma.

CD44 splice variants, especially those containing the v6 domain, are assumed to play a critical role in the malignant progression of many human tumors. This concept was based on (i) the aberrant expression of CD44v6 in malignant cells, often encoded by alternatively spliced transcripts, and (ii) the absence of CD44v6 expression in corresponding normal tissues. Remarkably, data on CD44v6 expression in squamous cells do not support this hypothesis: the v6 domain is highly expressed in normal squamous tissues and down-regulation has been described in the majority of squamous-cell carcinomas of the head and neck (HNSCC). In this study, we have compared the expression of v6 in normal oral mucosa and HNSCC in a qualitative and quantitative way. Immuno-histochemistry was performed with 3 different anti-v6 antibodies (U36, U39 and VFF18) on a large panel of HNSCC cell lines and tumors. The v6-encoding splice variants were characterized by screening a cDNA library of a human HNSCC cell line and by RT-PCR on HNSCC cell lines, microdissected normal mucosa and primary as well as metastatic HNSCC tissue. The results revealed that there is no, or only marginal, down-regulation of CD44v6 in HNSCC. About 97% of the primary HNSCC tumors exhibited a high and homogeneous staining pattern (U36, 270/277; U39, 268/277 tumors with more than 50% positive cells). Furthermore, the v6-containing CD44 splice variants present in HNSCC primary tumors and metastases were identical to those expressed in normal mucosa. Our data indicate that v6-containing CD44 splice variants do not play a role in the malignant progression of HNSCC.

Perspectives of combined radioimmunotherapy and anti-EGFR antibody therapy for the treatment of residual head and neck cancer.

Rhenium-186 based radioimmunotherapy (RIT) may have potential for the treatment of minimal residual disease in patients with squamous cell carcinoma of head and neck (HNSCC). In an effort to enhance the efficacy of RIT, we evaluated the combination of RIT and anti-epidermal growth factor receptor (EGFR) therapy in nude mice bearing established HNSCC s.c. xenografts. For this purpose we used the EGFR-blocking monoclonal antibody (MAb) 425. Treatment of HNSCC-bearing mice with the combination of a single administration of 200 microCi 186Re-labeled MAb U36 as well as 1.1 mg unlabeled MAb 425 showed an enhanced efficacy in comparison to the single treatments. When 500 microCi 186Re-labeled MAb U36 were administered, all tumors eventually regressed completely. The combination of this RIT treatment with multiple injections of MAb 425 significantly increased the rate of tumor regression. Although RIT with 186Re-labeled MAbs appears to be very efficient on HNSCC xenografts, the combination with anti-EGFR MAb 425 may enhance the efficacy.